UC San Diego UC San Diego Electronic Theses and Dissertations Title Insights from reconstructing cellular networks in transcription, stress, and cancer Permalink https://escholarship.org/uc/item/6s97497m Authors Ke, Eugene Yunghung Ke, Eugene Yunghung Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Insights from Reconstructing Cellular Networks in Transcription, Stress, and Cancer A dissertation submitted in the partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioinformatics and Systems Biology by Eugene Yunghung Ke Committee in charge: Professor Shankar Subramaniam, Chair Professor Inder Verma, Co-Chair Professor Web Cavenee Professor Alexander Hoffmann Professor Bing Ren 2012 The Dissertation of Eugene Yunghung Ke is approved, and it is acceptable in quality and form for the publication on microfilm and electronically ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ Co-Chair ________________________________________________________________ Chair University of California, San Diego 2012 iii DEDICATION To my parents, Victor and Tai-Lee Ke iv EPIGRAPH [T]here are known knowns; there are things we know we know. We also know there are known unknowns; that is to say we know there some things we do not know. But there are also unknown unknowns; there are things we do not know we don't know. Donald Rumsfeld v TABLE OF CONTENTS SIGNATURE PAGE .............................................................,............................... iii DEDICATION .......................................................................................................iv EPIGRAPH ........................................................................................................... v TABLE OF CONTENTS .......................................................................................vi LIST OF FIGURES ............................................................................................. xiv LIST OF TABLES ............................................................................................... xix ACKNOWLEDGEMENTS ....................................................................................xx VITA .................................................................................................................. xxii ABSTRACT OF THE DISSERTATION ............................................................. xxiii INTRODUCTION .................................................................................................. 1 Bioinformatics and Systems Biology ................................................................. 1 Experimental Methods ...................................................................................... 2 Microarrays. ................................................................................................... 2 Next-Gen Sequencing. .................................................................................. 3 Computational Methods .................................................................................... 4 Preprocessing. .............................................................................................. 4 Normalization. ............................................................................................... 5 vi Summarization. ............................................................................................. 5 Differentially expressed genes. ..................................................................... 6 Multiple Testing, ............................................................................................ 7 Functional annotation. ................................................................................... 7 Experimental Design ....................................................................................... 11 Curse of Dimensionality .................................................................................. 12 CHAPTER 1 NF-kB BINDING ............................................................................ 14 Introduction ..................................................................................................... 14 Representations of Response Elements. .................................................... 15 NF-kappaB family. ....................................................................................... 17 Secret Word Problem ...................................................................................... 19 Historical Perspective. ................................................................................. 19 Predictive Power. ........................................................................................ 19 Conservation. .............................................................................................. 20 Expression ...................................................................................................... 22 Target genes. .............................................................................................. 22 Expression. .................................................................................................. 23 Guilt By Association. ....................................................................................... 24 Cog in the machine. ........................................................................................ 25 vii Discussion ....................................................................................................... 26 CHAPTER 2 OXIDATIVE STRESS .................................................................... 29 Introduction ..................................................................................................... 29 Hydrogen Peroxide Induces Oxidative Stress in Primary Endothelial Cells .... 31 Results ............................................................................................................ 33 Hemichannels and Connexins. .................................................................... 33 Cellular Sources of ROS. ............................................................................ 34 ROS clearance. ........................................................................................... 37 MAPK/P38 signaling. ................................................................................... 40 NRF Pathway. ............................................................................................. 42 DNA repair pathways. ................................................................................. 43 BCL2 family. ................................................................................................ 45 Loss of Mitochondria. .................................................................................. 46 Caspase Cascade. ...................................................................................... 47 TP53, ROS clearance, and MTOR. ............................................................. 49 Heme function, synthesis and degradation.................................................. 51 Reproducibility Issues ..................................................................................... 53 Verification of microarray trends. ................................................................. 53 Testing of H202 and HMVEC-L. .................................................................. 56 viii Calibration of Assays. .................................................................................. 57 Loss of Hydrogen Peroxide in Media. .......................................................... 59 Assay conditions. ........................................................................................ 60 Real Time-PCR data. .................................................................................. 61 Discussion ....................................................................................................... 63 Heme. .......................................................................................................... 64 Methods .......................................................................................................... 65 Cell culture. ................................................................................................. 65 Cell Staining. ............................................................................................... 65 Spectrophotometry. ..................................................................................... 66 Hydrogen Peroxide Colormetric kit. ............................................................. 66 LDH assay. .................................................................................................. 66 Caspase Activity. ......................................................................................... 66 Total RNA preparation. ................................................................................ 66 RT-PCR. ...................................................................................................... 66 Microarray analysis. .................................................................................... 66 Data analysis. .............................................................................................. 67 Pathways.