UNIVERSITÀ DEGLI STUDI DI PADOVA Dipartimento di BIOMEDICINA COMPARATA E ALIMENTAZIONE Corso di dottorato di ricerca in SCIENZE VETERINARIE Ciclo XXIX TRANSCRIPTOMIC APPROACHES TO STUDY THE EFFECTS OF XENOBIOTICS IN RUMINANTS Tesi redatta con il contributo finanziario della Commissione Europea – Programma europeo ERASMUS MUNDUS Action 2 (AL FIHRI) Coordinatore: Ch.mo Prof. (Alessandro ZOTTI) Supervisore: Ch.mo Prof. (Mauro DACASTO) Dottorando: RAMY ELGENDY 2017 To my parents, sister and brother.. Even if you don’t fully understand what I am doing, and probably won’t read this thesis ever, Thank you for being always proud of me. شكر ًا لكل ما فعلتموه من أجلي To all the obstacles and hardships.. You have made me stronger Acknowledgements ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my advisor – and friend – Prof. Mauro Dacasto for the continuous support of my PhD study and related research, for his guidance and motivation. His encouragement and oppeness allowed me to grow both scientifically and professionally. I couldn’t have imagined having a better advisor. Besides my advisor, I would like to thank Dr. Mery Giantin who taught me many things from the scratch. Without her knowledge and precious support it would not be possible to conduct this research. She has been a caring and supportive big sister to me. I would also like to thank both Prof. Juan Loor and Dr. Massimo Bionaz for acting as external evaluators of my thesis and for providing me with valuable comments and observations. This thesis represents not only my work in the lab or at the computer’s keyboard, it is a collective representation of more than three years of team work at the Pharmacogenetics and Toxicogenomics laboratory of Prof Dacasto. That’s why I want to thank my fellow labmates for everything we encountered together. Thanks to Vanessa who helped me in the lab during my first year of the PhD. Thanks to Rosa, Eleonora, Roberta, and Silvia for the long days (and evenings) we were working together, and for all the fun we have had in the last three years. Also, I thank all my friends here in Agripolis. Thanks to Federico, Marianna, Maria Elena, Max, Enrico, Rosella, Davide,Valentina, Alessandro, Lisa, Cincia, Giulia, Roberta, Jessica, Maria Laura, Massimo, Serena, Matteo, and Morgan for all the smiles and coffees we shared together. Thanks to Francesco - my friend, flatmate and colleague - for all the days and nights we cycled together to and from work, and for all the nice adventures we encountered together. I would like to thank my friends Joy, Ibrahim, Eslam, and Basma for accepting nothing less than excellence from me. Thanks to Biljana for believing in me and for being supportive and caring – definitely life is more colorful now. Last but not the least, I would like to thank my Family; my parents, my brother and sister for supporting me spiritually throughout my life. Table of contents TABLE OF CONTENTS LIST OF ABBREVIATIONS ........................................................................................................................................ iii SUMMARY ................................................................................................................................................................... iv 1. BACKGROUND ....................................................................................................................................................... 1 1.1. Growth promoters in food-producing animals .................................................................................................. 3 1.2. Transcriptomics ................................................................................................................................................. 7 1.3. Nutrigenomics ................................................................................................................................................. 10 2. AIMS ........................................................................................................................................................................ 13 3. REFERENCES ....................................................................................................................................................... 14 I. Transcriptomic analysis of skeletal muscle from beef cattle exposed to illicit schedules containing dexamethasone ................................................................................................................................................................ 22 Abstract ......................................................................................................................................................................... 23 Introduction .................................................................................................................................................................. 23 Materials and methods .................................................................................................................................................. 25 Animals and experimental design ............................................................................................................................. 25 Sample collection and RNA extraction ..................................................................................................................... 26 RNA amplification, labeling and hybridization ........................................................................................................ 27 Normalization of the microarray data ....................................................................................................................... 28 Quantitative Real time PCR ...................................................................................................................................... 29 Statistics and Principal Component Analysis (PCA) ................................................................................................ 32 Results .......................................................................................................................................................................... 33 Animal health status and growth performance ......................................................................................................... 33 Confirmatory qPCR analysis .................................................................................................................................... 35 Clustering and principal component analyses ........................................................................................................... 38 Discussion ..................................................................................................................................................................... 42 Conclusions .................................................................................................................................................................. 47 Supplementary materials............................................................................................................................................... 47 References .................................................................................................................................................................... 48 II. The transcriptome of muscle and liver is responding differently to a combined trenbolone acetate and estradiol implant in cattle............................................................................................................................................... 55 Abstract ......................................................................................................................................................................... 56 Introduction .................................................................................................................................................................. 56 Materials and methods .................................................................................................................................................. 58 Animals and experimental design ............................................................................................................................. 58 Sample collection and RNA extraction ..................................................................................................................... 59 RNA amplification, labeling and hybridization ........................................................................................................ 59 Normalization of microarray data and identification of DE genes ........................................................................... 60 Functional enrichment analysis................................................................................................................................. 60 Quantitative Real time RT-PCR (qPCR) validation ................................................................................................. 60 Statistics and Principal Component Analysis (PCA) ................................................................................................ 62 Results .......................................................................................................................................................................... 63 Phenotypic measures ...............................................................................................................................................