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Josip Juraj Strossmayer University of Osijek University of Dubrovnik Ruđer Bošković Institute, Zagreb Josip Juraj Strossmayer University of Osijek University of Dubrovnik Ruđer Bošković Institute, Zagreb University Postgraduate Interdisciplinary Doctoral Study of Molecular Biosciences Petra Marić ECOTOXICOLOGICAL CHARACTERIZATION OF CYANOBACTERIA AND INVASIVE ALGAE FROM THE GENUS CAULERPA USING THE CONCEPT OF EFFECTS-DIRECTED ANALYSES (EDA) DOCTORAL THESIS Osijek, 2015. TEMELJNA DOKUMENTACIJSKA KARTICA Sveučilište Josipa Jurja Strossmayera u Osijeku Doktorska disertacija Sveučilište u Dubrovniku Institut Ruđer Bošković Poslijediplomski interdisciplinarni sveučilišni studij Molekularne bioznanosti Znanstveno područje: Prirodne znanosti Znanstveno polje: Biologija Ekotoksikološka karakterizacija cijanobakterija i invazivnih algi roda Caulerpa primjenom koncepta učincima usmjerenih analiza Petra Marić Disertacija je izrađena u: Institut Ruđer Bošković, Zagreb Mentor: dr.sc. Tvrtko Smital, znanstveni savjetnik Kratki sažetak doktorske disertacije: Koncept učincima usmjerenih analiza predstavlja suvremeni multidisciplinarni dijagnostički alat znanosti o okolišu koji omogućava identifikaciju, karakterizaciju i prioritizaciju toksikanata prisutnih u složenim okolišnim uzorcima. U ovom istraživanju koristili smo ovaj koncept za provedbu detaljne ekotoksikološke karakterizacije dvaju tipova bioloških uzoraka, invazivne tropske alge roda Caulerpa i više cijanobakterijskih sojeva. U oba tipa uzoraka otkriveni su biološki relevantni mehanizmi toksičnosti sekundarnih metabolita na fazu 0. i I. staničnog detoksikacijskog mehanizma. Broj stranica: 186 Broj slika: 68 Broj tablica: 13 Broj literaturnih navoda: 352 Jezik izvornika: engleski Ključne riječi: učincima usmjerene analize, Caulerpa, cijanobakterijski sojevi, ekotoksikološka karakterizacija, biološki testovi, stanični detoksikacijski mehanizam Datum obrane: 30. lipanj 2015. Stručno povjerenstvo za obranu: 1. Branimir Hackenberger Kutuzović, prof. dr. sc. izvanredni profesor, Sveučilište J.J. Strossmayera u Osijeku, predsjednik 2. Tvrtko Smital, dr.sc. znanstveni savjetnik, Institut Ruđer Bošković, mentor i član 3. Vera Cesar, dr.sc. redovita profesorica, Sveučilište J.J. Strossmayera u Osijeku, član 4. Marijan Ahel, dr.sc. znanstveni savjetnik, Institut Ruđer Bošković, zamjena člana Disertacija je pohranjena u: Nacionalnoj i sveučilišnoj knjižnici Zagreb, Ul. Hrvatske bratske zajednice 4, Zagreb; Gradskoj i sveučilišnoj knjižnici Osijek, Europska avenija 24, Osijek; Sveučilištu Josipa Jurja Strossmayera u Osijeku, Trg sv. Trojstva 3, Osijek. BASIC DOCUMENTATION CARD Josip Juraj Strossmayer University of Osijek, Croatia PhD thesis University of Dubrovnik, Croatia Ruđer Bošković Institute, Croatia University Postgraduate Interdisciplinary Doctoral Study of Molecular biosciences Scientific Area: Natural sciences Scientific Field: Biology Ecotoxicological characterization of cyanobacteria and invasive algae from the genus Caulerpa using the concept of effects-directed analyses (EDA) Petra Marić Thesis performed at: Ruđer Bošković Institute, Zagreb, Croatia Supervisor: Tvrtko Smital, PhD, Senior Scientist Short abstract: The concept of effects-directed analyses (EDA) is a modern multidisciplinary diagnostic tool in the environmental science that enables the identification, characterization and prioritization of toxicants present in complex environmental samples. In this study we used the modified EDA concept for detailed ecotoxicological characterization of two types of biological samples, invasive tropical algae from the genus Caulerpa and cyanobacterial strains. Relevant mechanisms of toxicity of secondary metabolites present in biological samples were discovered to phase 0 and I of the cellular detoxification pathway. Number of pages: 186 Number of figures: 68 Number of tables: 13 Number of references: 352 Original in: english Key words: effects-directed analyses, Caulerpa, cyanobacterial strain, ecotoxicological characterization, biotests, cellular detoxification mechanism Date of the thesis defense: June 30, 2015 Reviewers: 1. Professor Branimir Hackenberger Kutuzović, PhD, Josip Juraj Strossmayer University of Osijek 2. Tvrtko Smital, PhD, Senior Scientist, Ruđer Bošković Institute, Zagreb 3. Professor Vera Cesar, PhD, Josip Juraj Strossmayer University of Osijek 4. Marijan Ahel, PhD, Senior Scientist, Ruđer Bošković Institute, Zagreb Thesis deposited in: National and University Library in Zagreb, Ul. Hrvatske bratske zajednice 4, Zagreb, Croatia; City and University Library of Osijek, Europska avenija 24, Osijek, Croatia; Josip Juraj Strossmayer University of Osijek, Trg sv. Trojstva 3, Osijek, Croatia. Research for this PhD thesis was performed in the Laboratory for Molecular Ecotoxicology, Division of Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, under the supervision of Dr. sc. Tvrtko Smital. This work was supported by the Ministry of Science, Education and Sport of the Republic of Croatia (Project 098-0982934-2745) and SCOPES joint research project supported by Swiss National Science Foundation (SNSF) (Grant SCOPES - IZ73ZO_152274/1). Acknowledgments First of all, I would like to thank my supervisor Dr. sc. Tvrtko Smital for giving me the opportunity to work in his laboratory during critical moment of my work in science. Also, I would like to thank Tvrtko for providing me the conditions to finish my PhD programme and to acquire all the necessary knowledge in my field of study. I thank him for support and help in challenging periods of my work in science. Secondly, I would like to thank my colleagues Jovica Lončar and Ivan Mihaljević for teaching me the experimental methods and for all good advice and help. I would like to thank Dr. sc. Jelica Simeunović and Dr. sc. Ante Žuljević for providing me with the valuable biological material without which this research could not be done. I am very much thankful to Dr. sc. Marijan Ahel for the knowledge and advice in chemistry but most importantly I thank him for conducting HPLC and LC-MS analyses and providing valuable chromatographic data that was of critical importance for this research. I thank my other chemist colleagues Iva Mikac, Sanja Koprivica and especially Ivan Senta who helped me with the HPLC analyses. Special thanks goes to my dear collegue Olivera Babić from the University of Novi Sad with whom it was a pleasure to work abroad and who taught me valuable lessons in science work. I hope you get all the best that you deserve. And most importantly, I would like to thank my mother Vinka, my father Ivan, my sister Ivona, and all my dear friends, especially Petra and Danijela for neverending support, care, patience, understanding and encouragement. I would never have been able to do this without your help and I cannot thank you enough. To my beloved uncle Šime who passed away…I hope you have found your peace! Petra Marić TABLE OF CONTENTS 1. INTRODUCTION 1.1. EFFECTS-DIRECTED ANALYSES .......................................................................... 1 1.1.1. Background ............................................................................................................... 1 1.1.2. EDA and TIE ............................................................................................................ 2 1.1.3. Principle of EDA ....................................................................................................... 5 1.1.4. Previous and current EDA studies ............................................................................ 6 1.1.5. EDA on biota samples .............................................................................................. 9 1.1.6. Advantages and limitations of EDA ....................................................................... 10 1.1.7. Modifications of the EDA protocol proposed in this study .................................... 13 1.2. BIOLOGICAL MATERIAL ...................................................................................... 14 1.2.1. Invasive algae from the genus Caulerpa................................................................. 14 1.2.1.1. Main features of C. racemosa and C. taxifolia ................................................ 15 1.2.1.2. Toxic secondary metabolites ............................................................................ 18 1.2.1.3. Spread and impact ............................................................................................ 22 1.2.1.4. C. racemosa and C. taxifolia in the Adriatic Sea ............................................. 23 1.2.2. Cyanobacterial strains ............................................................................................. 26 1.2.2.1. General ............................................................................................................. 26 1.2.2.2. Cyanotoxins ...................................................................................................... 28 1.2.2.3. Harmful algal blooms ....................................................................................... 31 1.2.2.4. Novi Sad Cyanobacterial Culture Collection ................................................... 33 1.3. CELLULAR DETOXIFICATION MECHANISM ............................................... 34 1.3.1. Current knowledge on Caulerpa and cyanobacterial interaction with the cellular detoxification mechanism ................................................................................................. 36 1.4. RESEARCH AIMS AND HYPOTHESES ............................................................... 38 2. MATERIALS AND METHODS 2.1. SAMPLING
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