The First Outstanding 50 Years of “Università Politecnica delle Marche”: Research Achievements in Life Sciences 9783030338329, 3030338320

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Sauro Longhi · Andrea Monteriù · Alessandro Freddi · Lucia Aquilanti · Maria Gabriella Ceravolo · Oliana Carnevali · Mario Giordano · Gianluca Moroncini   Editors

The First Outstanding 50 Years of “Università Politecnica delle Marche” Research Achievements in Life Sciences

The First Outstanding 50 Years of “Università Politecnica delle Marche”

Sauro Longhi Andrea Monteriù Alessandro Freddi Lucia Aquilanti Maria Gabriella Ceravolo Oliana Carnevali Mario Giordano Gianluca Moroncini •

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The First Outstanding 50 Years of “Università Politecnica delle Marche” Research Achievements in Life Sciences

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Editors Sauro Longhi Department of Information Engineering (DII) Marche Polytechnic University Ancona, Italy

Andrea Monteriù Department of Information Engineering (DII) Marche Polytechnic University Ancona, Italy

Alessandro Freddi Department of Information Engineering (DII) Marche Polytechnic University Ancona, Italy

Lucia Aquilanti Department of Agricultural, Food and Environmental Sciences (D3A) Marche Polytechnic University Ancona, Italy

Maria Gabriella Ceravolo Ospedali Riuniti di Ancona Ancona, Italy

Oliana Carnevali Department of Life and Environmental Sciences (DISVA) Marche Polytechnic University Ancona, Italy

Mario Giordano Department of Life and Environmental Sciences (DISVA) Marche Polytechnic University Ancona, Italy

Gianluca Moroncini Ospedali Riuniti di Ancona Ancona, Italy

ISBN 978-3-030-33831-2 ISBN 978-3-030-33832-9 https://doi.org/10.1007/978-3-030-33832-9

(eBook)

© Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

Università Politecnica delle Marche (UNIVPM in short) celebrates its 50th anniversary as an institution in 2019. A young, dynamic, and active University on multiple fronts, which in the first half-century of its life has been able to achieve important goals in many scientific areas. Hence, the idea of proposing a book celebrating the first 50 years, “UNIVPM50”, and the natural choice to divide the book into three thematic volumes: “Social Sciences and Humanities”, “Physical Sciences and Engineering”, and “Life Sciences”. The contents of these three volumes document the research activities of our University, both fundamental, applied and clinical. “The ability to think together and to produce research, through differentiated thought and culture collaboration, is the main reason for the existence of an academic structure”—from “L’Università di Ancona, 1969/1989”, a book celebrating the first 20 years of our University. The objective of “UNIVPM50” is therefore to present the most important research results achieved so far by our scientific community, with particular attention to the current frontiers of research and what future prospects might be. The contributions collected in the book summarize the main research efforts undertaken by the current faculty members and represent the outcome of a constant effort in the present, supported by past experiences, with the perspective to new emerging ideas in the three scientific areas in which UNIVPM plays a significant role. The book, however, is not to be considered only as a collection of research works: the contributions also represent a sample of the knowledge which is daily taught in our classrooms and laboratories. Indeed, from the research, the University constantly draws inspiration for high-quality teaching, and has the great responsibility of preparing young people for the world of professions. To train professionals, it is necessary that the link between academia and professional world is strong. The relationship with the territories is strengthened with actions to boost research and innovation, the so-called third mission, made up of technology transfer, patents, and entrepreneurship. These activities have led to the development of a contamination lab, followed by an incentive and promotion policy for university spin-offs that draw ideas from the best research results.

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Founded 50 years ago as “University of the territory”, UNIVPM must now confront itself in a perspective as broad and international as possible. Our research was and, still is, inspired by the territories, with the aim of increasingly open to the world. Internationalization is therefore a key to the present and future development of our University: international research, active student/teacher mobility policies, and the start of international degree courses, even as double degrees, are all measures aimed at projecting the University into an international dimension. In conclusion, “UNIVPM50” presents the main goals achieved in the first 50 years of story of our University, and imagine a future of which UNIVPM will, hopefully, be an integral part. Ancona, Italy

Sauro Longhi Rector of the Università Politecnica delle Marche

Preface

Università Politecnica delle Marche (UNIVPM in short) celebrates its 50th anniversary as an institution in 2019. Historically, UNIVPM was first established in Ancona in 1969 as Faculty of Engineering, followed by the Faculty of Medicine the next year. In 1971, the University was granted the status of State University, with the name of “University of Ancona”. The Faculties of Engineering and Medicine were later merged with the Faculty of Economics and Commerce in 1982. Further on, in 1988 and in 1991, respectively, the Faculty of Agriculture and the Faculty of Sciences were established. In 2003, the name of the University was changed to “Università Politecnica delle Marche”. Focusing on the present, the Faculty of Engineering is composed of four Departments: “Construction, Civil Engineering and Architecture”, “Materials, Environmental sciences and Urban planning”, “Information Engineering”, and “Industrial Engineering and Mathematical science”. The Faculty of Medicine and Surgery has four Departments: “Biomedical science and Public Health”, “Experimental and Clinical Medicine”, “Molecular and Clinical Sciences”, and “Odontostomatologic and Specialised Clinical Sciences”. The Faculty of Economics is composed of two Departments: “Management” and “Economics and Social Sciences”. Finally, the Faculties of Agriculture and Sciences count a Department each, “Agriculture, Food and Environmental Sciences” and “Life and Environmental Science”, respectively. UNIVPM placed 5th in the 2018/2019 Censis Italian University Ranking among the medium-sized Public Universities. Being a “young university” this must be considered as a good result. On the other hand, there is still a lot of work to do in order to position UNIVPM at higher levels of research, but thanks to our growing faculty’s expertise remarkable progress is being made each year. This book is dedicated to the celebration of 50 years of UNIVPM and has been motivated by the desire to present the most representative research results of our scientific community achieved so far, with particular attention to the current frontiers of research and to what the future perspectives could be, in order to build a better society providing a dynamic road map for the future. The contributions vii

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collected in the book summarize the main research efforts undertaken by current faculty members, from a vast range of researches. They represent the outcome of a constant effort in the present, supported by past experiences, with the perspective to new emerging ideas in the three major cores where UNIVPM plays a significant role, namely, Life Sciences, Physical Sciences and Engineering and Social Sciences and Humanities. Science is applicable to all living organisms on Earth, as such the Volume “Life Sciences” includes not only fundamental researches about genomes, evolution, and similar, but also researches about medical issues, food, health, the entire mind and body, as well as the environmental issues of Earth, where human beings live. Especially in recent decades, Life Science researches have enabled enormous breakthroughs regarding our understanding of fundamental, molecular, and cellular processes and mechanisms of life. In the present Volume, the Scientists working at the UNIVPM both in basic and applied Life Science branches describe the major results obtained in their research work and the expected future progresses. The chapters dealing with the research work carried out in the “Biomedical science and Public Health”, “Experimental and Clinical Medicine”, “Molecular and Clinical Sciences”, and “Odontostomatologic and Specialised Clinical Sciences” Departments emphasize the understanding of mechanisms that are relevant to all aspects of human diseases, the molecular, cellular, and functional basis of therapy, and its transfer to patients; recent developments of the research, like those in the field of forensic identification, are discussed as well. In more detail, the functioning of living cells and living systems has been in-depth investigated, with respect to the influence of key molecules and ions such as lipids, glutathione, nitric oxide, gamma-aminobutyric-acid, and Ca++. Histological studies were carried out, from ultrastructure to stem cell biology, whereas, as the study of complex systems is concerned, the results of the research on the so-called “gut-skin axis” are also reported, as well as those concerning the “adipose organ” and their secretes. A great research effort has been dedicated in the different fields of medicine, in order to achieve improved comprehension of the pathogenic mechanisms of relevant diseases that have enormous medical and social consequences, such as lung, rectal, liver, head/neck tumors; autoimmune disorders like Celiac disease; hematological disorders; vascular and musculoskeletal diseases; metabolic diseases, including Diabetes I; professional diseases, like asbestosis, as well as less known pathologies, like obstructive sleep apnea syndrome. Significative progress has been achieved in gerontology, anatomic pathology and molecular diagnostic pathology, and in Neuroscience, in particular, with respect to Cognitive Deterioration and Parkinson Disease, whereas, in the same field, a different approach has been devoted in the last years to study the Personality Organizations. Clinical research has been translated in clinical practice, as reported, for example, in obstetrics and gynecology, in the treatment of critically ill patients, as well as in regenerative dentistry, whereas with respect to therapy, of the outmost importance is the research work carried out for many years on the antibiotic-resistance emergence representing today one of the most serious public health problems.

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A great part of the Research at the Department of “Life and Environmental Science” has been, and is, devoted to face the challenges related to the aquatic environment, through the research of efficient countermeasures toward traditional and emerging pollutants, as well as through the study of the complex connections between marine biodiversity and ecosystems functioning, whereas other studies are dealing with alternative ingredients for aquaculture feed and for human foods, through the sustainable exploitation of insects, vegetables, and materials from marine sources. The contribution of the Scientists of this Department to the great debate on new models and adaptation strategies for safeguarding humans’ survival in our fast-changing Earth is also reported, as well as the evidences obtained on the relationship between organismal function and ecosystem in remote times, by analyzing ancient DNA. Thanks to the use of powerful experimental and computational techniques, relevant results have been achieved also in the research concerning the synthesis, structural characterization and activity of different classes of biomolecules, and the study of the molecular basis of antibiotic resistance. As stated in its name, the Department of “Agriculture, Food and Environmental Sciences” has three “souls” living and working together. The research for sustainable crop production is considered as a mandatory goal to face the climate changes and the erosion of genetic biodiversity, while promoting soil fertility, food security, and ecosystem services; particular attention is devoted to the achievement of a sustainable use and a reliable economic management of forests given their fundamental importance as multipurpose, complex, and self-regenerating ecosystems. The study of biodiversity is carried out at the level of genetics, species, communities/ecosystems, and landscapes, through the use of different disciplines/ approaches, such as phytosociology, vegetation mapping, ethnobotany, geomatics, robotics, etc. Last but not least, the research on food is dealing with traditional foods, especially fermented foods, that represent an inestimable heritage, as well as with emerging, novel foods, such as edible insects. Innovative foods are developed/studied with the particular aim of enhancing quality and functionality, intended as the presence of bioactive/nutraceuticals compounds. Ancona, Italy

Sauro Longhi Andrea Monteriù Alessandro Freddi Lucia Aquilanti Maria Gabriella Ceravolo Oliana Carnevali Mario Giordano Gianluca Moroncini

Contents

History and Scientific Production of Clinica Medica and Clinica Ematologica in Ancona . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Armando Gabrielli, Attilio Olivieri, Gianluca Moroncini, Antonella Poloni and Elena Marinelli Busilacchi

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Antimicrobial Resistance: A Challenge for the Future . . . . . . . . . . . . . . Pietro Emanuele Varaldo, Bruna Facinelli, Patrizia Bagnarelli, Stefano Menzo, Marina Mingoia, Andrea Brenciani, Andrea Giacometti, Francesco Barchiesi, Lucia Brescini, Oscar Cirioni, Giorgio Scalise, Pamela Barbadoro, Francesco Di Stanislao, Emilia Prospero and Marcello Mario D’Errico

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Interventional Pulmonology: Past, Present and Future . . . . . . . . . . . . . Stefano Gasparini, Martina Bonifazi and Lina Zuccatosta

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The Experimental Pathology at Ancona: 50 Years of Exciting and Pioneering Research on Human Pathology . . . . . . . . . . . . . . . . . . . Fabiola Olivieri, Maria Rita Rippo, Laura Graciotti, Armanda Pugnaloni, Francesca Fazioli and Antonio Domenico Procopio Histological Contamination in Clinical Research—from Ultrastructure to Stem Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monica Mattioli Belmonte, Monia Orciani, Antonio Gigante, Guendalina Lucarini, Giancarlo Balercia, Giorgio Arnaldi and Roberto Di Primio Half-Century Research and Teaching Activity in Anatomic Pathology Throughout the History of Technologic Innovation . . . . . . . . . . . . . . . . Gaia Goteri and Alessia Cimadamore Past, Present and Future in Forensic Human Identification . . . . . . . . . . Federica Alessandrini, Valerio Onofri, Chiara Turchi, Loredana Buscemi, Mauro Pesaresi and Adriano Tagliabracci

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From Clinical Research to Clinical Practice in Obstetrics and Gynecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stefano Raffaele Giannubilo, Giovanni Delli Carpini and Andrea Ciavattini

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Thirty Years of Parkinson’s Disease Management: From a Symptom-Based Approach to a Holistic Perspective . . . . . . . . . 107 Marianna Capecci and Maria Gabriella Ceravolo Cognitive Deterioration: Looking for a Better Clinical Characterization and a Definition of the Determining Factors . . . . . . . . 123 Mauro Silvestrini and Leandro Provinciali A 25 Years-Long Journey with GABA Transporters . . . . . . . . . . . . . . . 137 Fiorenzo Conti A 25-Year Long Journey into the World of NO . . . . . . . . . . . . . . . . . . . 155 Laura Mazzanti, Arianna Vignini and Monica Emanuelli The Adipose Organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Saverio Cinti and Antonio Giordano Vascular, Metabolic and Musculoskeletal Diseases: From Experimental to Clinical Research . . . . . . . . . . . . . . . . . . . . . . . . 185 Riccardo Sarzani, Rossella De Angelis, Marica Bordicchia, Marco Di Carlo, Emilio Filippucci, Federico Giulietti, Francesco Spannella and Walter Grassi Celiac Disease: A Journey Through Time and Space . . . . . . . . . . . . . . . 203 Carlo Catassi and Elena Lionetti Research Strands in Dermatology and Gastroenterology Units of Department of Clinical and Molecular Sciences in Polytechnic Marche University . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Antonio Benedetti, Anna Campanati, Emanuela Martina, Oriana Simonetti, Emanuele Bendia, Antonio Di Sario, Luca Maroni, Marco Marzioni, Irene Pierantonelli, Paola Sassaroli, Laura Schiadà, Gianluca Svegliati-Baroni, Giuseppe Tarantino and Annamaria Offidani Obstructive Sleep Apnoea Syndrome and Arrhythmia: Results of 10 Years’ Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Laura Cipolletta, Alessia Urbinati, Maria Vittoria Matassini, Marchesani Francesca, Stefano Gasparini and Alessandro Capucci Advances in Hepatobiliary Surgery: The Ancona’s Experience with ALPPS Procedure for Extended Liver Resections . . . . . . . . . . . . . 265 Federico Mocchegiani and Marco Vivarelli

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Evolution and Future Perspectives of Rectal Cancer Treatment with Minimally Invasive Surgical Techniques . . . . . . . . . . . . . . . . . . . . 285 Mario Guerrieri and Roberto Ghiselli The Formidable Metamorphosis of the Salamander’s Wool: Asbestos from Eternal Material to Awful Pathologies . . . . . . . . . . . . . . 303 Lory Santarelli, Marco Tomasetti, Massimo Bracci, Monica Amati, Matteo Valentino, Ernesta Pieragostini, Silvia Rinaldi and Rossana Berardi A New Era in Restorative Dentistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Giovanna Orsini, Vincenzo Tosco, Riccardo Monterubbianesi, Giulia Orilisi and Angelo Putignano Biomarkers in Head and Neck Oncology: From Early Diagnosis to Tailored Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Andrea Santarelli, Marco Mascitti, Monica Emanuelli and Maurizio Procaccini The Adaptive Evolutionary Post-Rationalist Approach: Epistemology and Biological Pattern of the Personality Organizations (POs) . . . . . . . . 349 Bernardo Nardi and Umberto Volpe Intracellular Calcium and Ischemic Damage: Dual Role of the Na+/Ca2+ Exchanger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Simona Magi, Pasqualina Castaldo, Vincenzo Lariccia, Silvia Piccirillo, Marta Maiolino and Salvatore Amoroso Evaluation of the Microcirculation in Critically Ill Patients . . . . . . . . . . 373 Elisa Damiani, Roberta Domizi, Claudia Scorcella, Andrea Carsetti and Abele Donati Methods in Childhood Health: Chronic Disease Epidemiology and Age Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Rosaria Gesuita, Luigi Ferrante, Edlira Skrami and Flavia Carle Multiple Roles of Membrane Lipids: Implications for Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Gianna Ferretti, Tiziana Bacchetti and Rosamaria Fiorini Glutathione, an Over One Billion Years Ancient Molecule, Is Still Actively Involved in Cell Regulatory Pathways . . . . . . . . . . . . . . 417 Tatiana Armeni and Giovanni Principato New Insights for Early Warning and Countermeasures to Aquatic Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 Oliana Carnevali, Maura Benedetti, Francesca Beolchini, Antonio Dell’Anno, Daniele Fattorini, Stefania Gorbi, Silvia Illuminati, Francesca Maradonna, Giuseppe Scarponi and Francesco Regoli

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Marine Biology. Biodiversity and Functioning of Marine Ecosystems: Scientific Advancements and New Perspectives for Preserving Marine Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Cecilia Maria Totti, Stefano Accoroni, Marco Barucca, Silvia Bianchelli, Maria Assunta Biscotti, Barbara Calcinai, Adriana Canapa, Cinzia Corinaldesi, Roberto Danovaro, Cristina Gioia Di Camillo, Emanuela Fanelli, Cristina Gambi, Stefania Puce, Tiziana Romagnoli and Carlo Cerrano Synthesis, Structural Insights and Activity of Different Classes of Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 Elisabetta Giorgini, Francesca Biavasco, Roberta Galeazzi, Giorgia Gioacchini, Eleonora Giovanetti, Giovanna Mobbili, Mario Orena, Maria Grazia Ortore, Samuele Rinaldi, Andrea Antonino Scirè, Francesco Spinozzi, Fabio Tanfani, Carla Vignaroli and Paolo Mariani Conservation and Management of Biodiversity and Landscapes: A Challenge in the Era of Global Change . . . . . . . . . . . . . . . . . . . . . . . 483 Simona Casavecchia, Marina Allegrezza, Edoardo Biondi, Andrea Galli, Ernesto Marcheggiani, Simone Pesaresi, Fabio Taffetani, Stefano Tavoletti, Silvia Zitti, Maurizio Bianchelli, Nello Biscotti, Jacopo Facchi, Diana Galdenzi, Marco Galié, Roberta Gasparri, Linda Iommarini, Andrea Lancioni, Lara Lucchetti, Giacomo Mei, Ambra Micheletti, Silvia Montecchiari, Massimiliano Morbidoni, Cecilia Ottaviani, Morena Pinzi, Michele Rismondo, Giulio Tesei and Liliana Zivkovic The Unobservability of the Temporal Scale in Biological Studies . . . . . . 505 Vincenzo Caputo Barucchi, Anna La Teana, Anna Sabbatini and Mario Giordano Alternative Ingredients for Feed and Food . . . . . . . . . . . . . . . . . . . . . . . 529 Tiziana Bacchetti, Anna Annibaldi, Francesca Comitini, Maurizio Ciani, Elisabetta Damiani, Alessandra Norici, Luca Tiano, Cristina Truzzi and Ike Olivotto Food Quality and Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Deborah Pacetti, Bruno Mezzetti, Francesca Balducci, Michele Balzano, Patricia Carloni, Sara Castiglioni, Michele Cianci, Pasquale Massimiliano Falcone, Natale Giuseppe Frega, Alessandra Giardinieri, Luca Mazzoni, Gabriele Minazzato, Nadia Raffaelli, Silverio Ruggieri and Federica Zamporlini

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Valorization of Foods: From Tradition to Innovation . . . . . . . . . . . . . . 565 Lucia Aquilanti, Andrea Osimani, Federica Cardinali, Francesca Clementi, Roberta Foligni, Cristiana Garofalo, Nino Loreto, Serena Mandolesi, Vesna Milanović, Massimo Mozzon, Simona Naspetti, Marina Pasquini, Andrea Roncolini, Sara Ruschioni, Riccardo Sabbatini, Francesco Solfanelli, Maria Federica Trombetta, Daniela Vairo and Raffaele Zanoli Sustainable Crop Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 Davide Neri, Oriana Silvestroni, Nora Baldoni, Matteo Belletti, Elisa Bellucci, Elena Bitocchi, Franco Capocasa, Paride D’Ottavio, Matteo Francioni, Danilo Gambelli, Vania Lanari, Tania Lattanzi, Francesca Massetani, Laura Nanni, Roberto Papa, Serena Polverigiani, Silvia Sabbadini, Marco Toderi and Laura Trozzo Innovation in Sustainable Management of Plant Diseases and Pests, and Effects on the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601 Gianfranco Romanazzi, Paola Riolo, Daniele Duca, Roberto Orsini, Elga Monaci, Sergio Murolo, Roxana Luisa Minuz, Chiara Mengarelli, Martina Perugini, Elisa Verdolini, Alessio Ilari, Valeria Mancini, Erica Feliziani, Lucia Landi, Ester Foppa Pedretti, Rodolfo Santilocchi, Costantino Vischetti and Nunzio Isidoro Forests and Soils: Sustainable Products and Ecosystem Services for Human Well-Being . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617 Giuseppe Corti, Carlo Urbinati, Stefania Cocco, Cristiano Casucci, Giuseppe Toscano, Adele Finco, Deborah Bentivoglio, Giorgia Bucci, Valeria Cardelli, Arianna De Bernardi, Marziyeh Hoseini, Francesco Malandra, Manuela Mancini, Giorgio Rossini, Dominique Serrani, Enrico Tonelli and Alessandro Vitali Homo Sapiens, Anthropocene and Disaster Risk Reduction . . . . . . . . . . 631 Fausto Marincioni and Alessandra Negri

History and Scientific Production of Clinica Medica and Clinica Ematologica in Ancona Armando Gabrielli, Attilio Olivieri, Gianluca Moroncini, Antonella Poloni and Elena Marinelli Busilacchi

Abstract Clinica Medica and Ematologica in Ancona stemmed some 30 years ago from one originator medical team dedicated to Internal Medicine practice with a special interest towards immunological and hematological studies. This original scientific interest has spread over a multitude of fellows, some of whom have reached outstanding academic and clinical results, maintaining and extending the excellent reputation of the founding group over the years and until the present time. The best scientific achievements have been obtained in the field of systemic sclerosis and chronic graft versus host disease, whereas the clinical activity has been successfully dedicated to the management of autoimmune diseases and hematological disorders, with particular attention to innovative immunomodulatory therapies and stem cell transplantation.

1 Clinica Medica The Division of Internal Medicine (Clinica Medica) of Università Politecnica delle Marche is part of Dipartimento di Scienze Cliniche e Molecolari and it has grown under the leadership of Prof. Giovanni Danieli who moved from the University of A. Gabrielli · G. Moroncini (B) Clinica Medica, Department of Clinical and Molecular Sciences, Università Politecnica Delle Marche, Via Tronto 10, 60126 Ancona, Italy e-mail: [email protected] A. Gabrielli e-mail: [email protected] A. Olivieri · A. Poloni · E. M. Busilacchi Clinica Di Ematologia, Department of Clinical and Molecular Sciences, Università Politecnica Delle Marche, Via Tronto 10, 60126 Ancona, Italy e-mail: [email protected] A. Poloni e-mail: [email protected] E. M. Busilacchi e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_1

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Bologna to join the newly established Faculty of Medicine of Università di Ancona (subsequently renamed Università Politecnica delle Marche) in 1974, with his young Collaborators Maria Montroni, Pietro Leoni, Angelo Corvetta and the present head of the Division Armando Gabrielli. Over the years the Division with the recruitment of several other eminent physicians and researchers advanced health through excellence in each of its core domains: education, patient care and discovery, through inspiring engaged members, encouraging innovative thinking, and building collaborative partnerships. In this respect, it is worthy remembering the contribution of the present, permanent staff which includes Maria Giovanna Danieli, Antonella Festa, Paolo Fraticelli, Michele Luchetti, Lucia Manfredi, Gianluca Moroncini, Giovanni Pomponio. The Division has also been the training ground for most of the Marche region’s physicians, nurses, therapists, and other health care professionals, and has become a Centre of excellence in the Country for its commitment to providing comprehensive education and training to students, residents and fellows and for its dedication to research, and patient care. The Schools of Specialization in Internal Medicine, in Emergency Medicine and Allergology and Clinical Immunology are based in the Division. The founding group gave then rise to the Division of Hematology with Pietro Leoni as chairman, and the Laboratory of Diagnostic Immunology with Maria Montroni as Head, who achieved great consideration and were highly renowned in the Italian Academic world. Research has been one of the core functions of the Division, spans a wide variety of topics and involves basic research, clinical research, clinical trials and studies of animal model systems in collaboration with other clinical and basic science departments, affiliated hospitals and organizations. Research, however, focused on three main areas: autoimmunity, both clinical and translational, rheumatic diseases, hematology, and immunodeficiencies. Key findings were made in the field of systemic sclerosis (scleroderma) that set the foundation for a large international effort, with the demonstration and characterization of stimulatory autoantibodies targeting PDGF receptor in scleroderma patients and patients with chronic Graft versus Host Disease (GVHD) and scleroderma-like features. These discoveries, for which the dedication and expertise of Silvia Svegliati and Gianluca Moroncini together with Tatiana Spadoni, Cecilia Tonnini, Antonella Grieco, and Chiara Paolini were instrumental, are covered by multiple international patents. These findings were the subject of several publications [1–11]. In addition, some of the molecular mechanisms involved in the pathogenesis of fibrosis with particular attention to Ras, reactive oxygen species and Wnt signalling were elucidated and published [7, 8, 12–17]. The information described above were used to design clinical trials in Systemic sclerosis and related disorders [18–22].

History and Scientific Production of Clinica Medica …

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However, scleroderma was not the only disease investigated. Recently, particular attention has been focused on inflammatory myositis and spondyloarthritis. In summary, the Division of Internal Medicine strives to provide outstanding care to patients and to develop new knowledge through innovative basic biomedical discovery and rigorous clinical research, in order to translate these discoveries into novel therapies.

1.1 Clinica di Ematologia The Division of Hematology (Clinica di Ematologia) of Università Politecnica delle Marche belongs to the Dipartimento di Scienze Cliniche e Molecolari and, like the Division of Clinica Medica, stems from the leadership of our mentors, Prof. Giovanni Danieli, and by Prof. Pietro Leoni, who was the founder of the Hematology school at Ancona University, with the fundamental support of Attilio Olivieri, the current Head of the Hematology Division, who has set up the Stem Cell Transplant (SCT) Unit in our center. During the last decades the Clinica di Ematologia expanded both the clinical skills and the scientific achievements, reaching a role of national leadership in several fields, first of all the transplant activity and the cellular biology. Today the Division of Hematology represents one of the most important Stem Cell Transplant Centers in our Country, with around 1500 transplant procedures performed in adults and it is currently certified for the international transplant activity, according to the JACIE organization. Prof. Attilio Olivieri has been the national coordinator of Autologous Stem Cell Transplant activity for 6 years and, since 2015 he is the chairman of the Graft Versus Host Disease (the main complication of allogeneic SCT) for the Italian Society for bone marrow transplantation (GITMO). Prof. Attilio Olivieri has developed several lines of research, not only in the transplant setting, but also in other fields, mainly stem cell biology, pathogenesis and therapy of myelodysplastic syndromes, treatment of multiple myeloma and cutaneous lymphoma, thanks to the active collaboration of the main coworkers who are currently embedded in the Clinica di Ematologia: Prof. Antonella Poloni [23–27], Dr. Massimo Offidani [28–31] and Dr. Serena Rupoli [32, 33]. The most relevant scientific line of research and development of Clinica di Ematologia is today represented by the SCT: Prof. Attilio Olivieri has been one of the Italian pioneers of stem cell mobilization [34–43] and he has contributed tremendously to developing the autologous transplantation activity, including the biology of stem cell mobilization and opening new modalities of autologous transplantation such as the outpatient SCT program and the use of cytoprotective agents [43–64] as well the new modalities of allogeneic SCT setting [65–71]. During the first years of activity he also contributed, together with Antonella Poloni, to develop original approaches in the setting of Stem cell manipulation and ex vivo purging [72–74]. Since 1992, besides the HSC and bone marrow microenvironment, Antonella Poloni deeply developed the knowledge on mesenchymal stromal cells (MSC), their

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differentiation potential, the role in regenerative medicine and the stromal support to hematological malignancies, MSC from chorionic villi and amniotic fluid [75–78]. Recently the experimental research has been focused on bone marrow adipocytes properties and characterization and relationships with tissue adipocytes [79–87]. In the last 15 years we concentrated our efforts in developing the knowledge in the graft versus host disease (GVHD) pathophysiology and treatment, particularly in the setting of chronic GVHD (cGVHD), in collaboration with the Lab Team of Clinica Medica, leaded by Prof. Armando Gabrielli. cGVHD is the leading cause of late non-relapse mortality in Hemopoietic Stem Cell Transplant (HSCT) survivors. Up to now a standard satisfactory treatment for these patients does not exist. cGVHD is an immune-mediated disease, resulting from a complex interaction between donor and recipient adaptive immunity, but its exact pathogenesis is still incompletely defined [88, 89]. Recent animal studies suggest that B cells might play an important role in the biology of cGVHD. Further circumstantial evidence for the involvement of B cells in cGVHD pathogenesis comes from reports of successful treatment of cGVHD with B-cell depletion [90]. Several lines of evidence clearly demonstrate that in addition to donor CD4+ T cells, donor B cells are required for the induction of cGVHD and many data confirm that cGVHD is associated with a perturbed B-cell homeostasis. Because a considerable number of circulating memory B cells express autoreactive antibodies, the deregulated inflammation during GVHD might lead to activation of these autoreactive B cells and subsequent autoantibody production. The development of these antibodies requires the presence of alloreactive CD4 T cells [91] and the appearance and titer of autoantibodies have been correlated with GVHD onset and activity [92]. Many cGVHD patients show skin and visceral involvement with fibrotic scleroderma-like features; our recent experimental data [11], showed that patients with cGVHD with fibrotic/sclerotic manifestations, have agonistic antibodies activating the platelet-derived growth factor receptor (PDGF-R), like patients with Systemic Sclerosis, suggesting a similar pathogenesis for these two diseases; indeed the up-regulation of the PDGF-R intracellular pathway leads to increased reactive oxygen species (ROS), with consequent increased collagen synthesis, which, in turn, contributes to the pathologic lesions observed in both cGVHD and SS [1]. Many data strongly suggest that, among the profibrotic cytokines, besides PDGF, also transforming growth factor β (TGF-β) can play a relevant role in the pathogenesis of fibrotic damage. Dual blockade of PDGF or TGF-β signaling has been shown to reduce the development of fibrosis in various experimental models [93, 94]. Recent in vitro data showed that the Tyrosine Kinase Inhibitor (TKI), Imatinib strongly inhibits both PDGF and TGF-β intracellular signaling [95]. Taking a cue from this background, Prof. Attilio Olivieri launched a phase 1– 2 prospective national study with a Tyrosine kinase, PDGF-R inhibitor (Imatinib) in patients with scleroderma-like cGVHD, achieving encouraging results [18]. The promising response rate observed in this preliminary cohort of patients with multirefractory disease, pushed the Italian Drug regulatory Agency (AIFA) to approve this drug for this new indication. Basing on this background Attilio Olivieri designed a second larger prospective translational study, in order to validate the NIH response

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criteria for cGVHD [96, 97] in a cohort of patients with multirefractory cGVHD. This project has been granted by AIFA and the study results have prospectively validated for the first time the NIH response criteria in cGVHD, confirming the role of anti-PDGF-R antibodies in this disease [98]. Following the publication of these results Attilio Olivieri has been embedded with other 4 European scientists in the US NIH board for the preparation of the new international consensus document for the response criteria in cGVHD. This document has been presented in Washington on June 2014 and published on Biology of Bone Marrow Transplantation in 2015 and represents today the benchmark for the new interventional studies in cGVHD [99]. Refractory cGVHD (SR-cGVHD) represents an unmet clinical need in the field of allogeneic transplantation, lacking effective treatments; however, research has been very active in this field, with more than 50 different interventions tested in more than 150 studies in the last years. Methodological issues have been long suspected as a cause for stagnant research in SR-cGVHD. Starting from this point we have performed a meta-analysis of the interventional studies in SRcGVHD setting. We have selected the 82 most representative studies, showing a pooled 66% response rate. These apparently satisfactory results have been evaluated for the methodological quality, according to 18 critical items extracted from a checklist from the NIH recommendations for response evaluation, that have been extensively applied in the 82 studies evaluated. We made a meta-regression in order to correlate the major methodological scores to the response rate in the 82 studies, showing a significant inverse correlation between the adherence to NIH recommendations and the response rate, suggesting that an improvement of the adherence to these recommendations, significantly lowered the response estimation. In conclusion we have demonstrated an unrealistically high response rate in most studies, suggesting a clear overestimation of the treatment efficacy, while a more stringent adherence to the NIH recommendations contributed to minimize this important bias improving over time the response evaluation [20]. During these last years Attilio Olivieri covered several aspects in the setting of the GVHD [90, 100–105], including the participation, as invited speaker and/or chairman, in several International Meetings: he recently gave an educational lecture on SR-cGVHD at the EBMT annual Meeting and was the coordinator of cGVHD sessions at the two recent ASH Meetings. He served has reviewer for several prestigious Journals such as: New England Journal of Medicine, Leukemia, British Journal of Hematology, Bone Marrow Transplantation, Biology of Bone Marrow Transplantation. He received a Grant of 292.000 Euros from AIFA for developing Imatinib treatment in cGVHD, and thanks to the results published this drug has now been approved in Italy for SR-cGVHD. He is one of the members founder of the GVHD-hub, a European international Academic organization for spreading the knowledge in this disease and he is the leader of the European working group for cGVHD response criteria in a COST European project.

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Attilio Olivieri has recently developed in collaboration with a software house (Bimind, Jesi, Italy) an original algorithm for evaluating automatically the response by different investigators, that is currently employed in the most important Italian centers for allogeneic transplantation and will be soon available in the Web for all the European countries.

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18. Olivieri A, Locatelli F, Zecca M et al (2009) Imatinib for refractory chronic graft-versus-host disease with fibrotic features. Blood 114:709–718 19. Fraticelli P, De Vita S, Franzolini N et al (2015) Reduced type I collagen gene expression by skin fibroblasts of patients with systemic sclerosis after one treatment course with rituximab. Clin Exp Rheumatol 33:S160–S167 20. Olivieri J, Manfredi L, Postacchini L et al (2015) Consensus recommendations for improvement of unmet clinical needs–the example of chronic graft-versus-host disease: a systematic review and meta-analysis. Lancet Haematol 2:e297–e305 21. Fraticelli P, Gabrielli B, Pomponio G et al (2014) Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study. Arthritis Res Ther 16:R144 22. Iudici M, Moroncini G, Cipriani P et al (2015) Where are we going in the management of interstitial lung disease in patients with systemic sclerosis? Autoimmun Rev 14:575–578 23. Maurizi G, Mattiucci D, Mariani M et al (2017) DNA demethylating therapy reverts mesenchymal stromal cells derived from high risk myelodysplastic patients to a normal phenotype. Br J Haematol 177:818–822 24. Poloni A, Maurizi G, Mattiucci D et al (2014) Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes. Leukemia 28:2241–2244 25. Poloni A, Serrani F, Berardinelli E et al (2013) Telomere length, c-myc and mad-1 expression could represent prognosis markers of myelodysplastic syndrome. Leuk Res 37:1538–1544 26. Poloni A, Goteri G, Zizzi A et al (2013) Prognostic role of immunohistochemical analysis of 5 mc in myelodysplastic syndromes. Eur J Haematol 91:219–227 27. Mattiucci D, Maurizi G, Leoni P et al (2018) Agingand senescence-associated changes of mesenchymal stromal cells in myelodysplastic syndromes. Cell Transplant 27:754–764 28. Offidani M, Bringhen S, Corvatta L et al (2007) Thalidomide-dexamethasone plus pegylated liposomal doxorubicin vs. thalidomide-dexamethasone: a case-matched study in advanced multiple myeloma. Eur J Haematol 78:297–302 29. Offidani M, Corvatta L, Maracci L et al (2013) Efficacy and tolerability of bendamustine, bortezomib and dexamethasone in patients with relapsed-refractory multiple myeloma: a phase II study. Blood Cancer J 3:e162 30. Nabissi M, Morelli MB, Offidani M et al (2016) Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration. Oncotarget 7:77543–77557 31. Morelli MB, Offidani M, Alesiani F et al (2014) The effects of cannabidiol and its synergism with bortezomib in multiple myeloma cell lines. A role for transient receptor potential vanilloid type-2. Int J Cancer 134:2534–2546 32. Rupoli S, Goteri G, Pulini S et al (2005) Long-term experience with low-dose interferon-alpha and PUVA in the management of early mycosis fungoides. Eur J Haematol 75:136–145 33. Rupoli S, Canafoglia L, Goteri G et al (2016) Results of a prospective phase II trial with oral low-dose bexarotene plus photochemotherapy (PUVA) in refractory and/or relapsed patients with mycosis fungoides. Eur J Dermatol 26:13–20 34. Olivieri J, Attolico I, Nuccorini R et al (2018) Predicting failure of hematopoietic stem cell mobilization before it starts: the predicted poor mobilizer (pPM) score. Bone Marrow Transplant 53:461–473 35. Olivieri A, Saraceni F (2016) Mobilization policy in multiple myeloma: minimum target or law of redundancy? Two different approaches by the two sides of the Atlantic Ocean. Bone Marrow Transplant 51:348–350 36. Saraceni F, Shem-Tov N, Olivieri A et al (2015) Mobilized peripheral blood grafts include more than hematopoietic stem cells: the immunological perspective. Bone Marrow Transplant 50:886–891 37. Pierelli L, Berto P, Accorsi P et al (2013) The costs of mobilisation and collection of peripheral blood stem cells in multiple myeloma and lymphoma in an European country: results from The Gruppo Italiano Trapianto Midollo Osseo (GITMO) and Societa Italiana di Emaferesi e Manipolazione Cellulare (SIdEM) survey. Transfus Apher Sci 49:615–622

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38. Lanza F, Lemoli RM, Olivieri A et al (2014) Factors affecting successful mobilization with plerixafor: an Italian prospective survey in 215 patients with multiple myeloma and lymphoma. Transfusion 54:331–339 39. Pierelli L, Perseghin P, Marchetti M et al (2012) Best practice for peripheral blood progenitor cell mobilization and collection in adults and children: results of a Societa Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO) consensus process. Transfusion 52:893–905 40. Attolico I, Pavone V, Ostuni A et al (2012) Plerixafor added to chemotherapy plus G-CSF is safe and allows adequate PBSC collection in predicted poor mobilizer patients with multiple myeloma or lymphoma. Biol Blood Marrow Transplant 18:241–249 41. Olivieri A, Marchetti M, Lemoli R et al (2012) Proposed definition of ‘poor mobilizer’ in lymphoma and multiple myeloma: an analytic hierarchy process by ad hoc working group Gruppo ItalianoTrapianto di Midollo Osseo. Bone Marrow Transplant 47:342–351 42. Olivieri A, Offidani M, Cantori I et al (1995) Addition of erythropoietin to granulocyte colonystimulating factor after priming chemotherapy enhances hemopoietic progenitor mobilization. Bone Marrow Transplant 16:765–770 43. Olivieri A, Offidani M, Ciniero L et al (1995) DHAP regimen plus G-CSF as salvage therapy and priming for blood progenitor cell collection in patients with poor prognosis lymphoma. Bone Marrow Transplant 16:85–93 44. Saraceni F, Bruno B, Lemoli RM et al (2017) Autologous stem cell transplantation is still a valid option in good- and intermediate-risk AML: a GITMO survey on 809 patients autografted in first complete remission. Bone Marrow Transplant 52:163–166 45. Martino M, Lemoli RM, Girmenia C et al (2016) Italian consensus conference for the outpatient autologous stem cell transplantation management in multiple myeloma. Bone Marrow Transplant 51:1032–1040 46. Capelli D, Chiarucci M, Poloni A et al (2014) Mobilization-driven postconsolidation therapy in elderly patients with acute myeloid leukemia: feasibility and efficacy of autologous stem cell transplantation versus low-dose gemtuzumab ozogamicin. Biol Blood Marrow Transplant 20:1399–1406 47. Martino M, Montanari M, Ferrara F et al (2014) Very low rate of readmission after an early discharge outpatient model for autografting in multiple myeloma patients: an Italian multicenter retrospective study. Biol Blood Marrow Transplant 20:1026–1032 48. Perseghin P, Marchetti M, Pierelli L et al (2014) A policy for the disposal of autologous hematopoietic progenitor cells: report from an Italian consensus panel. Transfusion 54:2353– 2360 49. Martino M, Olivieri A, Offidani M et al (2013) Addressing the questions of tomorrow: melphalan and new combinations as conditioning regimens before autologous hematopoietic progenitor cell transplantation in multiple myeloma. Expert Opin Investig Drugs 22:619–634 50. Martino M, Montanari M, Bruno B et al (2012) Autologous hematopoietic progenitor cell transplantation for multiple myeloma through an outpatient program. Expert Opin Biol Ther 12:1449–1462 51. Lemoli RM, D’addio A, Marotta G et al (2010) BU/melphalan and auto-SCT in AML patients in first CR: a ‘Gruppo Italiano Trapianto di Midollo Osseo (GITMO)’ retrospective study. Bone Marrow Transplant 45:640–646 52. Olivieri A, Capelli D, Troiani E et al (2007) A new intensive induction schedule, including high-dose Idarubicin, high-dose Aracytin and Amifostine, in older AML patients: feasibility and long-term results in 42 patients. Exp Hematol 35:1074–1082 53. Olivieri A, Santini G, Patti C et al (2005) Upfront high-dose sequential therapy (HDS) versus VACOP-B with or without HDS in aggressive non-Hodgkin’s lymphoma: long-term results by the NHLCSG. Ann Oncol 16:1941–1948 54. Olivieri A, Lucesole M, Capelli D et al (2005) A new schedule of CHOP/rituximab plus granulocyte-macrophage colony-stimulating factor is an effective rescue for patients with aggressive lymphoma failing autologous stem cell transplantation. Biol Blood Marrow Transplant 11:627–636

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55. Olivieri A, Scortechini I, Capelli D et al (2004) Combined administration of alphaerythropoietin and filgrastim can improve the outcome and cost balance of autologous stem cell transplantation in patients with lymphoproliferative disorders. Bone Marrow Transplant 34:693–702 56. Olivieri A, Brunori M, Capelli D et al (2004) Salvage therapy with an outpatient DHAP schedule followed by PBSC transplantation in 79 lymphoma patients: an intention to mobilize and transplant analysis. Eur J Haematol 72:10–17 57. Montanari M, Capelli D, Poloni A et al (2003) Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation: a comparison between controlledrate freezing and uncontrolled-rate freezing at 80 degrees C. Transfusion 43:42–49 58. Olivieri A, Capelli D, Montanari M et al (2001) Very low toxicity and good quality of life in 48 elderly patients autotransplanted for hematological malignancies: a single center experience. Bone Marrow Transplant 27:1189–1195 59. Capelli D, Santini G, De Souza C et al (2000) Amifostine can reduce mucosal damage after high-dose melphalan conditioning for peripheral blood progenitor cellautotransplant: a retrospective study. Br J Haematol 110:300–307 60. Bordignon C, Carlo-Stella C, Colombo MP et al (1999) Cell therapy: achievements and perspectives. Haematologica 84:1110–1149 61. Lemoli RM, Martinelli G, Olivieri A et al (1999) Selection and transplantation of autologous CD34+ B-lineage negative cells in advanced-phase multiple myeloma patients: a pilot study. Br J Haematol 107:419–428 62. Offidani M, Corvatta L, Olivieri A et al (1999) Infectious complications after autologous peripheral blood progenitor cell transplantation followed by G-CSF. Bone Marrow Transplant 24:1079–1087 63. Aglietta M, Bertolini F, Carlo-Stella C et al (1998) Ex vivo expansion of hematopoietic cells and their clinical use. Haematologica 83:824–848 64. Olivieri A, Offidani M, Montanari M et al (1998) Factors affecting hemopoietic recovery after high-dose therapy and autologous peripheral blood progenitor cell transplantation: a single center experience. Haematologica 83:329–337 65. Olivieri A, Offidani M, Ciniero L et al (1994) Optimization of the yield of PBSC for autotransplantation mobilized by high-dose chemotherapy and G-CSF: proposal for a mathematical model. Bone Marrow Transplant 14:273–278 66. Corradini P, Tarella C, Olivieri A et al (2002) Reduced-intensity conditioning followed by allografting of hematopoietic cells can produce clinical and molecular remissions in patients with poor-risk hematologic malignancies. Blood 99:75–82 67. Chiusolo P, Bug G, Olivieri A et al (2018) A modified post-transplant cyclophosphamide regimen, for unmanipulated haploidentical marrow transplantation, in acute myeloid leukemia: a multicenter study. Biol Blood Marrow Transplant 24:1243–1249 68. Eder S, Labopin M, Finke J et al (2017) Safety and efficacy of thiotepa-based conditioning for allogeneic transplantation in AML: a survey from the ALWP of the EBMT. Bone Marrow Transplant 52:238–244 69. Olivieri J, Mancini G, Goteri G et al (2016) Reversal of poor graft function with iron-chelating therapy after allogeneic transplantation for severe aplastic anemia. Leuk Lymphoma 57:965– 968 70. Corradini P, Vitolo U, Rambaldi A et al (2014) Intensified chemo-immunotherapy with or without stem cell transplantation in newly diagnosed patients with peripheral T-cell lymphoma. Leukemia 28:1885–1891 71. Mariotti J, Maura F, Spina F et al (2014) Impact of cytomegalovirus replication and cytomegalovirus serostatus on the outcome of patients with B cell lymphoma after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 20:885–890 72. Corradini P, Zallio F, Mariotti J et al (2005) Effect of age and previous autologous transplantation on nonrelapse mortality and survival in patients treated with reducedintensity conditioning and allografting for advanced hematologic malignancies. J Clin Oncol 23:6690–6699

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73. Poloni A, Leoni P, Buscemi L et al (2006) Engraftment capacity of mesenchymal cells following hematopoietic stem cell transplantation in patients receiving reduced-intensity conditioning regimen. Leukemia 20:329–335 74. Poloni A, Leoni P, Curzi L et al (1999) Ex vivo pharmacological purging of leukapheresis collections with nitrogen mustard: amifostine pretreatment improves both early and late peripheral blood progenitor cell recovery. Exp Hematol 27:1548–1556 75. Olivieri A, Poloni A, Montanari M et al (1997) Pharmacologic bone marrow purging: is there any place for etoposide? In vitro comparison with mafosfamide. J Hematother 6:137–144 76. Poloni A, Maurizi G, Serrani F et al (2012) Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate. Cell Prolif 45:66–75 77. Poloni A, Maurizi G, Babini L et al (2011) Human mesenchymal stem cells from chorionic villi and amniotic fluid are not susceptible to transformation after extensive in vitro expansion. Cell Transplant 20:643–654 78. Poloni A, Maurizi G, Rosini V et al (2009) Selection of CD271(+) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow. Cytotherapy 11:153–162 79. Poloni A, Rosini V, Mondini E et al (2008) Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta. Cytotherapy 10:690– 697 80. Poloni A, Maurizi G, Leoni P et al (2012) Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells. Stem Cells 30:965–974 81. Maurizi G, Della Guardia L, Maurizi A et al (2018) Adipocytes properties and crosstalk with immune system in obesity-related inflammation. J Cell Physiol 233:88–97 82. Maurizi G, Poloni A, Mattiucci D et al (2017) Human white adipocytes convert into “rainbow” adipocytes in vitro. J Cell Physiol 232:2887–2899 83. Poloni A, Maurizi G, Mattiucci D et al (2015) Biosafety evidence for human dedifferentiated adipocytes. J Cell Physiol 230:1525–1533 84. Poloni A, Maurizi G, Anastasi S et al (2015) Plasticity of human dedifferentiated adipocytes toward endothelial cells. Exp Hematol 43:137–146 85. Poloni A, Maurizi G, Foia F et al (2015) Glial-like differentiation potential of human mature adipocytes. J Mol Neurosci 55:91–98 86. Poloni A, Maurizi G, Serrani F et al (2013) Molecular and functional characterization of human bone marrow adipocytes. Exp Hematol 41(558–566):e552 87. Mattiucci D, Maurizi G, Izzi V et al (2018) Bone marrow adipocytes support hematopoietic stem cell survival. J Cell Physiol 233:1500–1511 88. Mattiucci D, Naveiras O, Poloni A (2018) Bone marrow “yellow” and “red” adipocytes”: good or bad cells? Curr Mol Biol Rep 89. Kansu E (2004) The pathophysiology of chronic graft-versus-host disease. Int J Hematol 79:209–215 90. Zaja F, Bacigalupo A, Patriarca F et al (2007) Treatment of refractory chronic GVHD with rituximab: a GITMO study. Bone Marrow Transplant 40:273–277 91. Zhang C, Todorov I, Zhang Z et al (2006) Donor CD4+ T and B cells in transplants induce chronic graft-versus host disease with autoimmune manifestations. Blood 107(7):2993–3001 92. Patriarca F, Skert C, Sperotto A et al (2006) The development of autoantibodies after allogeneic stem cell transplantation is related with chronic graft-vs-host disease and immune recovery. Exp Hematol 34:389–396 93. Abdollahi A, Li M, Ping G et al (2005) Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis. J Exp Med 201:925–935 94. Daniels CE, Wilkes MC, Edens M et al (2004) Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis. J Clin Invest 114:1308– 1316 95. Distler JH, Jungel A, Huber LC et al (2007) Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis. Arthritis Rheum 56:311–322

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96. Filipovich AH, Weisdorf D, Pavletic S et al (2005) National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 11:945–956 97. Pavletic SZ, Martin P, Lee SJ et al (2006) Measuring therapeutic response in chronic graftversus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. Response Criteria Working Group report. Biol Blood Marrow Transplant 12:252–266 98. Olivieri A, Cimminiello M, Corradini P et al (2013) Long-term outcome and prospective validation of NIH response criteria in 39 patients receiving imatinib for steroid-refractory chronic GVHD. Blood 122:4111–4118 99. Lee SJ, Wolff D, Kitko C et al (2015) Measuring therapeutic response in chronic graft-versushost disease. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: IV. The 2014 Response Criteria Working Group report. Biol Blood Marrow Transplant 21:984–999 100. Pierelli L, Bosi A, Olivieri A (2018) “Best practice” for extracorporeal photopheresis in acute and chronic graft-versus-host disease by Societa’ Italiana di Emaferesi and Manipolazione Cellulare and Gruppo Italiano Trapianto Midollo Osseo: a national survey to ascertain its degree of application in Italian transplant centers. Transfusion 58:217–222 101. Giaccone L, Mancini G, Mordini N et al (2018) ‘Real-life’ report on the management of chronic GvHD in the Gruppo Italiano Trapianto Midollo Osseo (GITMO). Bone Marrow Transplant 53:58–63 102. Perseghin P, Marchetti M, Messina C et al (2013) Best practice recommendations in: (1) Peripheral blood stem cell mobilization and collection and (2) acute and chronic GvHD treatment using extracorporeal photopheresis. A joint effort from SIdEM (Societa Italiana di Emaferesi e Manipolazione Cellulare) and GITMO (Gruppo Italiano Trapianto di Midollo Osseo). Transfus Apher Sci 48:195–196 103. Poloni A, Sartini D, Emanuelli M et al (2011) Gene expression profile of cytokines in patients with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with reduced conditioning. Cytokine 53:376–383 104. Patriarca F, Sperotto A, Damiani D et al (2004) Infliximab treatment for steroid-refractory acute graft-versus-host disease. Haematologica 89:1352–1359 105. Marinelli Busilacchi E, Costantini A, Viola N et al (2018) Immunomodulatory effects of tyrosine kinase inhibitor in vitro and in vivo study. Biol Blood Marrow Transplant 24:267–275

Antimicrobial Resistance: A Challenge for the Future Pietro Emanuele Varaldo, Bruna Facinelli, Patrizia Bagnarelli, Stefano Menzo, Marina Mingoia, Andrea Brenciani, Andrea Giacometti, Francesco Barchiesi, Lucia Brescini, Oscar Cirioni, Giorgio Scalise, Pamela Barbadoro, Francesco Di Stanislao, Emilia Prospero and Marcello Mario D’Errico Abstract The global emergence of antibiotic-resistance, together with the lack of/reduced development of new antibiotic molecules, currently represents a serious public health problem as it can mean the return to a pre-antibiotic era in which infections caused by multiple-resistant pathogens are intractable. Since the beginnings, the interest of the Institutes of Microbiology, Hygiene and Public Health, and Infectious Diseases was focused on antibiotic resistance: from molecular mechanisms, through epidemiology and clinical issues, to prevention. Future perspectives include the search of new strategies and/or new compounds for prevention and control of difficult-to-treat pathogens in a multidisciplinary approach.

P. E. Varaldo · B. Facinelli · M. Mingoia (B) · A. Brenciani (B) Microbiology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche Medical School, Ancona, Italy e-mail: [email protected] A. Brenciani e-mail: [email protected] P. Bagnarelli · S. Menzo (B) Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche Medical School, Ancona, Italy e-mail: [email protected] A. Giacometti (B) · F. Barchiesi · L. Brescini · O. Cirioni · G. Scalise Infectious Diseases Clinic, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche Medical School, Ancona, Italy e-mail: [email protected] P. Barbadoro (B) · F. Di Stanislao · E. Prospero · M. M. D’Errico Hygiene and Public Health Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche Medical School, Ancona, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_2

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1 From Insights to Future Challenges The global emergence of antibiotic-resistance, together with the reduced development of new antibiotic molecules, currently represents a serious public health problem as it can mean the return to a pre-antibiotic era in which infections caused by multiple-resistant pathogens are intractable. In fact, WHO estimated that in 2050, if no action is taken, there will be more deaths from resistant infections than cancer. To tackle such problems, the scientific community is investing substantial resources in the search for alternative strategies for the control of difficult-to-treat pathogens.

1.1 In Microbiology Since the beginnings, the interest of the Institute of Microbiology (now the Microbiology Section of the Department of Biomedical Sciences and Public Health) was focused on bacterial antibiotic resistance. The main research fields on antibiotic resistance concerned: (1) corsivo antibacterial activities of new antibiotics; (2) surveillance of antibiotic resistance and population structure analysis of antibiotic resistant pathogens; (3) characterization of mobile genetic elements (MGE) carrying resistance genes; (4) genome sequencing. A large part of our studies has contributed to a greater knowledge of the elements of important multidrug resistant (MDR) human pathogenic streptococcal species, such as Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus suis [92–94, 128]. A unique recombination-mediated genetic plasticity is a distinctive feature of S. pneumoniae, the most common cause of communityacquired pneumonia and responsible for a plethora of invasive infections. In this context, a cause for serious concern was the emergence and spread of MDR clinical pneumococci where multiple resistance has been associated with MGE. Our research has contributed greatly to expand the features related to pneumococcal MGE, with the characterization of several new elements and/or new combinations of resistance genes [22, 32–34, 86, 88, 90, 99, 100]. S. pyogenes (Group A streptococci, GAS) is another human pathogen with a high prevalence worldwide. Clinical manifestations range from non-invasive, self-limiting purulent infections of the pharynx and skin to severe, invasive infections; sequelae include acute rheumatic fever, and glomerulonephritis. Its multiple virulence factors enable it to attach to host tissues, evade the host immune response and invade cells. Despite β-lactams are the drugs of choice to treat GAS pharyngotonsillitis, macrolides are largely used, thus a significant increase in the percentage of erythromycin resistant GAS was reported in Italy in the mid-1990s. Several MGEs carrying antibiotic resistance genes have also been characterized in the GAS [13, 21, 22, 43, 55–57, 87, 106, 123]. S. agalactiae (Group B streptococci, GBS), is both a normal component of the human microbiota and a leading cause of neonatal infections and a pathogen of growing importance

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in adults with underlying medical conditions. In addition, resistance to macrolides and clindamycin is increasing worldwide, as well as resistance to fluoroquinolones [121]. Several features have a major role in the diversification of GBS lineages and are driving the evolution of its resistoma [87, 89, 91, 95, 97]. S. suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Several MGE carrying resistance genes are similar to those reported in major streptococcal pathogens [98, 102]. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci [65, 98, 101, 106]. Listeria monocytogenes is widely distributed in the environment and is frequently isolated from a variety of sources, including soil, vegetation, food and water. As intracellular pathogen, L. monocytogenes escapes host defenses and antibiotics that cannot enter eukaryotic cells. Listeriosis is most often transmitted through food and primarily affects older adults, pregnant women, newborns, and adults with weakened immune systems [39–42, 44, 45, 72, 74]. Mycobacterium abscessus, an environmental, nontuberculous, rapid growing Mycobacterium, is an emerging human pathogen causing serious lung infections and one of the most difficult to treat, due to its multidrug resistance and biofilm-forming ability [71]. Linezolid (LZD) is the first member of oxazolidinones introduced into clinical use in the early new century to treat serious infections by Gram-positive organisms, including MRSA and VRE. For some years after its introduction, LZD-resistant isolates were only sporadically detected, the reported resistance mechanisms being confined to spontaneous ribosomal mutations. Later, transferable LZD resistance has also emerged. Multiple studies have shown that the use of antimicrobials in livestock leads to increased antibiotic resistance and that drug resistance determinants can be transferred from animal to human. In recent years, our studies have focused on LZD-resistance of both species, of human and animal origin, especially on genetic characterization of conjugative plasmids involved of the spread of acquired genes responsible for linezolid resistance [3, 4, 23]. Future perspectives include research activities for antibiotic resistance breakers (ARBs) (acting in synergy with conventional drugs), anti-virulence compounds (impairing virulence) of natural origin and phage therapy. ARBs have little or no antibiotic activity but co-administered with the antibiotic they either (i) block the main bacterial resistance mechanisms or (ii) enhance the antimicrobial action of the drug. From the drug discovery point of view, this combined drug therapy has the advantage that it is not necessary to expend effort in the challenging identification of new targets, essential for bacterial survival. Targeting microbial virulence rather than survival seems to be an exciting strategy, since the modulation of virulence factors might lead to a milder evolutionary pressure for the development of resistance. Additionally, anti-infective chemotherapies may be successfully achieved by combining antivirulence and conventional antimicrobials, extending the lifespan of these drugs. Phage therapy offers many advantages over chemotherapy: (a) it is effective against resistant bacteria because the mechanisms of bacteriolysis differ completely

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from those of antibiotics; (b) it has high specificity for target bacteria; (c) it can fast react to the appearance of phage-resistant mutants because the phages themselves are able to mutate; (d) the cost of developing a phage system is cheaper than that of developing a new antibiotics. The search for new strategies started few years ago and already yielded several significant scientific papers [2, 35, 36, 61, 71, 73, 96, 107]. New investigations will involve studies on: (i) intracellular pathogens, interaction of pathogens with human cells, including the antagostic activity of prebiotics/probiotics and phytocompounds; (ii) biofilm formation; (iii) plant components: antimicrobial activity, synergy with antibiotics, antivirulence activity; (iv) antimicrobial and antivirulence activity of honeybee; (v) phage therapy. The laboratory of Microbiology plans to investigate necrobiome in collaboration with forensic medicine. The study of microbiomes has enormous potential for forensic science because microbes are ubiquitous and specific microbial communities are often associated with selected processes or environments. Microbiomes have excellent potential as a forensic tool for estimating postmortem interval. “Microbial clock” could become a powerful tool for crime scene investigators looking to estimate the time and even the circumstances of death.

1.2 Virology The Institute of Microbiology (currently Microbiology unit of the Department of Biomedical Sciences and Public Medicine) has been also very active in virologic research. Three main research areas have been developed since the foundation in 1978 of the virology section and laboratory: (1) HIV infection/AIDS, (2) Chronic hepatitis viruses (HBV and HCV) and (3) Human papillomaviruses.

1.2.1

HIV Infection

Since quite soon after its discovery in 1983, HIV pandemic, for its huge clinical impact on the world population, has become one of the main research areas for the Institute of Microbiology. In this context it has pioneered research in fundamental molecular aspects of the infection in patients [7, 8]. Such research led to the introduction and development of the first seminal quantitative molecular assay for the measurement of HIV viral load in the blood [10, 11, 31, 77], and HIV patients in Ancona were the first in the world for whom HIV viral load was measured (in collaboration with the Immunology and Infectious Diseases wards). Subsequently, the same technology was applied to a group of non-progressor HIV-1 patients, in collaboration with the research group led by Giuseppe Pantaleo and Anthony Fauci at the NIH (Bethesda MD, USA), [103]. In addition to quantitative molecular aspects, the Institute of Microbiology dedicated its research efforts, based on genotypic and innovative recombinant phenotypic assays, to the study of HIV drug resistance against

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antiretroviral drugs and viral tropism [5, 9, 78, 84, 108, 129]. These kinds of studies are still currently part of the mainstream research of the Microbiology unit. As the clinical interest has shifted towards integrase inhibitors, viral evolution towards resistance to these compounds will be one of the main research topics in the next few years. Another study currently ongoing concerns the pathogenic and epidemic potential of transmitted resistance (i.e. patients initially infected with resistant viruses), a puzzling phenomenon with potential clinical consequences that still involves around 10% of new infections, despite the much lower circulation of resistant viruses in chronically infected patients.

1.2.2

Viral Hepatitis

The development of quantitative molecular methods initially developed for HIV was also applied to the study of viral hepatitis [62, 63, 67]. In addition, further molecular aspects of HBV molecular biology (in vitro hepatoma cell models, x protein expression and function, in vivo precore mutations) and their role in cell transformation were investigated [29–31, 64, 66, 80]. More recently research on HBV, in collaboration with the National Institute for Infectious Diseases L. Spallanzani in Rome, was focused on antiviral drug resistance and on acute infection [82, 85, 122, 130]. Other studies on Hepatitis E, allowed the identification of Region Marche as a national hot-spot for this emerging zoonosis [1, 124]. Current and future research on HBV will focus on the investigation of the intrahepatic replication dynamics of HBV. The adequate knowledge of the virus-host interplay, viral evolution and the extent of liver colonization in chronic hepatitis patients will be the key to the development of successful strategies for a functional cure of the infection. By these strategies, patients could be weaned from “lifelong” antiviral drug administration typical of current therapeutic interventions. The studies will concern the relationships between host immune response, cccDNA concentration, viral mutations and their compartmentalization in the liver, to identify peripheral molecular and serologic markers for a non-invasive evaluation of intrahepatic viral dynamics.

1.2.3

HPV Infection

Early after the foundation of the Institute of Microbiology, HPV infection and its relationship with cancer has also been one of the main research topics. A very accurate Molecular assay for HPV DNA detection in clinical samples was developed, capable of detecting virtually all mucosal HPV types (in addition to many cutaneous ones) in a single test. Sensitive HPV detection allowed also accurate genotyping by an original restriction endonuclease analysis [46, 79]. By this technology several new (at the time) unknown HPV types were detected. Of these, HPV 87 was completely characterized at the Institute: the complete genome was cloned and sequenced

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and the oncogenic properties of its E6 and E7 proteins were assessed, allowing its classification as a low risk type [83].

1.2.4

Other Viruses

Despite the absence of continuous research projects history on these topics, other viral infections have also been occasionally investigated, mostly for their pathogenetic or epidemiological aspects: HTLV [81], HHV6 [125], BKvirus [70], Influenza virus [58, 116, 126, 127], RSV [104], West Nile Virus [6]. Current research is ongoing on measles virus and on infection by enteroviruses. The former because vaccine coverage in Italy is still insufficient and the current epidemic is involving, with increasing incidence, immunocompromised patients with fatal pulmonary or neurologic complications. As for enteroviruses, these picornaviruses, for which no vaccine or specific antiviral treatment is available, are occasionally responsible for serious pediatric infections, ranging from neonatal sepsis, myocarditis or meningitis, and surveillance monitoring their circulation and the potential introduction of new genotypes may be useful.

1.3 Through Clinical Management Clinical management of antibiotic resistant infection has been one of the most important areas of research of the Infection Disease Unit, including either in vitro, and in vivo, and clinical studies carried out.

1.3.1

In Vitro Studies

The in vitro studies were performed to evaluate the antimicrobial activity of several traditional antibiotics and new molecules against a wide range of both control bacterial strains (ATCC strains) and multi-resistant nosocomial isolates. Interaction studies have also been performed to demonstrate the presence of synergism between drugs currently in clinical use and experimental molecules. The experimental molecules that have been used in these studies are called antimicrobial peptides and can derive from microorganisms, plants, fish, amphibians, reptiles, birds or mammals [53, 59, 118]. Some molecules seem to represent an important and inducible antibacterial defense system in insects, which are known to lack lymphocytes and immunoglobulins. Others would seem to be responsible for the unusual capacity of animals used in the laboratory, such as amphibians, to remain free of infections even after surgical experiments performed without antibiotic coverage. Most of these peptides could be therapeutically useful due to the low molecular weight and the hypothetical antimicrobial activity. In fact, some studies, although

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limited, have shown an appreciable in vitro activity towards bacteria, fungi and protozoa. The peptides would act by disrupting the membrane functions responsible for osmotic equilibrium. It has been hypothesized that the modalities of action of these molecules on the bacterial, fungal or protozoal membrane and on artificial lipid membranes could be similar, involving the formation of ion channels through the layers, without requiring a specific target. At the same time, some of our preliminary experiments have shown that various molecules originating from vertebrates do not provoke hemolysis of human erythrocytes or perform hemolytic action only at high concentrations. As part of this project, various molecular mechanisms responsible for the onset of resistance by pathogenic microorganisms against current-use antimicrobial molecules were also evaluated.

1.3.2

In Vivo Studies

Several compounds, primarily antimicrobial peptides and quorum sensing inhibitors have been tested in animal models to verify their effectiveness in preventing vascular prosthesis infection even after contamination by antibiotic-resistant microbial strains [24–26, 28, 48–52, 119, 120]. Some peptides have proved to be extremely effective even when used only topically, with a consequent reduction in costs and unwanted pharmacological effects. During these years some experiments have been centered on a molecule identified by the acronym RIP (RNAIII inhibiting peptide). This is an heptapeptide originally isolated from the supernatant of S. xylosus cultures. Studies conducted in collaboration with the University of Tel Aviv (Israel) and Davis (California, USA) have shown that this molecule in vitro, using cells or plastic material, shows an evident ability to counteract the adhesion of S. aureus and S. epidermidis. Its mechanism of action differs from that of common antibiotics because, instead of carrying out bactericidal activity, it inhibits communication between cell and cell, ultimately hindering the virulence of staphylococci and their ability to adhere to different materials forming biofilms. The RIP competes with an effector of that complex system that has been called quorum sensing, because it is the basis of the ability of the staphylococcal cells to communicate with each other and to self-regulate. Studies in animal models have demonstrated the real efficacy of positively charged peptide molecules in determining a significant reduction in lethality during septic shock. The molecules used, some new and synthesized in collaboration with researchers from other countries (University of Gdansk-Poland, Tufts-USA University, University of Negev-Israel, Nanjing-China University), have shown double activity: anti-endotoxin and antibiotic. The experiments were centered on gramnegative shock therapy in animal models (Wistar rats) where the septic shock was experimentally reproduced according to various methods (injection of purified lipopolysaccharide extracted from Escherichia coli, endoperitoneal injection of high inoculum of standard strains (ATCC) of gram-negative microorganisms, induced peritonitis by ligation and puncture of the cecum). Regardless of the model used, the peptide molecules have shown a capacity to influence the survival of the animal and

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the serum levels of endotoxin and cytokines (TNF, IL-1, IL-6) significantly higher than that controls. During these years, various animal models (Wistar rats, Balb-c mice) of surgical wound infection were made in which several molecules were evaluated both alone and in association with commonly used antibiotics. These studies have shown that some of these compounds (tigecycline, RIP, cationic peptides) had not only an antimicrobial activity (expected) but also other polyfunctional properties. It has been highlighted that these molecules were able to promote tissue regeneration and therefore a faster recovery. Also, of note is the activity of chemo-tactical recall demonstrated by some antimicrobial peptides.

1.3.3

Clinical Studies

Effectiveness of antiretroviral drugs in patients with HIV infection and new protocols for the treatment of HCV and HBV infection in outpatient activities have been evaluated and are still under investigation. The activity of our center and the large amount of medical assistance performed is demonstrated by the participation to several international multicentric studies concerning the effectiveness of new drugs in the treatment of HIV infection and viral hepatitis. The results of these studies were presented at various scientific conferences or published in international journals [12, 20, 27, 37, 47, 76, 97]. During these years various protocols have been produced for the rationalization of economic resources in the management of the patient with HIV infection, such as protocols of simplification in the therapeutic field, counseling activities through the administration of specific questionnaires, optimization of the examinations of laboratory and timing of follow-up, monitoring of the response to therapy and the appearance of side effects in the context of better management, implementation of “retention in care” projects for HIV-positive patients.

1.4 To Prevention and Infection Control Antimicrobial resistance and healthcare-associated infections (HAIs) are among the most serious public health problems, globally and in Europe, since they represent an important threat to patient safety; the increasing problem of antibiotic resistance is complicating the prevention and treatment of such infections, even further. European Centre for Disease Prevention and Control ECDC estimated that approximately 4 million patients acquire a HAI each year in all EU Member States and that deaths attributable to multidrug-resistant bacteria are currently estimated at 25,000. During the years, research activities in this area at the Hygiene Unit have been focused on: surveillance, prompt-response and infection control, and training. Surveillance represent the first step in infection control, leading to evaluation of the burden of disease, highlight of outliers and outbreaks, analysis of risk factors to

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control events, and monitoring interventions. The interest of the Section of Hygiene in surveillance, traces back to the 70 s when it was possible to enquiry the whole antibiotics utilization at the Ancona Regional Hospital [38]. Risk of contracting a HAI is higher in Intensive Care Unit patients, where important invasive procedures are performed, and antibiotics are widely used. The intensity of care may be associated to adverse events such as sepsis, catheter associated urinary tract infections and ventilator associated pneumoniae. The Section of Hygiene, in collaboration with the Hospital Hygiene Unit of the Ancona Associated Hospitals has contributed to the knowledge of the epidemiology of such infections with a focus on ventilator associated pneumonia [109, 115]. Nevertheless, attention has been given to specific patient populations like premature babies, especially vulnerable to infections, potentially leading to serious adverse outcome. Research has been focused on neonatal sepsis by Group B Streptococcus, and to gestational age as a single risk factor for the events [15, 16]. Risk assessment in healthcare setting has included the surveillance of surgical wound infections, from definition of their microbial ecology [54], to studies evaluating risk factors such as the presence of drains, or prolonged duration of surgery [17, 110, 114]. Moreover, the threat of Multidrug-Resistant A. baumannii has been evaluated in cardiac surgery patients [105]. Particular attention has been also given to quality improvements of surveillance systems, including the characteristics of surgical wound infections identified during the post-discharge period [114], and the importance of the definition of an appropriate cut off in the identification of duration of surgery as a factor associated to an increased risk of infection [110]. These efforts have been subsequently focused on continuous quality improvement of the perioperative antibiotic prophylaxis [112], taking into consideration the variables associated with inappropriate administration and measuring the impact on surgical site infection rates. Moreover, antibiotic stewardship in surgery has been focused on patients with complicated intra-abdominal infections caused by multi-drug-resistant organisms, where instruments have been identified to assist in the prompt identification of these patients, to optimize empiric therapy [60]. The problem of defining a model for antimicrobial stewardship in surgery has been evaluated at the global level, also, in the context of the participation to The Global Alliance for Infections in Surgery [117]. Continuous surveillance has been carried out also with the utilization of laboratory methods, which has given the opportunity to control contamination of medical products as potential source of HAIs [69, 113]. The analysis of factors associated to the development of healthcare infections have included the focus on one of the most important determinants of infectious risk and use of antibiotics, such as prolonged duration of catheterization and place of catheter insertion [14]. Moreover catheter-associated urinary infections (CAUTI) have been studied in elderly patients [131] where they represent the most common cause of HAIs, and an important cause of mortality. As already introduced, surveillance is the basis of intervention planning; during the years, efforts have been focused on the primum movens of CAUTI (i.e. catheterization itself) with the implementation of educational programs [68]. In this context, thanks

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to the implementation of a surveillance system and continuous training, a successful reduction of catheterization rate was achieved at our hospital. The attention towards cross-transmission, has been evaluated with a focus on hand hygiene, studied by means of a comparison of surgical hand preparation agents [19], but also in clinical practice, dealing with the effects of hospital-wide interventions including checking rounds for isolation precautions in the control of multidrugresistant organisms [18]. Moreover, a multimodal infection control program with monitoring of alcohol-based hand rub, and molecular surveillance was performed in ICU to control horizontal transmission of the same PFGE genotype [111]. The fight of antibiotic resistance is not limited to the hospital setting, with an interest in its control in a one health perspective that represents one of the most important challenges for the future: does the animal matrix influence resistance in humans? Does the environment play a role in the game? The occurrence of antibiotic resistance at the animal-human interface has been already investigated, with respect to the prevalence of Livestock-Associated Methicillin-Resistant S. aureus among farm and slaughterhouse workers [75], and further research is in progress. At a primary prevention level, efforts should be directed towards the understanding of knowledge gaps in antibiotic resistance awareness and inappropriate drugs utilization, to implement educational interventions targeted to citizens and healthcare professionals in the context of antibiotic stewardship. Research activities are needed with the objective of identifying risk factors for cross-transmission in the healthcare and community setting by means of epidemiologic and molecular instruments, and to test alternative strategies for prevention (i.e.: clinical testing of probiotics’ potential to prevent resistance). Antibiotic resistance is an important menace to the health and safety of patients within all the range of healthcare networks as well as of citizens in the community setting in Italy, and worldwide. With the increasing resistance to last-line antibiotics, research is needed to face a future where infectious diseases could once again become largely life threatening.

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Interventional Pulmonology: Past, Present and Future Stefano Gasparini, Martina Bonifazi and Lina Zuccatosta

Abstract Over the last decades, there have been huge progresses in most of fields of medicine, due to a deeper comprehension of pathogenic mechanisms of diseases along with outstanding advance in technology, suitable for diagnostic and therapeutic purposes. This applies also to respiratory medicine, in particular to the area of interventional pulmonology, where the development of sophisticated technological tools has revolutionized diagnostic work-up and therapeutic management of relevant pathologies, such as lung cancer, interstitial lung diseases, obstructive lung diseases (COPD) with advanced emphysema, and severe asthma. Most of these innovative approaches have been tested and adopted for the first time in Italy (sometimes in Europe) in our Pulmonary Diseases Unit, currently recognized as one of the most important centers of interventional pulmonology over the world, and daily visited for training by foreigners coming from several countries. In this review, we summarize the leading lines of research carried out by our team over the last decades, in both diagnostic and therapeutic fields of interventional pulmonology, with the subsequent clinical implications.

1 Introduction Respiratory diseases have become a global health issue, due to the increasing exposure to environmental risk factors and ageing of general population. The lungs, indeed, are the largest internal organ in the body constantly and directly exposed S. Gasparini (B) · M. Bonifazi Department of Biomedical Sciences and Public Health, Università Politecnica delle Marche, Via Tronto 10/A, 60126 Ancona, Italy e-mail: [email protected] M. Bonifazi e-mail: [email protected] S. Gasparini · M. Bonifazi · L. Zuccatosta Respiratory Diseases Unit, Azienda ospedaliero-universitaria Ospedali Riuniti, Via Conca 71, 60126 Ancona, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_3

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to the environment, such as outdoor and indoor air pollution, occupational toxics, tobacco smoke and infectious agents. According to the latest epidemiological estimates, around 235 million people suffer from asthma, more than 200 million people have chronic obstructive pulmonary disease (COPD), of whom 65 million with moderate-to-severe form, more than 100 million people experience sleep disordered breathing, 8.7 million people develop tuberculosis (TB) annually, and more than 50 million people struggle with occupational lung diseases. Lung cancer is currently the leading cause of cancer death around the world, with an overall 5-year survival rate lower than 15% [1]. The huge progresses in the field of respiratory medicine over the last decades, in particular in the area of interventional pulmonology by development of sophisticated technological tools, have revolutionized diagnostic work-up and therapeutic management of most of lung diseases, such as lung cancer, diffuse parenchymal lung diseases, chronic obstructive lung diseases (COPD) with advanced emphysema and severe asthma. Most of these innovative approaches have been tested and adopted for the first time in Italy (sometimes in Europe) in our Pulmonary Diseases Unit, currently recognized as one of the most important centers of interventional pulmonology over the world, and daily visited for training by foreigners coming from several countries. This successful result has been built, step by step, through a hard job, started almost 40 years ago by our group and witnessed by several papers, published in relevant peer-reviewed journals. In this review, we summarize the leading lines of research carried out by our team over the last decades, in both diagnostic and therapeutic fields of interventional pulmonology, and research projects, currently ongoing or due to start, that are expected to further extend the landscape of management of major respiratory diseases.

2 Diagnostic Work-Up of Thoracic Diseases The flexible fibreoptic bronchoscope, since its first introduction in the late 1960s, has become the milestone of interventional pulmonology. This is an invaluable tool for diagnosis of many thoracic disorders, with an excellent safety profile, characterized by low morbidity (0.1–2.5%) and negligible mortality (0.05%). The main revolution in this context was represented by the introduction of transbronchial aspiration techniques, that have extended pulmonologists’ perspective beyond the airways [2]. In particular, the adoption of flexible transbronchial needles (TBNA) has led to successfully biopsy hilar/mediastinal lesions, as well as submucosal, peribronchial, and peripheral lung nodules/masses (PPN/Ms), avoiding more invasive or risky approaches [3]. Regarding the diagnostic work-up of PPN/Ms, these may be approached both percutaneously and transbronchially. Percutaneous needle aspiration (PCNA), performed under fluoroscopic or CT guidance, plays a relevant role in the management of PPN/Ms, showing a sensitivity that ranges from 70 to 97%. However, concerns

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have risen about the safety profile of this technique, due to the relatively frequent occurrence of hemorrhage and pneumothorax. The endobronchial approach using standard flexible bronchoscopy with transbronchial needles, although characterized by a more heterogeneous diagnostic yield, is a safer procedure and it offers the advantages to provide, during a single examination, a pathological diagnosis of nodules, information on mediastinal staging and airways involvement, as well as to identify potential synchronous lesions. Due to the lack of studies directly comparing these two procedures, an evidencebased recommendation on the best choice between them has yet to be established, but a relevant contribution in this context was published by our group, reporting one of the largest experience (more than 1.000 consecutive patients) on combination of these techniques in the PPN/Ms management [4]. In particular, a single diagnostic team (a pulmonologist, radiologist, and cytopathologist), performed both the procedures under fluoroscopic guidance in a sequential algorithm, in which flexible bronchoscopy with TBNA was carried out first, due to the safer profile and the possibility to obtain a simultaneous mediastinal staging, and PCNA after, in case of inconclusive results at immediate cytologic assessment performed by the cytopathologist, present in the bronchoscopy suite. We concluded that transbronchial and percutaneous approaches must be considered complementary, rather than alternative, as their integrated use not only increased the diagnostic yield but also provided important information for the staging of lung cancer. Another relevant key message of this paper was focused on the utmost importance of the multidisciplinary approach in order to optimize the diagnostic management of PPN/Ms with a reduction in hospitalization time and consequent cost saving. Since the diffusion of this report in 1995, other papers have been published on this content by our team over the following two decades, including further experiences, qualitative and systematic reviews [5–8]. In most recent years, new technologies have been proposed for the bronchoscopic approach to PPN/Ms, including ultra-thin bronchoscopes, able to penetrate more distally into the bronchial tree, and innovative guidance systems such as virtual bronchoscopy, endobronchial ultrasound (EBUS) and electromagnetic navigation, currently used in our center. The main advantage of new guidance techniques is related to the possibility of approaching even small lesions that are not visible by fluoroscopy. In particular, the use of electromagnetic navigation bronchoscopy (ENB) has gained popularity as a complementary guidance tool during bronchoscopic biopsy of peripheral nodules. These systems utilize preoperative CT data and an electromagnetically tracked guide for procedure planning and guidance, but is not a real-time procedure. To overcome this limitation, our main perspective over the next years, will be to adopt the cone-beam CT (CBCT), that enables 3-dimensional (3D) fluoroscopic image guidance, with high sensitivity, low complication rates and acceptable amount of radiation dose (comparable with CBCT-guided percutaneous needle interventions and considerably lower than conventional CT guidance). As above mentioned, TBNA is an invaluable sampling technique also for diagnostic work-up of mediastinal adenopathies/masses, since allows to diagnose and stage malignancies during the same procedure, avoiding some unnecessary surgical

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approaches. This advantage was proven and quantified in a large prospective international multi-institutional clinical study, involving selected community hospitals and academic medical centers worldwide, including the ours. In this study, TBNA, in a complementary approach with CT scanning, precluded additional thoracic surgery in almost one third of patients, known to have significant cardiopulmonary and vascular comorbidity, confirming the essential role of transbronchial procedure in this context [9]. This concept was further underlined in subsequent reports and comprehensive reviews [7, 10–12]. However, against very high specificity (96–100%), the sensitivity of TBNA varies greatly in the published literature, being influenced by selected clinical and procedural aspects. In order to better define the role of such factors, out group has carried out a systematic review, the only available in the literature so far, providing an extensive description and synthesis of the main results from all published studies evaluating TBNA yield predictors for the diagnosis of mediastinal lymphadenopthies/masses [13]. In summary, major predictors in unselected population, as well as in suspected/known lung cancer clinical setting, included an increasing lymph node size, the presence of abnormal endoscopic findings, underlying malignant conditions, station 4R and 7 as site of samples, and the use of histological needle by an “experienced” bronchoscopist, although the type and duration of educational interventions evaluated varied widely among studies. With reference to the number of needle passes, although data were limited, we concluded that it is necessary to perform at least three needle passes up to a maximum of five to obtain the best accuracy. Other predictors in patients with suspected/known lung cancer included selected features of primary tumor, as the presence of small cell lung cancer (SCLC) subtype rather than non small cell lung cancer NSCLC and right-side location. There was also evidence on the role of rapid on-site cytological evaluation (ROSE) [13]. The immediate cytological assessment plays, indeed, an important role during sampling procedures, as it allows to assess quality of specimens collected in the diagnostic suite, providing relevant information to the operator, who could stop the exam if samples are adequate for diagnosis, as well as modify the technique or target site, if not. Furthermore, the operator could require to collect additional material for ancillary studies to further characterize the lesions, such as immunocytochemical stain, electron microscopy and microbiological evaluation. The evaluation of cytological sample adequacy has become even more important in the last years, with the advent of targeting lung cancer therapy, that requires specimens with enough material for performing immunocitochemestry and biomolecular assessment. Despite these advantages, ROSE is not routinely performed in all interventional pulmonology centers, because of the lack of cytopathologists available to join the procedures. To overcome this problem, our group conducted a study to evaluate whether a trained pulmonologist could be able to on-site assess the adequacy of TBNA specimens for the diagnosis of hilar/mediastinal adenopathies after an extensive educational intervention, including a training period alongside a board-certified cytopathologist and theoretical in-depth studies. One pulmonologist of our Pulmonary Diseases Unit underwent a three months training, taking part in the weekly diagnostic cytophatological session (3 h per session, for a total of 18 sessions) under the guidance and the

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supervision of a board-certified pathologist. This pulmonologist and the cytopathologist, deemed as gold standard, performed ROSE of 361 TBNA on 84 patients with by hilar/mediastinal lymphadenopathies and classified specimens into five diagnostic categories in a blinded fashion. There was an overall substantial agreement between observers (kappaw = 0.73, 95% CI 0.61–0.86; p < 0.001), that became excellent in cases of malignant disease. The diagnostic accuracy of ROSE performed by pulmonologist was not statistically different from that provided by cytopatologist. Our study provided the first evidence worldwide that a trained pulmonologist may be able to on-site evaluate the adequacy of cytological specimens, allowing to obviate most of the difficulties related to the involvement of the cytopathologists in daily diagnostic activities, and to reduce the costs, further optimizing the cost-effectiveness profile of TBNA [14]. However, TBNA is a blind technique without guidance system, and this has represented the rationale to develop a tool that uses reflected sound waves to visualize structures surrounding the airways. The endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA), by means of echoendoscope, offers the operator a real-time visualization of lymph nodes and surrounding structures, enabling to direct the tip of the needle to the target area. Overall, in the hands of experienced operators, EBUS provides an excellent diagnostic accuracy, leading to avoid more invasive procedures, and serious complications are extremely rare. As a result, EBUS practice has rapidly increased, especially after the advent of linear technology, and a large amount of experiences worldwide have been published over the last years, including our contributes [15–17]. Although a superiority of the imaging-guided over the conventional procedure could be reasonably supposed by pooled estimates from literature, evidence-based data on a direct comparison between the two procedures in randomized fashion had been lacking and the choice between them had been based more on empirical evaluations of operators rather than on established diagnostic algorithms. In this context, our group performed the first randomized controlled trial to test the superiority of EBUS-TBNA over c-TBNA for the diagnosis of hilar/mediastinal lesions of unknown origin We aimed also to evaluate the cost-effectiveness profile of a staged strategy, including c-TBNA as initial test, immediately followed by EBUS-TBNA, in case of inconclusive results at rapid on-site evaluation in the same bronchoscopy session. Two hundred and fifty-three patients were randomized to either EBUS-TBNA (n = 127) or c-TBNA (n = 126). Thirty-one patients of c-TBNA group subsequently underwent EBUS-TBNA. The sensitivity EBUS-TBNA was higher, but not significantly superior to that of c-TBNA (respectively 92%, [95%, CI 87–97%] and 82% [95%, CI 75–90%], p > 0.05). The sensitivity of the staged strategy was 94% (95%, CI 89–98%). No major adverse events occurred. We showed that EBUS-TBNA was the single best diagnostic tool, although not significantly superior to c-TBNA. Due to the favorable cost-effectiveness profile of their sequential combination, we proposed to introduce this staged strategy into clinical practice [18]. Another huge revolution in the field of interventional pulmonology has been the introduction of transbronchial lung criobiopsy (TBLC) in the diagnostic work up of Interstitial Lung Diseases (ILDs). For instance, advances in comprehension of ILDs pathogenesis has coupled with exciting evolutions in technologies related to

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tissue sampling, no longer exclusive domain of thoracic surgeons. The term “ILDs” includes a wide spectrum of heterogeneous entities with different prognosis, as well as treatment options. Due to the recent progresses in therapeutic landscape of ILDs management, the distinction between idiopathic pulmonary fibrosis (IPF), the most prevalent and severe form, and other diseases, has become even more important. Nowadays, two oral anti-fibrotic drugs, pirfenidone and nintedanib, are available in the market for the treatment of IPF, although the long-term prognosis of these patients remain poor. An accurate diagnosis of IPF is a challenging process that requires an integrated multidisciplinary approach involving pulmonologists, radiologists, and, when lung tissue is needed, also pathologists. In this context, the role of conventional transbronchial lung biopsy is limited to the exclusion of specific disorders (i.e. sarcoidosis, carcinomatous lymphangitis, organizing pneumonia), since the small sample size, the rate of crush artifacts and the high likehood to sample mostly centrilobular areas do not allow to properly identify more complex and spatially heterogeneous morphological patterns. This is the reason why the current guidelines recommend surgical lung biopsy (SLB), when a pathological assessment is needed to establish a diagnosis. However, SLB is characterized by appreciable costs and risks, with a mortality rate of 2–4% within 90 days. Moreover, many subjects are not elegible because of a combination of advanced stage, age, comorbidities, respiratory failure, and pulmonary hypertension. More recently, TBLC has been proposed as a valuable alternative tool for the pathologic assessment of ILDs. Actually, the use of cryoprobes for bronchoscopic procedures was firstly described as early as 1977 for therapeutic purposes in case of airways occlusions. The ingenious novelty consists in using a flexible cryoprobe through a flexible bronchoscope to obtain parenchymal lung tissue. Our center was one of the first center in the world to adopt TBLC, and, later, the growing amount of data on successful experiences has led to its routine adoption in selected interventional pulmonology centers worldwide. In this context, our group, in collaboration with an another Italian center, reported the first retrospective comparison on DY and safety between SLB and TBLC in a large population, and provided the first systematic review and meta-analysis of studies published on this topic [19, 20]. Overall, 150 patients underwent SLB and 297 underwent TBLC. The median time of hospitalization was 6.1 days (SLB) and 2.6 days (TBLC; p < 0.0001). Mortality due to adverse events was 2.7% (SLB) and 0.3% (TBLC) of the patients. Pneumothorax was the most common complication after TBLC (20.2%) and No severe bleeding was observed. TBLC was diagnostic for 246 patients (82.8%), SLB for 148 patients (98.7%, p = 0.013). The meta-analysis of 15 investigations including 781 patients revealed an overall DY of 0.81 (0.75–0.87); the overall pooled probability of developing a pneumothorax, as retrieved from 15 studies including 994 patients, was 0.06 (95% CI 0.02–0.11). Based on these data, we concluded that TBLC is an effective and safe procedure, with lower complication and mortality rates compared to SLB. We also proposed an algorithm according to which TBLC should be considered the first diagnostic approach for obtaining tissue in ILDs, reserving the surgical approach for cases in which TBLC is not feasible. The ‘final’ risk of mortality, considering TBLC and SLB together in a “staged” approach, proves to be 0.3–1.4%, is significantly lower than the overall mortality with SLB alone.

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We are currently the regional tertiary referral center for diagnosis and management of ILDs, and we have a strict network of collaboration with the other national and international centers. In our center, more than 200 patients per year are currently diagnosed and managed and a patients’ association has been recently founded. Moreover a dedicated care pathway (“percorso diagnostic terapeutico-PDTA”) for ILD management has been developed to optimize timing and resources. Several clinical research activities in ILDs are currently ongoing, and some of them are focused on tissue phenotyping/characterization, as the advent of TBLC, with its favourable risk/benefit profile, has hugely improved our chances of sample collection, suitable for clinical as well as for research purpose.

3 Therapeutic Management of Respiratory Diseases Over the last fifty years, interventional pulmonology’s role has become increasingly established also in the field of therapeutic management of selected respiratory diseases, such as pleural disorders and chronic obstructive lung diseases. The term “Pleural Diseases” includes a wide spectrum of heterogeneous entities, as a pleural involvement may occur in several neoplastic and non-neoplastic conditions with different etiologies, prognosis, as well as treatment options. Pleural effusion is commonly faced by clinicians in daily practice, and, due to the increasing burden of pleural diseases worldwide, a personalized cost-effective management of these conditions is essential to optimize the healthcare sources. To date, more than 50 causes of pleural effusions have been identified and include local diseases, systemic conditions, organ dysfunction and drug reactions. Therefore, a multidisciplinary approach from different specialties is highly recommended to ensure the best management, which lies, first of all, in a tailored treatment or, at least, control of the underlying cause. Pleurodesis, intended to produce a pleural symphysis via thoracoscopic (poudrage) or chest tube (slurry) insufflation of a sclerosing agent, is still considered as the first-line therapy, except for patients with ‘trapped lung’, which significantly decreases the probability of a successful procedure. A recent network meta-analysis of studies on interventions for the management of malignant pleural effusion led to conclude that talc poudrage is the more effective technique, as it resulted in a significantly lower rate of failures than the other methods. Our group took part in one of the most important and large study in this context, that was a multicentre, open-label, prospective cohort study of 558 patients with malignant pleural effusion who underwent thoracoscopy and talc poudrage with 4 g of calibrated French large-particle talc in 13 European hospitals, and one in South Africa. The primary endpoint was the safety profile of talc pleurodesis, that was overall favourable, as no acute respiratory distress syndrome occurred [21]. Such report had a huge impact on malignant pleural effusion management, and talc pleurodesis has definitely become the gold standard in this context. A substantial proportion of neoplastic pleural involvement is caused by pleural mesothelioma (PM), that is

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a rare, devastating tumor, strongly related to asbestos exposure. The variable incidence of PM worldwide mostly reflects asbestos utilization and exposure. Due to the extremely long latency period between first exposure and disease occurrence, epidemiological projections of PM incidence in Western Europe indicate a peak around 2020, and a trend towards reduction only afterwards. The prognosis of PM is extremely poor, with 5-years survival around 10%. Available data from clinical trials on its management are limited and controversial. Therefore, treatment approaches, that include chemotherapy (pemetrexed or raltitrexed and platinum-based agents as the only approved drugs), radiotherapy and surgery, either in combination or as single treatment, are highly heterogeneous worldwide. The different treatment strategies adopted by clinicians represented the rationale for a study recently published by our group in collaboration with the department of Epidemiology of University of Milano, aiming to assess treatment and prognosis of PM in a large real world cohort of patients from Lombardy, the largest Italian Region (about 10 million inhabitants) through a record linkage between healthcare administrative databases [22]. Out of 1326 patients with PM identified in the study period 2006–2011, 754 (56.9%) received any treatment for PM: 205 (15.5%) underwent surgery, and 696 (52.5%) used chemotherapy. Surgery was spread across several hospitals, and most patients diagnosed in nonspecialized centres (70%) underwent surgery in the same centres. Age at diagnosis was a strong inverse determinant of surgery. Determinants of receiving chemotherapy were younger age, a more recent first diagnosis, and first diagnosis in a specialized centre. OS was 45.4% at 1 year, 24.8% at 2 and 9.6% at 5 years (median 11 months). OS decreased with age, and was higher for who underwent surgery, but not for those treated with chemotherapy. We confirmed that management of PM varied widely in clinical practice and significant predictors of treatment were younger age and recent diagnosis, though a high proportion of patients were not treated. Patients were treated in various hospitals indicating the importance of concentrating serious rare neoplasms in Comprehensive Cancer Centers. Due to our clinical and research experience on pleural diseases, we were invited by a peer-review journal to summarize the latest insights regarding this field in a comprehensive qualitative review, recently published [23]. Chronic obstructive pulmonary disease (COPD) is a common respiratory disease, characterized by persistent airflow limitation associated with chronic inflammation of the airways and lungs, in response to noxious particles and gases. COPD is a protean disease that includes different phenotypes, and therapeutic strategy should be personalized and tailored to specific patient features. In particular, patients with advanced emphysema gain and perceive limited benefits from existing pharmacological treatment options, due to the lack of efficacy in reducing hyperinflation, that is the key physiopathological mechanism in this context. Lung hyperinflation is defined as an abnormal increase of the amount of gas in the lungs and airways at the end of the tidal (spontaneous) expiration, highly influencing respiratory dynamics. Therefore, lung hyperinflation plays a central role in the perception of dyspnea and poor exercise tolerance and it contributes to a worse disease prognosis irrespective of the lung function. Such a relevant physiopathogenic mechanism offered the rationale for the development of a targeted treatment option, the lung volume reduction

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(LVR). This procedure is intended to reduce hyperinflation by excluding the most damaged area of lung parenchyma, in order to optimize respiratory mechanics of the remaining tissue, reducing the work of breathing. The first attempts of surgical lung volume reduction (LVRS) were performed in the 1950s, but it was only in 2003, with the publication of the results of the National Emphysema Treatment Trial that this procedure demonstrated to significantly improve clinical and functional status in selected emphysematous patients. However, the questionable cost-effectiveness profile, as well as the considerable mobility and mortality associated with LVRS, prompted reflection on alternative, less invasive, approaches to achieve LVR, such as endoscopic techniques (BLVR). In the past 20 years, the ambitious purpose of achieving the same clinical benefits with reduced risks and costs has permeated the world of interventional pulmonology and several different techniques have been successfully developed and labeled in the market. According to the underlying mechanism of action and reversibility, they can be divided into 2 main groups: blocking reversible devices, and nonblocking irreversible devices. The first group, represented by unidirectional valves, relies on occlusion and atelectasis of the most hyperinflated lobe. Nonblocking devices are designed to exclude the targeted lobe by inducing an irreversible parenchymal reaction, and include coils and sclerosis agents, such as sealants and thermal vapor ablation. Our center was one of the first worldwide, in which any of these devices was tested. Unidirectional valves are the most widely investigated devices in the context of BLVR therapy, with the largest series of patients treated so far. We took part of the first international randomized trials [24–26], including the Endobronchial Valve for Emphysema Palliation Trial (VENT), conducted in the US (321 patients in 31 centers) [24] and in Europe (171 patients in 23 sites) [25, 26], that showed a statistically significant, improvement in lung functions, exercise tolerance and quality of life compared to controls subjects on best medical therapy. The growing evidence on efficacy and safety of this treatment over years has allowed to include it in the current guidelines as a valuable option for patients with advanced emphysema and selected morphological features, identified as predictors of successful outcomes. In our center, so far, more than 100 patients have been treated with valves and followed-up over time to assess long-term benefits of this procedure. Moreover, a research study on other aspects that might impact the success of treatment, such as the presence of a predominant small airways involvement, is currently ongoing. Nonblocking devices, such as coils, sealants and thermal vapor ablation were also tested and adopted in our center for the first time in Italy, and since then, several patients have been studied and treated over years, allowing to become one of the groups with the large experience in Europe, as witnessed also by reviews published on this topic [27, 28].

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References 1. European Lung WhiteBook. The burden of lung disease. https://www.erswhitebook.org/ chapters/the-burden-of-lung-disease/ 2. Shure D (1989) Transbronchial biopsy and needle aspiration. Chest 95(5):1130–1138 3. Tsuboi E, Ikeda S, Tajima M et al (1967) Transbronchial biopsy smear for diagnosis of peripheral pulmonary carcinomas. Cancer 20(5):687–698 4. Gasparini S, Ferretti M, Secchi EB et al (1995) Integration of transbronchial and percutaneous approach in the diagnosis of peripheral pulmonary nodules or masses. Experience with 1,027 consecutive cases. Chest 108(1):131–137 5. Mondoni M, Sotgiu G, Bonifazi M et al (2016) Transbronchial needle aspiration in peripheral pulmonary lesions: a systematic review and meta-analysis. Eur Respir J 48(1):196–204 6. Gasparini S, Bonifazi M, Wang KP (2015) Transbronchial needle aspirations vs. percutaneous needle aspirations. J Thorac Dis 7(Suppl 4):S300–S303 7. Gasparini S (1997) Bronchoscopic biopsy techniques in the diagnosis and staging of lung cancer. Monaldi Arch Chest Dis 52(4):392–398 8. Gasparini S, Zuccatosta L, Zitti P et al (1999) Integration of TBNA and TCNA in the diagnosis of peripheral lung nodules. Influence on staging. Ann Ital Chir 70(6):851–855 9. Harrow EM, Abi-Saleh W, Blum J et al (2000) The utility of transbronchial needle aspiration in the staging of bronchogenic carcinoma. Am J Respir Crit Care Med 161(2 Pt 1):601–607 10. Gasparini S, Silvestri GA (2005) Usefulness of transbronchial needle aspiration in evaluating patients with lung cancer. Thorax 60(11):890–891 11. Herth FJ, Rabe KF, Gasparini S et al (2006) Transbronchial and transoesophageal (ultrasoundguided) needle aspirations for the analysis of mediastinal lesions. Eur Respir J 28(6):1264–1275 12. Trisolini R, Patelli M, Ceron L et al (2011) Transbronchial needle aspiration. Monaldi Arch Chest Dis 75(1):44–49 13. Bonifazi M, Zuccatosta L, Trisolini R et al (2013) Transbronchial needle aspiration: a systematic review on predictors of a successful aspirate. Respiration 86(2):123–134 14. Bonifazi M, Sediari M, Ferretti M et al (2014) The role of the pulmonologist in rapid on-site cytologic evaluation of transbronchial needle aspiration: a prospective study. Chest 145(1):60– 65 15. Tremblay A, McFadden S, Bonifazi M et al (2018) Endobronchial ultrasound-guided transbronchial needle aspiration with a 19-G needle device. J Bronchol Interv Pulmonol 25(3):218–223 16. Tremblay A, Myers R, Beaudoin EL et al (2018) Initial clinical experience with a flexible peripheral 21-G needle device. J Bronchol Interv Pulmonol 25(4):346–348 17. Herth FJ, Schuler H, Gompelmann D et al (2012) Endobronchial ultrasound-guided lymph node biopsy with transbronchial needle forceps: a pilot study. Eur Respir J 39(2):373–377 18. Bonifazi M, Tramacere I, Zuccatosta L et al (2017) Conventional versus ultrasound-guided transbronchial needle aspiration for the diagnosis of hilar/mediastinal lymph adenopathies: a randomized controlled trial. Respiration 94(2):216–223 19. Ravaglia C, Bonifazi M, Wells AU et al (2016) Safety and diagnostic yield of transbronchial lung cryobiopsy in diffuse parenchymal lung diseases: a comparative study versus video-assisted thoracoscopic lung biopsy and a systematic review of the literature. Respiration 91(3):215–227 20. Gasparini S, Bonifazi M (2016) Cryobiopsy for interstitial lung diseases. J Bronchol Interv Pulmonol 23(1):4–6 21. Janssen JP, Collier G, Astoul P et al (2007) Safety of pleurodesis with talc poudrage in malignant pleural effusion: a prospective cohort study. Lancet 369(9572):1535–1539 22. Carioli G, Bonifazi M, Rossi M et al (2018) Management and survival of pleural mesothelioma: a record linkage study. Respiration 95(6):405–413 23. Gasparini S, Bonifazi M (2017) Pleural diseases. Curr Opin Pulm Med 23(3):269–274 24. Sciurba FC, Ernst A, Herth FJ et al (2010) A randomized study of endobronchial valves for advanced emphysema. N Engl J Med 363(13):1233–1244

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25. Herth FJ, Noppen M, Valipour A et al (2012) Efficacy predictors of lung volume reduction with Zephyr valves in a European cohort. Eur Respir J 39(6):1334–1342 26. Valipour A, Herth FJ, Burghuber OC et al (2014) Target lobe volume reduction and COPD outcome measures after endobronchial valve therapy. Eur Respir J 43(2):387–396 27. Gasparini S, Zuccatosta L, Bonifazi M, Bolliger CT (2012) Bronchoscopic treatment of emphysema: state of the art. Respiration 84(3):250–263 28. Stratakos G, Emmanouil P, Gasparini S (2013) Novel modalities and agents in bronchoscopic lung volume reduction. Curr Drug Targets 14(2):253–261

The Experimental Pathology at Ancona: 50 Years of Exciting and Pioneering Research on Human Pathology Fabiola Olivieri, Maria Rita Rippo, Laura Graciotti, Armanda Pugnaloni, Francesca Fazioli and Antonio Domenico Procopio

Abstract Half century ago, a few academic pioneers founded the laboratories of experimental and ultrastructural pathology in Ancona. From this origin, a new phase of experimental studies developed aimed at translational and clinical research up to the present, when our group is internationally recognized for its fundamental contributions in gerontological research and molecular diagnostic pathology. Since the desire of immortality and of eternal youth seems to be as old as mankind, in the future we plan to focus our scientific research on Regenerative Medicine and Rejuvenation strategies. This is the most ambitious aim in the framework of the world aging population. We do not know whether we would achieve these results by ourselves. We are confident that, as in the past, new generations of scientist of the school of experimental pathology at Ancona will get the baton by the older one and lead the future with the same enthusiasm, love and commitment.

1 The Beginnings of Experimental Pathology at the UNIVPM: The Basic and Ultrastructural Research The history of Experimental Pathology at Ancona began in the early ‘70s, when Professor Andrea Corsi was appointed Chairman of General Pathology at the newly created Faculty of Medicine and of which he became Dean. Member of the Accademia dei Lincei, Prof. Corsi had been trained scientifically at the University of Padua where he had conducted pioneering studies on the role of the thymus in the immune system [13, 14, 31]. Prof. Corsi and his collaborators, including Prof. Anna Luisa Granata, Prof. Cesare Vecchi, and Dr. Laura Graciotti, created the first Institute of General F. Olivieri (B) · M. R. Rippo · L. Graciotti · A. Pugnaloni · F. Fazioli · A. D. Procopio Section of Experimental Pathology and Occupational Medicine, Department of Clinical and Molecular Sciences (DISCLIMO), Università Politecnica Delle Marche, Via Tronto 10/A, Torrette, Ancona, Italy e-mail: [email protected] F. Olivieri · A. D. Procopio Center of Clinical Pathology and Innovative Therapy, National Institute, IRCCS INRCA, Ancona, Italy © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_4

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Pathology and the first Laboratory of Experimental Pathology in Ancona, including a facility for ultrastructural studies equipped with a Philips CM10 electronic microscope. Their efforts allowed the activation of new lines of research on skeletal muscle [15, 16] and obtained the first European Community funded research program at our University (MOLECULAR BASIS OF CELL MOVEMENT: Investigation of the fundamental molecular mechanisms of ameboid cell motility and transport using a combination of molecular, cellular and structural biology methods CE-Human and capital mobility CHRX-CT93-0250. 1993–1996). The group was active also on clinical diagnostics. The experimental group founded by Prof. Andrea Corsi at Ancona obtained important results in the study of microcirculation with particular regard to the relationship between capillary blood flow and skeletal muscle function [17, 26, 35, 68] and in the study of Duchenne Muscular Dystrophy [32]. These later studies demonstrated that cardiac muscle has peculiar molecular defects arising from lack of Dystrophin that suggests different pathogenesis with respect to skeletal muscle. Dystrophin absence lead to a delocalization of KATP channel complex and of Creatine Kinase M the enzymes necessary for the channel correct functioning. Alterations due to dystrophin lack was also investigated in the hippocampus of mdx mice showing alteration in term of cellular and synaptic composition that underlies other muscle unrelated pathogenic mechanism [33].

2 The Phase of Consolidation: The Translational and Molecular Research After Prof. Corsi retired, in 2001 the Faculty appointed Full Professor of General Pathology Professor Antonio Domenico Procopio, a cancer immunologist with a strong background in experimental and clinical pathology. Before reaching Ancona, Prof. Procopio had trained at the La Sapienza University of Rome with Prof. Luigi Frati and at the NCI, Frederick, MD with Dr. Ronald Herberman. He had worked at the NIH, Bethesda, MD where he had conducted pioneering studies on SV40 and mesothelioma and directed the Laboratory of Clinical Pathology at the G. D’Annunzio University of Chieti, Italy. To promote translational and molecular research, short after his arrival to Ancona Prof. Procopio founded the first Department of the Faculty of Medicine (Department of Molecular Pathology and Innovative Therapies, today DISCLIMO), which he directed for many years. More recently, he founded the Center of Clinical Patology and Innovative Therapy at the National Institute on Aging (INRCA), devoted to basic and clinical research, and established the Courses of Clinical Pathology and the Clinical Pathology Residency Program of our Faculty, considered a national excellence. In those years, Prof. Maria Rita Rippo, a leading immunologist experienced in the mechanisms of programmed cell death, joined the group moving from Rome “La Sapienza”. She had worked with Prof. Roberto Testi and Prof. Ruggero De Maria, publishing pioneering studies on the role of FasL and mitochondria in apoptosis

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[20, 21, 64]. Later, other leading scientists permanently joined the Ancona group, including Prof. Francesca Fazioli (molecular oncology) [22, 23] and Prof. Armanda Pugnaloni (cancerology). Our group refocused its activity on translational research of neoplastic pathologies of mesodermal tissues. Since the first moment of his transfer to Ancona, in the late 90’s, Prof. Procopio has continued his studies on pleural malignant mesothelioma (MM). MM, an insidious and still lethal cancer, originates from the mesothelium, which covers the pleura and the others serous cavities such as pericardium, peritoneum, vaginal testis. It is the most common malignant neoplasm of these structures, characterized by a poor prognosis, as it is little responsive to current treatments. MM was found a disease strictly related to asbestos, a harmful natural material endowed with exceptional persistence [40, 41]. During 2000s our group achieved very important results concerning the molecular mechanism responsible of MM progression and the identification of novel diagnostic and prognostic markers. Using different approaches ranging from molecular biology to computational analysis, we have identified several mechanisms of the resistance of MM to chemotherapy, to pro-apoptotic cytokines-induced cell death, and to toxicity of asbestos (Part of the results arise from an Italian Research Project of National Interest—PRIN 2011: “In Vitro Biological Activity and Mechanisms of Lung and Pleural Cancers Induced by Mineral Fibers” [40]. Finally the epigenetic mechanisms involved in MM development and progression has been elucidated [39]. In his early studies on mesothelioma, Procopio and co-workers demonstrated that SV40 (Simian virus-40) plays a role as oncogenic virus and that it is expressed in tissue, urine and blood samples from patients with malignant and non malignant pleural disease [8, 9, 73]. Accordingly, he demonstrated that SV40 sequences are negative prognostic cofactor in patients with malignant pleural mesothelioma [60]. These pioneering researches have paved the way for subsequent studies that have been funded by the Italian Ministries of Health and of Scientific Research and the Italian Association on Cancer Research (AIRC) and have allowed our group to build a network of collaboration with the world’s most recognized experts in this field. The following years were therefore rich in discoveries that helped international scientific research to develop and try new strategies for controlling the progression of this cancer, which is still incurable today, and for the therapy of other less aggressive ones. The results obtained prompted AIRC to fund a permanent position of Professor in General Oncology, that has been covered by competitive national examination by Prof. Francesca Fazioli. The most interesting and cited studies demonstrated that human malignant mesothelioma cells produce autocrine factors promoting their growth and multidrug resistance. Early transforming proteins of SV40, the large tumor antigen (Tag) and the small tumor antigen (tag) induce in mesothelioma cells Vascular endothelial growth factor (VEGF) expression which in turn acts as an autocrine growth factor enhancing their proliferation [11, 73]. In addition to this proliferative advantage, autocrine production of tyrosine kinase receptor c-Kit and its ligand stem cell factor SCF induces the zinc finger transcription factor Slug and chemoresistance [12]. Therefore, MM

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cells develop a strong resistance to chemotherapy- and immune system-induced programmed cell death because of the overexpression of some anti-apopototic proteins that act at different levels of the extrinsic pro-apoptotic pathway induced by the cytokine FasL/CD95L and to a lesser extent TNF-related apoptosis-inducing ligand (TRAIL) and of the intrinsic one. In particular, we showed that the protein FLIP (FLICE-Inhibitory Protein), which inhibits apoptosis by competing with the procaspase-8 binding to Fas receptor complex, is constitutively expressed in all MM cell lines and is more expressed in primary MM cells than in normal mesothelial cells [65]. We also demonstrated that MM cells overexpress Bcl-2 and telomerase activity due, at least in part, to Methionine aminopeptidase-2 (MetAP2) expression and function [10]. However, we showed that α-tocopheryl succinate (α-TOS), a strong pro-apoptotic agent, can synergize with TRAIL to kill MM via mitochondrial pathway, whereas both of them are nontoxic to normal mesothelial cells [76]. Accordingly, subsequent studies, conducted by other international groups, have shown that FLIP expression is potently downregulated in MM cells in response to the histone deacetylase inhibitor Vorinostat (SAHA), and that this compound overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma [19]. In the last ten years our research group, which has grown not only in the number of participants but also in the expertise, has expanded the research lines, focusing on the role of epigenetic mechanisms in the development of diseases associated with aging, including tumors. In this context, the study of the role of small non coding RNAs and in particular of miRNAs, as modulators of proteins important in the development and progression of the tumor and markers for the diagnosis and prognosis of malignant mesothelioma, has prevailed over the other epigenetic mechanisms. An interesting research was conducted by our new unit of computational pathology, showing how to manage big miRNA-related data generated by recent technological advances, especially high-throughput sequencing. A number of bio-informatic tools and databases have been devised to manage this growing body of data in diverse areas of miRNA research, to assist investigators in choosing the most appropriate tools for their needs [1]. Since miRNAs have established diagnostic value in cancer and pollution exposure and, furthermore, minimally invasive, specific and sensitive biomarkers for early and effective diagnosis of MM in high-risk patients are urgently needed, computational pathology unit identified high-confidence miRNAs that can serve as biomarkers of asbestos exposure and MM by a systematic review and a qualitative meta-analysis. In particular, two miRNA signatures of deregulated circulating (miR-126-3p, miR-103a-3p, and miR-625-3p) and tissue (miR-16-5p, miR-126-3p, miR-143-3p, miR-145-5p, miR-192-5p, miR-193a-3p, miR-200b-3p, miR-203a-3p, and miR-652-3p) miRNAs were identified with potential early diagnostic value and designated as “mesomiRs” [39]. A large-scale, standardized validation studies are ongoing to assess their clinical relevance, so as to move from the workbench to the clinic.

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3 The New Program on Aging and Age-Related Diseases (ARDs): Identification and Characterization of Innovative Biomarkers of Aging and ARDs Due to the increased lifespan, the demographic patterns and the ageing of the European and Italian population is changing. A new line of research focused on the identification of innovative genetic and epigenetic biomarkers of aging and age-related human diseases (ARDs) was launched in 2007, in the framework of the research activity of the Experimental Pathology team. Under the leadership of Professor Fabiola Olivieri the group have pioneered this topic. Prof. Olivieri, before joining our Group in Ancona as a leading gerontologist, was trained scientifically at “Alma Mater Studiorum” University of Bologna, and worked at INRCA, the national Italian Institute on Aging, with Prof. C. Franceschi. Her guide successfully finalized our efforts to achieve the objectives of this ambitious project, requiring a comprehensive knowledge of ageing processes not only at the medical-clinical level, but also at the level of the biological and molecular mechanisms (genetic, epigenetic, hormonal) that underlie healthy ageing. Applying a multidisciplinary approach, integrating the expertise in different areas, such as clinical pathology, oncology, cardiology, molecular biology, genetic, biostatistics and computational analysis, our research group was involved in a number of national and international collaborative projects, supported by Operative Regional Program (POR) Marche, Italian Ministry of Education-Universities and Research (MIUR), Italian Ministry of Health and projects co-founded by European countries. Within this framework, in the last 10 years, we have been able to publish more than 150 articles (that received more than 10,000 citations, Scopus, 2018) in peer-reviewed top-ranking international journals. The conceptualization that underlies these studies is that “ageing” is not a static but rather a dynamic phenotype that changes over time; a continuous interaction between individuals’ genetic makeup and environmental factors results in a spectrum of states that range from healthy ageing to ARDs. This dynamic interaction drives an age-related remodelling of a number of pathways/systems, providing the chance to reach the extreme limit of human life in healthy state, which is reflected in the ever-increasing number of centenarians [6, 28, 70]. If a continuum between unhealthy and healthy ageing exists, the major chronic ARDs should be conceptualized and studied within the framework of the ageing process and its basic molecular and cellular mechanisms. This conceptualization implies that aging process per se and the development of the most common ARDs are somewhat separate but must share somehow common set of basic biological mechanisms, mainly the development and progression of an inflammatory state named inflammaging, an increased burden of senescent cells and a defective cellular cross-talk [27]. Some years ago we advanced a general hypothesis that the ageing process and the development of ARDs could be fostered by a low-grade, chronic inflammatory process that was designated inflammaging [24]. A number of data suggest that this process is sustained by pro-inflammatory cells and circulating compounds [25, 58, 59]. Notably, the rate

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of inflammaging progression is currently recognized as the main force driving aging and one of the main risk factors for clinical morbidity and mortality in the elderly [27]. Since ageing involves the entire organism, it is conceivable that preservation of a health state during ageing is ensured by efficient tissue and organ crosstalk. Based on this hypothesis age-related health deterioration would stem from defective tissue and organ crosstalk. Molecules circulating in the bloodstream would be the main culprits: studies of their expression and of the pathways they target have the potential to provide information on the ageing process and healthy/non-healthy ageing trajectories [43]. Circulating extracellular nucleic acids were first described in the human bloodstream more than 50 years ago. However, quite recent evidence suggests that circulating RNA/DNA and their shuttles (exosomes and protein/lipoproteins) constitute a new efficient system for inter-tissue and inter-organ crosstalk as well as an integrated reservoir of information relating to all body tissues and organs. These biomarkers are expected to be informative, easily accessible, and cost-effective candidate biomarkers of the ageing process, enabling assessment of the health status of individuals both at the level of specific tissues/organs and at the systemic one [44]. Importantly, their different shuttles could affect the efficiency of delivery to target cells, hence their biological effects [57]. Rather than a single miRNA or cf-DNA sequence, combinations of molecules associated with specific shuttles (signatures) are expected to be relevant to the pathogenesis, prediction, diagnosis and prognosis of the major human ARDs. Based on these hypotheses and the integrated and complementary expertise of the General Pathology team, an integrated series of studies was conducted to test the ability of miRNA/cf-DNA signatures and their shuttles to be robust predictive/prognostic biomarkers of the most common ARDs, i.e. type 2 diabetes (T2DM), cardiovascular disease (CVD), tumors and neurodegenerative diseases [46]. We analysed existing sample collections from a number of case-control studies nested in cohort studies and longitudinal studies, including healthy individuals of different ages ranging from healthy young subjects to centenarians and their offspring, and non-healthy individuals ranging from obese adults to patients affected by CVD [47, 49, 50], T2DM [45, 58], and neurodegenerative diseases (AD, NAD). One of the clinically relevant result on circulating miRNAs concerns the diagnostic performance of miR-499-5p in acute non-ST elevation myocardial infarction (NSTEMI) in the elderly. Circulating miR-499-5p exhibited a diagnostic accuracy superior to that of troponin in patients with modest elevation at presentation [49]. Admission levels of circulating miR-499-5p were also associated with the risk of death in elderly patients after acute NSTEMI [47]. A recent systematic review confirmed the clinical relevance of miR-499-5p for diagnosis of cardiovascular disease [42]. Finally, we also investigated the effect of anti-aging treatments on miRNAs expression, both in vivo and in vitro. A study on postmenopausal monozygotic twin pairs showed that hormone replacement therapy was able to enhance IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions [48]. The antiinflammatory effect of ubiquinol-10 was analysed in endothelial cells showing that miR-146a modulation was induced by this treatment [51].

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Prof. Massimo Re, otolaryngology Unit, also made collaborative studies on expression levels and clinical significance of specific miRNAs (miR-21-5p, miRlet-7a, and miR-34c-5p) in Laryngeal Squamous Cell Carcinoma [61, 62], Ex vivo studies were paralleled to in vitro data on cellular models [52]. Our results suggest that miRNAs involved in the modulation of inflammatory processes and in the acquisition of “senescence phenotype” in senescent endothelial cells, fibroblasts and circulating cells may play a role in propagating cell senescence and inflammaging, thus being biomarkers of risk to develop the most common ARDs. In a paper published in 2013 we named these circulating miRNA-based signatures as “inflamma-miRs” [53]. In addition to miRNAs, we analysed a number of senescence associated biomarkers, i.e. telomere length [5, 7, 38, 53, 55, 56, 74], telomerase activity [55], RNAseH2 activity [71], beta-galactidase activity [69], DNA methylation status [4], M1/M2 circulating balancing [18] and n-glycomic changes in serum proteins [75]. The results of these studies, conducted on healthy subjects of different age and in patients affected by ARDs, support the view that the most common ARDs are characterized by an “accelerated aging phenotype”. Increasing attention is currently devoted to vesicles released by living cells in the bloodstream, that can vary in size, origin and content. The best characterized vesicles are those derived from plasmatic membrane blabbing, named micro-vesicles (MVs) and those derived from multi-vesicular bodies (MVBs), named exosomes (EXs). EXs and MVs can represent a cleaner source of information, since circulating MVs-packaged information (i.e. miRNAs, proteins, DNA fragments) in addition to their role as biomarkers, act as functional mediators being involved in the cellular crosstalk. The informative potential, role and specificity of circulating miRNAs/cfDNA, and their shuttles required a comprehensive experimental design involving a highly integrated ex vivo and in vitro approach [54, 57]. We are currently studying the cargos composition of exosomes and microvescicles circulating in plasma of patients affected by the most common age-related diseases (i.e. cancer, CVD and T2DM) and comorbidities (hypertension, frailty), as well as vesicles-based signatures released by senescent cells (endothelial cells, cancer cells, fibroblasts), in order to identify “senescence-associated vesicle profiles” that could represent an innovative approach of “liquid biopsy” in the framework of aging. Another recently intriguingly hypothesis is that cytoplasmic (cy) and cell-free (cf) DNA pools trigger inflammation and innate immunity at local and systemic level. In particular, cyDNA could play a crucial role in the phenomenon of cell senescence and in the cognate pro-inflammatory secretome [72]. Since epigenetic modifications are reversible, affecting gene expression without altering the DNA sequence, recently we activated a line of research aimed to assess the impact of lifestyles, such as exercise and diet, on aging processes [2, 3, 34, 63]. Although in vivo detection and targeting of senescent cells are still being investigated, it is likely that therapeutic strategies based on antioxidant and anti-inflammatory compounds would involve generalized anti-aging effects [59]. Although considerable work is still required to explore the toxicity profile and the extent of the benefits

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provided by natural and synthetic anti-aging compounds, nutritional approaches are expected to provide new tools to combat ARDs. Under the guidance of Prof. Maria Rita Rippo, the research group has also highlighted mechanisms of resistance to programmed cell death of senescent cells and on those that regulate the differentiation of mesenchymal stromal cells (MSCs) which show alterations in their proliferative and differentiating capacity in elderly [77]. In particular, for the first time we suggested and demonstrated that mito-miRs (mitochondria resident or associated to mitochondrial membranes) are modulated and can control expression of Bcl-2 family members and the oxidative and energetic status in aging cells thus contributing to their resistance to pro-apoptotic stimuli, pro-inflammatory phenotype and altered autophagy [29, 30, 67]. Furthermore, our group demonstrated that FasL, whose circulating levels decrease progressively during aging [36], is modulated in post-menopausal women [37] and play a pivotal role in bone homeostasis inhibiting MSCs differentiation in adipocytes and inducing their proliferation [66]. These data are interesting in the light of the imbalance between adipocytes, osteoblasts and MSCs existing within the bone marrow of aged people suffering of osteoporosis.

4 Future Perspective The desire of eternal youth seems to be as old as mankind, as documented in ancient literature. Organ and tissue loss through disease and injury motivate the effort to develop therapies that can regenerate tissues and decrease reliance on transplantations. Regenerative medicine can potentially restore diseased and injured tissues and whole organs. We plan first to slow the aging processes and postpone the development of the most common age-related diseases, and after to push forward humanity lifespan. We do not know where the next fifty years would bring our scientific research. We already made the choice to track our technological path through Regenerative Medicine, Rejuvenation, Robotics, and Computational Pathology, and we are developing the skills needed for that. What is very clear to us is that in the past 50 years it was possible to build up a free, positive, and creative environment which our young colleagues begun to refer as “the school of Ancona of experimental pathology”. We are confident that within this environment, the new generation of scientists that here and abroad is blooming will very soon get the baton by the older one and lead the future with the same enthusiasm, love and commitment.

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68. Rivero F, Albrecht R, Dislich H, Bracco E, Graciotti L, Bozzaro S, Noegel AA (1999) RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes. Mol Biol Cell 10:1205–1219 69. Spazzafumo L, Mensà E, Matacchione G, Galeazzi T, Zampini L, Recchioni R, Marcheselli F, Prattichizzo F, Testa R, Antonicelli R, Garagnani P, Boemi M, Bonafè M, Bonfigli AR, Procopio AD, Olivieri F (2017) Age-related modulation of plasmatic beta-galactosidase activity in healthy subjects and in patients affected by T2DM. Oncotarget 8(55):93338–93348 70. Spazzafumo L, Olivieri F, Abbatecola AM, Castellani G, Monti D, Lisa R, Galeazzi R, Sirolla C, Testa R, Ostan R, Scurti M, Caruso C, Vasto S, Vescovini R, Ogliari G, Mari D, Lattanzio F, Franceschi C (2013) Remodelling of biological parameters during human ageing: evidence for complex regulation in longevity and in type 2 diabetes. Age (Dordr). 35:419–429 71. Storci G, De Carolis S, Papi A, Bacalini MG, Gensous N, Marasco E, Tesei A, Fabbri F, Arienti C, Zanoni M, Sarnelli A, Santi S, Olivieri F, Mensà E, Latini S, Ferracin M, Salvioli S, Garagnani P, Franceschi C, Bonafè M (2019) Genomic stability, anti-inflammatory phenotype, and up-regulation of the RNAseH2 in cells from centenarians. Cell Death Differ. https://doi. org/10.1038/s41418-018-0255-8 72. Storci G, De Carolis S, Olivieri F, Bonafè M (2018) Changes in the biochemical taste of cytoplasmic and cell-free DNA are major fuels for inflamm-aging. Semin Immunol 40:6–16 73. Strizzi L, Catalano A, Vianale G, Orecchia S, Casalini A, Tassi G, Puntoni R, Mutti L, Procopio A (2001) Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol 193:468–475 74. Testa R, Olivieri F, Sirolla C, Spazzafumo L, Rippo MR, Marra M, Bonfigli AR, Ceriello A, Antonicelli R, Franceschi C, Castellucci C, Testa I, Procopio AD (2011) Leukocyte telomere length is associated with complications of type 2 diabetes mellitus. Diabet Med 28:1388–1394 75. Testa R, Vanhooren V, Bonfigli AR, Boemi M, Olivieri F, Ceriello A, Genovese S, Spazzafumo L, Borelli V, Bacalini MG, Salvioli S, Garagnani P, Dewaele S, Libert C, Franceschi C (2015) N-glycomic changes in serum proteins in type 2 diabetes mellitus correlate with complications and with metabolic syndrome parameters. PLoS ONE 10:e0119983 76. Tomasetti M, Rippo MR, Alleva R, Moretti S, Andera L, Neuzil J, Procopio A (2004) Alphatocopheryl succinate and TRAIL selectively synergise in induction of apoptosis in human malignant mesothelioma cells. Br J Cancer 90:1644–1653 77. Wilson A, Shehadeh LA, Yu H, Webster KA (2010) Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells. BMC Genom 11:229

Histological Contamination in Clinical Research—from Ultrastructure to Stem Cell Biology Monica Mattioli Belmonte, Monia Orciani, Antonio Gigante, Guendalina Lucarini, Giancarlo Balercia, Giorgio Arnaldi and Roberto Di Primio Abstract Histological studies born in Ancona as morphological ultrastructural investigations. The ability to correlate structure and function concomitantly with the development of bio-molecular technologies was an important step forward for the scientific growth of the histological group. Starting from studies on tissue regeneration, which focused on cells, engineered material biocompatibility and appropriate biochemical factors interrelationship, researches were directed both to the study of wound healing and to deepen our knowledge on adult mesenchymal stem cells behaviour in normal and in pathological conditions. Knowledges of the group were important starting point for fruitful collaborations with clinicians.

1 Histology at Glance Histology is a science on development, structure and functions of cells, tissues and organs, and is a fundamental part of medical education. Originally born as a morphological science, at present it consists of different interrelated aspects like: the study of development, structure and functions of cells (cytology), the investigation of rules and mechanisms of embryonic development (embryology), the studies how to make and explore histological preparations with help of a microscope, and the studies of development, structure, functions and reactive changes in tissues (histology) as well in various organs (microscopic anatomy). Microscopy allows the study of an organism on different levels: (i) Subcellular—studying ultramicroscopic features of cellular structures with Transmission and Scanning Electronic Microscopy; (ii) Cellular—evaluating structure and reactive M. M. Belmonte (B) · M. Orciani · G. Lucarini · R. Di Primio Section of Histology, DISCLIMO, Università Politecnica Delle Marche, Ancona, Italy e-mail: [email protected] A. Gigante Clinic of Orthopedic, DISCLIMO, Università Politecnica Delle Marche, Ancona, Italy G. Balercia · G. Arnaldi Division of Endocrinology and Metabolic Diseases, DISCLIMO, Università Politecnica Delle Marche, Ancona, Italy © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_5

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changes of cells with light microscope techniques; (iii) Tissue—studying structures, functions and development of a whole tissue; and (iv) Organ—analyzing microscopic structure and functions of various organs. The father of Italian Histology is Giulio Bizzozero (1846–1901) who attracted around him many collaborators, including Camillo Golgi (1843–1925).

2 Histological Contamination at UNIVPM In Ancona, the teaching of Histology born as an extension of the Histological and Anatomical School of the University of Bologna represented by Prof. Carlo Rizzoli, Dean of the Alma Mater from 1976 to 1985, and his eminent pupil Prof. Francesco Antonio Manzoli. Since the opening of the Medical School, several Professors have succeeded in teaching Histology and, starting from 1991, the section of Histology has been definitively established, with full Professor, Researcher and Technician. From then, several PhD students, Research fellows and Research Assistants have scientifically grown within the group, letting them to find suitable placements within University and/or in different research areas. Histological expertise provided important cues for different Institutions within the School of Medicine of Ancona, with particular reference to the Orthopedic Clinic and the Division of Endocrinology and Metabolic Diseases. The staff of the Clinic of Orthopedics is a branch of the Catholic University in Rome. Prof. Francesco Greco has been the head of the academic School in Ancona from 1991 to 2012. Then Prof. Luigi De Palma first and, at present, Prof. Antonio Gigante and Prof. Nicola Specchia have succeeded in clinical and teaching activities as well as in research focusing on shoulder surgery, joint reconstructive surgery, orthopaedic trauma and fractures, ankle surgery and spine surgery. Since the opening in 1992 of the Division of Endocrinology and Metabolic Diseases and of the School of Specialization in Endocrinology, several Professors (Prof. Roberto De Pirro, Prof. Franco Mantero, Prof. Marco Boscaro and, at the present, Prof. Giorgio Arnaldi and Prof. Giancarlo Balercia) have succeeded in teaching, clinical activities and research focusing on hypothalamic-pituitary-adrenal axis and andrology. One of the most important chapters was written in October 2002, when a workshop was held in Ancona to reach a Consensus Statement on the diagnosis and management of Cushing’s syndrome, a complex clinical condition resulting from prolonged and inappropriately high exposure of tissues to glucocorticoids [1].

3 Histology and Research The research activity of the histological group of Ancona was initially based on ultrastructural evaluations of several different pathologies in collaboration with the clinical institutions of the University of Ancona and Bologna.

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One of the topics that gradually took hold in the research was related to Tissue Engineering. This discipline using a combination of cells, engineering materials, and suitable biochemical factors to improve or replace tissues and organs biological functions, well fits with the foundations of histology as well as with the skills of the research group. The interdisciplinary of the tissue engineering approach enhanced the collaborations with other national and international Research Institutions and the obtainment of Grants from the Italian Ministry for Research and University, as well as from the European Community. Up to now, the scientific activity of the histological group has increased, evaluating different aspects of cell and tissue behaviours. From the initial studies on tissue engineering, biomaterials and biocompatibility, mainly related to musculoskeletal system, researches were directed to the study of adult mesenchymal stem cells derived from different sources, both in normal and in pathological conditions. Moreover, in order to deep the knowledge in appropriate regenerative approaches, histological analyses on wound healing assessment have been exploited.

3.1 Bone Tissue Regeneration The rising incidence of bone disorders and the increase in ageing population caused the need of more effective strategies to meet this request. Bone tissue engineering approaches, combining biomaterials, cells and signaling factors, represent alternatives to conventional bone grafts for repairing or rebuilding bone defects. It must be underlined that skeletal tissue engineering has not yet achieved complete translation into clinical practice because of several challenges. Bone has the capability to self-repair. Postnatal bone preserves an intrinsic ability for well-ordered growth, remodeling to satisfy mechanical needs and renewal after damages. However, in large bone defects this ability can fail, resulting in long-lasting defects that can determine a loss of function and bone regenerative ability declines with age. In bone defect treatments, the “gold standard” remains bone grafting even if this strategy may show advantages and disadvantages related to the different nature of bone grafts. When using new therapeutic options in tissue engineering strategies, the inherent reparative capacity of bone grafts represents the natural model to reproduce [36]. Suitable scaffolds, growth factors [16, 26], and/or cells, has, in some cases, improved grafts incorporation, osteoconductivity, osteoinductivity and osseointegration [13]. Scaffolds must provision cell colonization, proliferation, migration and differentiation as well as possess appropriate physicochemical properties crucial for tissue formation [24]. As material scaffold, biological polymers (e.g. collagen and hyaluronic acid) were studied for bone tissue engineering, since they furnish to cells innate biological informational guidance thus favoring cell adhesion and promoting their responses in term of proliferation and differentiation. Ultrastructural and immunohistochemical investigation evidenced that cell behavior is affected by scaffold material chemical

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Fig. 1 Ultrastructural images of cell-biomaterial interaction

properties [16] (Fig. 1). Since concern exists on the potential risk of disease transmission and biological polymer weak mechanical properties, synthetic polymers have been suggested as valid alternatives. Among these, polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (PLGA) [27], and polycaprolactone (PCL) [19] have been studied. Different traditional techniques have been used to generate a range of three-dimensional scaffolds with different porosities and surface and tested by means of in vitro cultural or co-cultural approaches. For mimicking the mineral phase of bone, bioactive inorganic materials (e.g. tricalcium phosphate, HA and bioactive glasses) have been integrated into the natural [42] or synthetic [15] organic phase and tested with different in vitro and ex vivo approaches, to evaluate their role in bone regeneration both in normal and in pathological conditions, such as osteoporosis [14]. Cells are commonly used to repair injured tissue, as they are physiologically involved in tissue development and homeostasis. Osteoblasts (or their precursors)

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and osteocytes, which are the main controller of bone deposition, modelling and remodeling, are an outstanding cell font for an effective cell-based skeletal treatment. The use of Mesenchymal Stromal Cells (MSCs) into bone tissue engineering strategies has been a crucial progress and apart from common sources of MSCs, such as bone marrow and fat, alternative fonts have been explored. A striking stem cell reservoir, meeting bone tissue engineering criteria, is the skin basal layer [30]: these cells, called skin-derived multipotent stromal cells (S-MSCs), are capable to become adipocytes, osteoblasts, chondrocytes, neurons, and pancreatic cells [38]. As the growth, development, and regeneration of bone as well as cartilage rest on periosteum presence, researches have also been devoted to the evaluation of MSCs derived from periosteum (PDSCs). Evaluation of their differentiation ability in response to mechanical (stiffness) and chemical (e.g. growth factors, hydroxyapatite-HA) stimuli [9, 12, 24] have been performed. Results of these experimentation highlighted the role of stiffness in triggering the expression of osteogenic genes in PDPCs and of HA in accelerating the process [24] (Fig. 1). As far cell use in bone tissue engineering strategies is concerned, an intriguing aspect of investigation was related to possible changes due to donor age and replicative senescence in term bone modeling and remodeling [11, 43]. The study on age-related changes in human periosteal precursor cells evidenced changes that need to be considered in the development of regenerative medicine strategies in bone aging and/or bone metabolic diseases [11]. Moreover, overall the findings suggested the use of only early-passage PDPCs for bone regenerative approaches based on the local recruitment of stem cells. On the contrary, the later cell passages, obtained by PDPCs subculturing, could represent a suitable in vitro tool to investigate new scaffolds intended for bone regeneration in elderly [43]. A further aspect to be considered when creating a viable bone construct is angiogenesis. In this respect, two different strategies were investigated: one explored the use of two cell types in a bioreactor [19], whilst the other looked to the use of vascular endothelial growth factor (VEGF), a master player in angiogenesis during bone development. Studies on synergistic or cumulative consequence of VEGF and of Insulin-like Growth Factor (IGF) evidenced that these factors determine a different MAPK or PI3K/AKT signaling pathway activation in relation to the origin of stem cells, thus causing dissimilar responses [9, 12]. These observations open new insight in the development of appropriate strategies in bone regeneration. Rapid Prototyping (RP), developed in the mid-1980s, has received increasing attention for the development of a high personalized and cost-effective medical therapy. RP provides a high level of control of the scaffold architecture, flexibility to scale-up fabrication, assure tuning and reproducibility of the manufacturing process. RP permits the set-up of 3D objects by means of data generated by computer-assisted design (CAD) software or imported from clinical 3D scanners such as X-ray computed tomography (CT), magnetic resonance imaging (MRI). The CAD model is then transformed to a standard tessellation language (STL) file that guides the 3D-printer computer system to generate layer-by-layer the object [39]. In this respect PressureActivated microsyringe (PAM) fabrication, a peculiar microextrusion technique, has been used to modulate different cell cytotype behavior in response of topological

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features [27] and to generate bioactive glass–poly(lactic-co-glycolic acid) (PLGA) scaffolds reflecting the topological characteristics of cancellous bone [26]. Based on the results of this study, composite PAM scaffolds could find applications for the development of in vitro bone tissue models as well as in tissue engineering and/or regenerative medicine approaches [37].

3.2 Mesenchymal Stem Cells In the last decades, the use of MSCs deserved increasing attention by researchers because of their pleiotropic properties. As mentioned above, MSCs can directly differentiate into specialized cells and be used to reconstitute damaged tissues or can induce the differentiation of other cell types by secreting active soluble factors able to modulate the cell fate and the microenvironment (paracrine effect). Microenvironment plays a crucial role in the behavior of MSCs and contradictory results have been reported about MSCs involvement in pathologies characterized by an inflamed microenvironment [35]. In some diseases, MSCs counteract inflammation by secreting anti-inflammatory cytokines and promoting the involvement of immune cells; in others, MSCs sustain inflammation (that becomes chronic), interfere with the host immune surveillance and enhance disease development. In this intricate scenario, the effect of inflammation on MSCs behavior in selected pathologies has been evaluated, allowing the creation of a strong collaboration with the clinics of Endocrinology, Plastic and Reconstructive Surgery, Dermatology, Obstetrics and Gynecology and Thoracic Surgery (Fig. 2). Psoriasis is an immune-mediated inflammatory disease (IMID) involving skin and is characterized by epidermal hyperproliferation, abnormal keratinocyte differentiation, angiogenesis and an imbalance between Th1/Th17 and Th2 cytokines [34]. While the involvement of keratinocytes was well established, little was known about MSCs participation. Their role has been assessed and specified during eight research articles that have gradually confirmed as MSCs are involved in psoriasis onset and development. MSCs from psoriatic skin secreted dysregulated amount of VEGF, iNOS and Th1–Th17 cytokines [2–4, 6] and were responsive to the biologic used drugs [4–6]. All these data suggested that MSCs probably represent the cells primarily enrolled in the “psoriatic march”. Interestingly, it was found that, in indirect co-culture system, healthy MSCs were able to restore the dysregulated cytokines profile of psoriatic MSCs, driving them towards a condition nearer to the physiological one, enforcing the evidence that psoriasis is not a skin disease but an immune pathology [7] (Fig. 2). The involvement of MSCs has been then evaluated in other cutaneous diseases, such as atopic dermatitis (AD) [33] and hidradenitis suppurative (HS) [8]. Mesenchymal stem cells isolated from the skin of patients with AD or HS were activated toward an inflammatory status and this imbalance between proinflammatory and anti-inflammatory activities of MSCs could favor the hypothesis of their pathogenic involvement in these diseases [10]. Inflammation is a distinctive marker also in leiomyoma onset [31] and in Cervical Intraepithelial Neoplasia (CIN) [32]. These

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Fig. 2 Isolation of MSCs from different adult tissues, their characterization and identification of dysregulated pathways in selected pathologies

pathologies affect the female reproductive system increasing infertility. Although the etiology of leiomyoma is unclear, a progenitor/undifferentiated cell population has been described whose dysregulation may be involved in the onset of uterine conditions under an inflamed stimulus. MSCs isolated from healthy myometrium and leiomyoma showed a differential expression of cytokines related to acute and chronic inflammation; in detail, it was observed an upregulation of cytokines related to chronic inflammation in leiomyoma progenitors that could promote the formation of a microenvironment suitable for leiomyoma onset and development. The effects of the paracrine effect exerted by MSCs were strongly evident on CIN, whose regression rate is age-related. MSCs from cervixes of young and old women secreted different

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amounts of cytokines related to acute/chronic inflammation; in addition, in an indirect co-culture system with cervix cancer cells, MSCs from young patients displayed a stronger anti-tumoral effect than MSCs from older women. The dark side of the MSCs is represented by Cancer Stem Cells (CSCs), undifferentiated cells able to drive the onset and development of tumors. At the beginning, the CSCs theory was referred only to cancer. Only many years later, it has been enlarged also to benign tumors [44]. In addition, by observing the presence of CSClike cells in tumors of epithelial origin, a new scenario has been proposed: MSCs in epithelial tumors may derive by Epithelial to Mesenchymal Transition (EMT). Recent studies have highlighted a link between EMT and cancer stem cells (CSCs) formation [20]. Using a mammary tumor progression model [27], it has been shown that cells possessing both stem and tumorigenic characteristics can be derived from human mammary epithelial cells by EMT induction [28]. In this light, the presence of MSCs and their response to drugs becomes of primary concern. In collaboration with the Clinic of Endocrinology, the involvement of MSCs in benign tumors as well as their response to traditional drug has been evaluated. As tumor model, pituitary adenoma (both GHoma and not-functioning pituitary adenomas, NFPA) were chosen. Interestingly, MSCs from GHoma and NFPAs expressed elevated amounts of genes/proteins refereed to EMT, suggesting the involvement of this mechanism in their onset. Since traditionally drugs often failure tumor healing because do not affect CSCs, the effect of somatostatin analogs (SSAs) on proliferation and receptor expression was evaluated in MSCs derived from GHoma and NFAPs [36]. SSAs, currently used in the management of human GHomas, exert antiproliferative effect also in MSCs that, because of their derivation from CSCs, may be a new meaningful target for drugs treatment [33].

3.3 Wound Healing The dynamics of wound healing are complicated. The in-depth understanding of the normal healing process represents an essential prerequisite to appreciate the pathology and develop suitable regenerative strategies. Several instruments have been described for clinical assessment of the wound but, as also evidenced by recent literature [17], the role of the histological examinations for grading of wounds as well as for the evaluation of possible therapeutic outcomes is emerging. Over time, several studies have been conducted and many wound healing models proposed to evaluate the healing process and standardize the semi-quantitative and quantitative evaluation of selected parameters of wound healing. Initial studies were performed since 1997, exploiting the role of chitosan and chitin derivatives [25, 29]. The use of these molecules was experienced both in experimental animal models and as wound medicaments in human ulcers. Obtained results were pioneering in this field.

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More recently, periodontal lesion restoration and cutaneous wound healing, with greater attention to burns, have been the subjects of investigations. Healing morphological features (basic components including angiogenesis, inflammation, fibroplasia and restoration of the connective tissue matrix, wound contraction and remodeling, epithelialization and differentiation) [18, 21] as well as the expression of tissue regeneration markers were assessed. The periodontal regeneration involves periodontal fiber development and angiogenesis to generate a new connective tissue; in addition, the tissue inflammation is a highly ordered process that significantly impacts wound healing outcomes. CD133 expression, a universal marker for tissue stem/progenitor cells and an important regulator of angiogenesis, proliferation, apoptosis and inflammation, was analyzed in gingival biopsies affected by chronic periodontitis before periodontal treatment with Demineralized Freeze-Dried Bone Allograft and correlated with periodontal tissue response in the same site at 12 months post-surgery. The findings evidenced that gingival CD133 expression could represent a predictive marker for promising periodontal healing since it correlated with clinical attachment level (CAL) gain achievable in intrabony defects management with bone substituted [22]. Many of the studies have also assessed the expression of some angiogenesis markers, commonly associated with inflammation in peri-implant mucositis and periimplantitis. As stated before, angiogenesis plays an important role in osseointegration process by contributing to inflammatory and regenerative phases of surrounding alveolar bone. Obtained results supported that peri-implantitis is characterized by unique and distinctive features and peri-implant pocket depth has a great impact, being closely related to the inflammation marker expression (VEGF, CD44). Overall the identification of specific biomarkers, by means of histological and immunohistochemical techniques might help in choosing an ad hoc individual treatment [23]. Age-related differences in cutaneous wound healing have been reported but few information is available for wound healing after burn injury. Histological and immunohistochemical studies evaluating changes in the expression of matrix metalloproteinase (MMP-9), collagen IV, K6 and CD44 in the burn wound healing of an experimental animal model, evidenced a delayed process in aged animals. It was hypothesized that the age dependent response to injury were controlled by these factors and therefore their appropriate modulation could lead to a better and faster recovery of skin damage in elderly [40] (Fig. 3). At last, histology can also be a powerful tool in the evaluation of the effect of novel drugs on wound healing, providing information on the usefulness of combination therapy. In particular, the histological investigations evaluated the effects of some new antimicrobial peptides on the healing of cutaneous wounds affected by methicillinresistant Staphylococcus aureus [41].

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Fig. 3 Wound healing histological evaluation and MMP9 immunohistochemical expression (chronic wound healing marker) in burn wounds of aged and young rats

4 Conclusions Ultrastructural morphological studies have been the fundament of first investigations. The start of the bio-molecular technologies represented an important step forward in the scientific growth of the research group. This allowed the understanding of the different mechanisms involved in the regeneration of tissues, in the disease onset and in wound healing. Knowledges obtained from the scientific activity of the histological group and represented by the integration of ultrastructural observations with biofunctional data, represented a key point for the integration with clinical research needs.

References 1. Arnaldi G, Angeli A, Atkinson AB et al (2003) Diagnosis and complications of Cushing’s syndrome: a consensus statement. J Clin Endocrinol Metab. https://doi.org/10.1210/jc.2003030871 2. Campanati A, Consales V, Orciani M et al (2017) A role of mesenchymal stem cells in the pathogenesis of psoriasis: current perspectives. Psoriasis (Auckl). https://doi.org/10.2147/ptt. s108311.25 3. Campanati A, Orciani M, Consales V et al (2014) Characterization and profiling of immunomodulatory genes in resident mesenchymal stem cells reflect the Th1–Th17/Th2

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imbalance of psoriasis. Arch Dermatol Res. https://doi.org/10.1007/s00403-014-1493-323 4. Campanati A, Orciani M, Ganzetti G, Consales V, Di Primio R, Offidani A (2016) The effect of etanercept on vascular endothelial growth factor production by cutaneous mesenchymal stem cells from patients with psoriasis. J Int Med Res 44(6–9):24 5. Campanati A, Orciani M, Gorbi S, Regoli F, Di Primio R, Offidani A (2012) Effect of biologic therapies targeting tumour necrosis factor-α on cutaneous mesenchymal stem cells in psoriasis. Br J Dermatol. https://doi.org/10.1111/j.1365-2133.2012.10900.x.27 6. Campanati A, Orciani M, Lazzarini R et al (2017) TNF-α inhibitors reduce the pathological Th(1)–Th(17)/Th(2) imbalance in cutaneous mesenchymal stem cells of psoriasis patients. Exp Dermatol. https://doi.org/10.1111/exd.1313926 7. Campanati A, Orciani M, Sorgentoni G et al (2018) Indirect co-cultures of healthy mesenchymal stem cells restore the physiological phenotypical profile of psoriatic mesenchymal stem cells. Clin Exp Immunol. https://doi.org/10.1111/cei.13141.28 8. Campanati A, Orciani M, Sorgentoni G, Consales V, Offidani A, Di Primio R (2018) Pathogenetic characteristics of mesenchymal stem cells in hidradenitis suppurativa. JAMA Dermatol. https://doi.org/10.1001/jamadermatol.2018.2516.30 9. Dicarlo M, Bianchi N, Ferretti C, Orciani M, Di Primio R, Mattioli-Belmonte M (2016) Evidence supporting a paracrine effect of IGF-1/VEGF on human mesenchymal stromal cell commitment. Cells Tissues Organs 201:333. https://doi.org/10.1159/000445346.14 10. Elinav E, Nowarski R, Thaiss CA, Hu B, Jin C, Flavell RA (2013) Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms. Nat Rev Cancer. https://doi.org/ 10.1038/nrc361121 11. Ferretti C, Lucarini G, Andreoni et al (2015) Human periosteal derived stem cell potential: the impact of age. Stem Cell Rev. https://doi.org/10.1007/s12015-014-9559-3.15 12. Ferretti C, Vozzi G, Falconi M et al (2014) Role of IGF1 and IGF1/VEGF on human mesenchymal stromal cells in bone healing: two sources and two fates. Tissue Eng Part A. https:// doi.org/10.1089/ten.13 13. Fini M, Giavarese G, Nicoli Aldini N et al (2000) The effect of osteopenia on the osteointegration of different biomaterials: histomorphometric study in rats. J Mater Sci Mater Med 11:1–7 14. Fini M, Giavarese G, Torricelli P et al (2001) Biocompatibility and osseonintegration in osteoporotic bone. A preliminary in vitro and in vivo study. J Bone Joint Surg 83:139–143 15. Gentile P, Mattioli-Belmonte M, Chiono V et al (2012) Bioactive glass/polymer composite scaffolds mimicking bone tissue. J Biomed Mater Res A. https://doi.org/10.1002/jbm.a.34205.8 16. Gigante A, Manzotti S, Bevilacqua C, Orciani M, Di Primio R, Mattioli-Belmonte M (2008) Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials. Eur J Histochem 52(169–74):6 17. Gigante A, Cappella M, Manzotti S et al (2010) Osteoinduction properties of different growth factors on cells from non-union patients: in vitro study for clinical application. J Biol Regul Homeost Agents 24:51–62 18. Gupta Akriti, Kumar Pramod (2015) Assessment of the histological state of the healing wound. Plast Aesthetic Res 2(5):239. https://doi.org/10.4103/2347-9264.15886238 19. Kyriakidou K, Lucarini G, Zizzi A et al (2008) Dynamic co-seeding of osteoblast and endothelial cells on 3D polycaprolactone scaffolds for enhanced bone tissue engineering. J Bioact Compatible Polym. https://doi.org/10.1177/0883911508091905.17 20. Liu X, Fan D (2015) The epithelial–mesenchymal transition and cancer stem cells: functional and mechanistic links. Curr Pharm Des 21(1279–91):34 21. Lucarini G, Tirabassi G, Zizzi A et al (2016) Uncoupling of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) in gingival tissue of type 2 diabetic patients. Inflammation 39(2):632–642. https://doi.org/10.1007/s10753-015-0288-9 22. Lucarini G, Zizzi A, Ferrante L, D’Angelo AB, Rubini C, Aspriello SD (2015) CD133 expression could be a predictive marker of periodontal regeneration. J Biol Regul Homeost Agents 29:663–669

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23. Lucarini G, Zizzi A, Rubini C, Ciolino F, Aspriello SD (2018) VEGF, microvessel density, and CD44 as inflammation markers in peri-implant healthy mucosa, peri-implant mucositis, and peri-implantitis: impact of age, smoking, PPD, and obesity. Inflammation. https://doi.org/10. 1007/s10753-018-0926-0.41 24. Mattei G, Ferretti C, Tirella A, Ahluwalia A, Mattioli-Belmonte M (2015) Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation. Sci Rep. https://doi.org/10.1038/srep10778.5-12 25. Mattioli Belmonte M, Gigante A, Muzzarelli R et al (1999) N, N-dicarboxymethyl chitosan as delivery agent for bone morphogenetic protein in the repair of articular cartilage. Med Biol Eng Comp 37(130–134):2 26. Mattioli-Belmonte M, De Maria C, Vitale-Brovarone C, Baino F, Dicarlo M, Vozzi G (2015) Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration. J Tissue Eng Regen Med. https://doi. org/10.1002/term.2095.20 27. Mattioli-Belmonte M, Vozzi G, Kyriakidou K et al (2008) Rapid-prototyped and salt-leached PLGA scaffolds condition cell morpho-functional behavior. J Biomed Mater Res A. https:// doi.org/10.1002/jbm.a.31483.19 28. Morel AP, Lie‘vre M, Thomas C et al (2008) Generation of breast cancer stem cells through epithelial–mesenchymal transition. PLoS One. https://doi.org/10.1371/journal.pone.0002888 29. Muzzarelli RAA, Mattioli-Belmonte M, Pugnaloni A, Biagini G (1999) Biochemistry, histology and clinical uses of chitins and chitosans in wound healing. EXS 87(251–64):39 30. Orciani M, Di Primio R (2013) Skin-derived mesenchymal stem cells: isolation, culture, and characterization. Methods Mol Biol. https://doi.org/10.1007/978-1-62703-330-5_21.10 31. Orciani M, Caffarini M, Biagini A et al (2018) Chronic inflammation may enhance leiomyoma development by the involvement of progenitor cells. Stem Cells Int. https://doi.org/10.1155/ 2018/1716246.31 32. Orciani M, Caffarini M, Lazzarini R et al (2018) Mesenchymal stem cells from cervix and age: new insights into CIN regression rate. Oxid Med Cell Longev. https://doi.org/10.1155/2018/ 1545784.32 33. Orciani M, Caffarini M, Sorgentoni G, Ricciuti RA, Arnaldi G, Di Primio R (2017) Effects of somatostatin and its analogues on progenitor mesenchymal cells isolated from human pituitary adenomas. Pituitary. https://doi.org/10.1007/s11102-016-0770-x.37 34. Orciani M, Campanati A, Caffarini M et al (2017) T helper (Th)1, Th17 and Th2 imbalance in mesenchymal stem cells of adult patients with atopic dermatitis: at the origin of the problem. Br J Dermatol. https://doi.org/10.1111/bjd.15078.29 35. Orciani M, Campanati A, Salvolini E et al (2011) The mesenchymal stem cell profile in psoriasis. Br J Dermatol. https://doi.org/10.1111/j.1365-2133.2011.10438.x.22 36. Orciani M, Davis S, Appolloni G et al (2015) Isolation and characterization of progenitor mesenchymal cells in human pituitary tumors. Cancer Gene Ther. https://doi.org/10.1038/cgt. 2014.63.36 37. Orciani M, Fini M, Di Primio R, Mattioli-Belmonte M (2017) Biofabrication and bone tissue regeneration: cell source, approaches, and challenges. Front Bioeng Biotechnol. https://doi. org/10.3389/fbioe.2017.00017. 1 38. Orciani M, Mariggiò MA, Morabito C, Di Benedetto G, Di Primio R (2010) Functional characterization of calcium-signaling pathways of human skin-derived mesenchymal stem cells. Skin Pharmacol Physiol. https://doi.org/10.1159/000270383.11 39. Quadrani P, Pasini A, Mattioli-Belmonte M et al (2005) High resolution 3D scaffold model for engineered tissue fabrication using a Rapid Prototyping Technique. Med Biol Eng Comput 43:1–4 40. Simonetti O, Lucarini G, Cirioni O et al (2013) Delayed wound healing in aged skin rat models after thermal injury is associated with an increased MMP-9, K6 and CD44 expression. Burns. https://doi.org/10.1016/j.burns.2012.09.013.42 41. Simonetti O, Lucarini G, Orlando F et al (2017) Role of daptomycin on burn wound healing in an animal methicillin-resistant Staphylococcus aureus Infection Model. Antimicrob Agents Chemother 61(9):43

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Half-Century Research and Teaching Activity in Anatomic Pathology Throughout the History of Technologic Innovation Gaia Goteri and Alessia Cimadamore

Abstract In the fifty years of our School of Medicine, the research activity in our Anatomic Pathology (AP) has successfully applied technological innovations in the continuous development of knowledge in the field of human diseases, focusing on neoplasms. This has allowed Anatomic Pathology to evolve from basic macroscopic and microscopic observation to a clinical science with diagnostic, prognostic and predictive role. Anatomic Pathology is nowadays a morpho-molecular discipline, in which the original approach is not lost, but is enriched by the current knowledge and is projected into the future. This review will cover the history of our scientific and teaching activity as seen throughout our technological evolution. The research activity that has allowed AP to obtain international recognition has been instrumental in our teaching of Pathology. Over the years, we have formed scores of pathologists and researchers. We are proud that some of our former students have achieved prestigious positions in international and national institutions.

1 The Role of Anatomic Pathology Anatomic Pathology (AP) distinguishes a myriad of diseases, encompassing degenerative, inflammatory, pre-neoplastic and neoplastic diseases, based on their appearance at macroscopic and histologic level. In health-care, the role of AP is crucial in the diagnosis and treatment of several diseases, in particular in tumors, as pathology is a unique specialty that successfully bridges the gap between basic science research and patient management. Research and teaching activities in the last fifty years in AP have been oriented to increase knowledge and to transmit new acquisitions to

G. Goteri (B) · A. Cimadamore Section of Anatomic Pathology, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Via Tronto 10/a, Torrette di Ancona, 60020 Ancona, Italy e-mail: [email protected] A. Cimadamore e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_6

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medical students, residents and doctorate students, in order to ameliorate the diagnostic approach of diseases, and consequently the therapeutic management and the definition of prognosis of patients. Subjectivity of the diagnostic interpretation is the major problem to efface in AP. We well know that morphologic evaluation alone is clearly subjective and prone to inter-observer and even intra-observer variability of interpretation. In some fields of pathology, diagnosis can be very complex. Diagnostic criteria derive from the experience accumulated in decades of shared discussion among expert pathologists. Pathologists have made great efforts during these years to reduce subjectivity by analyzing morphologic criteria, and creating reliable diagnostic algorithms and classification schemes. The efforts were also to update classification systems periodically, accordingly to the new acquisitions. With time, pathologists have described new provisional entities and they have studied collected series in order to confirm the reproducibility of the diagnosis. The research has focused on the continue revisions of the classifications of the old and new diseases. History of research activity in AP is, in other words, a history of a number of tempts to reduce subjectivity of interpretation adding objective features to a descriptive terminology, by using techniques from basic sciences and transforming the Surgical Pathology of last century to a modern Molecular Pathology of the future. On the other hand, diagnosis should be not only reproducible, but also clinically relevant, as subtle differences in morphology may not have a clinical significance. This aspect is particular important in health-care where the diagnosis derives often from an efficient clinicopathologic correlation. In peculiar fields of pathology, clinical and morphological features need to be integrated for the final diagnosis. In cutaneous lymphoproliferative disorders evaluation of clinical lesions together with the histologic features is essential for the correct interpretation (Fig. 1).

2 Technological Innovation in Anatomic Pathology In this chapter, we intended to cover our research and teaching work in the last fifty years through the history of the technological innovation we experienced; at the same time, through this analysis, we will explore the possibility of future developments of our discipline. The first technical resource we applied in our research activity was Computational Analysis, followed by antigen expression evaluated by Immunohistochemistry (IHC) and later on by analysis of genetic abnormalities revealed by Molecular Techniques. All these techniques allowed us to produce objective and quantitative data useful for disease diagnosis and prognostication. They have been the methods of hundreds of research papers. This research activity has allowed our institution to obtain international recognition and to improve our teaching activity. Over the years, we have formed scores of pathologists and researchers. We are proud that some of our former students have achieved prestigious positions in international and national institutions.

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Fig. 1 Clinico-pathologic features if early mycosis fungoides. Clinical presentation with cutaneous patches with variable dimension in the trunk (a); cerebriform lymphocytes invading the epidermis (b); immunoreactivity for CD4 (c) and negativity for CD8 (d)

2.1 Computational Analysis Knowledge of histologic features in Anatomic Pathology has traditionally been based on visual examination of microscopic images, and traditionally been communicated by descriptive, linguistic terms and by photographic materials. There has always

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been a weak link between visual knowledge and linguistic description, due to the uncertainties involved in their correspondence. Prof. R. Montironi and collaborators performed several studies by applying computational analysis as an integration of diagnostic imagery, computed image information and linguistic descriptive terms, and became an international leader in this field [1, 2]. The studies were founded on the hypothesis that a computer graphic enhancement in the image—e.g. in color in the image, could clearly show what the linguistic assessment of a diagnostic feature would mean; moreover, a very large image database of examples could be instantly retrieved and displayed for comparison. This certainly could convey a comprehensive impression of what the linguistic assessment encompassed in terms of appearance in the image. In the very same manner, a quantitative numeric definition of a diagnostic clue could be established such that the traditional visual diagnostic knowledge, the delineation of diagnostic clues—including their variance—and the corresponding numeric characterization were integrated, all to reduce to a minimum the uncertainties associated with linguistic description. In practice, the symbolic information used in diagnostic decisionmaking was systematically ordered, compared, numerically assessed in its probability, and combined such that a conclusion could be drawn. The framework for the processing of such symbolic information was an expert system, an inference network or a case-based reasoning system. Automated reasoning was implemented by the use of a rule base and information flow control modules. Automated reasoning allowed decision support systems to follow highly adaptive decision sequences, capable of handling contradictory evidence, exceptions in diagnostic clue expression, and non-monotonic decision-making. Computational analysis allowed the construction of Bayesian belief networks useful for distinction of morphologically blurred or not completely defined categories. For example, one of them appeared useful for the differential diagnosis of neoplastic and atypical prostatic glandular proliferations [3]. From other studies some morphologic features appeared to be relevant, as nuclear and nucleolar enlargement in prostate expressed by morphometry. From these studies, we acquired the knowledge that normal-looking ducts and acini from prostate harboring pre-neoplastic and neoplastic lesions showed morphological nuclear abnormalities that were not seen by the human eyes, but that can be detected with image analysis. Such changes might be of diagnostic importance, especially in cases where clinical suspicion for cancer prevails after a negative biopsy [4]. Important data regarding differences in morphometric features between incidental and clinically detected prostatic carcinomas supported their biological differences [5]. In renal clear cell carcinomas, nuclear features analyzed by morphometry were related to the four Fuhrman grades used for the classification of these neoplasms [6]. Scores of medical students, residents and doctorate students received accumulating knowledge in computational analysis in genito-urinary pathology, when actively participated to training and in research finalized to their academic career.

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2.2 Immunohistochemistry Starting from the eighties, immunohistochemistry (IHC) has been fundamental to acquire knowledge of tissue expression of molecules recognized by an immune reaction between cellular components and specific antibodies conjugated with enzymatic systems. The unique feature that makes IHC standing out among many other laboratory tests is that it does not destroy histologic architecture, and thus the assessment of an expression pattern of the molecule is possible in the context of a microscopic examination. Initially IHC was applied only to frozen tissues, limiting its use to samples received fresh. When antibodies working on formalin-fixed and paraffinembedded tissues became available, the technique was largely applied to archive material allowing large studies on series with long follow-up. Our laboratories were equipped with instruments regularly updated, capable of automated staining and with significant high-throughput. These technical achievements allowed us to study extensively the archive material in retrospective studies, enlarging the number of our case series and the relevance of achieved results. These technologic achievements have contributed to develop successfully different pathology subspecialties—in particular, in neoplasms from the genito-urinary, central nervous system, head and neck, breast, and hematopoietic organs. Technological update of IHC has improved significantly in the last years staining interpretation. Through this continuously updated technique, we have been able to acquire a better understanding of the pathological processes observed in the tissues under the microscope as IHC specifically visualizes distribution and amount of a certain molecule in the tissue using specific antigen-antibody reaction. Investigations on tissue antigens aimed to find correlation useful for diagnosis and prognosis in several tumors. Several different types of antigens were object of analysis, i.e. related to cell proliferation, cell differentiation, stromal invasion, development of vascular support to tumor cells and to apoptotic death. The applications of IHC have expanded explosively as basic science discovered more and more molecules related to the pathogenesis, diagnosis, and treatment of diseases. Evaluation of proliferative activity through Ki67 antigen expression has been used for prognostication in several malignant tumors [7–9]. In tumors of the hematopoietic and lymphatic systems, IHC has been crucial for the differential diagnosis among lymphomas sharing many similarities in clinical presentation and morphology, but substantially different in biologic course and prognosis [10]. It has also been applied to study the microenvironment cell associated with lymphomas, exploring the role of immune escape and immune control in lymphomas (Fig. 2) [11, 12]. Nowadays, as we entered in the era of precision medicine, the pathologist has the potential to analyze precisely both the target molecule with IHC and its subcellular, cellular, and intercellular relation, and these parallel analyses are particular important in biomedical research fields, such as new drug development and prognostic/predictive biomarker investigation. Survival data obtained from clinical trials shape the cut-offs and IHC scoring that serve as recommendations for patient selection both for targeted and conventional therapies. Examples of this important

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Fig. 2 Evaluation of CD1a+ dendritic cells in early mycosis fungoides by immuno-histochemistry as prognostic parameter [11]

application are the studies on breast carcinoma, where assessment of Estrogen and Progesterone Receptors along with HER2 status has been among the first approved immunostaining assays revolutionizing breast cancer treatment [13]. In more recent years, Programmed Death Ligand 1 (PD- L1) IHC assays have been approved as companion or complimentary diagnostic tools predicting the response to checkpoint inhibitors. Anti-PD-L1 and anti-PD-1 monoclonal antibodies have inaugurated a new period in the treatment of advanced cancers, but the path to approval of these biomarkers is filled with immuno-histochemical challenges [14]. Many students who participated actively to research projects during their academic career, received information regarding the importance of immunohistochemistry in AP and became familiar with both the morphologic and phenotypic features of pathological tissues.

2.3 Molecular Technologies Currently, we have entered in the era of molecular technology, applied mainly to nucleic acids. Nucleic acids can be extracted easily from archival pathology tissue specimens, cytological samples, liquid biopsies, and to patient-derived tumor models.

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There are currently a number of tissue-based molecular tests used in pathology practice, which had been under our investigations before the introduction in routine pathology. In some subspecialties of Pathology, molecular diagnostics has progressed more rapidly than in others. This is particularly true for research in Hematopathology. Investigations on gene translocation by Fluorescent In situ Hybridization (FISH), on B-cell and T-cell clonality, and on gene-expression profiling were performed in research projects before the introduction in clinical routine practice [15–18]. In particular, one of the hot topics actually is the molecular evaluation of diffuse large Bcell lymphomas, one of the most frequent lymphoma with potential aggressive course. According to the putative cell of origin, these lymphomas can be subclassified into three molecular subtypes by gene expression profiling (GEP): the germinal center B-cell type (GCB), the activated B-cell type (ABC) and the unclassifiable type (UC). NanoString (NS) 20-genes assay has emerged as a feasible technique for molecular classification with a high concordance with GEP. Molecular classification of diffuse large B cell lymphomas with these technologies has shown prognostic impact, since clinical outcome of non-GCB type is inferior if compared to GCB type in patients (pts) receiving CHOP or Rituximab-CHOP. Moreover, double or triple hit involving MYC, BCL6 and BCL2 genes has shown to be a negative prognostic factor, also in primary cutaneous lymphomas [16].

3 Future Directions in Anatomic Pathology We will probably experience in a next future a revolutionary complete reshape of tissue-based and cell-based molecular testing, as next generation sequencing is beginning to show its full potential for the reclassification of diagnosis, therapeutic decision-making beyond the classic organ-specific treatment options and the detection of standard-of-care mutations. In this new era, the pathologists would experience an expanding molecular expertise within their own laboratories and integrate more and more genomics in tissue and cellular research. This will contribute to identify more and more specific molecular biomarkers relevant for diagnosis, prognosis and prediction of response to therapy. Embracing effectively morphology with the ample available genomic information will increase the importance of AP in the future, both in health-care system and in clinical trials. The best premises are that AP would have a central role in oncologic research and this will have effects on the teaching activity, as future pathologists would deliver morpho-molecular diagnoses more than simple morphologic descriptions. These considerations are crucial in rethinking our teaching activity in Anatomic Pathology, particularly for residents in Pathology. Molecular pathology rotations will be a mandatory part of the residency training program; this is the key for better preparation of pathology residents to meet this increasing role of molecular testing in patient care and management. Careful attention would be paid to several issues, as future pathologist should deeply understand the technologies that generated the

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results, make reliable judgement as to whether a specific test is technically satisfactory or suboptimal, treat and store optimally the clinical samples, manage complex data with informatic devices, and finally understand the clinical relevance of test results. Interestingly, according to several scientists we are dealing with another revolution, that of digital pathology and artificial intelligence (AI) [19]. In pathology, AI will elicit the automated interpretation of pathological images with AI algorithms both on Hematoxylin and eosin stained sections and on immunostained tissue sections. AI would improve the diagnostic efficiency in the distinction between benign tissue and tumours and enable a more precise grading of dysplasia and in-situ lesions. It would be possible to evaluate more precisely the presence and the extent of invasion or identify easily the presence of micrometastases in lymph node samplings, which are time-consuming. The hope is that also the scoring of multiple biomarkers by immunohistochemistry and the evaluation of the immune response to cancer would be facilitated. AI would help pathologists in the evaluation of percentage of tumor cells and overall cellular content in samples driven to molecular analysis. Moreover, it will be possible to create a next-generation morphology by extracting new pattern from the digital images and clinical correlates. Last, but not least, pathologists will probably dismiss traditional microscopes. In conclusion, we feel that pathologists will continue to be at the forefront of translational research if they accept the challenges of genomic medicine. Our choices in pathology at the present time will dictate the extent and quality of pathology and of medicine over the next decades.

References 1. Hamilton PW, Bartels PH, Thompson D et al (1997) Automated location of dysplastic fields in colorectal histology using image texture analysis. J Pathol 182:68–75 2. Bartels PH, Thompson D, Montironi R et al (1996) Automated reasoning system in histopathologic diagnosis and prognosis of prostate cancer and its precursors. Eur Urol 30:222–233 3. Montironi R, Bartels PH, Hamilton PW et al (1996) Atypical adenomatous hyperplasia (adenosis) of the prostate: development of a Bayesian belief network for its distinction from well-differentiated adenocarcinoma. Hum Pathol 27(4):396–407 4. Montironi R, Filho AL, Santinelli A et al (2000) Nuclear changes in the normal-looking columnar epithelium adjacent to and distant from prostatic intraepithelial neoplasia and prostate cancer. Morphometric analysis in whole-mount sections. Virchows Arch 437(6):625–634 5. Montironi R, Mazzucchelli R, Santinelli A et al (2005) Incidentally detected prostate cancer in cystoprostatectomies: pathological and morphometric comparison with clinically detected cancer in totally embedded specimens. Hum Pathol 36(6):646–654 6. Montironi R, Santinelli A, Pomante R et al (2000) Morphometric index of adult renal cell carcinoma. Comparison with the Fuhrman grading system. Virchows Arch 437(1):82–89 7. Lopez-Beltran A, Luque RJ, Alvarez-Kindelan J et al (2004) Prognostic factors in stage T1 grade 3 bladder cancer survival: the role of G1-S modulators (p53, p21Waf1, p27kip1, Cyclin D1, and Cyclin D3) and proliferation index (ki67-MIB1). Eur Urol 45(5):606–612

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8. Bruni P, Conti C, Giorgini E et al (2004) Histological and microscopy FT-IR imaging study on the proliferative activity and angiogenesis in head and neck tumours. Faraday Discuss 126:19–26 9. Garzetti GG, Ciavattini A, Goteri G et al (1995) Ki67 antigen immunostaining (MIB 1 monoclonal antibody) in serous ovarian tumors: index of proliferative activity with prognostic significance. Gynecol Oncol 56:169–174 10. Dogan A, Burke JS, Goteri G et al (2003) Micronodular T-cell/histiocyte-rich large B-cell lymphoma of the spleen: histology, immunophenotype, and differential diagnosis. Am J Surg Pathol 27(7):903–911 11. Goteri G, Filosa A, Mannello B et al (2003) Density of neoplastic lymphoid infiltrate, CD8+ T cells, and CD1a+ dendritic cells in mycosis fungoides. J Clin Pathol 56(6):453–458 12. Cencini E, Fabbri A, Rigacci L et al (2017) Evaluation of the prognostic role of tumourassociated macrophages in newly diagnosed classical Hodgkin lymphoma and correlation with early FDG-PET assessment. Hematol Oncol 35(1):69–78 13. Santinelli A, Baccarini M, Colanzi P et al (2002) Immunohistochemical evaluation of HER2/neu expression in infiltrating breast carcinoma: a study of re-producibility. Anal Quant Cytol Histol 24(1):54–62 14. Lopez-Beltran A, Henriques V, Cimadamore A et al (2018) The identification of immunological biomarkers in kidney cancers. Front Oncol 8:456 15. Goteri G, Lucarini G, Zizzi A et al (2011) Comparison of germinal center markers CD10, BCL6 and human germinal center-associated lymphoma (HGAL) in follicular lymphomas. Diagn Pathol 6:97 16. Lucioni M, Berti E, Arcaini L et al (2016) Primary cutaneous B-cell lymphoma other than marginal zone: clinicopathologic analysis of 161 cases: comparison with current classification and definition of prognostic markers. Cancer Med 5:2740–2755 17. Goteri G, Simonetti O, Rupoli S et al (2007) Differences in survivin location and Bcl-2 expression in CD30+ lymphoproliferative disorders of the skin compared with systemic anaplastic large cell lymphomas: an immunohistochemical study. Br J Dermatol 157(1):41–48 18. Rusconi C, Re A, Bandiera L et al (2018) Cell-of-origin identification and prognostic correlation in HIV-associated diffuse large B-Cell lymphomas: results of an Italian multicentric study. Blood 132:5294 19. Salto-Tellez M, Maxwell P, Hamilton P (2019) Artificial intelligence—the third revolution in pathology. Histopathology 74(3):372–376

Past, Present and Future in Forensic Human Identification Federica Alessandrini, Valerio Onofri, Chiara Turchi, Loredana Buscemi, Mauro Pesaresi and Adriano Tagliabracci

Abstract The Institute of Legal Medicine of Polytechnic University of Marche has been working in the field of forensic identification since 1980s, contributing actively to the development of the research and the application of new techniques. Before the DNA era, human identification was based on the analysis of surface polymorphic antigen systems of blood groups and HLA complex and then on the analysis of serum proteins and red cell polymorphic isozymes. The era of forensic DNA analysis began in 1985, when Alec Jeffreys described a genetic polymorphism in the human myoglobin gene. In 1987 Kary Mullis developed the polymerase chain reaction (PCR) and the revolutionary power of this technology was immediately perceived and the lab was quickly switched towards this new approach. The lab of the Institute of Legal Medicine is equipped with the most recent and high throughput instruments and technologies for DNA typing, has achieved the ISO/IEC 17025 accreditation for the participation to the National DNA Database of genetic profiles, and is a partner of the European network of forensic genetic labs. The lab is now one of the Italian leading centers in forensic genetics and it is actively involved in the most innovative research fields in forensic genetics.

F. Alessandrini · V. Onofri · C. Turchi (B) · L. Buscemi · M. Pesaresi · A. Tagliabracci Department of Biomedical Sciences and Public Health, Università Politecnica delle Marche, Via Tronto, 60126 Ancona, Italy e-mail: [email protected] F. Alessandrini e-mail: [email protected] V. Onofri e-mail: [email protected] L. Buscemi e-mail: [email protected] M. Pesaresi e-mail: [email protected] A. Tagliabracci e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_7

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1 Pre-DNA History Before the DNA era, human identification was based on forensic serology. Serology is a term used to describe laboratory tests that examine specific antigen-antibody interactions and immunological assays enable rapid screening for particular antigens with monoclonal antibodies. The genetic markers typing in forensic serology can be divided into two groups based on differences in biochemistry and method of detection. Although these early methods had a very low power of discrimination, they were still quite capable of excluding individuals who did not match. The first group of genetic markers comprised the polymorphic antigen systems found on red cell and other cell surfaces, while the second one was represented by the polymorphic soluble protein markers. The discovery of AB0 blood groups by Karl Landsteiner in 1900 at University of Vienna marked the first demonstration that normal individuals could be differentiated at the biochemical level [32]. His work, which resulted in the 1930 Nobel Prize in Medicine, identified four blood types: 0, A, B and AB and each type of blood groups is differently widespread in the populations. Blood groups are determined by the presence of specific antigen polymorphisms on the surface of red blood cell. The antigens are inherited from an individual’s parents and therefore they can be used to test relationships. The AB0 system was the first genetic evidence used in court in 1915 by the Italian professor Leone Latters of the Institute of Forensic Medicine in Turin [35–37]. Another early genetic blood typing system developed was the MN system in 1927. Ten year later, Alexander Weiner discovered the Rh factor while studying the Rhesus monkeys [33]. Overall about sixteen red cell surface antigen polymorphisms were used in forensic identification in those years. The discovery of the system of histocompatibility antigens found on white cells, termed as HLA system, was more recently, around 1960s. The HLA was the most extensive of the recognized human polymorphisms [24]. In Ancona, at the Institute of legal Medicine, the first studies for individual identification were based on the analysis of red cell blood group systems by using immunological assays. In particular, the MNSs, ABO, Rh and Kell systems were analyzed in population samples of Marche region [14, 15]. The further type of serological markers was represented by the genetically polymorphic soluble proteins and most of them have been discovered since about 1965. Amino acid sequences in some proteins vary among individuals in the human population. The use of multiple protein polymorphisms can produce modest power of discrimination, however, before the introduction of DNA testing, protein profiling was widespread performed in forensic biology laboratories for identification purposes. The polymorphic soluble proteins can be subdivided in intracellular enzymes and proteins found in blood plasma and/or other body secretions. The human red and white blood cells as well as the blood serum present different number of isozymes, which are multiple forms of a protein enzyme that can catalyze the same biochemical reaction in spite of slightly different amino acid sequences. Overall a large number, about sixty, of soluble protein were used in forensic identification and they found

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broad application in forensic lab due primarily to the development and utilization of electrophoresis, a technique which separates molecules according to their electrical charge features into distinguishable alleles. By the early 1980s many labs began using isoelectric focusing (IEF) polyacrylamide gel electrophoresis, which is capable of higher resolving power than protein electrophoresis. Our research group has been working for several years on the soluble isozymes founded in red blood cells, by typing the Acid Phosphatase (ACP1), Adenylate Kinase (AK), Esterase D (ESD), Glyoxylase I (GLO), Phosphoglucomutase-1 (PGM1) [16, 17, 21, 58, 61] and on the soluble isozymes founded in blood serum, by typing Immonoglobulins (Km) and Immonoglobulin G (Gm), alfa 1 Antitrypsin (Pi), Coagulation factor XIII, Group Specific Component (Gc) and Transferrin (Tf) [57, 59, 60, 63, 64]. Our studies contributed to collection and implementation of frequency data of cell surface and soluble protein markers in Italian population, suitable for parentage testing and casework analysis [62].

2 DNA Analysis The era of forensic DNA analysis began in 1985 to resolve an immigration dispute, when Alec Jeffreys at the University of Leicester, UK, found extraordinarily variable and heritable patterns from repetitive DNA analyzed with multi-locus probes [27, 28]. These simple tandem-repetitive regions of DNA or minisatellites were found dispersed in the human genome and was assessed to be hypervariable between people but conserved throughout the cells of an individual. Thus was born the DNA fingerprinting, completely specific to an individual (or to his or her identical twin), one of the most great discoveries of the late 20th century, which would revolutionize the process of forensic human identification. In DNA fingerprinting, radio-labeled DNA probes containing minisatellite were hybridized to DNA previously digested with a restriction enzyme, separated by agarose electrophoresis and immobilized on a membrane by Southern blotting. After exposure to X-ray film, it was possible to visualize these variable fragments and their profiles compared between individuals [29]. Two years later, Kary Mullis, a chemist prized with the Noble for his discovery, developed a reliable molecular biology technique to amplify specific DNA sequences in vitro, named PCR (polymerase-chain-reaction) [43]. The method consisted of repetitive cycles of specific short stretches of preselected DNA, able to produce approximately 100 billion copies of one molecule of DNA in a few hours, starting with tiny amounts of biological sample and oligonucleotide primers derived from sequence data. In contrast to conventional serological methods, any human tissue or body fluid containing nucleated cells can be analyzed for DNA profiling. Since the technique was fast and easy, PCR has replaced the Southern-blotting technology in forensic genetics laboratories, revolutionized the research opening new horizons in forensics, and influenced criminal and legal policies.

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The Institute of Legal Medicine of Univpm immediately understood the innovative power of this technology and switched towards this new approach to the forensic identification as leading technique in its laboratory. The majority of DNA loci studied in forensic identification derived from “noncoding” portions from the human genome and are located either in the vicinity of expressed (coding) genes or in stretches of DNA sequences interspersing with the genes. They are spread over the whole genome and typing results do not contain any useful information which might reveal genetic traits or predispositions for inherited disease about the individual studied. The technology applied to forensic sciences goes hand in hand with scientific discoveries in the biomedical field. In genetics, the methods of analysis and the instruments for detection have immediately pursued the discovery of the new polymorphisms. The new methods, always improved compared to the previous ones, have been developed over the years in order to increase the analytical sensitivity and the speed of execution. These two objectives are at the base of genetic investigations. The increase of analytical sensitivity aims to raise the discriminative power of a new method or marker, so that a smaller degree of uncertainty could weight on a test in which one individual must be distinguished from another. The second goal is to reduce the response time, making possible investigations faster and facilitate the identification of individuals leaving a biological stain on the crime scene. One of the first widely used PCR-based test was a kit for detecting sequence variation at the Human Leukocyte Antigen (HLA) DQA1 gene. This kit combined the PCR technique with dot blot hybridization, allowing direct investigation of the region, and could distinguish up to nine different alleles [65]. Moreover, other types of forensic marker, the VNTR (variable-number-tandem repeat) have been successfully typed by PCR. Our research group, in the early 90s, studied the allelic and genotypic frequencies of different VNTR markers, such as ApoB, MCT118, YNZ22 and COL2A1 [6, 23, 63, 66, 67]. Since the mid-1990s, the golden research age of DNA fingerprinting, the highly efficient of microsatellite DNA, the so called short-tandem-repeat (STR) markers, containing originally four base pair repeat sequences, have been used routinely for human identification [38]. The STR loci used in standard DNA profiling are all inherited in a Mendelian manner, are not genetically linked, and therefore are inherited independently. Polyacrylamide gel electrophoresis was the original method for separating STR alleles: DNA was added to a gel matrix, and in the presence of an electric field, the negatively charged DNA was separated based on the size allowing to distinguish alleles of the STR loci [4]. Capillary electrophoresis (CE) was a major breakthrough, as the separating polymer is much safer to use, and the detection of the STR fragments is based on laserinduced fluorescence of a dye attached to the 5 end of one of the primers used in the PCR [9]. A large number of STRs could be detected in a single capillary electrophoresis injection generating for each individual a unique genetic code. In 1996, our laboratory, among the first in Italy, was equipped with the one-capillary

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sequencer ABI Prism 310 and the analysis of autosomal and Y-chromosome multiallelic STR multiplexes was introduced in the routinely identification analysis. This step has incredibly increased both the sensitivity and the speed of execution for the analyses, allowing to obtain a genetic profile within few hours. For this reason, STR analysis has been the standard in forensic DNA examinations for almost 20 years and has became the standard method in forensic stain typing. Validation studies and interlaboratory proficiency testing to demonstrate as comprehensively as possible the robustness and reproducibility of the tests were undertaken in worldwide laboratories because of the significance of DNA evidence in any criminal case [22]. Relevant publications of University Ancona team gained special merits in this field [7, 8, 53, 68, 69]. DNA frequency databases were generated for all populations to ensure an accurate (and frequently conservative) estimate of the frequency of the DNA profile [11]. Within the last decade, a series of STR systems located on the Y chromosome have been developed and demonstrated to be suitable for a variety of forensic applications, where mixtures of male and female DNA are analyzed, as happens in cases of rape or other sexual crimes [3, 31, 54]. The Y chromosome is inherited as a complete chromosome from father to son, so all male siblings share the same Y chromosome [12, 34, 47, 52]. It does not recombine with any partner and hence all the loci remain in the same order and linked from one generation to the next. The genetically linked DNA loci on the Y chromosome are termed as haplotype. The largest forensic Y chromosome haplotype database is the YHRD (http://www.yhrd.org) from the Institute of Legal Medicine and Forensic Sciences in Berlin, Germany. Also the STR loci on the X chromosome were introduced in forensic kinship testing, mainly in solving of complex cases or in the identification of the victims of a mass disaster in which the parents of the offspring were not available [5, 56]. At the same time, capillary electrophoresis allowed to start studies on the hypervariable regions of mitochondrial DNA (mtDNA) using the Sanger sequencing technique which allows the analysis of ancient, degraded samples or hair shafts, where the STRs typing fails. The characteristics of high copy number and maternal inheritance makes mtDNA a further powerful tool for forensic identification [10, 26]. MtDNA is particularly useful in cases of missing persons when maternal relatives can act as reference samples. Most of quickly evolving sites in the mitochondrial genome that are relevant for the discrimination of haplotypes in forensics are in the control region (CR). Traditional protocols have targeted the three hypervariable segments of the CR (HVS-I, HVS-II, and HVS-III) using independent amplification and different combinations of primers. This restricted approach has a number of implications, first among all an increased risk of chimeric haplotypes or so-called “artificial recombinants”, due to the inadvertent mix-up of mtDNA segments from different individuals. For this reason in the last ten years laboratory protocols have been established and improved to allow the amplification of the entire CR in a single reaction. Important contributions to the study of mitochondrial DNA polymorphisms were provided by the UNIVPM forensic lab [70, 72, 73, 75]. To improve the current use of mitochondrial DNA in forensic science the EDNAP Mitochondrial DNA Population Database (EMPOP; www.empop.org) was established, that was designed to

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serve as a reference population database for use in the evaluation of mtDNA evidence worldwide, but also as quality-control tool for scientists in forensic genetics by the implementation of a quality assurance check of the data. Sanger sequencing has also been used for the characterization of new allelic variants of microsatellites and for the characterization of “DNA barcoding”, with main application to forensic entomology [2, 41]. The research and development of further markers named single-nucleotidepolymorphisms (SNPs) started in our laboratory in 2001. A SNP is a base variation at a single position (A, C, G, and T) in a DNA sequence among individuals. If more than 1% of a population does not carry the same nucleotide at a specific position in the DNA sequence, then this variation can be classified as a SNP. If a SNP occurs within a gene, then the gene is described as having more than one allele. In these cases, SNPs may lead to variations in the amino acid sequence. SNPs, however, are not just associated with genes but they can also occur in noncoding regions of DNA. Autosomal SNPs useful for individual identification and Y-SNPs able for inferring geographical affiliation of male individuals were studied by our group [1, 46, 71]. Furthermore, this technique has also been applied to the research on SNPs involved in individual susceptibility to alcoholism [48, 74]. To this purpose a novel technique based on primer extension by fluorescently labeled dideoxyterminators, named minisequencing, was introduced in our lab. Besides, thanks to a 4 capillaries sequencer acquired on 2005 and a second 8 capillaries sequencer on 2013, in these years the laboratory routinely adopted multiplex PCRs with 17 autosomal and 15 STRs Y-chromosome STRs, as well as SNPs and mtDNA. At the present the laboratory analyzes in less than 2 h up to 27-locus validated multiplex PCR assay, reaching a discriminatory power of 10−32 . The sequencing techniques have evolved in the second half of 2010s and the “next generation” sequencing or massive parallel sequencing technique (MPS) was developed. The Ion Torrent technology was introduced in Ancona’s lab in 2013. This technology provides fast and high-throughput collection of short sequences of DNA. At first the MPS was used for studies on the so-called molecular autopsies, mainly in the study of cardiomyopathies, and subsequently for studies on new markers for personal identification such as mtGenome and microhaplotypes [76]. Beyond the typing methods, DNA extraction and quantification methods are issues on which our research activity has been focused. DNA extraction is a crucial moment of individual identification in the case of forensic traces. The evolution in this field has been less tight; the laboratory has validated increasingly effective methods for the recovery of larger amounts of DNA by chelating resins, methods with phenol chloroform, silica based membrane up to paramagnetic beads. The latter technique was automated in 2007 with the introduction of a dedicated robot. DNA quantification has been essential since the 2000s, as it allows scientist to plan the subsequent analysis steps appropriately. Beginning with agarose gel with low resolution power, we then moved on to spectrophotometric detection. This method allows the quantification of high molecular weight DNA, but is not human specific and the eluted is affected by the salts and protein residues. Then our laboratory adopted a method based on the hybridization of human-specific alpha satellite probe on locus D17Z1, followed

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by the detection of streptavidin-biotin conjugate, a more sensitive method able to detect up to 150 pg of DNA and to show the degree of degradation of DNA, but very difficult and not so reproducible. In 2004 the laboratory finally introduced the real-time PCR for the qualitative assessment (gender of the subject, presence of PCR inhibitors) and quantitative (up to a few picograms) of the DNA, still representing the gold standard for this application. In 2012 this step was automated with a robot to standardize repeatability and thus increase the accuracy of the analysis. In the last few years, because the increasing number of disputes over DNA tests in the courts worldwide, the issue of quality has become decisive and international consensus was arise to make the forensic genetics a strong and reliable science. So one of the greatest changes to have occurred is the introduction of ISO accreditation to all operational laboratories [40]. For forensic genetics laboratories the ISO/IEC 17025 was adopted as standard to provide uniform technical criteria for developing a quality management system. This standard sets in place standard operating procedures, as well as competency tests. Examples of competency tests are proficiency tests where known samples are provided by a testing agency to whom the correct answers should be returned. These types of tests demonstrate confidence in the method and the ability of the staff. Moreover, the Italian law 85/2009 (institution of national criminal DNA database) required the ISO/IEC 17025 accreditation for the laboratories which enter DNA profiles for matching comparison. The forensic genetic laboratory of Univpm has achieved the accreditation in 2016 with the aim of increasing the lab’s DNA testing quality and feed the national database with criminal and unknown subjects profiles.

3 The Future of Forensic Genetic Intelligence The Olympic motto by Pierre de Coubertin “Citius, Altius, Fortius” (which is Latin for “Faster, Higher, Stronger”) could be borrowed to describe where forensic genetics is heading thanks to the advancement in knowledge and technologies: faster results, higher sensitivity and information content, stronger conclusions, mostly with challenging samples. The introduction of rapid and portable fully automated DNA profiling systems allow DNA testing protocols to get faster, providing swab-into profile-out results in less than 90 min, directly on the crime scene (Rapid DNA Analysis). The potential impact of rapid DNA technology is evidenced by the fact that FBI plans to approve Rapid DNA systems for buccal swab use in accredited labs first and then approve systems for booking station use based on the experience gained from accredited lab use and lessons learned from the pilot testing in 2019 [25]. The future of higher information content from forensic DNA analysis depends on improved detection sensitivity, higher information content from expanded sets of core STR loci [44] and deeper information from sequence analysis of alleles [49] and possibly supplemental genetic markers, related not only to personal identification and parental testing, but also to other important issues such as body fluids identification

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[13, 19, 55], post mortem interval (PMI) estimation [39] and investigative purposes (forensic DNA phenotyping [30], age [50] and biogeographic prediction [51]). A novel type of molecular markers, useful in forensics for individual identification, ancestry inference, estimating relationships and deconvoluting mixtures, are microhaplotype loci (microhaps, MHs), defined by two or more closely linked SNPs associated in multiple allelic combinations [45]. The value of these markers is enhanced by massively parallel sequencing (MPS), which allows the sequencing of both parental haplotypes at each of the many multiplexed loci [76]. When a DNA profile is obtained from a crime scene and there is no match on a national DNA database or there is not any suspect to compare the DNA profile to, forensic DNA phenotyping may be used to predict a suspect’s appearance, to narrow down from a population to a pool of possible suspects for investigation. Tests can currently predict sex, hair, skin and eye colour. But these tests are not accurate at 100% level and are still in development [30]. Knowledge about the genetic basis of any other physical traits (height and facial structure) is not yet advanced enough for them to be predicted from a DNA sequence. Predicting an individual’s biogeographic ancestry, i.e. the broad geographic region their biological ancestors originated from is useful and can be achieved by analysing a number of ancestry informative markers (AIMs) [51]. For now, the available tests that are suitable for crime sample analysis can only reliably predict to which of the major continental groups a person belongs (African, Western Eurasian, East and South Asian, or Native American); they cannot say which country someone comes from. However human genetic variation does not allow all forensically relevant questions to be answered. Some questions may instead be addressable via epigenomics, in particularly DNA methylation, as the epigenome acts as an interphase between the fixed genome and the dynamic environment. Current progress in forensic epigenetics are helping in discovering what type(s) of cells are present in a stain, the age of an unknown trace donor (epigenetic clock), discriminating between identical twins [77], and in the future epigenomic is expected to predict lifestyle habits (smoking and drinking status, diet, physical activity status, body size/shape, etc.). All these pieces of information are useful to lead investigations and increase the ability of police to find unknown perpetrators [77]. Also non-human DNA has been demonstrating its utility in the forensic field. For example, microbes have potential to be used as physical evidence for forensic science, because they are ubiquitous and have predictable ecologies [20]. Microbioma analysis may aid investigations, allowing discrimination of environmental samples (e.g. soil), postmortem interval estimation (PMI) based on the microbial succession during decomposition [42], geolocation, not-sterile body fluids and tissues characterization and also human identification, through inter-individual variations of the microbial communities living on and in the human body (microbial fingerprint). Last but not the least, improving the ability to decipher and interpret all the data obtained from DNA analysis provides probably the greatest challenge but also the largest opportunity for future advances in forensic DNA analysis. The forensic genetic evidence does not give a “yes or no” answer: it can only ever be expressed in terms of probability. Conclusions that are stronger, can be drawn in many cases

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with probabilistic approaches that are constantly under development, not only for the statistical evaluation of a genetic profile match and a complex DNA mixture [18], but also for familial searching, useful for finding close relatives, and for the prediction of age, external visible traits and PMI estimation. Forensic genetics continues to be an innovative, dynamic and evolving field of research, and the information that can be gleaned from the smallest traces of biological material continues to grow. It is important to correctly understand these increased possibilities and address the challenges that enhanced DNA analysis could bring. Accurate communication between scientists and non-scientists are pivotal, both to ensure that their expectations of the technology are realistic, and its limits are properly understood. These are the challenges that await our laboratory for the near future.

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65. Tagliabracci A, Giorgetti R, Agostini A et al (1992) Frequency of HLA DQA1 alleles in an Italian population. Int J Legal Med 105(3):161–164 66. Tagliabracci A, Giorgetti R, Agostini A et al (1992) Analysis of D1S80 (pMCT118) locus polymorphism in an Italian population sample by the polymerase chain reaction. In: Advances in forensic haemogenetics, vol 4, p 109 67. Tagliabracci A, Buscemi L, Cucurachi N et al (1994) PCR typing of the COL2A1 system: allelic frequencies in two population samples from North and Central Italy. In: Advances in Forensic Haemogenetics, vol 5, p 590 68. Tagliabracci A, Buscemi L, Bianchi F et al (1998) Polymorphism and sequence variations of the HumCD4 pentameric microsatellite in an Italian population sample. J Forensic Sci 43(4):841–844 69. Tagliabracci A, Buscemi L, Sassaroli C et al (1999) Allele typing of short tandem repeats by capillary electrophoresis. Int J Legal Med 113(1):26–32 70. Tagliabracci A, Turchi C, Buscemi L et al (2001) Polymorphism of the mitochondrial DNA control region in Italians. Int J Legal Med 114(4–5):224–228 71. Turchi C, Onofri V, Alessandrini F et al (2006) Multiplex genotyping of 22 autosomal SNPs and its application in the forensic field. Prog Forensic Genet 11(1288):40–42 72. Turchi C, Buscemi L, Previderè C et al (2008) Ge.F.I. Group. Italian mitochondrial DNA database: results of the Ge.F.I-ISFG collaborative exercise and proficiency testing. Int J Legal Med 2008 122(3):199–204 73. Turchi C, Buscemi L, Giacchino E et al (2009) Polymorphisms of mtDNA control region in Tunisian and Moroccan populations: an enrichment of forensic mtDNA databases with Northern Africa data. Forensic Sci Int Genet 3:166–172 74. Turchi C, Piva F, Solito G et al (2012) ADH4 intronic variations are associated with alcohol dependence: results from an Italian case-control association study. Pharmacogenetics Genomics 22:79–94 75. Turchi C, Stanciu F, Paselli G et al (2016) The mitochondrial DNA makeup of Romanians: a forensic mtDNA control region database and phylogenetic characterization. Forensic Sci Int: Genet 24:136–142 76. Turchi C, Pesaresi M, Tagliabracci A (2017) A microhaplotypes panel for forensic genetics using massive parallel sequencing. FSIGenet Suppl Ser 6:e117–e118 77. Vidaki A, Kayser M (2017) From forensic epigenetics to forensic epigenomics: broadening DNA investigative intelligence. Genome Biol 18:238

From Clinical Research to Clinical Practice in Obstetrics and Gynecology Stefano Raffaele Giannubilo, Giovanni Delli Carpini and Andrea Ciavattini

Abstract The research activity of the Obstetrics and Gynecology Section of our Department (Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche—Università Politecnica delle Marche) was characterized from strong interest to clinical application of our findings, with the aim of improving prevention, diagnosis and therapy both in Obstetrics and in Gynecology. Here we present the history and the current developments of our main research areas: benign and preneoplastic diseases of the female reproductive system (uterine fibroids, cervical intraepithelial neoplasia, endometriosis), gynecological oncology, pre-eclampsia, and obstetric complications.

1 Gynecology 1.1 Uterine Fibroids The clinical research on uterine fibroids has been mainly conducted on the evaluation of the effectiveness of different surgical techniques. More specifically, we reported that the ultra-minilaparotomic approach to myomectomy seems to be a safe and effective minimally invasive alternative to standard open myomectomy in the treatment of large myomas, with low rates of intraoperative and postoperative complications [1]; however, when compared to the laparoscopic approach, a significantly lower recovery time was found in women who underwent laparoscopic myomectomy [2]. S. R. Giannubilo · G. Delli Carpini · A. Ciavattini (B) Clinica di Ostetricia e Ginecologia a prevalente indirizzo ostetrico e gravidanza ad alto rischio, Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Università Politecnica delle Marche, Via Corridoni 11, 60123 Ancona, Italy e-mail: [email protected] S. R. Giannubilo e-mail: [email protected] G. Delli Carpini e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_8

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As an alternative to traditional surgical approaches, the effectiveness of laparoscopic cryomyolysis has been evaluated, reporting a decrease of myoma volume of 60.3% and complete symptom relief in 83.6% of patients after a 12-month follow-up from cryomyolysis [3]; moreover, women subjected to cryomyolysis presented good pregnancy outcome in the subsequent pregnancies, with low incidence of preterm birth, cesarean section or other adverse obstetric outcomes [4]. More recently, the surgical outcomes of laparoscopic uterine artery bipolar coagulation in addition to myomectomy were compared with respect to myomectomy alone, showing that patients who underwent this combined approach presented a lower intraoperative blood loss, with better recovery after surgery [5]. The therapeutic approach to uterine fibroids has been also studied from a nonsurgical point of view; in particular, we focused on the relation between hypovitaminosis D and uterine fibroids, reporting that women affected by uterine fibroids who underwent vitamin D supplementation therapy were less likely to need surgical treatment and did not present size variation of fibroids during 12 months of therapy [6]. A specific interest in the relation between uterine fibroids and pregnancy has been developed from our research group. In details, we highlighted how women affected by uterine fibroids presented a higher risk of adverse obstetric outcomes, in particular in term of preterm birth, preterm premature rupture of membranes, and cesarean section; the effect seems to be more relevant in the presence of multiple fibroids, rather than large (>5 cm in diameter) fibroids [7]. We also reported the growth trend analysis of pre-pregnancy diagnosed uterine fibroids in early pregnancy, demonstrating a significant growth up to 12 gestational weeks, with a slow-down by mid-pregnancy; these size variations were related to the serum levels of human chorionic gonadotropin in the same gestational period [8].

1.2 High-Risk Human Papillomavirus Infection and Cervical Intraepithelial Neoplasia The diagnosis and treatment processes of high-risk human papillomavirus infection (HPV) and cervical intraepithelial neoplasia (CIN) had been extensively researched by our group, in line with a conservative approach to the pathology, to the preservation of the anatomical and functional integrity of the reproductive system, to the effect of pregnancy on the natural history of cervical intraepithelial neoplasia, and to the effect of treatments on the outcome of subsequent pregnancies. The research activity was carried out both at the local level and coordinating research groups at national level. In particular, we evaluated the pathogenic potential of underdiagnosed HPV types in determining the progression of infection to low-grade and high-grade CIN and we have identified six new putative novel HPV types at low to intermediate risk [9, 10].

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The effectiveness of conservative approach to low-grade CIN, with prolonged follow-up up to two years, was proven in a study from 2017, indicating that after 24 months from diagnosis, 88.5% of lesions will undergo to regression, 10.8% to persistence, and only 0.7% to progression to high-grade CIN. The risk of persistence or regression was reported to be higher in the presence of high-grade referral cytology or in case of tobacco use [11]. Regarding the risk of adverse obstetric outcomes in the subsequent pregnancies, we reported a higher risk of early spontaneous miscarriage in women subjected to loop electrosurgical excision procedure (LEEP) for high-grade CIN, particularly if the time interval between the procedure and the pregnancy was lower than 12 months [12]. In the controversial field of the relation between cervical excisional procedures and risk of preterm birth, we highlighted how only the length of the excised specimen seems to be a predictor of preterm birth, rather than the transverse diameter or the volume, determining higher risks for increasing cone lengths [13]; the detrimental effect of excisional procedures on the anatomo-functional integrity of the reproductive system was also confirmed by the findings of a reduced healing (sonographicallyevaluated lower levels of tissue regeneration) of uterine cervix after six months from LEEP in the case of larger cones [14]. The increased awareness of the operators about the importance of containing cone dimensions at equal clinical efficacy was highlighted from a national multicentric survey that reported a trend of decreasing cone length specimens in the last ten years in five Italian institution, without causing a global increase in positive endocervical margins [15]. Regarding the effect of pregnancy on CIN lesions, we found a higher expression of glycodelin and a lower expression of Ki67 in CIN in pregnant women, with a likely less proliferative activity of CIN during pregnancy, probably due to the effect of the different hormonal status [16]. In addition, we demonstrated that pregnancy status does not seem to affect the diagnostic performance of colposcopy for the diagnosis of CIN and invasive cervical cancer, but the reliability of colposcopy seems to be greater if performed during the first half of pregnancy [17]. Numerous aspects of the diagnosis and clinical management of vaginal intraepithelial neoplasia (VAIN) have been the subject of our research, conducted mainly at national level, with coordination of numerous Italian institutions. More specifically, we reported that the outcome of excisional procedures for VAIN, reporting a risk of occult invasive disease in 10% of the cases, with increasing risks for tobacco users, for biopsy diagnosis of VAIN3 and for previous hysterectomy for HPV-related diseases [18]. Moreover, the CO2 -laser approach to excisional procedures for VAIN seems to be a safe approach with low-risk of complications (7.0% minor complications and 0.8% major complications) [19].

1.3 Endometriosis The research conducted by our study group about endometriosis started initially with the analysis of natural killer cell activity. We showed that endometriosis-affected

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patients presented a significant lower level of natural killer cell activity in relation to disease stage, with a negative correlation with serum estradiol levels [20] and prolactin levels [21]. Moreover, in addition to natural killer cell activity reduction, we also found a decreased peripheral blood polymorphonuclear leukocyte chemotactic index in advanced stages of disease [22]. Natural killer cell activity was also demonstrated to be positively influenced during therapy with GnRH agonist within the first 12 weeks of medical treatment. However, the values of natural killer cell activity were constantly lower in patients who had disease recurrence [23]. We have reported a crucial role of VEGF in the pathogenesis of endometriosis, especially in the presence of large and bilateral cysts; neoangiogenesis seems to affect the outer cyst wall, contributing to the fibrosing process of adhesion formation through an interaction with capsular fibroblasts [24]. Those new vessels that arise from the capsule seems to be stimulated to proliferate by VEGF and protected from apoptosis by endothelial cell surviving [25]. More recently, we focused on vitamin D serum levels in women affected by endometriosis cysts, showing that a relatively high rate of women with ovarian endometriosis presented an hypovitaminosis D, with a linear correlation between 25-OH-D3 serum levels and the diameter of ovarian endometrioma [26]. Analyzing the role of unfolded protein response (UPR) genes (ATF6, GRP78, CHOP, and XBP1) in the pathogenic process of endometrioid ovarian carcinoma and endometriosis, we found that those genes are involved in the neoplastic progression of endometrioid ovarian cancer, with a significantly higher expression of ATF6 and GRP78, and a significantly lower expression of CHOP and XBP1. Those alterations seem to be acquired following ovarian localization of ectopic endometrial cells [27].

1.4 Gynecological Oncology In the field of gynecological oncology, the research focused mainly on the clinical significance of Ki67 antigen expression, natural killer cell activity, and 72 KDa metalloproteinase analysis in gynecological malignancies. In particular, the role of Ki67 antigen expression as prognostic factor in ovarian and cervical cancer was investigated. Concerning serous ovarian cancer, a higher expression of Ki67 antigen was shown to be negatively related to disease stage and disease progression and directly related to vascular endothelial growth factor (VEGF) expression, which a negative association with disease-free survival [28]. In cervical cancer, Ki67 antigen expression was demonstrated to be higher in locally advanced cervical cancer of young (50% of events). The apnoea hypopnea index (AHI) is the severity marker of SDB and is represented by the number of apnoea and hypopnea per hour of sleeping time. An AHI of ≥15/h was chosen as a pathological cut-off because an at least moderate form of SDB were considered as clinically relevant in term of effects on study endpoints. General characteristics of the population, divided by presence or absence of SBD, are shown in Table 2. According to the cardiorespiratory monitoring testing performed at baseline, 36 patients (55.4%) had significant SDB, 18 patients (27.7%) had OSA, and the remaining 18 patients (27.7%) had CSA. The mean AHI value was 32 ± 11 for OSA patients, 45 ± 24 for CSA ones and 10 ± 3 for those without SDB. Heart failure medication prior to enrolment is shown in Table 2. No significant differences were seen among the three groups regarding major therapeutic drug classes along the follow-up. Biventricular stimulation percentage was comparable between groups at 6 months (96 ± 3% in OSA group, 98 ± 2% in CSA group and 97 ± 4% in patients without sleep apnoea, p = 0.567) and 12 months (96 ± 4% in OSA group, 95 ± 3% in CSA group and 96 ± 3% in patients without SDB, p = 0. 344). Repeated measurements showed a statistically progressive significant reduction of LVEDV in general population, which decreased from 225.2 ± 57.7 ml at baseline to 191.9 ± 58.2 ml at 6-month to 186.6 ± 68.4 ml at 1-year (p within groups 50% of the phospholipids in most eukaryotic membranes. Most PC molecules have one cis-unsaturated fatty acyl chain, which renders them fluid at room temperature. 1,2-Diacyl-sn-glycerols (DAG) are minor components of cell membranes (about 1 mol% of the lipids) however they are potent regulators of both the physical properties of the lipid bilayer and the catalytic behaviour of several membrane-related enzymes. Among minor lipids, there are phosphoinositides (PI) which are important in cell signal transduction. Sterols are the major non-polar lipids of cell membranes: cholesterol predominates in mammals. Lipid composition of different intracellular organelles can vary substantially, suggesting that different lipids are required for different functions. Membrane lipids directly control biophysical parameters such as curvature, order and fluidity. The fatty acyl composition (ratio saturated/unsaturated fatty acids, ratio cis/trans unsaturated fatty acids) also influences lipid geometry and affects membrane thickness and fluidity in concert with cholesterol and fatty acyl chain length. In biological membranes, in addition to structural roles, lipids play functional key roles in different biological processes. According to the fluid mosaic membrane model of Singer and Nicolson formulated in 1972 [43, 49] the biological membranes are lipid fluid bilayers where proteins can diffuse (Fig. 1). Under physiological conditions membrane lipids are organized in a liquid-crystalline phase (fluid phase), an essential physico-chemical state, which is required to assure a correct protein conformation and thus an optimal membrane function. One of the main research interest of our laboratory is the study of the physico-chemical state (fluidity, order) of artificial and natural membranes by spectroscopy techniques. Membrane fluidity can be modulated by different endogenous factors (lipid composition, cholesterol, proteins, temperature, etc.) and exogenous ones such as anesthetics [38], coenzyme Q [27] and polyunsaturated fatty acids [50]. It can also be modified by pollutants, as reported by Valentino et al. [51], who have shown a decrease of membrane fluidity using the Electron Spin Resonance technique in erythrocytes isolated from the blood of workers occupationally exposed to lead. Also signal transduction mechanisms can affect membrane fluidity in activated human polymorphonuclear leukocytes (PMNs) and platelets as shown in the in vitro studies by Fiorini et al. using fluorescence spectroscopy technique [24, 34]. PMN plasma membranes showed a time limited decrease of fluidity during the stimulation with various agents known to activate the respiratory burst and a transient decrease of membrane fluidity has been observed in platelets activated with Platelet Activating Factor (PAF), which triggers a cascade of biochemical events at the plasma membrane level. Membrane fluidity has also been studied by fluorescence spectroscopy in subjects affected by various syndromes, such as trisomy 21, primary ciliary dyskinesia (PCD) and chronic diseases. Modifications in erythrocyte membrane fluidity have been shown at different levels of the bilayer in children with trisomy 21 and this alteration could be in part attributed to an increased oxidative damage [35]. PMNs from subjects with PCD demonstrated an impaired chemotactic activity with an increase of plasma membrane fluidity as reported by Fiorini et al. [26].

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Alterations of membrane fluidity have been observed also in erythrocytes of obese children and were related to differences in lipid composition with respect to control subjects [11]. After more than 40 years, many experimental data support the fluid mosaic membrane model, adding more and more new concepts on the structure and dynamic of the membranes. The traditional model of the plasma membrane as a homogeneous fluid lipid bilayer has been extended in recent years, as it has become clear that the plasma membrane consists of thousands of different lipids and is a much more complex structure than previously thought [43, 49]. One property of lipids that has fascinated scientists is their phase behavior. Phospholipids adopt defined phases depending on their molecular structure and the physical conditions. Mixtures of phospholipids can coexist in two fluid phases with different physical properties: liquid-disordered and liquid-ordered. Some developments and refinements were brought to the fluid mosaic model especially in terms of composition and molecular organization. The most important evolution of this model was obtained in 1997 with the works of Simons et al. and of Brown et al. [4]. They suggested that membrane lipids are organized into phase-separated microdomains, called “lipid rafts”, with both a specific composition and a molecular dynamic that are different to the ones of the surrounding liquid crystalline phase. Lipid “rafts”, are small and transient and become stabilized during signaling and vesicle budding. Proteins contribute to phase separation and preferentially distribute into one of the two different phases. Although only the liquid-crystalline lamellar phases had been considered as functionally relevant, a number of other lipid phases, e.g. liquid-ordered, cubic, and others have been shown of physiological interest. Moreover lipid-lipid and lipid-protein interactions appear to be much more dynamic than first appreciated. Cell membrane proteins exist either free or membrane-bound. Specific lipids play crucial roles in signal transmission across membranes and in the modulation, regulation and membrane-association of proteins. Several proteins, previously described as soluble cytoplasmic proteins, appear to interact both specifically with lipids and directly with the hydrophobic part of membranes, indicating a reversible association of these proteins with membranes under appropriate conditions. Such proteins belong to a new class of membrane proteins defined as the ‘amphitropic’ proteins. One of the new view of membranes is the presence of microdomains of different physico-chemical phase which can regulate protein activity and thus modulating cell functions. The presence of this membrane microheterogeneity has been studied by us in model and natural membranes by fluorescence spectroscopy. The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene in artificial multilamellar vesicles showed a lifetime distribution that depends on different locations of DPH along the membrane normal, characterized by different dielectric constants [22]. In erythrocyte membranes DPH fluorescence decay distributions revealed the pivotal homogenizing effect of cholesterol on the membrane microheterogeneity [23]. It has been shown that the interactions of cholesterol with certain phospholipid classes leads to a liquid ordered lamellar phase [31], where the lipids are fluid, but with the fatty acyl chains ordered. The coexistence of membrane microdomains of different phases (liquid-crystalline and liquid ordered) can generate nanometer or micrometer separate compartments (or patches) which are very important for modulating protein

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functions. As aforementioned, particular kinds of dynamic domains are the so called “lipid raft”, formed by sterols and sphingolipids, which can be of small size (20– 200 nm) [48] and with a lifetime from 102 to 103 s [36, 46]. Rafts have been shown to have an important role in signal transduction, being able to form clusters of required proteins for cell signaling [30, 45]. “Caveolae” are characterized by the abundance of three isoforms of membrane proteins: Caveolin-1, -2 and -3. Caveolin-1, a 22 kDa protein, is the main component of caveolae and has been implicated in several signalling cascades. The importance of this protein was established by the development of caveolin-1-knockout mouse models and the observation of impaired nitric oxide signaling and cardiovascular abnormalities in these mice [6, 47]. Alterations of cholesterol content reflect in perturbations of lipid rafts and disruption of many of the cell signaling pathways dependent upon this membrane architecture. Alterations of lipid and protein composition in membrane rafts have been described in human diseases and the importance of lipid raft signaling in the pathogenesis of a variety of conditions, such as Alzheimer Disease, Parkinson Disease and prion diseases, systemic lupus erythematosus, HIV and cardiovascular disease [8, 45]. Specific membrane domains represent an interesting target for pharmacological approaches in the cure and prevention of these diseases.

2 Plasma Lipoproteins and Their Physio-pathological Relevance Isolation of circulating lipids associated to proteins was described in equine serum in 1929. Following studies demonstrated lipoproteins in human blood (Fig. 2). Lipoprotein exert a key role in transport of lipids. They transport triglycerides, free cholesterol, esterified cholesterol, phospholipids and bioactive molecules (e.g., tocopherols, carotenoids, coenzyme Q) from cells involved in their synthesis to cells where they are catabolized. The discovery, classification, and characterization of lipoproteins and some of their key metabolic determinants have represented foundational accomplishments in research on atherosclerosis and other chronic diseases.

Fig. 2 Development in understanding plasma lipoprotein in medicine

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Five lipoprotein classes (chylomicrons, very low density lipoproteins (VLDL); low density lipoproteins (LDL); high density lipoproteins (HDL); lipoprotein (a) (Lp(a)) have been isolated in human plasma and serum and their lipid and protein (apoprotein) contents have been characterized [21, 40]. The study of plasma lipoprotein structure and their physio-pathological relevance represents one of our main research topics. The common function of all apoproteins is to help solubilize neutral lipids in the circulation. The apolipoproteins bind readily to phospholipids–water interfaces and, under appropriate conditions, can spontaneously form discrete particles with phospholipids. In vivo, the assembly of apolipoproteins with lipids to form lipoproteins may require the assistance of cellular proteins. Proteins, phospholipids and free cholesterol are localized at the lipoprotein surface formed by a monolayer of phospholipids. Triglycerides and cholesteryl esters are contained in the lipoprotein core. Dynamic properties of the lipid monolayers depend largely on the nature of the constituent lipids. For example, the surface lipids of LDL are more condensed and rigid than those of HDL because of the presence of more saturated fatty acids, a higher sphingomyelin-to-phosphatidylcholine ratio and a higher unesterified cholesterol-to-phospholipid ratio. In addition to transport of lipophilic molecules, plasma lipoproteins exert several other physiological roles and modulate cellular functions and signal transduction through interactions with cell receptors. Furthermore, they exert regulatory role such as modulation of cell proliferation and apoptosis [41]. Lipoprotein particles isolated from human cerebrospinal fluid (CSF) have been called HDL-like lipoproteins because they have density and chemical composition similar to HDL of human plasma. HDL-like lipoprotein particles and apoproteins in human CSF and surrounding vasculature have a role in lipid metabolism in central nervous system (CNS). In the circulation, lipoproteins are highly dynamic. They undergo enzymatic reactions of their lipid components, facilitated and spontaneous lipid transfers, transfers of soluble apoproteins. Conformational changes of the apoproteins related to compositional changes of lipoprotein lipids have been described. A conformational role of lipids has been demonstrated in HDL. Negatively charged phospholipids modulate cellular cholesterol efflux, antioxidative and anti-inflammatory properties of HDL. Furthermore, both core neutral lipids in HDL (cholesteryl esters and triacylglycerols) and surface lipids can impact surface fluidity and, in turn, antioxidative and other functions of HDL [5, 53]. Liver, kidney, and peripheral tissues are involved in lipoprotein catabolism via receptor-mediated endocytosis. Interactions between lipid-apolipoproteins modulate lipoprotein functions. Inborn errors of lipid metabolism and/or environmental factors modulate lipoprotein composition and functions. The presence and levels of lipid species in biological membranes or lipoproteins can change in response to diet, physiological and environmental factors. Alterations of membrane and/or lipoprotein lipid composition, that have been associated either with adaptive responses or with the aetiology of the disease, have been described in several human pathologies such as in obesity, diabetes, autoimmunity or inborn errors of lipid metabolism. Alterations of lipid composition and physico-chemical properties of HDL and LDL have been demonstrated in our previous studies in lipoproteins isolated from

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patients affected by obesity, diabetes and psoriasis [10, 16, 18–20]. During their life span, compositional changes of lipoproteins realize in vivo due to the reaction with several molecules (glucose, free radicals, homocysteine-thiolactone) lipid and protein components at the lipoprotein surface. Both circulating lipoproteins and long-lived structural proteins are non-enzymatically glycated in diabetes and are associated with diabetic complications. It was shown in vivo that LDL and HDL apoproteins in diabetes contain a higher proportion of lysine-bound glucose when compared with lipoproteins isolated from non diabetic individuals. The formation of glycated lipoproteins is markedly accelerated in diabetes because of the increased availability of blood glucose. In diabetes, also compounds derived from glucose, such as glycolaldehyde or methylglyoxal, reversibly condense with the amino groups on proteins, forming products, which may result in oxidative fragmentation to yield heterogeneous compounds referred to as advanced glycation end products (AGEs). We thought of interest to investigate structural and functional properties of modified lipoproteins. We have demonstrated that the modifications of lipoproteins glycated by glucose or methylglyoxal, are reflected in alterations of structure and functions of HDL. In detail glycated HDL have a lower antioxidant effect [1, 12]. Previous studies have demonstrated that plasma lipoproteins are sensitive to homocysteinylation in vivo [33] and that N-homocysteinylated forms of LDL and HDL also occur in human blood [32]. The reaction is favourited by high concentrations of homocysteinethiolactone (Hcy-thiolactone), a reactive homocysteine metabolite [32, 33]. During their lifespan, HDL interact with vascular endothelial cells and circulating cells that generate Hcy-thiolactone; the molecule is very reactive and diffuses through the cell membrane to the extracellular fluids, where can modify proteins. Using HDL homocysteinylated by Hcy-thiolactone, we demonstrated that HDL are sensitive to homocysteinylation and we confirmed a relationship between homocysteinylation of HDL proteins and modifications of HDL physico-chemical properties (polarity and order) and functions [13]. Moreover using LDL homocysteinylated by Hcy-thiolactone, we have demonstrated structural and functional alterations of Hcy-LDL and we suggested that homocysteinylation could increase atherogenicity of LDL [14].

3 Lipid Oxidation in Biological Membranes and Lipoproteins In biological membranes and lipoproteins, polyunsaturated fatty acid chains (PUFAs) and cholesterol are particularly susceptible to oxidation triggered by reactive oxygen species (ROS) and reactive nitrogen species produced by cells and enzymes such as lipoxygenases and cyclooxygenase. In PUFA, the molecular rearrangement forms a conjugated diene structure and then reacts with oxygen to form peroxyl radicals. In

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the absence of antioxidants and other stable proton donors, oxidative propagation occurs. Several products of lipid peroxidation are formed in vivo during free radicalinduced lipid peroxidation. The possibility of quantifying and monitoring these products is an attractive tool for assessing the oxidative/inflammatory state of human diseases. Among lipid oxidation products, oxidized cholesterol, lipid hydroperoxides have been detected in human blood and cerebro-spinal fluid. Membrane lipids can also be oxidized modifying membrane physico-chemical state and thus affecting biological functions as reported by the studies of Kantar et al. [35] and Fiorini et al. [25]. Other markers are isoprostanes produced by non-enzymatic, free radical-induced oxidation of arachidonic acid. Measurements of levels of lipid peroxidation can be made in serum, plasma, urine and other body fluids, and their abundance correlates well with the oxidative state in vivo. Elevated levels of isoprostanes and other markers of lipid peroxidation are associated with many diseases in which free radicals have been implicated. Another group of lipids that appear to contribute to chronic disease in several ways are oxidized phospholipids which were found to be present in mildly oxidized LDLs (ox-LDLs) and atherosclerotic lesions [52]. Oxidized LDL acts as a ligand for a specific receptor on macrophages termed the “scavenger receptor”, and thus are internalized by cultured macrophages much more rapidly than normal LDL [7, 42]. Lipid peroxidation is thought to be common to many chronic inflammatory diseases. The role of lipid peroxidation of plasma lipoproteins in the development of human chronic-degenerative diseases has been widely investigated by us [9, 15, 19, 20, 44]. Particular attention has been focused to human LDL and HDL for their key role in cholesterol transport. Recent studies have quantified circulating levels of ox-LDL and ox-HDL in human blood supporting the susceptibility of lipoproteins in vivo. An increase of the levels of ox-HDL and ox-LDL has been reported by us in patients after stroke, in multiple sclerosis [9, 15, 17] and Alzheimer Disease [2].

4 Perspectives in Lipid Research Over the past few years, new types of applications of lipids in medicine have been described. Liposomes, nanocarriers comprised of lipid bilayers encapsulating an aqueous core, have the ability to encapsulate a wide variety of diagnostic and therapeutic agents. Therefore, a growing interest is devoted to utilizing liposomes as nanocarriers for therapeutic application [39]. Liposomes were first reported by Bangham and his co-workers in 1964 (Fig. 1). The cell like structure of liposomes persuaded scientist to investigate liposomes as a tool to carry potent drug molecules into human body. In 1971, a first report was published on the liposomal encapsulation of a therapeutic agent [28].

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More recently extensive research has been carried out to optimize encapsulation, stability, circulation time and target specific drug delivery. Furthermore, chemical composition of liposomes has been modified by selective ligands to obtain multifunctional liposomes. After 50 years, next generation of liposomes, their preparation methods and progress in clinical applications have been improved and their potential applications are in the field of cancer, gene therapy, immunotherapy and infectious diseases as reviewed by Madni et al. [39]. Potential clinical applications of drug transport systems apply concepts on structure/function and physiological role(s) of high density lipoprotein nanoparticles. The ability of HDL to accommodate highly water insoluble constituents in their core regions enables HDL type nanoparticles to effectively transport hydrophobic drugs subsequent to systemic administration. The primary focus is on the application of HDL type drug delivery agents in cancer chemotherapy [37].

References 1. Bacchetti T, Masciangelo S, Armeni T et al (2014) Glycation of human high density lipoprotein by methylglyoxal: effect on HDL-paraoxonase activity. Metabolism 63(3):307–311 2. Bacchetti T, Vignini A, Giulietti A et al (2015) Higher levels of oxidized low density lipoproteins in Alzheimer’s disease patients: roles for platelet activating factor acetyl hydrolase and paraoxonase-1. J Alzheimers Dis 46:179–186 3. Bangham AD, Horne RW (1964) Negative staining of phospholipids and their structural modification by surface active agents as observed in the electron microscope. J Mol Biol 8:660–668 4. Brown RE (1998) Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J Cell Sci 111:1–9 5. Camont L, Lhomme M, Rached F et al (2013) Small, dense high-density lipoprotein-3 particles are enriched in negatively charged phospholipids: relevance to cellular cholesterol efflux, antioxidative, antithrombotic, anti-inflammatory, and antiapoptotic functionalities. Arterioscler Thromb Vasc Biol 33:2715–2723 6. Drab M, Verkade P, Elger M et al (2001) Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293:2449–2452 7. Esterbauer H, Wäg G, Puhl H (1993) Lipid peroxidation and its role in atherosclerosis. Br Med Bull 49:566–576 8. Fantini J, Garmy N, Mahfoud R et al (2002) Lipid rafts: structure, function and role in HIV, Alzheimer’s and prion diseases. Expert Rev Mol Med 20:1–22 9. Ferretti G, Bacchetti T (2011) Peroxidation of lipoproteins in multiple sclerosis. J Neurol Sci 311:92–97 10. Ferretti G, Alleva R, Taus M et al (1994) Abnormalities of plasma lipoprotein composition and fluidity in psoriasis. Acta Derm Venereol 74:171–175 11. Ferretti G, Dotti M, Bartolotta E et al (1998) Changes of erythrocyte membrane fluidity associated with childhood obesity: a molecular study using fluorescence spectroscopy. Biochem Med Metab Biol 40:101–108 12. Ferretti G, Bacchetti T, Marchionni C et al (2001) Effect of glycation of high density lipoproteins on their physicochemical properties and on paraoxonase activity. Acta Diabetol 38(4):163–169 13. Ferretti G, Bacchetti T, Marotti E, Curatola G (2003) Effect of homocysteinylation on human high-density lipoproteins: a correlation with paraoxonase activity. Metabolism 52(2):146–151

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Glutathione, an Over One Billion Years Ancient Molecule, Is Still Actively Involved in Cell Regulatory Pathways Tatiana Armeni and Giovanni Principato

Abstract Glutathione is a very ancient molecule widely distributed in aerobic cells and organisms, either prokaryotes or eukaryotes. Since glutathione in not found in anaerobic cells it could have evolved in the course of the adaptation to the presence of oxygen in the atmosphere. Glutathione is the major non-protein low molecular weight antioxidant and the most important cellular thiol reducing agent. Glutathione biosynthesis occurs in the cytosol from its constituent amino acids; GSH is present also in the most important cellular districts like mitochondria and nucleus to indicate its central role in several metabolic pathways and protective mechanisms. There are several glutathione dependent enzymes involved in various steps of cell metabolism. GSH is a key antioxidant that modulates various cellular processes and therefore is determinant for redox signaling, xenobiotics’s detoxication, regulation of cell proliferation, apoptosis and immune functions. Glutathione concentration and redox state is due to a complex interaction between biosynthesis, utilization, degradation, and transport. All these factors are of great importance for understanding the significance of cellular redox balance and its correlation with pathological conditions.

1 Glutathione, Oxygen and Evolution of Life Glutathione is a molecule made up of three amino acids joined by two peptide bonds. In order, the three amino acids are glutamic acid, cysteine and glycine. The first peptide bond is between the γ-carboxyl group of glutamic acid and the amino-group of cysteine, the second peptide bond is between the carboxyl group of glycine and the amino-group of glycine. The presence of the γ-glutamyl bond protects glutathione from degradation by intracellular proteases. T. Armeni · G. Principato (B) Department Odontostomatologic and Specialized Clinical Sciences, Section of Biochemistry, Biology and Physics, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy e-mail: [email protected] T. Armeni e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_28

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The tripeptide glutathione (L-γ-glutamyl-L-cysteinyl-glycine; molecular mass 307 daltons) has two negatively charged carboxyl groups and a positively charged amino group at physiological pH values. The overall form of the molecule is Yshaped and the common name, GSH, highlights the sulfhydryl group from which the particular redox properties of glutathione depend. When the sulfhydryl group is in reduced form, the GSH molecule behaves as a reducing agent, forming mixed disulfide with proteins (GSSP) or with sulfhydrylcontaining molecules XSH (GSSX), or reacting with another GSH to form GSSG. When glutathione is in the oxidized form, GSSG, acts as an oxidant by reducing to GSH, and eventually PSH or XSH if it reacts with a mixed disulfide. The −SH function in the glutathione molecule has a quite different reactivity from the −SH function of free cysteine or in proteins [26, 32, 54]. While cysteine is easily oxidized to cystine, and the −SH groups of proteins spontaneously tend to form mixed disulfides, the −SH of glutathione has a slower reactivity and therefore remains longer in the reduced form, available to react with molecules or free radicals that could damage the cell. Glutathione is a small and inexpensive molecule present in almost all eukaryote cells, except those few that lack mitochondria like Entamoeba histolytica [22], and in all tissues of eukaryote organisms. Because of its high intracellular concentration, several millimolar (mM), and its stable redox function is the main molecule ready to use in defense against oxidative stress. In the central part of the molecule, the sulfhydryl group (−SH) of the cysteine can serve as electron donor endowing glutathione with reducing properties and ability to remove free radicals through the formation of the thiyl radical, GS°, which can reacts with another GS° to form glutathione disulfide (GSSG). Glutathione play key functions in cells and in organisms, like scavenger of free radicals, in the mechanisms of xenobiotic’s detoxication, also playing a role in cell proliferation, growth and death. In mammals, transcription factors such as NF-E2, Nrf2, AP-1 and NFkB regulate the expression of the genes for glutathione synthesis [33]. The dysregulation of glutathione synthesis contributes to the pathogenesis of several diseases such as diabetes mellitus, pulmonary and liver fibrosis, alcoholic liver disease, cholestatic hepatic injury, endotoxemia and drug resistance of some tumor cells [33]. High glutathione concentrations are also in several prokaryotes, especially gramnegative bacteria [21, 52], and in at least one Haloarchaea in which only γ-glutamylcysteine is present (Sundquist and Fahey [53], Halobacterium halobium is now recognized as archaea, rather than bacteria). In all organisms in which is present, glutathione biosynthesis occurs in the cytosol but not by ribosomes, enzymatically with the catalysis of two different enzymes and hydrolysis of ATP [33]. The first, rate-limiting, is γ-glutamyl-cysteine synthetase, which catalyzes the synthesis of γ-glutamyl-cysteine starting from cysteine and glutamic acid; the second, glutathione synthetase, catalyzes the synthesis of glutathione by addition of a glycine to the C-terminal of γ-glutamyl-cysteine.

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In eukaryotes and prokaryotes that have glutathione, GSH derive by the sequential action of the same enzymes coded by separate genes, gsh1 and gsh2 in eukaryotes and gshA and gshB in prokaryotes [21]. However in some bacteria, only one protein accounts for γ-glutamyl-cysteine synthetase and glutathione synthetase activities [28]. The enzymes for glutathione synthesis are therefore ancient molecules that have evolved diversifying their amino acid sequences but keeping their mechanism unaltered. These enzymes are completely lacking in many prokaryotes and in some rare eukaryotes that do not have glutathione, like the anaerobic Entamoeba histolytica that lacks mitochondria. The finding that it grows without glutathione and other evidence support the hypothesis that a primary function of glutathione in eukaryotes involves protection against oxygen toxicity associated with mitochondria and suggests that eukaryotes may have acquired glutathione metabolism at the time that they acquired mitochondria [22]. Because glutathione performs vital functions in the organisms in which is present, how can organisms survive in which it is not present? Can they replace it with another molecule or do not need it? What makes the presence of glutathione essential in some prokaryotes and in almost all eukaryotes? Organisms in which glutathione is present have in common an aerobic metabolism; in anaerobic organisms, glutathione is never present [56], and the halophilic archea that produces γ-glutamyl-cysteine is an organism that can live in both anaerobic and aerobic conditions. In this archaea, γ-glutamyl-cysteine and bis-γ-glutamylcystine reductase appear to function in a way analogous to GSH and glutathione reductase in other cells. By comparing the effect of salt on the autoxidation rates of γ-glutamyl-cysteine, GSH, and cysteine, has found that in the absence of added salt, cysteine is oxidized more rapidly than γ-glutamyl-cysteine, which in turn oxidized more rapidly than GSH. In the presence of 4,3 M chloride (K+ and Na+) γ-glutamyl-cysteine resulted at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize γ-glutamyl-cysteine rather than GSH [53]. The ubiquitous presence of high concentrations of glutathione in aerobic cells indicates a key role of GSH in the mechanisms of adaptation to the presence of oxygen. In anaerobic organisms, both prokaryotes and eukaryotes, depletion of glutathione in cells leads to death in the presence of oxygen. Thus, GSH is an essential thiol antioxidant produced in the cytosol of all aerobic cells and plays a key role in protecting against oxidative stress by neutralizing free radicals and reactive oxygen species (ROS). Glutathione has a central role in the antioxidant mechanisms of aerobic cells; without the presence of glutathione, other antioxidants (for example vitamins such as ascorbic acid or vitamin E) are not sufficient to keep the cell alive under aerobic conditions.

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When the above-mentioned vitamins react they pass to an oxidized form which can be restored to a reduced state by glutathione. If the levels of glutathione decrease too much in an organ, this cannot used for transplantation. During ageing there is a decrease in the intracellular levels of GSH and a recent review [23] focuses “on the suitability of treatment with exogenous γ-GC to raise GSH levels by circumventing the age-related dysregulation of the rate-limiting step of GSH, providing promise for future research for the treatment of chronic oxidative stress-related diseases”. Thus, the glutathione molecule is very old, a molecular fossil that has gone through some billions of years of evolution, perhaps remaining unaltered. The appearance of photosynthesis and therefore the progressive establishment of the presence of oxygen in the atmosphere seems to date back to about two billion years ago [59]. Photosynthesis allowed life to produce very large amount of glucose directly from sunlight, carbon dioxide and water, brought life to the surface of the oceans, freeing life from the limited resources of geochemically derived reductants. The possibility to produce large amount of food greatly increased global primary productivity and triggered an incredible proliferation of prokaryotes. Photosynthesis provided an infinite amount of glucose that triggered an incredible proliferation of prokaryotes, and probably the oceans were green due to the presence of myriads of photosynthetic organisms. At the beginning, probably, the inevitable increased production of oxygen as a by-product of photosynthesis did not create problems as it reacted with transition metal ions present in the waters of the oceans leading to the production of oxides transformed into hydroxides and precipitated. In fact, large layers of precipitated transition metal oxides have been found and described dating back about two billion years ago. The composition of ions in oceans changed, and when transition metal ions have been no more available, oxygen started to accumulate and dramatically altered the redox state of Earth’s atmosphere and oceans, permanently changing biogeochemical cycles. In a further few hundred million years, oxygen contributed to produce a great mass extinction as demonstrated by the big stromatolites (fossils of prokaryotes) still visible today as high structures several meters. If we think about the size of a prokaryote, the presence of such fossil structures indicates the presence of a quantity of prokaryotes extraordinarily large to the point of leaving fossils arrived up to us. Since also the dating of those gigantic stromatolites is about 2 billion years, they would have formed in some tens of millions of years and then not formed again. Organisms proliferated because of oxygen and started to die because of oxygen. The life did not disappeared from the surface of Earth because some cells discovered antioxidant molecules, like glutathione, and were able to evolve respiration. In this way the oxidative power of oxygen was used to produce carbon dioxide, water and energy, aliquots of the energy of the light of sun. At this point, the availability of

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oxygen allowed for the evolution of aerobic respiration and biosynthetic pathways, and life started again to grow giving rise to an extraordinary evolution of organisms, prokaryotes and eukaryotes, up to the appearance of complex multicellular organisms.

2 Glutathione and Glutathione-Dependent Enzymes Glutathione is the main low-molecular-weight thiol, the major non-protein defence against oxidative stress and the most important cellular thiol redox buffer [51]. Typically, cysteine supply derives from the diet, from protein catabolism and in the liver from trans sulfuration of methionine, thus permitting the use of methionine for GSH synthesis. Outside the cell cysteine is not stable and it quickly autooxidizes to cystine, which is recruited by some cells and is converted again to cysteine intracellularly [47]. Cysteine and glutamate are bind through the γ-carbonil group of glutamate instead of the typical α-carboxyl group. This particular bond confers to glutathione a high stability since only very specific enzymes in particular conditions may operate on its degradation. One of these enzymes is γ-glutamyl-transpeptidase (GGT), able to hydrolyze this unusual peptide bond, forming glutamate and Cys-Gly [60]. The cytoplasmic compartment is the main source of GSH, where it is “de novo” synthesized and transported into subcellular organelles. In these compartments GSH participates in many cellular processes that are mainly connected to the thiol’s redox state. In last years cell redox state has been correlated to many regulative pathways and recently the importance of a real redox code has been revealed [31]. In this scenario glutathione plays a critical role in protecting cells from oxidative stress and xenobiotics as it has been highlighted that the glutathione redox potential (GSH:GSSG ratio) is fundamental for maintaining antioxidant defense, both in detoxifying pathways and in redox signaling [16, 50]. Glutathione is used into the cells as a redox buffer, as an antioxidant, as sulphur storage and it is involved in a variety of cellular pathways including redox signalling, post-translational modifications and xenobiotics’s detoxification [2]. The major and best-known function of GSH regards its role in cellular antioxidant defense. The thiol group of glutathione is a potent reducing agent, rendering GSH able to scavenge many dangerous molecules both through direct or indirect enzymatic reactions. In particular, GSH is the cofactor for main antioxidant enzymes belonging to the glutathione peroxidases and the glutathione S-transferases families [12] The glutathione peroxidase system has a crucial role in the protection mechanism against hydrogen peroxide and other peroxides, including lipid hydroperoxides. When the glutathione peroxidase-mediated detoxification is taking place, GSSG is produced and the GSH pool can be regenerated from GSSG via glutathione reductase [57].

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The glutathione-S-transferases (GSTs), a superfamily of Phase II detoxification enzymes, use GSH as a cofactor that reacts with electrophiles or oxidizing species such as xenobiotics and their metabolites to form conjugates, representing another important cellular defense mechanism of protection from reactive substances. After conjugation with electrophiles, the resulting GSH-adducts are actively excreted from the cell The detoxification process continues by a series of enzymatic steps aimed to convert a water-soluble conjugate into a mercapturic acid. GSH conjugation irreversibly depletes intracellular reduced glutathione and when the consumption is severe, cellular defenses based on GSH conjugation are impaired [11]. Another important detoxification pathway in which GSH takes part as a cofactor is the glyoxalase system. It comprises two enzymes, namely glyoxalase 1 (Glo 1) and glyoxalase 2 (Glo 2), that sequentially catalyse the conversion of methylglyoxal to D-lactate via the intermediate S-D- lactoylglutathione. The glyoxalase system is utilized by the cell to remove toxic α-oxoaldehydes, especially methylglyoxal (MG), in order to protect cells from α-oxoaldehydesmediated formation of advanced glycation end-products (AGEs) [10]. This system uses GSH to convert dangerous methylglyoxal to S-Dlactoylglutathione, a thioester of glutathione, which is then hydrolyzed to D-lactate and GSH that is again available as reduced form or used for glutathionylation of specific proteins [27]. The glyoxalase system is found throughout biological life and is thought to be ubiquitous. Studies performed by our research group has led to several results clarifying different aspects of glyoxalase system in particular on the Glo 2 enzyme. Glo 2 has been isolated and studied from plants [34, 42, 43] and several different vertebrates [44–46] and subsequently characterized [49]. The second most important main function of GSH is about the thiol status of proteins. In cell metabolism, the mechanism of thiol-mediated redox control is due to the ability of cysteine residues to change reversibly their redox state with consequent modifications in conformational, catalytic or regulatory functions of a protein. Proteins can be post-translationally modified through disulfide linkages between GSH and redox-sensitive cysteine residues within proteins by S-glutathionylation (P-SSG) [18]. The formation of protein-SSG mixed disulfides can often occur under oxidative stress and it serves to prevent irreversible oxidation of protein thiols, but can also operate as a biological redox switch in the regulation of signalling and metabolic pathways. Indeed, GSH is used in the S-glutathionylation of many proteins involved in regulation of metabolism, as kinases and proteins of the signalling pathways that regulate cell cycle or cell death, or proteins involved in glycolysis/energy metabolism as well as antioxidant enzymes, cytoskeletal proteins and chaperones [7]. Whereas glutathionylation mediates protection from oxidative stress and the redox regulation, this alteration in protein thiols must be removed when the signaling event or the cellular oxidative status is over. The process of de-glutathionylation is mainly catalyzed by glutaredoxin (Grx) under appropriate redox conditions and GSH plays a fundamental role in the catalytic mechanism of Grx. When glutaredoxin reacts

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with an S-glutathionylated protein, the target disulfide thiol is exposed to a nucleophilic attack that breaks the mixed disulfide and transfers the glutathione moiety to glutaredoxin itself. In this reaction, Grx gets glutathionylated (Grx-SSG) and the previously S-glutathionylated protein is released in its free thiol form. Then, GSH reduces the Grx-SSG to produce glutathione disulfide, recycling the reduced enzyme [40]. Previously, S-glutathionylation was considered as a nonspecific response to cellular oxidative status, in order to protect cysteines from irreversible oxidation, while recently it has been proposed as a regulatory and selective post-translational protein modification in response to specific physiological redox signaling. In the last few years, many proteins have been found to undergo post-translational modification by S-glutathionylation [58]. In this context we reported proteinprotein interaction between Glo 2 and specific proteins targets involved in the S-glutathionylation. Glo 2 is a metal dependent β-lactamase enzyme that shows a characteristic Zn2+ -binding motif, conserved in all known sequences, and a Zn(II) Fe(II) center [1]. The reaction catalysis, analyzed in different papers, hypothesizes the release from the Glo 2 active site of the product (D-lactic acid) and one molecule of unprotonated glutathione (GS−), which will be easily protonated by a water molecule depending on the acidity of the solution [14] or will be used for glutathionylation of specific target proteins. In our own work, using the natural substrate of Glo 2, S-D-lactoylglutathione (SLG), association complexes of Glo 2 with target proteins (actin and malate dehydrogenase) have been observed, leading to S-glutathionylation of analyzed proteins [20, 25]. Other functions of glutathione include the conjugation with nitric oxide to produce an S-nitrosoglutathione (GSNO), the physiological storage form of nitric oxide (NO) in tissues. S-nitrosoglutathione reductase (GSNOR) catalyzes the breaking of GSNO thus modulating the availability of reactive nitric oxide (°NO) in the cell. GSNO may have many roles in the cellular regulation patterns because it can release °NO in tissue or it can convey °NO in plasma contributing to increase the physiological levels of nitric oxide. The dysregulation of GSNOR can lead to drastic changes in protein Snitrosylation, an important post-translational modification (PTM), and can have numerous other downstream consequences. Many studies have shown as NO and GSNO play a critical role in smooth muscle relaxation, cardiopulmonary regulation, and neuronal signaling [24]. It is important to mention that nitrosative stress can cause S-glutathionylation of certain target proteins. An important study of Chen et al. showed that S-glutathionylation of endothelial nitric oxide synthetase reversibly decreased NOS activity. This reversible protein modification led to loss of NO and gain of O2 °- both associated with impaired endothelium-dependent vasodilatation [15]. The multifaceted involvement of glutathione makes it important in numerous physiological functions. Appropriate GSH concentrations are required for spermatogenesis, activation of T-cells and white blood cells, cytokines production, and also in

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the activation of immune responses [5, 6, 29]. It has also been shown that glutathione is involved in defence against environmental stress, infection, drugs, aging, radiation, burns and unhealthy diet [30]. Our own study have shown as GSH depletion was correlated with aging and diet in a model of rats fed by caloric restriction diet able to prolonging life [3, 4]. Glutathione other than in the cytosol is present also in the most important cellular districts like mitochondria, nucleus and endoplasmic reticulum to indicate its central role in several metabolic pathways. Mitochondrial glutathione (mtGSH) is of great importance to maintain redox balance status [39]. Mitochondria lack the enzymes required for glutathione biosynthesis, therefore the GSH pool in the matrix has to derive from the cytoplasm. mtGSH is mainly present in its reduced form, acts as an independent redox pool, and represents 10–15% of the total cellular GSH. Cytosolic GSH can pass the outer mitochondrial membrane via porin channels but its anionic properties at physiological pH, and the highly negative membrane potential, does not allow GSH to diffuse into the matrix. Consequently, GSH enters the matrix by carrier-mediated transport across the inner membrane. Among the various known carriers identified in the inner mitochondrial membrane, two of them (dicarboxylate, DIC Slc25a10; and 2-oxoglutarate, OGC Slc25a11) play a key role in the passage of GSH from the cytoplasm into the matrix. Nevertheless, it has been observed in rat liver mitochondria that, if dicarboxylate and 2-oxoglutarate carriers are blocked by inhibitors, the uptake of GSH is lower (45–50%), which means that also other transporters are involved in mitochondrial GSH uptake [61]. On the other hand, a recent alternative way for supplying GSH mitochondrial pool has been described by our group. In that work has been demonstrated that S-Dlactoylglutathione, a thioester of glutathione and an intermediate of the glyoxalase system, enters into mitochondria and is hydrolyzed by glyoxalase II to D-lactate and GSH thus restoring the mtGSH pool [8]. Mitochondria are considered the major source of ROS production, as a small amount of the consumed oxygen is subjected to reaction with electrons that get away from the electron transport chain (ETC), generating dangerous reactive species. Indeed, the concentration of superoxide anion in the mitochondrial matrix has been evaluated to be 5- to 10-fold higher than in the cytosol [41]. The constant production of ROS make mitochondria the target of the damaging effects of oxygen radicals, but the functionality of mitochondria is preserved by a defense system that repairs the oxidative damage generated [19, 36]. In this contest, GSH plays a key role in antioxidant defense. Mitochondria lack catalase so detoxification against ROS, as hydrogen peroxide, is possible mostly through glutathione peroxidases (Gpxs) and peroxyredoxins (Prxs). Through this system reducing equivalents of NADPH are used by glutathione reductases (GRs) and thioredoxin reductases (Trxs) to regenerate, respectively, mtGSH/Gpx or Prx/Trx antioxidant systems.

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If the activity of Gpx exceeds the capacity to remove all the GSSG by GRs, GSSG accumulates in mitochondria, as it cannot be exported into the cytosol. Thus, an increased production of GSSG can contribute to spontaneous mitochondrial glutathionylation of target proteins [38]. In fact, if the GSH:GSSG ratio decreases up to ~1 and an oxidant environment is present, it is known that non-enzymatic glutathionylation occurs in order to protect the irreversible oxidation of available cysteine thiols of proteins. Immediately after the levels of reduced glutathione return to normal conditions, due to the contribution of Grs, the reversal of glutathionylation by thiol– disulfide exchange takes place. S-glutathionylation that occurs non-enzymatically is non-specific and is connected to oxidative and pathological situations. By contrast, S-glutathionylation that takes place enzymatically is specific, fast, reversible, and is also necessary for modulating particular cellular processes [37]. In mitochondria some different proteins have been shown to be glutathionylated. This post translational modification is connected to ROS levels, H2 O2 production, mitochondrial environment, redox balance, protein microenvironment and can deeply affect mitochondrial function and redox signaling [48]. As an example, Complex I, which is sensitive to the redox environment, is a possible target of S-glutathionylation. The mitochondrial enzyme Grx2 is responsible for its reversible glutathionylation, and is greatly influenced by the GSH:GSSG ratio. In fact, a high GSH:GSSG ratio leads to a greater Complex I de-glutathionylation favoring its activation, whereas a low GSH:GSSG ratio leads to the reverse effect [55]. Glutathionylation of malate dehydrogenase favoring its dimerization and activation. Glo 2 is responsible for its reversible glutahionylation [20]. Generally, the whole electron transport chain (ETC) is sensitive both to changes in redox environment and to fluctuations in the GSH:GSSG ratio, as it has been observed that almost all of the components of ETC are subjected to glutathionylation [13]. Moreover, also uncoupling proteins (UCPs) are controlled by glutathionylation, therefore the electrochemical proton gradient is closely connected to the mitochondrial redox balance [35]. Another important role of mitochondria involves the control of cell death, including apoptosis and necrosis. Apoptosis can be induced by the death receptor (extrinsic) pathway or by the mitochondrial (intrinsic) pathway [17]. Exhaustion of mtGSH has been linked to apoptosis, either preparing for cell death or by modulating the permeability of mitochondrion and activating apoptotic effector caspases. GSH depletion, indeed, might influence other redox-sensitive steps in the apoptosis cascade such as increased ceramide production or enhanced Jun N-terminal Kinase (JNK) activation or inhibition of NF-kB transactivation of survival genes [9]. These observations suggest a critical role of mtGSH in the defense of cells from the two main apoptotic pathways. Basically, GSH oxidation or GSH efflux generate the impairment of the cellular glutathione redox couple GSH:GSSG, contributing to initiate apoptosis. Therefore, recovery and maintenance of the mtGSH pool is important to counteract oxidative stress and regulate cell death.

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3 Conclusions Because of its unique properties, glutathione has been selected and maintained for allowing life in the presence of oxygen produced by photosynthesis. The ubiquitary high levels of glutathione in aerobic prokaryotes and eukaryotes supports further its important role. A severe depletion of cell glutathione is incompatible for life. Glutathione dependent enzymes are widely spread in aerobic cells, and maintain signs of an evolutionary origin ancient more than one billion years. Glutathione is one of the most investigated antioxidants. The redox couple GSH:GSSG is known to be very important for the cell, playing crucial roles in the antioxidant system. Moreover, glutathione is involved in many metabolic and regulatory processes like gene expression, modulation of DNA synthesis, cell proliferation and apoptosis, signal transduction, immune system, detoxication of endogenous substances and xenobiotics, S-glutathionylation of proteins. The lack of adequate levels of glutathione contributes to severe oxidative stress that may evolve to aging or to the development of several diseases. Indeed, neurodegenerative disorders like Alzheimer’s and Parkinson’s, or hepatitis, cystic fibrosis, inflammation, cardiovascular disease and diabetes are all linked to oxidative damage and glutathione deficiency. For each of these pathological conditions it is not always easy to establish if depletion of GSH is either the cause or effect. Of course, for some pathological conditions caused by hereditary diseases or by external pathogens, depletion of GSH is the effect, whereas for other pathological conditions deriving from environmental conditions it is possible that GSH decrease could be one among the many causes of the onset and evolution of the illness. In both cases, it is desirable to bring glutathione levels to normal ranges in order to improve health conditions. Recently, many studies on glutathione in normal physiological functions and in human disease have started to lay emphasis on the importance of the incorporation and compartmentalization of GSH into distinct subcellular pools. Also, the factors that could influence the relationships between different intracellular pools of GSH have still to be better investigated, in order to identify the biochemical mechanisms responsible for the compartmentalization process and then be able to modulate or modify specific GSH level to prevent or reduce the risk of disease progression.

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New Insights for Early Warning and Countermeasures to Aquatic Pollution Oliana Carnevali, Maura Benedetti, Francesca Beolchini, Antonio Dell’Anno, Daniele Fattorini, Stefania Gorbi, Silvia Illuminati, Francesca Maradonna, Giuseppe Scarponi and Francesco Regoli

Abstract In the last few decades, challenges of the aquatic environment have been dramatically changed in terms of magnitude of disturbances or types of hazards, along with the rise of new scientific and technological opportunities. In addition to the traditional (legacy) chemicals like metals or organic chemicals (including PAHs, PCBs and dioxins), emerging pollutants such as endocrine disruptors (EDCs), pharmaceuticals and microplastics are examples of worrying stressors for health of aquatic organisms and ecosystems. These compounds, introduced by different environmental sources and having a wide distribution, are absorbed by several species causing potential deleterious effects. The challenges of such pollutants will be discussed focusing on (i) new detection strategies, (ii) mode of action, (iii) ecological risk assessment and (iv) implementation of mitigation actions, to reduce the contaminant impact in terms of health care costs and maintenance of biodiversity. Sustainable and ecologically compatible methods and strategies for the reclamation of aquatic sediments contaminated with organic pollutants and toxic metals will also be described. Such knowledge will foster the reduction of use or release of many hazardous substances, together with providing insights on the early warning detection of chemical harmful agents within an environmental monitoring and security application program. Based on this knowledge, in this review we attempted to describe the approaches that are taken by research groups with different backgrounds operating at Università Politecnica delle Marche.

O. Carnevali (B) · M. Benedetti · F. Beolchini · A. Dell’Anno · D. Fattorini · S. Gorbi · S. Illuminati · F. Maradonna · G. Scarponi · F. Regoli (B) Dipartimento di Scienze della vita e dell’Ambiente, Università Politecnica della Marche, Via Brecce Bianche, 60131 Ancona, Italy e-mail: [email protected] F. Regoli e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_29

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1 Legacy and Emerging Contaminants in Aquatic System One of the consequences of the current state of industrialization, growing demand for modern conveniences and improved quality of life is the increased exposure to several chemicals from industrial activities, traffic and energy production. Several studies worldwide have shown the potential of synthetic and natural chemicals to enter aquatic ecosystems causing adverse effects on aquatic organisms and food supplies [54].

1.1 Legacy Contaminants in Aquatic System Traditional contaminants, such as heavy metals and organic chemicals (e.g. polycyclic aromatic hydrocarbons PAHs, polychlorinated biphenyls PCBs, dioxins, pesticides) are chemical stressors which are further referred as Legacy Contaminants (LCs) because of their persistent, long-term effects in the environment and organisms. LCs are considered to be strongly associated with industrial activities. Even though their concentrations are seen to have an increase near urban centers, evidences of LCs have been also found in remote areas like Antarctic ecosystems [27]. Metals have both natural and anthropogenic sources. Some of them (e.g. Fe, Cu, Co, Mn, Zn) are essential nutrients for organisms, playing key roles in the functioning of enzyme systems. However, high concentrations induce detrimental effects in organisms and in the environment. Other non-essential metals (e.g., Cd, Hg, Ni, Pb) have no biological role in an organism’s physiology and are toxic even at trace levels. Human activity affects the natural, geological and biological cycling of metals through pollution of the air, water, and soil. It alters the chemical forms (i.e. speciation) of metals released in the environment affecting toxicity, distribution, bioaccumulation and biomagnification (e.g. methyl mercury) along the food chain [1, 15, 38]. Organic pollutants (e.g. PAHs, PCBs, dioxins, pesticides) are of great concern because of their toxicity and in some cases, their tendency to biomagnify along the food webs. The main source of these pollutants is anthropogenic such as industrial activities, municipal incinerations, sewage sludge, coal and oil combustion. Once released into the environment they resist to photolytic, chemical and biological degradation. Due to their physical and chemical properties, many of these chemicals are defined as Persistent Organic Pollutants (POPs) and are subjected to long-range transport and biomagnification, resulting in a widespread distribution even in regions where they have never been used [10]. The global Stockholm Convention on POPs, which entered force on 2004, is one of the most important actions of the international community aimed to reduce and eliminate the production, use and releases of these substances.

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1.2 Emerging Contaminants (ECs) in Aquatic System Beside Legacy Contaminants, emerging pollutants e.g. endocrine disruptors (EDCs), pharmaceuticals, microplastics, natural toxins, represent examples of worrying stressors for health status of aquatic organisms and ecosystems. EDCs were first identified as emerging contaminants in the late 60s. From a chemical point of view, EDCs include compounds such as alkyl phenols, bisphenols, phthalates, hormones, pharmaceuticals, PAHs and flame retardants. Long-term effects of EDCs have been reported in several species and wild populations including the onset of intersex, embryo mortality, alteration of sex ratio, reduction of reproductive success, malformations, immunosuppression, and mortality [52]. In addition, the last two decades witnessed remarkable worldwide increase of medicine consumption paralleled by the continuous input of pharmaceuticals in aquatic ecosystem with ubiquitous detection of concentrations ranging from ng/L to µg/L [35]. Low levels of non-steroidal anti-inflammatory drugs, antidepressants, antiepileptics and antibiotics have been found to occur in marine mussels along coastal areas resulting able to impair cellular and physiological processes [34]. As additional emerging pollutants, marine species are also affected by plastic contamination [51]: besides entanglement and ingestion of macro debris by large vertebrates, microplastics (particles < 5 mm) are accumulated by planktonic and invertebrate organisms, and are further transferred along food chains (Fig. 1).

Fig. 1 Microplastics collected on beaches and ingested by organisms

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Microplastics ingestion is a widespread phenomenon in marine trophic webs with typically almost 25% of organisms containing 1 or more particles [2]. In addition, plastics contain chemical additives and efficiently adsorb several environmental contaminants, thus representing a potential source of exposure to such compounds after ingestion [23, 39]. Harmful algal blooms and algal metabolites are further challenging hazards for both ecological impacts and risk to human health during massive blooms. Palytoxins and ovatoxins produced by the benthic dinoflagellate Ostreopsis ovata instigate mass mortality of benthic organisms. Recent studies highlighted their role in immunological and cellular impairment induced in mussels Mytilus galloprovincialis [24]. On the other hand, the non-toxic alkaloid caulerpin (CAU) contained in the invasive green alga Caulerpa cylindracea is seen to be accumulated in fish species feeding on this alga [25]. It causes molecular, cellular and physiological effects which alter lipid metabolism by interactions with Peroxisomal Proliferator Activated Receptor (PPAR) [53].

2 Analytical Methods for LCs and ECs Quantification Several LCs and ECs can distribute in water, sediments and biota with high potential threat to aquatic organisms and humans. The identification and quantification of these contaminants in different environmental matrices have become a major scientific task. Above all, proper sampling procedures are a vital step in ensuring that monitoring results stand as representative of the investigated condition. Since many contaminants are present in low concentrations, attention should be paid during sample manipulation and analysis to avoid any possible contamination or interferences. Laboratory, apparatus, reagents and all materials used for samples collection and storage should be properly selected and treated as recommended by European Directives on the good practice of laboratory and quality assurance [16]. Accuracy of pollutant measurements must be checked by analyzing certified reference materials (available for all matrices) or spiked samples. Sample pretreatment is another significant analytical step to get accurate measurements of chemical pollutants. Metal determination usually includes a microwave digestion of the samples with the previous addition of different acid mixtures according to the matrix to be analyzed. This procedure allows the destruction of the organic matter and the release of organically bound metals. For POPs and ECs extraction, most studies reported the application of liquid-liquid extraction and the solid phase extraction (SPE). In recent years, Pressurized Solvent Extraction (PSE) which is an automated rapid extraction technique utilizing common solvents at elevated temperature and pressure, is largely practiced. Specific extraction procedures based on the density gradient separation or enzymatic digestion can be applied for microplastics in biota.

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The main analytical tools available for these contaminants are Atomic Absorption Spectrophotometry (AAS), Inductively Coupled Plasma Mass Spectrometry (ICPMS), Inductively Coupled Plasma—Optical Emission Spectrometry (ICP-OES), voltammetry and chromatography (either gas or liquid) coupled to mass spectrometry (GC-MS, HPLC-ICP-MS). The FTIR and micro-FTIR are the elective analytical approaches to characterize polymer typology of meso- and microplastics. Optimization, set-up and improvement of traditional and newly advanced methodologies are needed to further reduce the lower detection limit (LOD) and the quantification limit (LOQ). Researchers can detect the pollutant concentrations in levels of ng or pg for most of the chemicals. In the field of ECs, more recent developments such as linear ion traps (LITs), quadrupole, triple quadrupole, quadrupole-time of flight (QqTOF) and quadrupole-linear ion trap (QqLIT) have been used for qualitative analysis and pollutant identification.

3 Biological Methods for LC and EC Effect Evaluation 3.1 Ecotoxicological Approach Although analytical methods play a key role in assessing contaminant contents, the eco-toxicological approach which is based on the bio-indicator organisms along with the use of bioassays or biomarkers are recommended to address the bioavailability as well as the biological effects of the diverse mixtures of legacy and emerging contaminants [3, 11, 40]. Laboratory bioassays are a common approach to evaluate toxicological endpoints at the organism level, in many test species from several taxa and representatives of the main ecological or trophic positions (i.e. from bacteria to fish and from decomposers to final consumers). Different biological endpoints can be evaluated with these tests such as death, development anomalies, behavioral modifications, changes in reproductive success, metabolic disorders, growth alterations and bioluminescence. However, bioassays are not reliable for chronic or long-term effects and controlled laboratory conditions are critically unsophisticated compared to the natural environment. A more comprehensive evaluation of the environmental quality includes sublethal effects of contaminants and measuring alterations at the molecular or cellular levels (biomarkers). A multi-biomarker approach could be used in environmental prognosis to predict long-term toxicological effects or changes in the higher levels of biological organization [3, 41, 42]. The more sensitive and widely used biomarkers in marine organisms include destabilization of lysosomal functions, induction of metallothioneins and of cytochrome P450 biotransformation system, alteration of antioxidant defences, neurotoxicity and DNA damages as loss of integrity and bases oxidation.

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3.2 Novel Biomarkers for Early Detection of Emerging Contaminants 3.2.1

Biomarkers for Reproductive Toxicology Early Warning

Fish reproduction is a complex process regulated by the tight crosstalk between hormones and receptors. Because of their chemical structures, chemical compounds are able to mimic and antagonize the action of endogenous hormones thus triggering the same biological functions. Identification of biomarkers for their early detection remains one of the main challenges for ecotoxicologists. These biological endpoints include molecular, biochemical and physiological markers which when integrated, clarify issues of contaminant bioavailability, bioaccumulation and physiological effects. Biomarker expression can be tissue or gender specific thus providing precise insights about the source of contamination or can be activated in response to a wide range of chemical stressors [28, 29]. For many years, the ability of estrogen-like compounds to induce the expression of two female sex specific molecular markers, vitellogenin and zona radiata protein, in the liver of male has been considered an early warning signal of environmental contamination [32, 36]. Recently, large focus was addressed to phthalate toxic effects and studies reported that zebrafish exposure to Di-(2-ethylhexyl)-phthalate (DEHP) and Di-isononyl phthalate (DiNP) severely affects a series of physiological reproductive endpoints including fertility and fecundity [48]. A set of reproductive biomarkers involved in ovarian growth and maturation such as bmp15, fshr, lhcgr, mpr and ptgs2 [12] and in steroidogenesis like star and cyp11a1 were significantly altered by phthalate exposure [48]. In males, DEHP induced a mitotic arrest during spermatogenesis along with an increase of DNA fragmentation [13], providing evidences of the detrimental effects of this class of plasticizer on reproductive functions of both genders.

3.2.2

Mode of Action (MoA) of Endocrine-Disrupting Chemicals with Focus on Metabolic Alterations, i.e. Obesity and Hepatosteatosis

Recently, the ability of DEHP to affect metabolism acting on five biological pathways: ‘foxA2 and foxA3 transcription factor networks’, ‘Metabolic pathways’, ‘metabolism of amino acides and derivatives’, ‘metabolism of lipids and lipoproteins’, and ‘fatty acid, triacylglycerol, and ketone body metabolism’, was demonstrated in the liver of male zebrafish [26]. Focusing on “metabolic pathways”, several studies reported the key role of endocannabinoids (ECs) in the onset of metabolic syndromes. The ability of phthalates to modulate the ECs has been demonstrated in zebrafish female, where the up-regulation of hepatic pparα, cb1 and srebp levels induced de novo fatty acid synthesis besides

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hepatic steatosis [50] and inhibited food intake stimulus [20, 22, 37, 46]. Likewise, in zebrafish exposed to bisphenol A (BPA), the onset of the steatotic liver was induced by an ECs mediated positive feedback loop [22, 33]. Moving to marine teleosts, in seabream fed with diets contaminated with BPA, nonylphenol (NP) or tert-octylphenol (t-OP) and their mixture, an alteration of liver morphology and transcript levels of genes involved in lipid metabolism and endocannabinoid system was observed. Surprisingly, the lowest dose of contaminant exerted the highest biological effects [11, 21, 30, 31].

3.2.3

Deregulation of Epigenome by Exposure to Endocrine Disrupting Chemicals

As stated above, exposure of zebrafish to BPA has severely impaired reproduction [47] by its upstream ability to deregulate epigenetic mechanisms. In the zebrafish ovary, BPA interferes with histone modification leading to the down-regulation of lhcgr mRNA levels, potentially affecting also global methylation and interfering with the dnmt 1,3,4,5,7 expression [47, 49]. Moreover, the 6,188 mRNAs and 15 miRNAs were differently expressed in the liver, with signatures associated with nonalcoholic fatty liver disease (NAFLD), oxidative phosphorylation, mitochondrial dysfunction and cell cycle suggesting that BPA has great potential to cause adverse health outcomes on metabolic process through miRNA control [44].

4 Weight of Evidence (WOE) Risk Analyses Multidisciplinary studies which combine chemical and biological measurements, represent an added value to monitoring and management protocols. Recent European Directives recommend the use of different quality indicators to evaluate the environmental status of aquatic ecosystems. However, the combination of multiple typologies of investigations (or lines of evidence, LOEs) is often hampered by the lack of standardized procedures for the interpretation and the integration of complex datasets of heterogeneous results, which characteristically require various expert judgements [40]. Additional critical issues are represented by development of qualitative and quantitative evaluations, the synthetic classification and, last but not the least, the communication of risk to stakeholders. In this respect, a quantitative “Weight Of Evidence” model (Sediqualsoft) has been recently developed to integrate and differently weight data from various lines of evidence, which include sediment chemistry, bioavailability of chemicals in key bioindicator species, sublethal effects measured through biomarkers, toxicological effects at organism level assessed by standardized laboratory bioassays and ecological indicators of benthic communities status. Different typologies of data are initially evaluated within single modules by logical flowcharts and mathematical algorithms which provide specific hazard indices for each of considered line of evidence, before their differential weighting

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and integration in a quantitative WOE evaluation [40]. The most relevant feature of this WOE model is the use of weighted evaluation criteria that abandon the logic of the tabular approach for chemical data or the worst result for biological evaluations, allowing a more accurate discrimination between samples and levels of hazard or risk. These criteria have been validated in numerous national and international case studies for environmental risk assessment associated with polluted sediments, harbor areas, complex natural and anthropic impacts on the marine environment, the rescue of Costa Concordia wreck [43]. The elaborations reported for chemical and ecotoxicological data have also been incorporated into the recent Italian legislation for quality characterization and classification of marine sediments subjected to the dredging activities (Ministerial Decree 173, 16 July 2016).

5 Bioremediation Strategies for Contaminated Sediments As described above, coastal marine sediments subjected to high anthropogenic inputs can accumulate large amounts of contaminants, which represent a major concern for their potential detrimental consequences on ecosystem health as well as on their provision of goods and services for human wellbeing. In sediments, contaminants such as metals and hydrocarbons undergo complex biogeochemical transformations which are largely dependent upon microbial activity. In particular, prokaryotic activity in sediments affecting the pH, redox potential and organic matter degradation processes can induce cascade effects on metal speciation, partitioning and bioavailability [18, 19]. At the same time, prokaryotes can degrade hydrocarbons, under both anoxic and oxic conditions, thus playing a key role in natural attenuation processes such as reduction of hydrocarbon contamination levels with time [14]. For these reasons, the exploitation of microbial assemblages is gaining prominence for their potential in the remediation of contaminated marine sediments. During the last decade, special attention was allotted to explore the potential of different bioremediation strategies for the remediation of marine sediments contaminated with organic pollutants (e.g. petroleum aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, organotin compounds) and toxic metals. The special focus of these strategies was not only on the remediation efficiency, but also on the comprehension of abiotic and biotic factors along with their interactions which can influence the overall bioremediation performance. The effects of biostimulation strategies carried out on marine sediments contaminated with hydrocarbons maintained in aerobic and anaerobic conditions and at two different temperature regimes on bioremediation performance in relation with changes of bacterial abundance and diversity have been investigated [14]. These findings revealed that temperature exerted the main effect on bacterial abundance, diversity and assemblage composition. In both aerobic and anaerobic conditions, biodegradation efficiencies of hydrocarbons were significantly and positively related with bacterial richness and evenness. This further lead to the hypothesis that bioremediation strategies, which can sustain high levels of bacterial diversity rather than the

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selection of specific taxa, may significantly increase the efficiency of hydrocarbon degradation in contaminated marine sediments. Hydrocarbon biodegradation has been additionally investigated in relation with microbial growth rates during biostimulation experiments on contaminated sediments in the presence of sand and inorganic nutrients as amendments [9]. Results of these experiments highlighted that inorganic nutrients significantly enhanced the biodegradation of low and high molecular weight hydrocarbons besides stimulated microbial growth, whereas sand addition increased only the removal of high molecular weight compounds. It was also observed that the simultaneous addition of inorganic nutrients and sand provided the highest biodegradation (>70% for aliphatic hydrocarbons and 40% for PAHs). A semi-empirical kinetic model successfully fitted the experimental temporal changes of hydrocarbon residual concentrations and microbial abundances proposing that this approach can be used as a support tool while designing bioremediation strategies on site. The reclamation of sediments contaminated with butyltin compounds (BTCs) represents a great challenge due to their high persistence and toxicity. Biostimulation and bioaugmentation strategies or a combination of both have been applied on harbor sediments contaminated with TBT [7]. Almost 50% of the TBT was degraded in 20 weeks after bio-treatment based on inorganic nutrients was added (i.e. biostimulation), whereas no degradation occurred by the addition of a microbial consortium (i.e. bioaugmentation). Conversely, bioaugmentation combined with biostimulation resulted in the decrease of about 50% of TBT content only after 4 weeks. A simple kinetic model fitted to experimental data of BTC concentration allowed to estimate that 10 weeks are required to decrease the contamination of BTCs by 90% using a strategy based on both biostimulation and bioaugmentation, and 29 weeks with biostimulation alone. Overall, this study indicates that strategies based on biostimulation coupled with bioaugmentation may be efficient to significantly reduce the concentration of organotin compounds as well in relatively shorter timescales. Although bioremediation can be effective for the removal of organic contaminants, this strategy can induce important changes on the partitioning and mobility of metals present in the sediment [14, 18]. As a such, care should be applied to monitor the potential changes in the mobility and bioavailability of heavy metals induced by bio-treatments, especially when applied on sediments displaying a mixed contamination (due to high concentrations of organic and inorganic contaminants). Changes in the partitioning and mobility of metals present in the sediment have been investigated in more detail during biostimulation experiments carried out on marine sediments contaminated by both hydrocarbons and metals in relation with changes in bacterial diversity [45]. This study highlighted that taxa not belonging to known hydrocarbonoclastic bacteria may be involved in the biodegradation of hydrocarbon and that archaea and bacteria may exert a synergistic effect to changes in metal partitioning. Further evidence of major changes in metal partitioning due to changes in bacterial diversity were acquired from biostimulation experiments carried out on anoxic marine sediments based on the addition of organic (lactose and/or acetate) and/or inorganic compounds [17]. Contaminated sediments supplied only with organic substrates were characterized by an increase of the relative importance

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of sulfate reducing bacteria belonging to the families Desulfobacteraceae and Desulfobulbaceae with a concomitant decrease of taxa affiliated with Flavobacteriaceae. An opposite effect was observed in the biotreatments supplied with inorganic nutrients too. The increase of bacterial metabolism coupled with the increase of bacterial taxa affiliated with Flavobacteriaceae were reflected in a significant decrease of Cd and Zn associated with sedimentary organic matter furthermore Pb and As associated with the residual fraction of the sediment. However, independently from the experimental conditions investigated, no dissolution of metals occurred which is providing an insight to a role of bacterial assemblages in controlling metal solubilization processes. Overall, these studies provided new insights for a better understanding of the potential consequences of bio-treatments on the metal fate in contaminated marine sediments [17]. Bioremediation strategies have been also explored for the ex situ reclamation of dredged sediments contaminated with metals, based on the addition of specific microorganisms which are able to mediate the solubilisation of metals and semimetals from mineral ores [19]. In particular, the efficiency of different bacteria has been investigated: (i) acidophilic chemoautotrophic, Fe/S-oxidising bacteria, (ii) acidophilic heterotrophic bacteria able to reduce Fe/Mn fraction, co-respiring oxygen and ferric iron and (iii) pool of the chemoautotrophic and heterotrophic bacteria reported above, since it was hypothesized that the two strains could cooperate through a mutual substrate supply [4]. This study provided evidence that strategies based on the combined addition of Fe-reducing bacteria and Fe/S oxidizing bacteria can significantly increase the removal of metals from contaminated sediments (up to more than 90%) additionally having the advantage to be independent from the requirement of sulphur. The efficiency of such microbes to solubilize metals from marine sediments characterized by different geochemical properties, metal concentrations and partitioning has been further investigated [6, 8, 18]. These studies provided evidence for a variable solubilisation efficiency of metals furthermore highlighting the role of autotrophic Fe/S oxidizing bacteria associated with the production and re-cycling of leaching chemical species into protons and ferric ions. Moreover, the results of these studies indicate that the interactions among the acidic and oxidative conditions, the chemical behavior in aqueous environment of each metal species and the geochemical characteristics of the sediment can greatly influence the metal solubilization efficiency (Fig. 2). Finally, the efficiency of acidophilic S-oxidizing bacteria isolated from sulphurrich deep caves (Frasassi, Italy) for the mobilization of (semi-)metals from contaminated sediments has been tested. These bio-treatments revealed that S-oxidizing bacteria isolated from Frasassi caves have a high potential in removing As from marine contaminated sediments, as never reported before [5]. To summarise, the achieved results open new perspectives to develop efficient bioremediation technologies for the removal of toxic metals from highly contaminated sediments.

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Fig. 2 Main factors and processes involved in metal solubilization from contaminated marine sediments: the interactions among the acid and oxidative conditions, the metal chemical properties and the geochemical characteristics of sediment can greatly influence metal solubilization efficiency (figure adapted from [18])

6 Conclusions and Future Perspective In addition to traditional pollutants, new emerging compounds are threatening aquatic environments and organism health condition. Novel methods are required to predict toxicity and fast reliable hazard identification of both already registered substances as well as novel chemicals. New analytical technologies have improved our capability to detect such compounds at ultra-trace levels and modes of action have been explained for several classes of chemicals. However, complex interactions occur in chemical mixtures or when pollutants act in combination with multiple stressors with different responses occurring at various levels, for instance, from transcriptional to post-translational or catalytic responses. The ecotoxicological approach has a key role in understanding the mechanisms of action of environmental pollutants providing new models and technological tools towards the more contemporary environmental challenges. The identification of a set of biomarkers can be used to screen potential hazardous chemicals for the environment and human health. Ecotoxicology has also a central role in multidisciplinary WOE studies for interpreting large datasets of heterogeneous data, thus allowing to better characterize environmental quality and to summarize ecological risk in an easy format for non-expert stakeholders. Bioremediation, being a low cost and environmental-friendly strategy, has a high potential for the remediation of chronically contaminated sediments since there are many novel harmful compounds widespread in the environment (Fig. 3).

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Fig. 3 The conceptual framework to assay the environmental risk of aquatic ecosystems

However, the feasibility and sustainability of bioremediation strategies for aquatic ecosystems have not been demonstrated yet and there is a need for scale-up investigations together with estimation of costs and environmental impact. These represent crucial aspects for the development of truly sustainable and ecologically compatible processes of bio-treatments of contaminated aquatic sediments. As shown in this review, the contribution of DiSVA researchers at UNIVPM to the study of aquatic organism and ecosystem health status is very relevant within an international scenario.

References 1. Annibaldi A, Illuminati S, Truzzi C et al (2011) SWASV speciation of Cd, Pb and Cu for the determination of the seawater contamination in the area of the Nicole shipwreck (Ancona coast, Central Adriatic Sea). Mar Pollut Bull 62:2813–2821 2. Avio CG, Gorbi S, Regoli F (2017) Plastics and microplastics in the oceans: from emerging pollutants to emerged threat. Mar Environ Res 128:2–11 3. Benedetti M, Giuliani ME, Regoli F (2015) Oxidative metabolism of chemical pollutants in marine organisms: molecular and biochemical biomarkers in environmental toxicology. Ann NY Acad Sci 1340(1):8–19 4. Beolchini F, Dell’Anno A, De Propris L et al (2009) Auto- and heterotrophic acidophilic bacteria interactions enhance the bioremediation efficiency of dredged sediment contaminated by heavy metals. Chemosphere 74:1321–1326 5. Beolchini F, Fonti V, Ozdemiroglu S et al (2017) Sulphur-oxidising bacteria isolated from deep caves improve the removal of arsenic from contaminated harbor sediments. Chem Ecol 3:103–113 6. Beolchini F, Fonti V, Rocchetti L et al (2013) Chemical and biological strategies for the mobilization of metal/metalloids in contaminated dredged sediments: experimental analysis and environmental impact assessment. Chem Ecol 29:415–426 7. Beolchini F, Rocchetti L, Dell’Anno A (2014) Degradation kinetics of butyltin compounds during bioremediation of contaminated harbour sediments. Chem Ecol 30:393–402

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Marine Biology. Biodiversity and Functioning of Marine Ecosystems: Scientific Advancements and New Perspectives for Preserving Marine Life Cecilia Maria Totti, Stefano Accoroni, Marco Barucca, Silvia Bianchelli, Maria Assunta Biscotti, Barbara Calcinai, Adriana Canapa, Cinzia Corinaldesi, Roberto Danovaro, Cristina Gioia Di Camillo, Emanuela Fanelli, Cristina Gambi, Stefania Puce, Tiziana Romagnoli and Carlo Cerrano Abstract The ocean is a complex three-dimensional world covering approximately 71% of the Earth’s surface offering a huge potential of new discoveries in all the fields of science. The hope is that these discoveries, if adequately supported and implemented, could lead to the finding of sustainable solutions inspiring new technologies and growth strategies. The continuous discovery of new species and their interactions, the increasing understanding of the complex connection between biodiversity and ecosystems functioning, and the dramatic relation between the loss of biodiversity and the spreading of non-indigenous species are clearly stating the importance to strengthen the effort on marine biology studies. The growing interest of young generations on these topics is ultimately confirming this urgent need.

1 Introduction The growth of our societies at global level is launching important challenges that are waiting for solutions. The International Union for Conservation of Nature (IUCN)

C. M. Totti (B) · S. Accoroni · M. Barucca · S. Bianchelli · M. A. Biscotti · B. Calcinai · A. Canapa · R. Danovaro · C. G. Di Camillo · E. Fanelli · C. Gambi · S. Puce · T. Romagnoli · C. Cerrano (B) Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy e-mail: [email protected] C. Cerrano e-mail: [email protected] C. Corinaldesi Dipartimento di Scienze e Ingegneria della Materia, Ambiente e Urbanistica, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_30

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defines all “actions to protect, sustainably manage, and restore natural or modified eco-systems that address societal challenges effectively and adaptively, simultaneously providing human well-being and biodiversity benefits” as Nature-based Solutions (NbS). The fast growth of human population suggests that the demand for food or energy will rapidly increase and, as a consequence, the intensification of oil and gas extraction from the ocean could represent a further pressure for the oceans. The ocean biodiversity is rapidly declining, habitats are being degraded, plastic and other wastes are accumulating, and, owing to CO2 rising, oceanic acidity and the temperature are rising. It is essential to take a holistic view to fully understand the drivers of and the potential solutions to the various problems. Decision makers are developing many strategies of investment and exploitation of marine resources triggering a general concern in the civil society. Especially young generations are demonstrating an unexpected awareness on the importance to change the way we are exploiting our ecosystems, and this new consciousness is mirrored in the increasing interest towards marine biology studies and the need to look for progress in a sustainable way. The sustainability of the blue economy is strictly related on our knowledge about how to mitigate the impacts of the multiple pressures on the ocean ecosystem, taking also into account that climate change is enhancing the effects of anthropogenic activities. The complex nature of marine sciences and the connectivity of the marine systems clearly demonstrates the importance to develop multidisciplinary competences, able to address strategic solutions towards collaborative efforts between public organisations and private sectors. At global level, there is a growing interest towards blue economies and at European level this approach has been developed in the frame of the Blue Growth Strategy. All these considerations are attracting an increasing number of young students to marine biological studies, with the hope to actively participate in the processes that would find and promote sustainable solutions.

2 Taxonomic Studies. The Fundamental Bricks Biodiversity is the variety of life on Earth from the genes within an organism right up to the ecosystem level: the different plants, animals and microorganisms, the genetic information they contain and the ecosystems they form. Our knowledge on biodiversity is covering a little percentage in respect of the estimated actual values, moreover, we are documenting a rapid decrease of biodiversity on the Planet with impressive rates. European nature legislation, in particular the Birds Directive and the Habitats Directive, forms the backbone of European strategy to face the biodiversity loss and the legal basis for the nature protection network (Natura 2000). Scientists that are able to identify species and describe new ones are called taxonomists. “Without taxonomy to give shape to the bricks, and systematics to tell us

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how to put them together, the house of biological science is a meaningless jumble [1]”. Indeed, conservation strategies are compromised by the absence of information on what species exist, their ecology, biogeography, and trends in abundance. Unfortunately, funding to support taxonomic studies are very scarce and the general alerts on biodiversity loss are paradoxically not adequately supported in term of economic investment. There is a growing attention toward molecular studies, which can only partially fill the gap of knowledge on the biodiversity [2]. The simple focus on species identification through the molecular approaches limits our understanding at the upper level of organization (species and ecosystems) compromising our understanding about relationships and functioning. Moreover, the tendency to automatize the species identification could lead to loss the occasion to exploit morphological characteristics for bio-applications. On the contrary, an integrative approach, based on morphological, eco-ethological and molecular analyses, would give a comprehensive overview of the biodiversity from a specific area [3]. The reason why an integrative approach must be pursued is that nature shapes the life on the Earth and offers to humankind the possibility to observe existing things and to be inspired towards effective and sustainable solutions. Biomimetics is the science that seeks innovative technologies by emulating nature’s time-tested ideas. Considering the living world arose from the mineral world, they are strongly connected and compatible [4]. This concept is the base of Biomineralogy focused on the way living organisms model the mineral world, but also on how the mineral world affects living organisms [5]. A lot of knowledge about the interaction between minerals and organisms is based on research performed with Porifera and Cnidaria including examples of species able to incorporate particles into their body. Minerals in the aquatic environment can trigger different responses to the growth and development of benthic organisms modulating gene expression [6, 7]. Moreover, the studies of marine natural products continue to expand with a steady increase in the annual number of new compounds described [8] and indicate in Porifera the most promising avenue for Blue Biotechnology [9], as they constantly rank first for the number of derived novel metabolites with pharmaceutical potential (e.g., anticancer, antiviral, anti-inflammatory). Porifera and Cnidaria, are phyla of relatively simple aquatic metazoans including about ten thousand of species each. Porifera comprehends the oldest metazoan species, characterized by simple organization, plasticity and great diversity and abundance; to date, about 9000 species are considered valid, but probably more than double species really exists. The importance of sponge functional roles in marine communities has been recently highlighted leading to a crescent consideration of this group in monitoring programs. Taxonomy of Porifera is mainly based on the morphology of the skeleton formed by siliceous or calcareous spicules or collagen diversely organized. The impressive plasticity of sponges increases the difficulties related to their identification but open important research questions on the drivers affecting biomineralization processes and the expression of genes involved [10].

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Cnidaria is a phylum of basal metazoans that includes more than 10,000 species. They play an important role both in benthic and planktonic communities, in fact their life cycle is usually characterised by the presence of the polyp (benthic) and the medusa (planktonic) stages. In this phylum the main challenge is represented by the huge plasticity of life cycles, strongly connected to trophic resources and with the possibility to reverse their development paths [11]. Generally speaking, we know that the biodiversity is greater than previously recognized, especially in remote and extreme habitats and that new species are waiting to be described. Moreover, the complexity of ecosystem structure and functioning is greater than previously known and the increasing wide spreading of alien species is continuously asking to redraw the complex interactions of trophic webs [12–15]. Evolution in benthic suspension feeders tends to favour organisms establishing mutual beneficial relationships. Interactions between organisms range from a simple epibiosis or commensalism to highly intimate associations, as in the case of obligate symbiosis or endosymbiosis. Facilitation processes enhance cooperation among species, especially in very stable habitats where long living ecosystem engineers structure the assemblage [16]. Also, the study of seasonal cycle of marine invertebrates is particularly important, especially in the frame of climate change. The biodiversity of the Mediterranean basin is the results of complex evolutionary histories that have been shaped by episodes of isolation and reconnection with the Atlantic [17], leading to the cohabitation of cold and warm-affinity species [18]. Thermal anomalies are affecting the survival and the distribution of many species altering the structure of benthic and pelagic realms and modifying the web of intra and interspecific interactions. This web plays a key role to maintain the homeostasis of the ecosystems at global level. Trophic interactions and both competitive and cooperative processes are the lines of this web and only a detailed knowledge of the actors building this structure can guarantee adequate measures to limit the alarming rate of loss of biodiversity at global level, due to cumulative anthropogenic impacts and climate change.

3 Molecular Phylogeny: A Powerful Tool for Investigating Biodiversity Since Charles Darwin laid the groundwork for the study of species evolution in the mid-1800s, the goal of every evolutionary biologist has been to graphically represent the evolutionary relationships of all species through a phylogenetic tree. For this purpose, for many years data obtained from the comparison of the morphological and physiological characteristics between different species were taken into consideration and a valid help was also provided by fossil finds, where they were available, of common ancestors. Although much of the evolutionary relationships of many taxa has long been clarified, there are still several disputes/controversies to be solved. Indeed, in some taxonomic groups, the difficulty is linked to the morphological

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characters that may have undergone variations, due to environmental adaptation (convergence and evolutionary parallelism), so complicated that they are no longer useful for tracing the evolutionary history of the organisms under study. Moreover, as far as invertebrates are concerned, the basic morphological characters for taxonomy are few and scarcely informative. Since the eighties of the last century, the advent of molecular techniques useful for obtaining nucleotide sequences has revolutionized the way of studying the evolutionary relationships between organisms. In fact, through the analysis of the nucleotide sequences it is possible to obtain hundreds or even thousands of useful characters which form a series of data independent from the morphological ones. Furthermore, the molecular characters do not undergo the relevant selection due to environmental parameters since this mainly acts on the functionality of transcription and on the gene product itself. The management of these data has been possible thanks to the parallel development of bioinformatics which, by applying mathematical models such as Maximum Parsimony, Maximum Likelihood, and Bayesian Inference on molecular data, allowed the development of software able to reconstruct the evolutionary history of organisms. Molluscs are a group of interest both for the high number of species and their relevance as a fishing resource; however, they present few and scarcely informative morphological characters that do not allow to establish the evolutionary relationships, making difficult their classification. At the beginning of the nineties molecular phylogeny was employed as tool to solve these issues. It should be noted that the first studies were pioneering considering that there were no databases and that, for these organisms, the nucleotide sequences available in the literature were counted on one hand fingers. In this context several studies have been focused on the systematic review of bivalves, such as clams and scallops, and of gastropods such as ovulids for which it was necessary to undertake a preliminary analysis to determine the most suitable molecular marker to solve the evolutionary relationships at various taxonomic levels in these classes. For this purpose, sequences of mitochondrial and nuclear genes were obtained to determine which of these had the variation rate commensurate with the evolutionary distance to be measured. To discriminate the evolutionary relationships between species of the same family, mitochondrial genes have proved to be the most suitable given their rate of evolution higher than nuclear one, while nuclear genes and in particular those of ribosomal RNAs were more suitable for higher taxonomic levels [19–25]. Molecular data have allowed to obtain the correct phylogenetic relationships among the species analyzed and have highlighted substantial inconsistencies with the classic systematic as the absence of a real phylogenetic distinction between the orders of the subclass Heterodonta [22], the supposed monophyletism of different genera of scallops and clams [19, 21, 23, 24] and the erroneous classification of different species in the subfamilies of the gastropod ovulids [25]. These results proved to be of the utmost importance so much that they gave way to a substantial revision of the systematic of these taxonomic groups by malacological experts who integrated their morphological data with molecular ones [26, 27]. Furthermore, the methodology implemented for these first studies was subsequently used to analyze a larger number of species with different features and geographical provenance [28, 29].

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This approach has also been used to investigate and understand the molecular bio-diversity of basal metazoans such as Porifera and black corals, characterized by a wide phenotypic plasticity within a single species that makes extremely difficult their classification. Even for these taxa, molecular phylogeny has been a key tool to reconstruct the phylogenetic relationships between the species [30–34]. Regarding vertebrates, although they have a high number of informative characters, molecular phylogeny has contributed to solve controversial issues. Evolutionary biologists have long debated whether at the origin of tetrapods there were coelacanths or lungfish. To clarify this long-running controversy, the International Consortium managed by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was established, and the most expert evolutionary biologists of the world were involved, including members of the molecular phylogeny laboratory of UNIVPM. Thanks to the employment of the next generation sequencing technologies, it was possible to obtain the genome of the Latimeria chalumnae and the transcriptome of its congeneric species L. menadoensis. These data, which represent an invaluable source of information, have finally allowed to establish that lungfish and not the coelacanths are the living organisms evolutionarily closer to the ancestor of tetrapods [35–37]. In addition, the data obtained in the framework of this international collaboration have also allowed to know genetic changes that permitted the transition from water to land, one of the most important step during vertebrate evolution [38–40]. The molecular approach together with the development of new sequencing technologies proves to be a powerful tool for investigating biodiversity. The UNIVPM always early catches up to the innovative aspects of biotechnological approaches to compete with the most important research groups in the world. Consequently, it is applying new molecular techniques for population studies such as the ddRAD-Seq in the framework of the research project on the protection of natural beds of the bivalve Chamelea gallina, which represents an important food and economic resource in the Adriatic Sea. In the last three decades there has been a constant and continuous decrease of this species, the application of this new technique will allow to assess the degree of genetic variability directly related to the health status of a population for a responsible management of the resource. Currently the molecular phylogeny laboratory of the UNIVPM is weaving a network of relationships with other research groups in the Mediterranean basin to expand the study on Chamelea gallina. For the next years, future studies will have as a target that of taking the opportunity provided by the high throughput sequencing technologies for DNA analysis and the new bioinformatic technologies to deeply understand the animal biodiversity both at the level of species and population.

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4 Coupling Biodiversity Studies with the Management of Marine Coastal Areas: Impact of Benthic HABs on Environmental and Human Health Algae play important roles in our life: they are the most important source of the organic carbon that is the basis for aquatic food chains, influencing the quality of seafood, and play a role comparable to that of land plants in producing atmospheric oxygen. Humans largely use algae for food and as source of natural products, including renewable biofuel. However, algae are involved in phenomena considered negative for humans and for the environment when they become invasive or produce harmful toxins. In this regard, the knowledge of the algal diversity is mandatory for the management of marine coastal areas, where such phenomena are more common. Shallow, well-illuminated coastal waters from tropical to temperate latitudes are attractive environments for humans. Unfortunately, these environments traditionally have been threatened, especially in tropical regions, by outbreaks of benthic dinoflagellates, such as Gambierdiscus, Ostreopsis and Prorocentrum involved in toxin production and seafood contamination (e.g. ciguatera, clupeotoxism, etc.) [41]. Environmental factors (mainly temperature and nutrients) affect both the bloom dynamics and the toxin production of these benthic dinoflagellates [42, 43]. Due to increasing ocean temperatures and eutrophication processes, there is cause for concern that tropical seafood poisoning events may spread into higher latitude areas, as the distribution of the implicated dinoflagellates moves poleward [44–46]. Some of these concerns have already been realised in several temperate areas. In the Mediterranean Sea, the dinoflagellates Gambierdiscus and Fukuyoa have recently been recorded in Cyprus, Greece and Spain, and have been associated with the occurrence of ciguatoxins in seafood in Israel [47–49]. In addition, in the last decade Ostreopsis blooms regularly occur in the Mediterranean Sea during summer-autumn [50]. In this area, to date three Ostreopsis species have been reported: O. cf. ovata, O. cf. siamensis [51, 52], and the new species O. fattorussoi, which has been described in the eastern Mediterranean basin [53]. O. cf. ovata is the most abundant and widely distributed benthic dinoflagellate in the Mediterranean coasts [54] and despite its origin is still unclear, it has been listed within the alien and invasive species [55]. Given the fast spread of these undesirable blooms in the last ten years, numerous field studies have been carried out [56–58]. O. cf. ovata produces a large array of palytoxin analogues, i.e. isobaric palytoxin (isobPLTX) and ovatoxins (OVTXs) namely OVTX–a to –h [59–61]. This toxic dinoflagellate has been often associated with many cases of suffering or mass mortalities of various marine organisms and human illness (e.g. fever, cough, dyspnoea, sore throat, rhinorrhoea, skin irritation, etc.) attributed to inhalation or cutaneous contact with cells or toxic aerosol [62–66]. A number of ecological, taxonomical and ecotoxicological studies highlighted that these benthic dinoflagellates grow on several types of benthic substrata (macrophytes, rocks, invertebrates, sands) often forming a brownish, mucilaginous mat that can be easily resuspended in the water column with maximum abundances typically recorded in summer-autumn [67, 68]. Abundances show a significant decrease

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with depth, most likely related to light intensity [56]. Substrate type and availability are also thought to influence blooms: living substrata often host lower abundances of epibionts than other substrates, suggesting colonization is possibly limited by allelopathic interactions between macroalgae and epiphytes [69]. Moreover, both laboratory and field studies suggest that allelopathic interactions likely take place also within the microphytobenthic communities influencing the dynamics of the blooms [70, 71]. The synergic effects of hydrodynamics, temperature and inorganic and organic nutrient availability are main factors affecting the blooms [72–74]. Based on the present findings, the Ostreopsis blooms seem to be maintained thanks to some adaptations that allow it to thrive in P-limited environments where organic P is the main source of P. Despite the number of studies on biology, ecology and toxin production of these benthic dinoflagellates, several aspects still need to be further investigated. For example, among the vectors of these intoxication i.e., marine aerosol, direct contact and per os ingestion, the latter needs certainly further studies given its possible implications on human health almost unknown in temperate areas nowadays.

5 The Deep Sea: Scientific Advancements and Challenges The deep ocean (waters and sediments beneath 200 m depth) is the largest biome of the global biosphere, encompassing 95% of the oceans’ volume. However, less than 0.0001% has been investigated so far, making it the least explored biome of Earth [75]. Deep-sea ecosystems are characterized by extreme conditions, including absence of light beyond 200–500 m depth, pressures’ range from 20 to >1100 atm (in the Marian trench) and temperatures’ range from −1.8 to 2 °C up to 450 °C (in areas closed to hydrothermal vents). Such conditions profoundly influence the physiological and adaptation mechanisms of organisms, which inhabit the water column and all seabed habitats down to 11,000 m depth, including microbes which dominate both in terms of biomass and abundance [76–78]. In the last decades, the deep-sea exploration has resulted in the discovery of tens previously completely unknown habitats and ecosystems, along with thousands of species of organisms inhabiting these remote systems [75, 79]. For instance, three species of the animal Phylum Loricifera (Spinoloricus cinziae, Rugiloricus sp. nov. and Pliciloricus sp. nov.), able to spend their life cycle entirely in anoxic sediments, have been discovered in L’Atalante Basin [80, 81]. Molecular methods and metagenomics have revolutionized deep-sea biology and ecology revealing a huge diversity of deep-sea animals and microbes (including bacteria, archaea, fungi and viruses), which are fundamental for the functioning of the global biosphere and for the comprehension of adaptation processes to the impact of global changes [76, 82–85]. Understanding how biodiversity varies at different spatial scales and the drivers behind these patterns is a crucial issue in ecology. The variability in biodiversity as species number and composition is high in deep-sea sediments and changes in food

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availability and habitat heterogeneity play a key role in driving their spatial distribution [86–89]. Biodiversity maintenance has been recognized as preeminent responsible for the ecosystem functioning, efficiency and, thus, provisioning of ecosystem services [90, 91]. The long-lived, late reproducing, and low fecundity life histories of many deep-sea organisms increase vulnerability to multiple human pressures and global climate change. Deep-sea ecosystems, indeed, are not exempt to anthropogenic impacts such as bottom trawling >200 m depth, deep-sea mining and extraction of polymetallic sulphides at hydrothermal vent chimneys, illegal disposal of litter and accumulation of chemical pollutants of industrial origin and plastics [92, 93]. In addition, the global footprint of climate change represents the single greatest concern for human impacts on ocean environments, largely through indirect effects. Available projections suggest that by 2100, temperatures under 3000–6000 m depth could increase by 1 °C and contribute to reductions in water column oxygen [94], which will decline for up to 3.7% or more. Values of pH will show the most significant reduction at bathyal depths (from 0.29 to 0.37 pH units), together with reduced flux of organic matter to the sea-floor. Because most deep-sea environments depend largely on surface production, climate change effects on surface processes will alter deepsea ecosystems globally [95, 96]. Such changes can significantly affect the growth rates, survival and recruitment of deep-sea organisms with severe consequences for potential recovery of deep-sea assemblages [97, 98]. In this context, there is an increasing evidence that deep-sea species, habitats and ecosystems require a sound monitoring strategy providing policy makers with appropriate tools to sustainably exploit and preserve deep-sea ecosystems [99]. At European level, the Marine Strategy Framework Directive (MSFD 2008/56/EC) represents the tool of the EU’s Integrated Maritime Policy to achieve Good Environmental Status (GES) of marine waters by 2020. The MSFD applies to the area of marine waters over which a Member state exercises jurisdictional rights in accordance with the UNCLOS. This includes also deep-sea waters, seabed and subsoil within European exclusive economic zone. However, MSFD is mostly focused on coastal habitats or those reached by fishery activities, while the huge dimensions of the deep sea and its variety of life in terms of species and habitats are rarely considered. An implementation of the MSFD for the deep sea is strongly invoked, both at Mediterranean (see “The Valletta declaration” at www.msfd-idem.eu) and European level [100], especially for the development of new criteria and indicators that can fully fit with the peculiarity of deep-sea ecosystems. In this regard, for the descriptor related to Biodiversity,—recent works evidenced peculiar habitats, as deep-sea canyons, represent a seascape unit deserving protection at different levels according to biogeographic and geomorphological features, also in a global biodiversity management perspective [101, 102]. Concerning descriptor 4 (D4)—food webs, current criteria and indicators can be informative on trophic functioning, but at their current state of development, they are insufficient to assess whether deep-sea food webs really are at GES. Moreover, existing information is fragmentary, being mainly focused on western Mediterranean Sea [103 and references therein cited]. Still, some

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compartments which play a fundamental role in deep-sea food webs such as macrozooplankton/micronekton or mesopelagic fishes, which act as top-down controllers are rarely considered [104, 105]. Monitoring and conservation need to be to fundamental aspects of deep-sea ecology. An ecosystem-based monitoring strategy is the first step to identify priority areas for conservation in the deep sea [99]. Finally, a challenging aspect is to incorporate the third dimension in marine systematic conservation planning, i.e. vertical conservation planning and zoning from the sea surface to the seafloor. This approach has the potential to revolutionize marine conservation research, practice and legislation [91]. At the same time, there is awareness that monitoring and conservation alone can be insufficient to reverse pervasive habitat degradation in some deep-sea ecosystems. Scientifically, deep-sea restoration actions are feasible although actual applications require a deeper understanding of resilience and recovery rates of deep-sea communities/habitats. Still advancements in technologies are needed also for reducing the elevated costs associated [106]. These efforts are fundamental for identifying the most effective restoration activities to maintain ecosystem goods and services provided by the deep sea, largest biome of the global biosphere.

6 New Perspectives in the Study of Marine Biodiversity The main problem that marine biologists have to face is the lack of baselines, the lack of historical series. The field of marine biology is heading toward a more global time-series approach as a function of recent discoveries largely in the photic zone of the oceans and in the deep ocean. The study of biodiversity asks for a modern approach merging novel tools (i.e., molecular biology, electron microscopy, synchrotron, photogrammetry, Georeferenced Information Systems, exploration of new areas) with traditional morphofunctional studies. Early stage researchers interested in marine biodiversity must acquire these specific competences and inspire and address the finding of sustainable solutions. Concerning the European scenario, the implementation or drafting of EU regulations in matter of aquatic biodiversity (Marine Strategy Framework Directive, Habitat Directive, Water Frame Directive, Integrated Coastal Zone Management), is offering unique opportunities to young marine biologists, leading the production of research outputs in line with the EU blue growth strategy. The urgent need to extend the spatial scale of our knowledge and to increase public awareness towards scientific questions and marine conservation is offering new perspectives to the next generation of marine scientists. Citizen science projects can represent a very effective tool to obtain data on marine diversity at a wide scale and to sensitize people on the feasibility of a sustainable development [107, 108] and scientists are required to drive this process.

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Acknowledgements The authors thank Prof. Ettore Olmo who founded the Faculty of Sciences in 1991, today Department of Life and Environmental Sciences, and led it as Dean for twenty years, encouraging and stimulating its development and research activity.

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Synthesis, Structural Insights and Activity of Different Classes of Biomolecules Elisabetta Giorgini, Francesca Biavasco, Roberta Galeazzi, Giorgia Gioacchini, Eleonora Giovanetti, Giovanna Mobbili, Mario Orena, Maria Grazia Ortore, Samuele Rinaldi, Andrea Antonino Scirè, Francesco Spinozzi, Fabio Tanfani, Carla Vignaroli and Paolo Mariani Abstract In this chapter, researches focusing on the synthesis, structural characterization and activity of different classes of biomolecules are reported. The covered topics range from the synthesis of new amino acids and foldamers to the structural and functional analysis of proteins, the preparation and characterization of nanosystems for drug delivery applications, the molecular bases of antibiotic resistance spread in Gram-positive bacteria and the study of topology and microstructure of tissues and cells. To this goal, different experimental and computational techniques are exploited, such as biomolecular assays, molecular dynamics, X-ray scattering and IR spectroscopy.

1 Introduction The Department of Life and Environmental Sciences (DiSVA) at UNIVPM was established in 2011 by merging a number of groups involved in a wide spectrum of fundamental and applied research topics in Biology. Today, about 60 permanent scientists conduct research in interdisciplinary areas such as analytical and organic biochemistry, biophysics, cellular and molecular biology, marine biology and ecology, biochemistry and genetics, microbiology and biotechnology. In this book, different chapters review activities developed at DiSVA, with the idea to show a few of the main results obtained in the last years and to delineate the future directions of the researches.

E. Giorgini (B) · F. Biavasco · R. Galeazzi · G. Gioacchini · E. Giovanetti · G. Mobbili · M. Orena · M. G. Ortore · S. Rinaldi · A. A. Scirè · F. Spinozzi · F. Tanfani · C. Vignaroli · P. Mariani (B) Department of Life and Environmental Sciences, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy e-mail: [email protected] P. Mariani e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_31

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At first, activities related to the preparation of conformationally restricted amino acids, e.g. synthetic peptides with definite conformations in solution biased by intramolecular hydrogen bonds, will be reported, as the obtained results exemplify the suggestive goal of using compact and specific conformations of the created unnatural oligomers (“foldamers”) to generate new biopolymer-like structural and functional behavior. The study on stability, structure, and functional activities of different proteins, also in connection with pathologies, will be reviewed in the following paragraphs. The use of X-ray and neutron small-angle scattering techniques, as well as of molecular modelling and FTIR spectroscopy will be discussed, with the scope of highlight how these techniques can be best exploited to derive information on protein folding/unfolding/misfolding mechanisms and on the correlated structural and dynamical properties. The subsequent paragraphs will report on the recent studies on biomolecular nanoparticles for biotechnological applications. The characterization, in vitro and in vivo testing and optimization of nanocarriers (from SLN to smart liposomes, cubosomes and biologically related hydrogels) will be described. The emphasis will be on proving that only by using a multidisciplinary approach (based on different physical techniques and exploiting in-house synthetic and computational skills) it has been possible to produce smart, efficient and stable nanoscaled drug delivery systems. Afterwards, studies on the emergence of antibiotic resistance through the acquisition of Mobile Genetic Elements in Gram-positive bacteria will be presented. In this case, the growing evidence that AR genes/MGEs are widely present in animal and environmental reservoirs and that the worrying human situation may be the tip of an iceberg will be underlined. Finally, studies on biological specimens will be discussed: FTIR microspectroscopy has been in fact exploited for analyzing the macromolecular composition and structure of tissues and cells to evaluate reliable spectral markers related to specific biomedical topics and pathologies.

2 From Conformationally Restricted Amino Acids to Foldamers In this paragraph, the preparation of novel conformationally restricted amino acids and synthetic peptides (“foldamers”), which display in solution definite conformation, is reported.

2.1 Conformationally Restricted Amino Acids Conformationally restricted peptidomimetics mimic a natural peptide or protein, producing the same biological effect without the problems associated with natural peptides.

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Fig. 1 Significant conformationally restricted amino acids and peptides prepared within this research

Cyclic analogs of GABA displaying three—1 [30] and four-membered rings 2 [31], were at first prepared, together with pyrrolidine acetic acid 3 [20] and the isostere of pregabalin, 4 [25] (Fig. 1). By using γ-lactam analogues, the mimics of FEG 5 [22], and RGDG 6 [21], were synthesized. Leu and Met-enkephalin were modified by insertion of the β-amino acid 7 in place of glycine [23], and the βmethyleneaspartic acid, 8, inhibitor of glutamate-aspartate transaminase [28], was prepared. Eventually, isosteres of β-homoserine, 9 [26, 27], aspartic acid, 10 [26, 27], α-methyl aspartic acid, 11 [13–15], and α-methyl homoserine, 12 [13–15], were synthesized.

2.2 Foldamers Synthetic oligomers that mimic the ability of peptides and proteins to fold in solution into robust secondary structures are named foldamers. Hexamer 16, obtained from βamino acid AOMPC, 13, folded into a 12-helix [46], and the subsequent introduction of one or two units bearing a Cα-Me (AMOMPC, 14) gradually changed the folding from the 12-helix to the 8-helix (Fig. 2). The AMOMPC hexamer, 17, displayed a stable helical 8-helix [24]. Eventually, starting from AOPIC, 15, the hexamer 18 was obtained displaying a zig-zag 8-helix [1].

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Fig. 2 AOMPC 13, AMOMPC 14 and AOPIC 15 monomers. Hydrogen bonds in the secondary structures assumed by AOMPC 16 and AMOMPC 17 hexamers. Minimum free energy Z8 structure of AOPIC hexamer 18 (in black C=Oi···H-Ni+2, in red Ni···H-Ni+2, in blue non-standard C=Oi···HCi-2)

3 Proteins: Function and Structure 3.1 Protein Structure: In-Solution Small-Angle X-Ray and Neutron Scattering In-solution small-angle scattering of X-rays or neutrons (SAXS and SANS, SAS) is a powerful technique for determining the structural features of biological molecules in solution. Concerning proteins, SAS provides information on size, shape, aggregation, stability and compactness [4, 53]. In the case of large complexes, contrast variation (accessed by simple mixing of water and deuterated water in the aqueous solvent or by using differentially deuterated components) can be used in SANS to highlight a single segment of a multi-part structure [61]. SAS can be also useful to monitor a

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protein-at-work or to follow the kinetics of conformational changes in time-resolved conditions [53]. At DiSVA, several protein structure problems have been addressed by SAS techniques at Large Scale Facilities (Synchrotrons and Nuclear Sources) also using new data analysis approaches [62] integrated in open-source platforms (GENFIT [63], QUAFIT [61]). Protein stability has been, for example, investigated. High pressures have been applied to lysozyme in solution [54]: results proved that the density of the hydration water has a discontinuity between 600 and 1000 bar, and that the global and local lysozyme dynamics change at a similar pressure. The role of pressure has been also investigated on amyloid fibrils formed by α-synuclein, a protein related to Parkinson’s disease [57]. Pressure-induced dissociation of the fibrils resulted from a negative activation volume, suggesting the existence of a hydration-mediated activated state. SAS has been also used to assess the protein structure predicted by bioinformatics tools or reconstructed according to a rigid-body modeling. For example, the superposition of the structure of tissue transglutaminase determined by homology modeling to the protein envelope reconstructed by SANS gives the conformational changes induced by Ca and GTP [42], while a thermodynamic model involving equilibria between monomer, also in a denatured state, oligomer and all possible intermediate dissociation products has been used to derive the dissociation/association pathway in hemocyanins [61]. Finally, the process of protein fibrillation has been also faced by SAS techniques, both determining the time-resolved aggregation pattern of a mutant apo-myoglobin [55] and testing potential therapeutic strategies on the amyloid-β peptide, linked to Alzheimer’s disease [5].

3.2 Modelling of Membrane Receptors: Application to Computer Aided Drug Design (CADD) 3.2.1

Serotonin Receptor 5-HT2C Antagonist and Inverse Agonist

The 5-HT2c receptor is a G protein-coupled receptor subtype of serotonin receptor family. Its antagonists are used for treating depressive and anxious states. Unfortunately, this class of antipsychotic drugs has variable efficacy and side effects that are particularly due to the difficulties to both design drugs targeting selectively 5-HT2c receptor and to the difficulties to predict the individual responses of the patients. The latter is because single nucleotide polymorphisms (SNPs) could alter the interaction between receptor and ligand.

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In a study aimed to shed light onto the activation/inactivation mechanism of 5HT2c, the binding mechanism of a group of known antagonists and inverse agonists of this receptor (Ritanserin, Fluoxetine, Methysergide, Amoxapine, Loxapine) has been considered [29]. The receptor-ligand complexes geometries were predicted, evaluating the drugs’ binding energies together with the molecular interactions considering both wild type 5-HT2c receptor and some of its mutated forms associated to some most common SNPs. The ligands differences in binding the 5-HT2c variants were carried out by docking associated to molecular dynamic simulations (MD) in membrane. Finally, the ligand positioning and evolution associated to the trans-membrane helices motions (TMs) during the MD simulations were observed, shedding light on the inhibition mechanism. As a conclusion, it has been assessed that the mutated forms of 5-HT2c give a different stabilization to the antagonists and inverse agonists binding, which in general results in a stronger decrease of the overall Gbind energy. Thus, the presence of these mutations in patients is expected to give rise to a stronger drug effect.

3.2.2

Fighting the Pseudomonas aeruginosa Efflux Pumps Mediated (MDR) Resistance: Combined In Silico/In Vitro Screening Protocol

Pseudomonas aeruginosa is mainly involved in Cystic Fibrosis (CF) lung colonization and it is resistant to a wide range of antibiotics. A major mechanism of resistance in this organism is represented by the efflux pumps, which selectively extrude antibiotics outside the bacterial cells. Among these pumps, MexAB-OprM and MexXYOprM mainly contribute to antibiotic resistance and thus they are election targets in developing new inhibitors (EPIs). These pumps are tripartite systems consisting of an integral membrane transporter with broad substrate specificity (MexB or MexY), an outer membrane channel (OprM), and a periplasmic protein adapter (MexA or MexX). In the last few years, we started a project aimed to the identification of compounds able to block the activity of P. aeruginosa efflux pumps thus overcoming antibiotic resistance [41]. An efficient in silico/in vitro protocol to find some promising leads was developed. Firstly, MexB was selected as protein target and a virtual screening (HTVS) of natural products was performed. As a result, two natural lead compounds (Morelloflavone and 3,6dyhydroxypregna-20-one) were identified and showed a very high binding strength as it can be seen from the predicted inhibition constant Ki (Fig. 3). The antibacterial activity of the natural compound/antibiotic associations was then tested and they result to act synergistically with ciprofloxacin at a concentration of 40 μg/ml causing a 16-times reduction of the MIC values. In P. aeruginosa C24, the addition of the two compounds caused a shift from a resistant (MIC 4 μg/ml) to a susceptible (MIC 0.25 μg/ml) phenotype. Time killing assays were also performed. The presence of both compounds at 40 μg/ml increased (1–2 log) the bactericidal effect of ciprofloxacin, with a decrease of the MBC to 2 (1/2 MIC) and 4 μg/ml (MIC),

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Fig. 3 Best two docking poses for morelloflavone (green) and 3,6-dihydroxypregnan-20-one (yellow) (MexB); in the focus images the binding energy and the corresponding predicted Ki are reported

respectively. Ethidium bromide accumulation assays confirm their inhibitory interactions with MexB. Their low toxicity by haemolysis tests on human red blood cells suggests a feasible use of these EPIs in combination therapies with ciprofloxacin, one of the most largely used antibiotics in P. aeruginosa CF infections treatment. Other studies are underway considering MexY as target to develop a synergic EPI to use with tobramycin.

3.3 FT-IR Spectroscopy as a Tool for Detection of Temperature-Induced Molten Globule-Like States in β-Sheet-Rich Proteins Protein folding has been the subject of major investigation during the past decades. Polypeptides were thought to exist in unfolded (U) or native (N) conformations. Ptistyn postulated the existence of folding intermediates, known as molten globules (MGs), which have a native-like secondary structure and a less compact tertiary structure [58]. Several biological roles have been attributed to MGs, including release of

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Fig. 4 a Infrared absorption spectra of a protein dissolved in H2 O (light grey) and D2 O (dark grey) at room temperature. White spectrum: a thermal denatured protein in D2 O. The chemical structures represent the peptide bond before and after H/D exchange. Reprinted with permission from Biomedical Spectroscopy and Imaging 1 (2012), 247–259, 247, https://doi.org/10.3233/bsi2012-0021, IOS Press. b IRDS calculated in the 20–90 °C temperature range for AGP dissolved in D2 O. IRDS are obtained by subtracting a spectrum recorded at a given temperature (e.g. 55 °C) from the one recorded at temperature 5 °C higher (e.g. 60–55 °C line). Reprinted with permission from Biochemistry 2005, 44, 15997–16006. Copyright American Chemical Society 2005

ligands by binding proteins [9], involvement in misfolding diseases [10], and translocation across biological membranes [65]. In the early nineties of the last century, a group of researchers at DiSVA began to study the structure-function relationships in proteins by FTIR spectroscopy and, in 1999, one of the group detected MGs in a study on thermal unfolding of porcine OBP [56]. This result was achieved thanks to the high sensitivity of IR spectroscopy towards β-sheets and the ability of IR difference spectra (IRDS) (Fig. 4b) to give at the same time information on the secondary structure and compactness/flexibility of the proteins [2, 3]. Here, the detection of MGs during heating of human alpha-1-acid glycoprotein (AGP), which structure is characterized by 8 antiparallel beta-strands, is described [2]. The absorption IR spectrum of a protein in H2 O displays the amide I (~1650 cm−1 ) and amide II (~1550 cm−1 ) bands (Fig. 4a, light grey spectrum). The amide I is composed of bands ascribable to the secondary structural elements of the protein [2]. In D2 O, the amide I and amide II bands shift to lower wavenumbers (dark grey spectrum, amide I and amide II ), due the exchange of amide hydrogens (Hs) with deuterium (Fig. 4a, chemical structures). For the same reason, the amide II band decreases in intensity (residual amide II): the higher the decrease in intensity the higher the accessibility of the solvent to the protein. At room temperature, the H/D exchange is never complete, due to the inaccessible Hs of the protein’s core (residual amide II). By increasing the temperature, the amide I band intensity decreases (white spectrum) owing to protein unfolding (denaturation) which in turn induces H/D exchange of the inaccessible Hs.

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In temperature-induced protein denaturation, IR spectra are recorded at regular intervals (e.g. 5 °C). The detection of MG-like states is based on the analysis of the spectral features of proteins prepared in D2 O and on the H/D exchange of inaccessible Hs. MGs can be identified when, during protein heating, β-sheet bands do not decrease in intensity, but shift to lower wavenumbers, which means that the secondary structure is maintained, and that β-sheets relaxed and underwent H/D exchange, as indicated by the decrease in intensity of the residual amide II band [3]. All this can be shown, at the same time, by IRDS. In these spectra, adjacent negative and positive peaks of similar intensities (see 60–55 °C spectrum) reflect a shift of β-sheet bands due to H/D exchange of buried Hs, as supported by the negative broad residual amide II band at 1545 cm−1 . Protein denaturation is testified by the presence of negative bands in the amide I region, as in the case of the 1629 cm−1 band in the 70–65 and 75–70 °C spectra. In conclusion, IRDS represent a valuable way to describe the sequence of events occurring during protein heating that in the case of AGP includes the formation of MGs in the 55–65 °C temperature range and thermal denaturation in the 65–75 °C interval. Further methods to detect MGs in β-sheet-rich proteins are based on different elaboration of IR spectra as described in [2, 3, 56], and references therein.

4 DNA Elements Responsible for the Spread of Antibiotic Resistance in Gram-Positive Bacteria The emergence and spread of antibiotic resistance (AR) in human bacteria have become a global public health issue. Horizontal gene transfer (HGT) is a major force driving the emergence of antibiotic-resistant strains through the acquisition of Mobile Genetic Elements (MGEs), like conjugative/transferable plasmids and phages from different, often unrelated, donors. Genomic islands (GIs), including chromosomal cassettes, Integrative and Conjugative Elements (ICEs), and Integrative Mobilizable Elements (IMEs), are major HGT mediators in prokaryotes; they can drive or have driven strain differentiation. At DiSVA, the spread of the AR to the most clinically important antibiotic classes, including glycopeptides [6], macrolides [7, 38, 66, 68], beta-lactams [51, 67–69], aminoglycosides [16, 50, 67, 68] and oxazolidinones [50] have been investigated, documenting AR genes/MGEs in bacteria from different reservoirs.

4.1 Plasmids and Phages The multidrug resistance (MDR) plasmids harbored by Enterococcus faecium strains isolated from marine sediment (pLAG) [16] (Fig. 5a) and human gut (pEf37BA) [51] were characterized. pLAG and pEf37BA were new elements transferable via

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Fig. 5 a pLAG, mobilizable by pHTβ17i48 (modified from Di Sante et al. [16]). b ICESp2905 (modified from Giovanetti et al. [38])

co-resident conjugative plasmids and the latter was the first pbp5-carrying plasmid transferable in vitro to a different genus. We also described pE35048-oc, the first E. faecium optrA-plasmid outside China [50]. Vancomycin resistance plasmids, all carrying Tn1546-like elements, were found in enterococci of different origin (human, animal, food) [6]. m46.1—a prophage-associated genetic element widespread in Streptococcus pyogenes and similar to the Siphoviridae family viruses—was found to carry the resistance genes mef (A) and tet(O) [7] and to be transferable in vitro to different Streptococcus species.

4.2 Genomic Islands ICEs mostly result from a backbone carrying a variable amount of cargo genes of variable origins. The genetic organization of ICESp2905 of S. pyogenes results from the insertion of erm(TR) and tet(O) fragments in a scaffold of clostridial origin [38]; whereas the latter was stably integrated, the erm(TR) fragment (IMESp2907) was excisable and cis-mobilized by ICESp2905 (Fig. 5b). A novel composite type IV (2B&5) SCCmec—resulting from the recombination of a type IV and a type V SCCmec—was detected in a single locus variant of an epidemic methicillin-resistant Staphylococcus aureus. Its similarity to livestockassociated SCCmec suggest genetic exchanges between human and animal strains [69].

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5 Nanosystems for Drug Delivery 5.1 Guanosine Hydrogels Guanosine nucleosides and nucleotides are known to self-associate in water to form supramolecular complex architectures [18] including hydrogels [11]. Such a characteristic is very attracting, as the design and development of materials exhibiting gelation properties and based on self-assembling is important for nanotechnology. Self-assembled hydrogels are physical gels: they form by the spontaneous ordering of monomeric units into polymer-like supramolecular aggregates. Their advantageous properties, as softness, assembling reversibility and self-healing, originate from the weak forces that stabilize the network. If monomeric units are biomolecules, they will also exhibit biocompatibility and biodegradability [11]. The characterization of guanosine properties at DiSVA dates from 90th, when it was observed that the guanosine 5 -monophosphate (GMP) in solution forms fourstranded helical structures [18]. These structures (G-quadruplexes) are long cylinders made by stacked tetrads, which arise from the association of 4 GMP by Hoogsteen hydrogen bonding. Because of the lateral interactions [43], G-quadruplexes show a lyotropic polymorphism, forming columnar cholesteric and hexagonal phases as a function of water concentration [18]. G-quadruplexes were characterized even in diluted solutions [45], and recently it was observed that binary mixtures of Guanosine (Gua) and GMP in excess water form stable and transparent gels over a temperature range that can be tuned by varying the Gua/GMP ratio [11]. An extended analysis by X-ray scattering and AFM showed that Gua/GMP quadruplexes are highly flexible and mutually stuck (Fig. 6). These properties are sufficient to guarantee the formation of a stable 3D network in excess water, opening possible applications in tissue engineering, wound healing and drug delivery.

Fig. 6 a Phase diagram of Gua/GMP at 97% w/w water. b AFM images of 3:4 Gua/GMP extra diluted. Triangle side sizes are 625 × 625 nm and 2.5 × 2.5 μm [10]

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5.2 Nanocarriers for Drug Delivery Lipid dispersions are very convenient matrices to dissolve active molecules and to control their delivery, improving bioavailability and reducing side-effects [8, 52]. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and monoolein aqueous dispersions (MAD) have been in particular studied at DiSVA. Indeed, SLNs are known to preserve the degradation of the included drugs and to modulate their release [52], NLCs allow to enhance the specificity towards cells and the drug bioavailability and MAD [44] are suitable to deliver both hydrophilic and lipophilic drugs. Since morphology has an influence on the delivery of encapsulated molecules, but also considering that the entrapped drug can influence the nanoparticle stability, an extensive structural characterization is of paramount importance. At DiSVA, X-ray diffraction and cryoTEM techniques have been successfully applied and [17] lists recent results.

5.3 Liposomal Delivery Systems Liposomes are spherical bilayered shells, encapsulating an aqueous core, easily prepared from natural or synthetic amphiphilic molecules through a self-assembly process. Since the discovery of their ability to entrap both lipophilic and hydrophilic molecules, numerous applications as nanoscale carriers have been found in literature; they are now recognized as a promising template for so called ‘smart’ nanosystems and rationally designed to efficiently entrap guests and deliver them in a controlled fashion. By taking advantage of synthetic and computational skills of our Molecular Modeling and Bionanotechnology group, drug and gene delivery systems have been developed where the phospholipidic structure has been modulated by inserting modified lipid, polymers or salts. Although cationic liposomes-DNA complexes are relatively efficient in delivering DNA into cells, they have the disadvantage of toxicity and low bioavailability. In the research of a possible alternative to cationic gene delivery system, the employment of neutral liposomes (NLs) which have shown a longer circulation time has been explored. Complexes of DNA with neutral liposomes promoted by divalent cations have been prepared in our laboratories. We developed liposomal gene delivery systems [19] which act as effective cationic vesicles; thanks to the inclusion of synthetic lipids carrying in the polar head a chelating group, they are able to form stable complexes with plasmid DNA in the presence of divalent metals. In the field of targeted drug delivery, we designed a lysosomotropic delivery system (Fig. 7) containing a M6P cholesteryl conjugate (Chol-M6P) and employed with DOPC or POPC in the preparation of functionalized vesicles [47]. Due to its affinity for the Cation-independent M6P Receptor (CIM6PR), M6P group enables liposomes

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Fig. 7 Employment of M6P-linked liposomes (MFL), prepared with the synthetic Chol-M6P, in targeting cells overexpressing CIM6PR, delivering C6-ceramide into lysosome and inducing apoptosis

to deliver bioactive molecules to lysosomes. We demonstrated that liposomes prepared with the synthetic Chol-M6P could specifically target MCF7 cells, overexpressing CIM6PR, respect to HDF normal cells; indeed, we successfully employed the M6P-linked liposomes in the selective delivering of C6-ceramide to breast cancer cells in vitro, which led to an enhanced efficacy of C6-ceramide apoptotic effect. Our next goal will be to optimize the strategy by using a Computer Aided Drug Design approach to modify the structure of M6P moiety and maximize the interaction with the target receptor. Our delivery vector can potentially be applied to other tumor types overexpressing M6P/IGF-II receptor. In the last years, we extended our interest to the antioxidant liposomes and different nanosystems have been synthesized and characterized [39, 40, 48, 49]. Among them, we obtained interesting results with nanoparticles encapsulating Epigallocatechin-3gallate (EGCG), a polyphenolic catechin from green tea, well known for being bioactive in age-associated pathologies. By a combined in silico-experimental approach we obtained a complete polyphenol encapsulation in an anionic liposome in the presence of MgCl2 and Poloxamer-407. Since oxidative stress is involved in numerous retinal degenerative diseases, the ability of these liposomes to contrast H2 O2 -induced cell death was assessed in human retinal cells.

6 FTIR Analysis of Cells and Tissues FTIR Microspectroscopy (FTIRM) is a well assessed analytical tool for the analysis of micrometric areas of tissues and cells, with minimal sample preparation and without the use of stains or probes, which let obtain on the same sample and at the same

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time an overview of the composition and structure of the most remarkable cellular components.

6.1 Vibrational Analysis of Oral Cavity Lesions The research team of IR spectroscopy of the Polytechnic University of Marche has applied the FTIRM technique to the vibrational characterization of several benign and malignant pathologies of the oral cavity (such as adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, squamous dysplasia, keratocystic odontogenic tumor, radicular cyst, residual cyst, unicystic ameloblastoma, ameloblastic fibroma, etc.) [34, 64]. A careful spectroscopic analysis was performed on the epithelial and connective layers of biopsy tissue samples with diagnosis of Oral Squamous Cell Carcinoma (OSCC) at different tumor grade (G1, G2, and G3). The spectral data were compared with those from dysplastic and healthy tissues and specific spectral markers likely correlated with carcinogenesis (such as modifications of proteins’ secondary structure and side chains’ length and of carbohydrates and nucleic acids conformations) were defined. The vibrational features of the different samples were also matched with the morphological traits identified by the histological analysis, and, in some cases, early stages of tumoral pathologies, not detectable with routinely diagnostic techniques, were identified [59]. FTIRM was also exploited to study the biochemical pathways activated by drugcell interaction. Human primary Oral Squamous Carcinoma Cells (OSCCs) were treated with cisplatin and 5-fluorouracil and analyzed, for the first time, under hydrated conditions by FTIRM, by using a specific microfluidic device for in vitro infrared analysis. In vitro FTIRM analysis of primary chemotherapy treated OSCCs evidenced a time-dependent drug-specific cellular response, also including apoptosis triggering. The univariate and multivariate analysis of IR data showed meaningful spectroscopic differences ascribable to alterations in cellular proteins, lipids and nucleic acids. These findings suggested different pathways and extent of cellular damage for the two drugs, not always provided by conventional cell-based assays [37].

6.2 Spectral Imaging Analysis of Oocytes and Follicular Cells: From Zebrafish to Humans In the last few years, an interesting research focusing on the spectral imaging analysis of female gametes and follicular cells has been successfully developed at DiSVA. The zebrafish, Danio rerio, is an experimental model in biomedical research. It provides several advantages in the study of folliculogenesis, because of its asynchronous ovaries. In 2009, for the first time, both zebrafish ovary sections and single

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Fig. 8 a 3D IR maps of III and IV-stages zebrafish oocytes. Reprinted with permission of Anal Bioanal Chem (2010) 398:3063. https://doi.org/10.1007/s00216-010-4234-2. Copyright 2010 Springer-Verlag. b FTIR Imaging analysis of a zebrafish ovary section. Reprinted with permission from J Mol Struct (2009) 938:207–213. https://doi.org/10.1016/j.molstruc.2009.09.029. Copyright 2009 Elsevier

oocytes at different development stage were spectroscopically analyzed (Fig. 8a), and specific spectral markers linked to the macromolecular changes occurring during oocytes growth identified. In addition, the spectral features of vitellogenin and yolk proteins were defined and their topographical distribution in ovaries highlighted (Fig. 8b) [12]. Zebrafish was also chosen as model to evaluate the effects on the reproductive capability of specific bioactive compounds, such as probiotic, melatonin and endocrine disruptors. The FTIRM analysis of thin ovary sections of fish administrated with probiotic highlighted a more homogeneous distribution of proteins and lipids, and higher relative amounts of helix-folded proteins and water. All these findings, together with the increase of cathepsin L, a lysosomal enzyme involved in the final oocyte maturation, already observed in vitellogenic oocytes from probiotic treated group, let hypothesize that this bioactive molecule induces an early maturation and up-take of vitellogenin [35]. A similar multidisciplinary approach was used to evaluate the effects of melatonin on zebrafish reproduction. The IR spectra of all classes of oocytes from zebrafish females treated with different melatonin concentration were analyzed by multivariate analysis, which let separate them into different clusters according to oocyte class and treatment. The vibrational analysis highlighted in oocytes deriving from melatonin treated females, modifications both in the protein pattern, and in the aliphatic chains and phosphate moieties [36]. Finally, the FTIRM analysis of oocytes from zebrafish exposed to five different doses of di-isononyl phthalate (DiNP), a high molecular weight phthalate commonly used as a plasticizer, was performed [60]. Regards human reproduction, for the first time, the FTIRM has been recently applied to study the “quality” of human oocytes, also related to aging, and their biochemical cross talking with Granulosa Cells (GCs). The spectral analysis carried out on two groups of consenting patients (A, 30 ± 2 years old; B, 39 ± 2 years old), showed a worsening in the quality of oocytes from elder women, in terms

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of a more permeable plasma membrane with acyl chains peroxidation processes. In addition, other detrimental processes, such as the occurrence of phosphorylative mechanisms, an altered protein pattern with higher amounts of unordered structures, changes in nucleic acids’ conformation, and the onset of epigenetic effects, were also detected [32]. Recently, the spectral features of Granulosa Cells were defined, and, for the first time, a close relation was found between the biochemical composition of human GCs and the fate of the companion oocyte, in terms of fertilization failure, embryo development failure, implantation failure and clinical pregnancy. In fact, the analysis of the spectral data revealed that GCs from the different experimental groups were characterized by specific spectral features, ascribable to different biological and metabolic characteristics. By applying several feature selection procedures, 17 spectral biomarkers were identified, validated also by FDA, PERMANOVA and CAP analyses [33].

7 Conclusions This chapter reviews several activities performed at DiSVA. In all cases, the fundamentals of a multidisciplinary approach have been underlined. The presented results also suggest that integrated—biological, biophysical, chemical, medical, veterinary, agricultural and environmental—methodologies and close collaborations with large scale facilities, international, national and local institutions as well as a broader use of the new omic approaches are needed to face critical areas and cutting edge researches. Keeping the competition going and expanding partnerships will be the challenge of the near future.

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Conservation and Management of Biodiversity and Landscapes: A Challenge in the Era of Global Change Simona Casavecchia, Marina Allegrezza, Edoardo Biondi, Andrea Galli, Ernesto Marcheggiani, Simone Pesaresi, Fabio Taffetani, Stefano Tavoletti, Silvia Zitti, Maurizio Bianchelli, Nello Biscotti, Jacopo Facchi, Diana Galdenzi, Marco Galié, Roberta Gasparri, Linda Iommarini, Andrea Lancioni, Lara Lucchetti, Giacomo Mei, Ambra Micheletti, Silvia Montecchiari, Massimiliano Morbidoni, Cecilia Ottaviani, Morena Pinzi, Michele Rismondo, Giulio Tesei and Liliana Zivkovic Abstract The study of biodiversity is commonly carried out through different approaches, such as those at the level of genetics, species, communities/ecosystems, and landscapes. The group of geneticists, geobotanists and landscape researchers of the Department of agriculture, food and environmental sciences has been dealing with S. Casavecchia (B) · M. Allegrezza · E. Biondi · A. Galli · E. Marcheggiani · S. Pesaresi · F. Taffetani · S. Tavoletti · S. Zitti · M. Bianchelli · N. Biscotti · J. Facchi · D. Galdenzi · M. Galié · R. Gasparri · L. Iommarini · A. Lancioni · L. Lucchetti · G. Mei · A. Micheletti · S. Montecchiari · M. Morbidoni · C. Ottaviani · M. Pinzi · M. Rismondo · G. Tesei · L. Zivkovic Department of Agricultural, Food and Environmental Science, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy e-mail: [email protected] M. Allegrezza e-mail: [email protected] E. Biondi e-mail: [email protected] A. Galli e-mail: [email protected] E. Marcheggiani e-mail: [email protected] S. Pesaresi e-mail: [email protected] F. Taffetani e-mail: [email protected] S. Tavoletti e-mail: [email protected] S. Zitti e-mail: [email protected] M. Bianchelli e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_32

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this topic for many years. In this chapter, the principal research lines followed during the last thirty years are briefly described in order to put the best results achieved and the future targets into evidence. For the research group in geobotany, the main fields of research involve phytosociology, syntaxonomy, vegetation mapping, species and habitat conservation, ethnobotany and the ecology of agroecosystems. Regarding genetic biodiversity, the research carried out in plant genetics and breeding has dealt with the collection, evaluation and molecular characterization of some populations of the grass pea in the Marche region. Concerning the study of biodiversity at the landscape level, the research carried out has focused on the rural landscape and has dealt with the role of geomatics, robotics and new technologies in analyzing spatial heterogeneity, the importance of a bionomics approach to the landscape and its interfaces, and the integration of the landscape into the economic development and sustainable growth.

N. Biscotti e-mail: [email protected] J. Facchi e-mail: [email protected] D. Galdenzi e-mail: [email protected] M. Galié e-mail: [email protected] R. Gasparri e-mail: [email protected] L. Iommarini e-mail: [email protected] A. Lancioni e-mail: [email protected] L. Lucchetti e-mail: [email protected] G. Mei e-mail: [email protected] A. Micheletti e-mail: [email protected] S. Montecchiari e-mail: [email protected]

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Fig. 1 The scheme shows the different levels in plant communities research, in space (geosygmeta) and in time (sygmeta)

1 The Role of Vegetation Science in Biodiversity Conservation and Management The knowledge of vegetation plays a key role in land planning and landscape management (Fig. 1). In Italy and in Europe, the most widely used classification approach for vegetation is that of the phytosociology of Braun-Blanquet [57]. This approach classifies the current vegetation hierarchically into 4 syntaxonomic levels (in bottom-up order): association, alliance, order, and class. Thus, the association is the fundamental unit of the syntaxonomic classification. Defined by Braun-Blanquet [57] and updated by M. Morbidoni e-mail: [email protected] C. Ottaviani e-mail: [email protected] M. Pinzi e-mail: [email protected] M. Rismondo e-mail: [email protected] G. Tesei e-mail: [email protected] L. Zivkovic e-mail: [email protected]

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Biondi [15], the association is represented by a typical combination of species with a precise geographical distribution and quantifiable ecological value [15, 30]. Therefore, the association (in addition to the other syntaxa) is an important bioindicator of the environmental quality and biodiversity. Phytosociology is a very holistic science as it integrates different environmental aspects. Indeed, in landscape phytosociology, the vegetation is studied not only in space but also in time. Moreover, the basic element of landscape phytosociology is the vegetation series [90]: a set of associations that succeed in time in a territory with homogeneous climatic, lithological and geomorphological characteristics (tessella) [39]. The different series of vegetation are organized in space according to the greatest environmental gradients (geological and climatic) that can be classified in the geoseries. These geoseries classify the evolutionary patterns of vegetation (associations that replace themselves over time) that occur according to different environmental conditions.

1.1 Phytosociology and Syntaxonomy Phytosociology has been one of the main areas of research in which the group of geobotanists of the Department of agriculture, food and environmental sciences (D3A) have been involved in recent decades. Indeed, several Italian and foreign areas have been investigated in order to describe the plant communities that compose the plant landscape. Several studies from the level of plant communities to that of the landscape were carried out in the main mountain groups of the central Apennines [2, 4, 6, 11, 22, 23, 29, 30, 45, 60, 87, 108], in the pre-Apennines [9, 13], in the subcoastal sector [10, 16, 17, 24, 94], and in coastal systems [18, 26, 58]. At the plant landscape level, the series and geoseries constitute models in which it is possible to integrate different environmental aspects (Fig. 2) that allow a complex and multidimensional reading of the plant landscape, as demonstrated by the research carried out at Campo Imperatore in the Gran Sasso d’Italia [30]. These models are also highly predictable, and therefore, they can play an important role in spatial planning and resource management. In recent studies conducted on ecotonal (herbaceous plants and shrubs) vegetation, a better understanding of the vegetation dynamic process following the abandonment of traditional anthropic activities was achieved [7, 8, 19–21, 43, 44]. These studies are of fundamental importance, as they are the basis for monitoring and planning conservation interventions, recovery and/or restoration of the grassland habitat. Since 2012, detailed multidisciplinary studies of applied plant ecology were carried out on the intra- and interspecific relationships to understand the coexistence of individuals in a community [55] and to explain dynamic processes at the landscape level [1]. The syntaxonomic revisions of the Italian vegetation were carried out in the course of several years [3, 5, 27, 31, 41, 83]. The most important results of these studies

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Fig. 2 Scheme of the geoseries of the subalpine bioclimatic belt of Campo Imperatore (Gran Sasso d’Italia) from [30]

led to the publication of the vegetation prodrome [33] that was adopted by the Italian Botanical Society as the reference for the classification of the whole of Italian vegetation.

1.2 Species and Habitat Conservation The 1992 EU Habitats Directive (92/43/EEC) adopted by the European Union to halt the loss of biodiversity and preserve its natural and seminatural terrestrial and marine habitats has, in fact, given considerable importance to the Braun-Blanquet classification of vegetation and then to vegetation maps. Approximately 60% of the habitats to be conserved and protected (Annex I) clearly refer to phytosociological syntaxa [88]. With the application of the Habitats Directive (92/43/EEC) in Italy, since the 1990s, research has focused on plant and habitat descriptions and their monitoring.

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During the late 1990s, research was focused on the recognition of habitat in the landscape in order to achieve a good understanding of the heritage of habitat biodiversity in the Italian territory. In those years, we collaborated with regional and national parks as our contribution to the correct management of habitats [28]. In the early 2000s, we concentrated on the definition of a new methodology for the design of the Ecological Network of the Marche Region [36, 42] (Fig. 3), as well as the development of habitat conservation with the proposal of new habitats [32], the enhancement of knowledge [34, 37, 38, 40] and the monitoring of habitats [71]. With our participation in a leading role, an important result was the drafting of the Italian Interpretation Manual in the 92/43/CEE directive [50], financed by the Italian minister for environment, land and sea. Regarding the restoration and conservation of biodiversity of grassland habitats, since 2004, multidisciplinary studies were carried out according to the methodology of applied plant ecology to evaluate the effects of different management methods on the biodiversity of grasslands in several sectors of central Italy within the Natura 2000 areas [12, 51–54, 117].

Fig. 3 Diagram showing the methodological process followed for the definition of the ecological network of Marche region

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With regard to species conservation, the Amphiadriatic Species Seed Bank (ASSB), a structure of the Botanical Garden of the Marche Polytechnic University and active since 2005, has a role of great importance. The main activity of ASSB is concerned with the seed collection, propagation and multiplication of rare and threatened species of the amphiadriatic area and of the main wild plant species for ecological restoration. Between 2005 and 2012, the ASSB took part in many ecological restoration projects such as the reintroduction of Anthyllis barba-jovis L. in the regional natural park of Mount Conero [79] and was a scientific partner for the reinforcement of populations of Moehringia papulosa Bertol. in the natural regional park of “Gola della Rossa e di Frasassi” [93]. Between 2013 and 2016, the ASSB carried out research about (i) the recovery of a landfill: “from waste collection center to Biodiversity Oasis” [46]; (ii) the recovery of the germplasm of native grasses of the secondary grasslands of the calcareous Apennines with an investigation of intra- and interspecies variation in seed behavior in native grasses [66, 67]; (iii) the restoration of wetlands [69]; and (iv) the seed germination behavior of many rare and threatened species [35]. Between 2016 and 2018, the ASSB carried out some studies about (i) the seed germination response and morphological assessment of some annual species for restoration projects of hypersaline environments [70]; (ii) the reproductive traits of some species endemic to the Apulia region; (iii) the seed germination behavior of coastal and dune species; and (iv) the conservation of germplasm belonging to crop wild relatives [95]. In the future, the ASSB, as a member of the Italian network of seed banks for native plant conservation (RIBES), will engage with the other banks of the RIBES in national and European projects aimed at the biodiversity conservation of target, rare and threatened species through seed collection, germination, multiplication, reintroduction, reinforcement and habitat restoration.

1.3 Vegetation Maps A vegetation map is the result of the spatialization of a vegetation classification (Fig. 3). A map of plant associations (Fig. 4) represents the spatial organization of the current vegetation, while a map of vegetation series represents the spatiotemporal variation of the plant association. These maps are fundamental for understanding and managing biodiversity patterns through space and time from local to global scales in the era of global change. During the 1980s and 1990s, the maps of Monte Conero, the basin of Gubbio (Umbria) [25], the regional parks of Taro [48] and Stirone [49] and the Campo Imperatore (Monte Gran Sasso) [47] were produced in a traditional way.

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Fig. 4 Maps of SAC ‘Monte Conero’ realized for REM project [42] and available in [64]. a Phytosociologic (plant associations) map. b Habitat (92/43/EEC) map

In contrast, in the early 2000s, the vegetation maps were produced in a modern way, adopting the use of geographical information systems (GIS). For the “Rete Ecologica Marchigiana” project (REM-Ecological Network of the Marche Region) [42], in collaboration with the University of Camerino, we produced a cartographic census of the entire territory of the Marche region consisting of phytosociological, geosynphytosociological and habitats maps for the Annex I of the EU Habitats Directive (92/43/EEC). All the information was stored in a vegetation information system [82] that today is embedded in an interoperable and interactive web GIS environment [64]. The vegetation information system is useful for biodiversity management and monitoring and for other different purposes of applied ecology, e.g., to map the high nature value farmland [65], to draft a proposal for the integration of Natura 2000 into the Pan-European Ecological Network [40], for a detailed biogeographical regionalization of the Marche region [59] and to contribute to the compilation of the third report on the conservation status of habitats in Italy [119]. The detailed maps produced to date are based on an ‘expert’ spatialization of the different vegetation types (associations, vegetation series, etc.) [61]. Future research is focused on developing a geostatistical mapping of plant associations and vegetation series that integrates the phytosociological field relevès (field observations) with multisource data predictors such as remote sensing vegetation indices, land-surface parameters derived from a DEM, and bioclimate data, considering, for the latter, the recent contributions of [81, 84] in the bioclimate mapping for Italy. We aim to establish a geostatistical mapping process that contributes to an effective and efficient vegetation monitoring system, both in terms of time and economy, and that allows the accuracy of the vegetation maps to be evaluated.

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1.4 Agroecosystems and Bioindication Since the late 1990s, our research began to focus on the study of the natural and seminatural vegetation of agroecosystems [75, 97, 98, 100, 104, 107, 118]. We concentrated on the definition of a method to assess environmental quality inside agroecosystems [89, 105, 106] based on a system of bioindicators that could be used as an analysis tool able to take advantage of all potential information brought by different biocoenoses. With the innovations of the phytosociological dynamic approach used as a starting point, the main objective was to integrate quantitative data with the results provided by the vegetational analysis concerning the description of land units and phytocoenotic mosaics. This kind of interpretation of landscape dynamics, especially inside agroecosystems, is coherent with the application of European agroenvironmental policies and with the main objective of biodiversity conservation. A knowledge base for the assessment of the effectiveness of policies is important, and such a knowledge base is also necessary to evaluate sustainable systems for environmental management inside both natural (protected areas) and agricultural systems where residual habitats need to be preserved. In particular, the residual woods of the hilly areas of the Marche region need evaluation, which show a loss of biodiversity probably due to the lack of forest management [99, 103, 118]. Within the study of agroecosystems, since 2010, we have been studying the traditional uses of wild plants in the rural communities of the Marche region in order to preserve the local traditional knowledge (TLK) [96]. The interest in the traditional uses of wild species led the working group to collaborate with the Conero regional park and some organic local farms in a local project that was cofounded by Rural Development Program (RPD) 2007–2013 of Marche region, with the aim to ideate fresh and processed products with local, edible wild plants [101, 102].

2 Genetic Biodiversity in Agriculture A primary role for the European community is to increase the biodiversity in agricultural systems. The main topics developed by the Plant Genetics and Breeding area of D3A include the following: – The collection, evaluation and molecular characterization of grass pea (Lathyrus sativus L.) populations of the Marche region. Grass pea is a very interesting grain legume crop for marginal areas and sustainable low-input agricultural systems, and to date, grass pea has not been deeply exploited for either animal or human nutrition [112–114]. – The reintroduction of grain legumes, such as fava bean (Vicia faba L.) and field pea (Pisum sativum L.), and forage crops for the development of animal production chains based on locally produced raw materials (concentrates and forages). The

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reintroduction of grain legumes into crop rotations would give more choices to farmers to differentiate crops, thereby increasing biodiversity, as an alternative to the classic durum wheat-sunflower pseudorotation, which is widely diffused in wide areas of central Italy. Moreover, with these legume crops, all positive effects will be reestablished due to nitrogen fixation and the reduction in chemical inputs in high-quality production systems [56, 109, 115, 116]. – The evaluation of the advantages, drawbacks and nutritional properties of old varieties of durum and bread wheat for organic farming systems. The recent attention of consumers to old wheat varieties, related to their nutritional characteristics due to the different gluten protein compositions, offers a great opportunity for farmers to develop more sustainable production systems, because the old varieties need very low nitrogen fertilization levels and due to the taller plants than those of the modern varieties, have a greater ability to compete with weeds [63, 111]. – The intercropping of cereals and grain legumes is considered a good strategy to increase biodiversity and reduce inputs in agricultural systems. Research is ongoing to verify the effectiveness of intercropping for the agricultural areas of central Italy, including the study of different combinations of cereals (barley, durum and bread wheat) and legumes (fava bean, field pea and grass pea) [110].

3 The Landscape as an Integrated (Holistic?) Project The landscape research team has widened its focus from the use of geomatics to agriculture to a wide range of research topics, such as strategic planning, rural development and general landscape monitoring for civil society. More recently, the interest of the group has further shifted to the primary use of ICTs and robotics in agriculture. In addition, the group has joined in some international experiences to help shed light on the role of open and green spaces (Cyberparks) in the innovative regeneration and planning of urban and countryside areas. Given the unresolved struggle on the sustainable use of land resources, which sheds light on how the landscape remains a combustible issue for international debate, the group has presented some research activities ranging from the ecological to the political and social aspects of daily human activity. Obviously, to tackle the ethical dimension of the landscape, the reading of the nature of the “landscape as a whole” is necessary, ranging from the ecological aspects (i.e., biodiversity, fragmentation, pollution, erosion, desertification, etc.) to its multifaceted inner meaning as a cultural element (identity, well-being, quality of life, etc.). The result remains an open and critical question in the relationship between man and nature. In this vision, the group joined many international scientific associations that were inspired by the European Landscape Convention (ELC). In particular, with the foundation of the Landscape Research Center-CIRP, the group led the UNIVPM affiliation to the UNISCAPE network in 2010.

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3.1 Building New Paradigms From theoretical and methodological points of view, an open debate with contributions from a large group of disciplines would ideally be a more effective and stimulating baseline. A significant idea of the landscape, able to incorporate not only the biophysical characteristics but also the mental, cultural, economic, social and other aspects, is needed to become the primary reference for integrated planning [14]. Such a reference will open up new governance models that consider the landscape as a whole [91]. The authors Profs. Galli and Marcheggiani [68] in collaboration with Prof. Hubert Gulinck of the KU Leuven University [76] delivered a reference framework. The work highlights the search for a constant balance among the aspects characterizing the multifaceted concept of landscape planning (Fig. 5). Recently, in collaboration with Prof. Hubert Gulink and the Forest Nature and Landscape unit at KU Leueven [72], the emerging concept was extended to the idea

Fig. 5 The iterative analytical framework represents our baseline model to account for our “ambitions” to read, understand and project landscapes whether valued outstanding or everyday trivial landscapes

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Fig. 6 Interacting waves of transformation and land use regimes. The third wave is an “awareness wave” rolling over the dominant regimes

of reclassifying open spaces in an innovative historical, categorical and spatial framework that is based on the concept of “waves and regimes” of landscape transformation (Fig. 6).

3.2 Adopting New Methods for Landscape Analysis An open question in the current debate is “how” to identify, analyze and evaluate the characteristics and values of each landscape (CLC, art, etc.). One way to shed light on the possibility of providing new sets of indicators by means of the mashup of geographic maps and the descriptor mix [77] has been to experiment starting from land use and land cover, landscape metrics and volunteering geographic data sets. In this context, the group contributed to broaden the original ideas of landscape bionomics by Ingegnoli [73] and of landscape interfaces by Gulinck et al. [72]. This approach has been applied [74] to a double set of two submetropolitan areas in Belgium (Asbeek) and in Lombardy (Bollate) where the key finding was the possibility of overcoming the dominant ideology in planning that opposes “form” and “content”, on the basis of enriching the analysis with a comparative method of landscape diagnosis (Fig. 7).

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Fig. 7 a Revision of integrated landscape planning. The explicit presence of analytic and diagnostic phases modifies the fluxes diagram with the strategic environmental assessment (dotted lines), which is able to change what could be wrong, even in upper scale plans. b Comparison between the movements of the landscapes of Asse (brown) and Bollate (blue) registered on the phase diagram representing the state of the complex system by the indicators HH (Human Habitat) and g-LM (general Landscape Meta-stability). Note that currently Bollate has a g-LM that is higher than that of Asse

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3.3 Boosting Participation of Communities In designing new landscape projects (prognosis) to encourage local sustainable development, a proactive involvement of the local people is also needed, as underlined in the CLC (Art 5). Regarding this topic, we experimented with the potentialities of the “ecomuseum” initiative [80] in an internal hilly area of the Esino catchment in the Marche region. Since traditional GIS and Web GIS solutions show considerable limitations regarding ITs and knowledge management, as well as in the ability to reconcile flexibility and interoperability, we proposed [78] a methodological reflection on the applicative potential of the Semantic Web GIS (SWGIS) in territorial resource management. More recently [62], considering that new sources of geotagged information derived from social media such as Twitter show great promise for geographic research on tourism, we investigated this topic in southern Italy.

3.4 Improving Knowledge and Access to Landscape Patrimony by ICT Tools A recent work we performed to demonstrate the benefits of standard data layer and augmented reality (AR) in watershed control outlined the guidelines of a novel approach for the health-check of linear buffer strips along river networks. A mobile environmental monitoring system for smart maintenance of riverbanks was designed [86], by embedding the AR technology within a geographical information system (GIS). Among others, public open spaces (POS), such as parks, gardens, and squares, would greatly benefit from this growing availability of relevant geolocated information [85]. Given the importance of these matters, the group contributed as secondary proposer to a successful COST Action (CyberParks TU 1306—Fostering knowledge about the relationship between information and communication technologies and public space [92]), financed by the network European Cooperation in Science and Technology COST. The significant outcomes of this phase of the project were referred to the Cardeto Park of Ancona (Italy), where a specific App combined with beacon technology was set up and experimented with by the local people (Fig. 8).

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Fig. 8 a The main objective of CyberParks is to create a research platform on the relationship between information and communication technologies (ICT) and the planning of public open spaces (the figure shows an example) and their relevance to sustainable urban development. b The impacts of this relationship are explored from social, ecological and urban design perspectives through ICT tools

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The Unobservability of the Temporal Scale in Biological Studies Vincenzo Caputo Barucchi, Anna La Teana, Anna Sabbatini and Mario Giordano

Abstract Evolutionary studies suffer of the impossibility of directly observing events that occurred far back in time, on a temporal scale much greater than human life. In our paper we describe and discuss the approaches that different branches of natural sciences have adopted to overcome this inherent and unsurpassable difficulty. We, for instance, explain how chemical features of geological stratigraphy can be used to deduce the conditions in which they formed, using known chemical and physical processes as models. We provide examples of how structural and compositional similarities are used by molecular biologists to infer ancestral traits. We describe how ancient DNA can be used to determine changes in genetic structure of organisms over evolutionary times. We provide instances of reconstruction of paleoenvironments to investigate the relationship between organismal functions and ecosystems in remote times. All these approaches allow to circumvent the unobservability of the past through rigorous approaches and solid assumptions, which, although they may not satisfy a strict Galilean method, constitute a scientifically sound way to investigate the biological past.

V. C. Barucchi · A. La Teana (B) · A. Sabbatini · M. Giordano (B) Dipartimento di Scienze della Vita e dell’Ambiente (DISVA), Università Politecnica delle Marche, Ancona, Italy e-mail: [email protected] M. Giordano e-mail: [email protected] V. C. Barucchi e-mail: [email protected] A. Sabbatini e-mail: [email protected] © Springer Nature Switzerland AG 2020 S. Longhi et al. (eds.), The First Outstanding 50 Years of “Università Politecnica delle Marche”, https://doi.org/10.1007/978-3-030-33832-9_33

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1 The Problem of the Unobservability of the Far Past in the Context of Biological Studies One of the most controversial aspects of biology concerns the impossibility of directly observing evolutionary change, especially at the scale of speciation or of the origin of the supra-specific taxa, and also with respect to metabolic processes and physiological responses to the environment. This “unobservability of the time scale” (sensu [66]) is often invoked by creationists to deny evolution, but also, with sounder arguments, by “Galilean” scientists, for whom a proper scientific approach should follow the hypothetical-deductive model: in a Galilean approach, we start from some propositions (postulates, axioms or premises) and from these we deduce (through logic) a series of hypotheses (conclusions, implications or predictions) that can be tested and therefore “falsified” through experimentation and observation. In the case of evolutionary studies, however, because they involve a temporal dimension, sometimes recourse to experimentation is inadequate. For example, the well-known ecological crisis that occurred at the end of the Mesozoic Era is a unique event and, as such, it is not possible to reproduce in the laboratory the extinction of non-avian dinosaurs or the disappearance of ammonites in the seas at the end of the Cretaceous. Similarly, the changes in algal physiology that derived from the modification of ocean chemistry are very hard to reproduce faithfully in the lab, since extant phytoplankters are probably different from those that were involved in the shift of dominance in the phytoplankton at the Paleozoic-Mesozoic boundary, and environmental reconstructions have a margin of uncertainty. This is not a good reason to consider biology as nonscientific. Similarly, to the historiographic approach, evolutionary biology adopts the heuristic method of the narrative explanation, which is subsequently checked (and possibly confuted) for its explanatory value [3, 79]. On the other hand, it has been pointed out that deep time does not allow a narrative that connects causes and effects beyond any shadow of doubt, as it is theoretically possible for more recent events, e.g. in the case of human genealogies, which can be traced back to a progenitor through a series of direct and concatenated ancestors. When a fossil emerges from deep time, it is impossible to say whether this is certainly a direct ancestor (a “missing link”) of other organisms. Each fossil represents in fact an isolated point that does not have any connection detectable with any other fossil and “all are immersed in a huge ocean of empty spaces” [41]. Although it may not be possible to link the fossils as rings of a long chain of being, it is possible to evaluate the state of diagnostic characters and identify those that persist in a primitive condition (plesiomorphic) and those in a derived state (i.e., apomorphic); through such analyses, the phylogenetic relationship between extant organisms can be derived [80]. If, by absurdity, the phenomena of metamorphism had erased all traces of past life, we could still be certain of evolution by observing morphological and molecular traits that unite the organisms in the great tree of life. Fossil remains that the organisms have left behind are not the only “facts” of evolution; also the similarities and the morpho-anatomical differences between the current forms, the metabolic pathways they use, the embryological processes through which they develop and the genetic,

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chromosomal and molecular characteristics that unite or separate them constitute solid milestones along evolutionary trajectories. These observable phenomena show conclusively that “nothing makes sense in biology except in the light of evolution” [32]. Darwinism explains the historical course of biology (“fact”) in terms of natural processes such as mutation, selection, genetic drift and migration, according to the rules of science. This approach is not different from the “fact” that an apple fell on Newton’s head: fact that he explained with the “theory” of gravitation. With this in mind, in this article we attempted to describe the approaches that are taken by research groups with different backgrounds operating at Università Politecnica delle Marche, with respect to the study of the past in biological contexts, from environmental, phylogenetic, molecular and functional perspective. We exemplify how the unobservability of the past can be circumvented through rigorous approaches and solid assumptions, which although may not satisfy the strict Galilean method, constitute a fully scientific way to investigate the biological past.

2 Evolution of the Environment Researchers studying the evolution of the environment and climate refer to the principle of “Uniformitarianism” of Sir Charles Lyell, who, in his Principles of Geology [76], wrote that “the present is the key to the past”. In other words, the geological past can be better understood through natural processes that we can still observe today (e.g. erosion, sedimentation, volcanic eruptions, earthquakes, glacier dynamics). At the time Lyell operated, geological dating was already based on Palaeontology, and conditions for an Earth history periodization were known. Darwin himself was inspired by Lyell’s work for his studies on the evolution of species. Uniformitarianism led Lyell’s contemporary, Jean-Baptiste Lamarck, to consider fossils as the representative of a non-extinct but progressively modified species. Even today, geologists and biologists study biogeochemical cycles with the awareness that such cycles bear traces of the past and consequences for the future. A full comprehension of the past requires a multidisciplinary approach and the development of “proxies” that can be used as cues for the deep-past. The word “proxy” derives from the latin “procuratio”, caring for, manage; it is used in evolutionary studies to indicate an indirect evidence that substitute for direct evidence which are unobtainable due to the unobservability of the past. Proxies can be biotic, geomorphic, geochemical, or geophysical; they can relate to environmental events that range in time from years to millennia. In most cases, proxies represent a way to reconstruct conditions in the course of Earth’s history by means of the preserved physical characteristics of the past that stand in for direct measurements. Typical examples of proxies are stable isotopic ratios (for instance of O, C, S), abundance and speciation of trace elements (e.g. Sr, Fe, Mo), ratios of trace elements (e.g. B, Ba, Mg, Sr, U, and Zn) to Ca of benthic and planktonic foraminiferal shell (Fig. 1), biomarkers (i.e. compound that are tightly linked to the presence of given organisms, such as alkenones, exclusively

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Fig. 1 Example of skeletal carbonates used as geochemical proxies a Benthic and planktonic foraminiferal assemblages of deep sediment from tropical Atlantic Ocean [82] (Scale bar = 700 μm); b Benthic Foraminifera: 1-3 Cibicidoides lobatulus, stereomicroscope images: dorsal, ventral and apertural side respectively; 4-6 C. lobatulus, SEM images: dorsal, ventral and apertural side respectively; c Planktonic Foraminifera: 1-3 Neogloboquadrina pachyderma, SEM images: ventral view and detail of diatoms near the aperture

present in haptophyte—see Pelusi et al. [90] and references therein—sterols and their derivatives, characteristic of different algal groups—see Kodner et al. [71]. The idea that chemical signals encoded in skeletal carbonates could be used to assess past environmental and climatic information dates back to the early part of the twentieth century [28]. However, only decades later, Harold Urey and his group at the University of Chicago were able to demonstrate the potential of oxygen isotope ratios (δ18 O) for paleothermometry [35, 36]. Recently, in paleoenvironmental reconstructions, researchers have been using a new approach based on “clumped” isotopes, which are related to the temperature-dependent ordering or “clumping” of 13 C–18 O bonds in carbonate minerals [34], to estimate water temperature. Carbonate clumped isotope thermometry is used to constrain the temperatures at the time of carbonate shell formation and estimate the temperature of ancient water masses independently from the isotopic composition of waters from which carbonates originated. Changes in

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stable C isotopes ratios (δ13 C) of carbonate shell (in particular benthic foraminifera) have been used as a proxy for export productivity and deep-water circulation [116]. The marine records of δ13 C provide information on the evolution of sources and sinks of carbon and are linked to the modulation of weathering in monsoonal regions [85] or to shifts in marine organisms, possibly linked to nutrients (also see Chap. 4). The ratio of strontium isotopes (87/86 Sr) in carbonate shells can be used to estimate the amount of fresh water supplied to the marine water in the basin in which the carbonate of the shell formed; it is also an indicator of paleosalinity [38]. In recent years, a variety of proxies have become available to infer past seawater carbonate chemistry; for instance, the boron isotopic composition (δ11 B) of marine carbonates reflects changes in seawater pH and trace elements (e.g. B, U) and the Zn–to-Ca ratio of benthic and planktonic foraminiferal shells records ambient [CO3 2− ] [59]. Also, the Mg/Ca ratio of foraminiferal tests has emerged as a promising temperature and salinity proxy and Mg/Ca ratios recorded in planktonic foraminiferal tests are frequently used to reconstruct past surface ocean temperatures and salinities [115]. In order to estimate paleoceanographic nutrient levels, and thereby explore links between nutrient availability, atmospheric CO2 and climate change, a range of nutrient proxies have been developed. The ratio of Cd to Ca in benthic foraminifera has been used as a proxy for dissolved phosphate, based on the observation that dissolved Cd and phosphate concentrations are linearly correlated in modern oceanic waters [19]. Molecular markers, i.e. compounds diagnostic of the presence of a certain organism or of the conditions in which they grew (e.g. long chain n-alkanes, n-alcohols, n-fatty acids used as indicator of the presence of terrestrial plants) and their specific carbon isotope signatures are excellent proxies for climate-controlled change in terrestrial vegetation and for input of vegetal remains from the weathering processes to the marine realm [21, 88]. Molecular markers from phototrophic primary producers (e.g. short chain n-alkanes, sterols) are very useful to estimate the organic carbon budget, especially when fossils are lacking. The TEX86 and the UK’37 lipid-based indices (GDGTs and alkenones, respectively) are proxies used to reconstruct the paleo-sea surface temperature [67]. Biomineralization [101], and especially calcification (by coccolithophores, foraminifera, molluscs, corals) has been thoroughly studied by means of stable isotopes and fossil analyses, providing clues on environmental conditions at the time of shell formation, also in connection to known physiological aspects of biomineralization. The metabolic cost of skeleton formation reflects the physical chemistry of an organism’s surroundings; cost-benefit of the skeletal biomineralization processes can also change as a function of Earth’s environmental history [69]. Over Earth history, members of many phyla secreted a mineralized skeleton (carbonate, silica and phosphate shells) and the study of the relationships of biomineralization, environment, and global biogeochemical cycles can help to achieve a better understanding of biological mineralization and life on Earth. For example, Hönisch et al. [63] suggest that the major biological crises in the history of the Earth correspond to the biocalcification crises. Extinction and radiation of calcareous fossils lineages (namely shallow reef builders, benthic and planktonic foraminifera, calcareous nannofossils) suggest events of major environmental change throughout the past 300

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My: as potential ocean acidification events, changing nutrient status and patterns of oceanographic circulation. Trends in the abundance of macro and trace element are studied also in sediments. Ti/Al, Si/Al and Zr/Al ratios are used to track aeolian transport of detrital material in the marine realm. The Ba/Al ratio is used as an indicator of marine primary productivity (e.g., [24]. Redox-sensitive trace elements (V, Cr, U, Ni, Cu, Mo, Co, Cd and Zn) are employed as paleo-redox proxies [123], to assess the occurrence of anoxic, suboxic or oxic bottom conditions. Redox conditions at the seafloor can also be estimated through the δ34 S of pyrite.

3 Evolution of Cellular Macromolecules The main approach to study the evolution of DNA, RNA and proteins is the comparative analysis of their sequences to establish the degree of sequence conservation and, consequently, the relationships among organisms. An investigation on the chronology of the acquisition of cellular components throughout evolution, however, requires not only the identification of sequences with a high degree of conservation, but also a detailed analysis of their distribution among extant organisms, in the assumption that a broad distribution is an indication of functions of pivotal importance and of early appearance during evolution. This type of analysis has produced a list of