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Type Your Frontispiece Or Quote Page Here Characterizing the spargel, nutcracker, PI31 and FBXO9 homologues as models of Parkinson Disease in Drosophila melanogaster by © Eric Maurice Merzetti A Thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland September 2016 St. John's, Newfoundland and Labrador ABSTRACT Parkinson Disease is a progressive neurodegenerative disorder resulting from the premature destruction or improper function of dopamine producing neurons in the striatum of the brain. Symptoms include resting tremor, bradykinesia, rigidity, postural instability, gait abnormality and additional severe cognitive impairment. Although Parkinson Disease has historically been thought of as a disease with sporadic origin, there are a number of genetic links and specific gene mutations found conserved across patients. These mutations are typically found in genes responsible for the proper functioning of proteasome activity or intracellular organelle homeostasis. The upkeep and repair of mitochondria involves a number of components including Pink1, Parkin, and the Peroxisome-proliferator-activated receptor coactivator (PGC) family of genes. The PGC family of genes have a single homologue in Drosophila melanogaster known as spargel. In Chapter Two, I characterized this gene in neuronal tissues and found that altered gene activity in dopaminergic neurons leads to a decrease in longevity and locomotor ability over time, indicative of a Parkinson Disease like phenotype. In Homo sapiens the PGC family genes are regulated through the activity of an intermediate protein, PARIS. In Chapter Five I identified three potential homologues of the PARIS gene in D. melanogaster and compared altered expression of them in neuronal tissues resulting in the identification of a strong PARIS candidate and two novel genes involved in neuronal development. The proteasome complex acts upon and destroys proteins targeted for destruction by addition of a ubiquitin moiety. This process is undertaken by ubiquitin ligase complexes. The target specific component of many of these complexes ii are the F-box genes. Chapters Three and Four characterize two F-box genes implicated in H. sapiens PD and identifies putative D. melanogaster gene homologs. The results of this thesis provide expanded knowledge of both confirmed and putative D. melanogaster homologues of H. sapiens disease related genes and new model systems with which future study of disease mechanisms may be carried out. iii ACKNOWLEDGEMENTS There are many people who have supported me throughout my thesis research. First and foremost, I thank my parents for all of their support and acceptance. Without their efforts I would not be here in a both a literal and figurative sense. I also wish to thank my supervisory committee members Dr. Tom Chapman and Dr. Kapil Tahlan for providing guidance and an open door whenever I had concerns throughout this process, your suggestions helped to ebb countless hours of stress. To my former and current lab mates in the Staveley lab I express my deepest thanks for helping to lighten the mood and keep the endless hours of work in the basement of the biotech building from being monotonous (mostly). Thanks to Dr. Daniel Jones, Dr. Ryan Mercer and Frankie Slade for editing and providing feedback on multiple drafts of multiple pieces of writing and sharing a pint both for successful and failed experiments alike. A special thanks must go out to Sheena Fry for not only editing and providing feedback on all aspects of this thesis but also for helping me maintain a bright, albeit sarcastic, outlook on life throughout my time writing it. To my supervisor Dr. Brian Staveley, I thank you for the opportunity to complete this degree in your lab and providing me with the tools and guidance necessary to do so. Finally, I would be remiss without thanking the past and present staff of Bitters Restaurant for all of the support, laughs and memorable evenings throughout my graduate degree. Research for this doctoral thesis has been supported by graduate student fellowships from the Memorial University School of Graduate Studies. Dr. Brian Staveley’s laboratory is supported by grants from the Natural Sciences and Engineering iv Research Council and a partnership grant from the Parkinson Society of Newfoundland and Labrador and Quebec. v Table of Contents ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iv Table of Contents ............................................................................................................... vi List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii List of Symbols, Nomenclature or Abbreviations ........................................................... xiv List of Appendices .......................................................................................................... xvii Appendix 1 – Supplemental material for Chapter 2 ................................................... xvii Appendix 2 – Supplemental material for Chapter 3 ................................................... xvii Appendix 3 – Supplemental material for Chapter 4 .................................................. xviii Appendix 4 – Supplemental material for Chapter 5 .................................................. xviii Appendix 5 – Additional Published Works .................................................................. xix Chapter 1 – Introduction and Overview .............................................................................. 1 1.1. Mediating Cell Survival and Cell Death ............................................................... 1 1.1.1. Premature Cell Death and Degenerative Disease .............................................. 2 1.1.2. Parkinson Disease .............................................................................................. 3 1.1.3. The Role of Mitochondria in Parkinson Disease ............................................... 7 1.1.4. The Ubiquitin Proteasome System in Parkinson Disease ................................ 10 vi 1.1.5. Drosophila melanogaster as a Model of Parkinson Disease ........................... 13 1.2. The PGC Family of Proteins ............................................................................... 18 1.2.1. PGC-1α, the Stress Responsive PGC Family Member .................................... 18 1.2.2. The Drosophila melanogaster PGC Family Homologue, spargel .................. 20 1.3. SCF Ubiquitin Ligase Complexes and F-box Proteins ....................................... 21 1.3.1. The FBXO7, FBXO9 and FBXO32 Sub-family of F-box Proteins ................. 22 1.3.2. Potential FBXO7 and FBXO9 Homologues in Drosophila melanogaster ....... 24 1.4. Research Goals ........................................................................................................ 28 1.5. References ............................................................................................................... 30 Co-Authorship Statement .................................................................................................. 38 Chapter 2 – spargel, the PGC-1α homologue, in models of Parkinson Disease in Drosophila melanogaster .................................................................................................. 40 2.1. Introduction ............................................................................................................. 40 2.2. Materials and Methods ............................................................................................ 43 2.2.1. Drosophila Media ............................................................................................ 43 2.2.2. Drosophila Transgenic Lines ........................................................................... 44 2.2.3. Scanning Electron Microscopy of Drosophila melanogaster Eye .................. 44 2.2.4. Ageing Analysis ............................................................................................... 45 2.2.5. Locomotor Analysis ......................................................................................... 45 vii 2.3. Results ..................................................................................................................... 46 2.4. Discussion ............................................................................................................... 52 2.5. References ............................................................................................................... 56 Chapter 3 – The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson Disease ..................................................................... 59 3.1. Introduction ............................................................................................................. 59 3.2. Materials and Methods ............................................................................................ 64 3.2.1. Fly Stocks ......................................................................................................... 64 3.2.2. Scanning
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