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Sirtuin 3 Expression and Acetylation of Three Downstream Targets in Painted Turtle Liver and Brain During Anoxia

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Sirtuin 3 Expression and Acetylation of Three Downstream Targets in Painted Turtle Liver and Brain During Anoxia By: Gabriele Nandal, B.Sc (Hons) A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Gabriele Nandal (2019) Sirtuin 3 Expression and Acetylation of Three Downstream Targets in Painted Turtle Liver and Brain During Anoxia Gabriele Nandal Master of Science Department of Cell and Systems Biology University of Toronto 2019 ABSTRACT The western painted turtle is a champion anaerobe; its anoxia tolerance is heavily enabled by metabolic depression. While cellular metabolism is highly regulated by reversible-acetylation of mitochondrial proteins; this phenomenon is relatively unexamined in the anoxia-tolerant turtle. Sirtuin 3 (SIRT3) is a mitochondrial global deacetylase involved in regulating many metabolic and stress-resistance processes, suggesting a potential role in the control of hypometabolism in the turtle. During early anoxia in the brain and liver, mitochondrial acetylation and SIRT3 protein levels increased. Cyclophilin D levels, a direct target of SIRT3, were also elevated during anoxia. Another target of SIRT3, NF-kB p65 exhibited increased deacetylation during anoxia and reoxygenation in the brain; while there was no difference in prevalence or activity of manganese superoxide dismutase, an antioxidant target. This thesis provides preliminary evidence that SIRT3 and mitochondrial protein acetylation may play a role in the regulation of anoxia in the painted turtle. ii ACKNOWLEDGMENTS The thesis cumulates all my work from the past two and a half years and although the entirety of all my work and efforts barely appear to evident, this report is what I have to show for it. Much of scientific research appears to involve extensive troubleshooting and I was not immune to this. I can confidently say Western blots work based on the gods, the weather, or some other mystical force, and not because you are performing the protocol perfectly or because you want them to. While I often said throughout my Masters, “It’s not science, if you’re not crying”, there are several essential people I would like to thank for helping to dissuade my tears. I would first like to thank my supervisor Dr. Les Buck for accepting me into his lab and letting me not only forge my own project but giving me the free reign to design it. His guidance helped me through the different avenues of my project as it evolved by connecting me with valuable resources. Also, as importantly, I would to thank Dr. Jim Eubanks for welcoming me into his lab and making me feel like own of his own. Dr. Eubanks was more than just a committee member, he provided me with essential advice, support, and wisdom that enabled the completion of my project and my thesis. Without Dr. Eubanks, I surely would still be struggling to obtain any data. I would also like to thank my other committee member Dr. Sergey Plotnikov for the guidance and critiques that pushed my project forward. I also would like to thank my work-wife, Elena Sidorova-Darmos, for not only her friendship but her unwavering assistance. From showing me how to perform Western blots, analyze data, and everything in between, I would absolutely not have completed my thesis without her. Elena’s constant support to answer late my night calls about what was wrong with my blots, where the multi-channel pipette was, or how to perform some statistical test, was invaluable. Next, I would like to thank my lab mate Farah Al-Dajani, for always being available to help me with dissections and whose company made it so much more pleasant. I couldn’t have asked for a better partner-in-turtle-crime. I also need to thank my last-minute thesis editor, Dene Ringuette, whose late-night conversations at Krembil made working at night a pleasure and whose presence brightened many evenings. Lastly, I could not have managed without the love and support from Mark Lutley who accompanied and encouraged me every weekend and every evening to Krembil. Without the motivation and emotional support he provided, there would have been many more tears. Finally, to my parents, thank you for your love and the foundation you provided me with to achieve great things in life. iii TABLE OF CONTENTS ABSTRACT ............................................................................................................................ ii ACKNOWLEDGMENTS .................................................................................................... iii LIST OF FIGURES AND TABLES ................................................................................... vii LIST OF APPENDICES ..................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................ ix CHAPTER 1: INTRODUCTION .......................................................................................... 1 1.01 General introduction ................................................................................................................... 1 1.02 Post-translational protein modification ...................................................................................... 1 1.03 Sirtuins ........................................................................................................................................ 2 1.04 Introduction to the mitochondrion .............................................................................................. 4 1.05 Mitochondrial ROS production and antioxidant defenses .......................................................... 5 1.06 Mitochondrial signaling .............................................................................................................. 6 1.07 Introduction to SIRT3 ................................................................................................................. 8 1.08 SIRT3 regulation of antioxidants and ROS ................................................................................ 9 1.09 SIRT3 regulation of mitochondrial metabolism ....................................................................... 10 1.10 SIRT3 influences cell survival and the mitochondrial permeability transition pore ................ 14 1.11 SIRT3 and hypoxia ................................................................................................................... 16 1.12 Expression and regulation of SIRT3 ........................................................................................ 16 1.13 SIRTs and hibernation .............................................................................................................. 18 1.14 The vitality of oxygen ............................................................................................................... 20 1.15 Surviving anoxia ....................................................................................................................... 21 1.16 Anoxic ATP production ............................................................................................................ 22 iv 1.17 Avoiding acidosis ..................................................................................................................... 24 1.18 Metabolic depression ................................................................................................................ 26 1.19 Molecular changes during anoxia ............................................................................................. 29 1.20 Anoxia intolerance .................................................................................................................... 31 1.21 Reoxygenation .......................................................................................................................... 32 1.22 SIRT3 and the painted turtle ..................................................................................................... 34 1.23 Objectives and hypotheses ........................................................................................................ 34 CHAPTER 2: MATERIALS AND METHODS ................................................................. 38 2.1 Animals ....................................................................................................................................... 38 2.2 Experimental protocol ................................................................................................................ 38 2.3 Tissue homogenization ............................................................................................................... 38 2.4 Mitochondrial isolation ............................................................................................................... 39 2.5 SIRT3 activity assay ................................................................................................................... 40 2.6 SOD activity assay ...................................................................................................................... 41 2.7 Antibodies and western blotting ................................................................................................. 42 2.8 Statistical Analysis ..................................................................................................................... 43 CHAPTER 3: RESULTS .....................................................................................................
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