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University of Florida Thesis Or Dissertation Formatting OXIDATIVE STRESS IN ARCHAEA: PROTEOME RESPONSES AND CHARACTERIZATION OF AN INORGANIC PYROPHOSPHATASE THAT DRIVES E1-LIKE ACTIVATION OF UBIQUITIN-LIKE PROTEIN MODIFICATION By LANA MCMILLAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Lana McMillan To my parents and fiancé for their constant encouragement and support ACKNOWLEDGMENTS I would like to express my deepest gratitude to my mentor, Dr. Julie Maupin- Furlow, for her guidance, kindness, understanding, and support. She has been an incredible mentor, allowing space for personal growth while keeping me on track. She encouraged and introduced some wonderful opportunities during my graduate school career such as mentoring undergraduates, teaching, and attending conferences. I would like to thank my committee members Dr. Linda Bloom, Dr. Sixue Chen, and Dr. Keelnatham Shanmugam for taking the time to provide advice and guidance. I would also like to thank the Genetics and Genomics Graduate Program, specifically Dr. Connie Mulligan, Dr. Wilfred Vermerris, Dr. John Davis, Dr. Doug Soltis, and Dr. Maurice Swanson for making the GG graduate students feel like we have a voice and for providing an excellent network of support. Thank you to the Department of Microbiology and Cell Science, especially the laboratories of Dr. Shanmugam, Dr. Romeo, Dr. Ingram, Dr. Preston, Dr. Rice, and Dr. Gurley for providing assistance and equipment which greatly helped my research projects. Thank you to Dr. Monika Oli for allowing me the wonderful opportunity to teach. Thank you to UF Interdisciplinary Center for Biotechnology Research (ICBR) Proteomics and Mass Spectrometry Core, especially Dr. Jin Koh for helping with the proteomic data analysis. I would like to acknowledge my parents for always making time to visit me from Missouri a few times a year and for fueling my curiosity for science from the beginning. I must give a special thank you to my Granny, for being my snail-mail pen pal throughout my time at graduate school and always sending positive and encouraging thoughts. I need to give the biggest thanks to my fiancé, Guido Pardi IV for escorting me to the lab for mid-night time points, writing programs for my proteomics project, and always being 4 there for me. I would like to thank my Gainesville family, Dr. Lara Ianov and her wife Okreatta Ianov for being an excellent support system and the very best friends. Thank you to past lab members who have been excellent role models and teachers, especially Dr. Jonathan Martin, Dr. Nathanial Hepowit, Dr. Xian Fu, and Dr. Shiyun Cao. I would like to thank my current lab members, especially Swathi Dantuluri and Sungmin Hwang, who keep the atmosphere in the lab light with their humor and wit. I also owe a big thank you to the dedicated undergraduate researchers who have worked with me over the years: Rawan Farah, Miguel Gomez, and Whinkie Leung. Being a mentor to undergraduate researchers has been an incredibly rewarding experience. Finally, I would like to thank my peers in the Genetics and Genomics and the Microbiology and Cell Science Graduate Programs, especially Winnie Hui and Anna Gioseffi, for their support and friendship. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 14 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 16 Introduction ............................................................................................................. 16 Extreme Living Conditions of Halophilic Organisms ............................................... 16 High Salt Stress ................................................................................................ 17 Desiccation and Starvation ............................................................................... 17 Oxidative Agents .............................................................................................. 18 Oxidative Stress ...................................................................................................... 18 Reactive Oxygen Species in the Cell ............................................................... 19 Archaeal Oxidative Stress Expression Studies ................................................. 21 Quantitative Proteomics .......................................................................................... 22 Sample Preparation .......................................................................................... 22 Mass Spectrometry .......................................................................................... 23 Label Free ........................................................................................................ 26 Stable Isotope Labeling of Amino Acids in Cell Culture .................................... 27 Isobaric Tags for Relative and Absolute Quantitation ....................................... 29 Absolute Quantification .................................................................................... 30 Selected Reaction Monitoring ........................................................................... 30 2-Dimesional Gel Electrophoresis .................................................................... 31 Limitations ........................................................................................................ 32 Archaeal Quantitative Proteomics .................................................................... 32 Inorganic Pyrophosphatase .................................................................................... 34 Commercially Available Inorganic Pyrophosphatases ...................................... 35 Archaeal Inorganic Pyrophosphatases ............................................................. 36 Halophilic Enzymes and Industrial Applications ............................................... 36 Objectives ............................................................................................................... 37 2 MATERIALS AND METHODS ................................................................................ 39 Materials and Methods for SILAC study.................................................................. 39 Materials ........................................................................................................... 39 Strains and Media ............................................................................................ 39 Generation of Mutant Strains ............................................................................ 39 6 Growth Assays for Minimal Amino Acid ............................................................ 40 Assay of Cell Survival after Exposure to Sodium Hypochlorite (NaOCl) .......... 40 Ellman’s Reagent Assay for Free Sulfhydryl Groups ........................................ 41 Isotopic Incorporation ....................................................................................... 42 SILAC Cultures, Sample Preparation, and Strong Cation Exchange Chromatography ........................................................................................... 43 Reverse Phase LC-Mass Spectrometry and Protein Identification ................... 44 NaOCl sensitivity assay .................................................................................... 46 Materials and Methods for HvPPA study ................................................................ 46 Materials ........................................................................................................... 46 Strains and Media ............................................................................................ 47 DNA Manipulations ........................................................................................... 47 Purification of HvPPA from Haloferax volcanii .................................................. 48 PPi Assay ......................................................................................................... 49 Coupled Assay of Ubiquitin-Like Protein Adenylation ....................................... 50 Protein Concentration Assay ............................................................................ 51 Protein Lyophilization ....................................................................................... 51 SDS-PAGE and Immunoblotting ...................................................................... 51 Dendrogram Analysis ....................................................................................... 51 3 DEVELOPMENT OF A MULTIPLEX SILAC-BASED APPROACH FOR QUANTIATIVE ANALYSIS OF A MODEL ARCAHAEAL PROTEOME UNDER DIFFERENT GROWTH CONDITIONS ................................................................... 55 Introduction for SILAC Study .................................................................................. 55 Results ...................................................................................................................
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