UNIVERSITY OF CALIFORNIA, SAN DIEGO Systems Analysis of Model Organisms in the Study of Human Disease Phenotypes A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioinformatics and Systems Biology by Merril Joy Gersten Committee in charge: Professor Shankar Subramaniam, Chair Professor Gabriel G. Haddad, Co-Chair Professor Christopher K. Glass Professor Trey Ideker Professor Wei Wang 2011 Copyright (or ©) Merril Joy Gersten, 2011 All rights reserved. The Dissertation of Merril Joy Gersten is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Co-Chair _____________________________________________________________________ Chair University of California, San Diego 2011 iii DEDICATION To my parents Who inspired me To my children Whom I try to inspire And to my husband Who makes all things possible iv TABLE OF CONTENTS Signature Page ............................................................................................................. iii Dedication ..................................................................................................................... iv Table of Contents .......................................................................................................... v List of Figures ............................................................................................................ viii List of Tables ................................................................................................................ ix List of Supplemental Figures ....................................................................................... x List of Supplemental Tables ....................................................................................... xi Acknowledgements ..................................................................................................... xii Vita ............................................................................................................................... xv Abstract of the Dissertation ...................................................................................... xvi 1. INTRODUCTION ................................................................................................... 1 2. AN INTEGRATED SYSTEMS ANALYSIS IMPLICATES EGR1 DOWNREGULATION IN SIVE- INDUCED NEURAL DYSFUNCTION .................................................................................................... 10 Abstract .................................................................................................................... 10 Introduction .............................................................................................................. 11 Results ...................................................................................................................... 13 Expression analysis of SIV-infected macaques ................................................... 13 Identification of transcriptionally active network modules ................................. 13 Functional enrichment of active network modules .............................................. 17 Network module analysis implicates EGR1 in SIVE-induced dementia ............. 18 EGR1 expression in neurons ............................................................................... 21 CCL8 downregulates EGR-1 in differentiated SK-N-MC cells ........................... 22 Discussion ................................................................................................................ 25 Materials and Methods ............................................................................................. 31 Ethics Statement .................................................................................................. 31 Human Protein Interaction Network ................................................................... 31 Rhesus Macaques ................................................................................................ 32 Microarray .......................................................................................................... 33 Immunohistochemistry ......................................................................................... 34 v Data Integration .................................................................................................. 35 Cell Culture ......................................................................................................... 36 Flow Cytometry ................................................................................................... 37 Macrophage Supernatant .................................................................................... 38 Western Blot ........................................................................................................ 38 RT-PCR ............................................................................................................... 39 Supplemental Figures ............................................................................................... 41 Supplemental Tables ................................................................................................ 43 Acknowledgements .................................................................................................. 67 3. GENOMIC SEQUENCING OF HYPOXIA-ADAPTED FLIES ...................... 68 Introduction .............................................................................................................. 68 Methods and Results ................................................................................................ 72 Notch Signaling Pathway .................................................................................... 72 Wnt Signaling Pathway ....................................................................................... 80 Discussion ................................................................................................................ 87 Supplemental Figures ............................................................................................... 92 Supplemental Tables ................................................................................................ 94 Acknowledgements ................................................................................................ 111 4. TRANSCRIPTIONAL ANALYSIS OF HYPOXIA-ADAPTED FLIES........ 112 Introduction ............................................................................................................ 112 Methods .................................................................................................................. 114 Results .................................................................................................................... 118 Discussion .............................................................................................................. 134 Supplemental Tables .............................................................................................. 140 Acknowledgements ................................................................................................ 145 5. EXPERIMENTAL VALIDATION OF WNT PATHWAY ACTIVATION IN TOLERANCE TO HYPOXIA IN FLIES .................................................... 146 Introduction ............................................................................................................ 146 Methods .................................................................................................................. 149 Results .................................................................................................................... 151 Neurons ............................................................................................................. 151 Hemocytes ......................................................................................................... 155 Discussion .............................................................................................................. 159 Supplemental Tables .............................................................................................. 162 Acknowledgements ................................................................................................ 166 6. CONCLUSIONS .................................................................................................. 167 vi REFERENCES ......................................................................................................... 170 vii LIST OF FIGURES Figure 2.1 Overview of analysis process .................................................................. 14 Figure 2.2 Highest scoring significant modules ....................................................... 16 Figure 2.3 Significant modules containing selected downregulated genes and RT-PCR confirmation ............................................................................. 19 Figure 2.4 EGR1 expression in hippocampal neurons ............................................. 22 Figure 2.5 CCL8 downregulated EGR1 in differentiated SK-N-MC cells ............... 23 Figure 2.6 Proposed model of EGR1 downregulation in SIVE ................................ 28 Figure 3.1 Selection of hypoxia-tolerant