GENE EXPRESSION PROFILING OF PERIPHERAL BLOOD LYMPHOCYTES FROM TYPE 1 DIABETES PATIENTS By CHRISTIN DAWN COLLINS 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 2005 Copyright 2005 by Christin Dawn Collins This dissertation is dedicated to my family, who have always supported and believed in me. ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Jin-Xiong She. Without his guidance and support, my graduate experience would have been bleak. His knowledge on so many topics and willingness to answer all my questions, no matter how frivolous, will always be appreciated. He has my endless admiration and respect. I can only hope to achieve the scientific excellence that he has. Next, I would like to offer many thanks to my committee members: Dr. Wayne McCormack, Dr. Richard McIndoe, Dr. Desmond Schatz, Dr. Laurence Morel, and Dr. Mark Yang. I am extremely grateful for their time, energy, and guidance. I would also like to thank Dr. Rob Podolsky whose tireless efforts to help me with my data analysis and statistical needs are so greatly appreciated. Without his hard work and willingness to explain things on a level that a statistically challenged person like me could understand, I doubt much sense could be made of this thesis. I want to extend a special thanks to Dr. Sarah Eckenrode who has been a mentor, friend, and confidante for my years in science. She has provided hours of support and guidance with her tireless enthusiasm and cheerful demeanor. Her help with my preparation of this paper cannot be fully expressed, but it will always be appreciated. And a special thanks to all the members of my laboratory who contributed to the completion of this project. From helping with patient blood draws, to showing me the ropes, to providing moral support and taking me to lunch, they will be cherished. Finally I would like to thank my fellow graduate students and friends. In particular, the iv friendship of Jessica Walrath and Nicole Tester has made my journey all the more enjoyable. v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi ABSTRACT..................................................................................................................... xiii CHAPTER 1 INTRODUCTION ........................................................................................................1 Medical Significance ....................................................................................................1 Epidemiology of Diabetes ............................................................................................2 Identification and Prediction of Type 1 Diabetes.........................................................3 Environmental Influences.............................................................................................5 Genetic Susceptibility of Type 1 Diabetes ...................................................................6 DNA Microarrays .........................................................................................................8 Significance ..................................................................................................................9 2 GENE EXPRESSION PROFILING OF TYPE 1 DIABETIC, AUTOANTIBODY POSITIVE, AND AUTOANTIBODY NEGATIVE INDIVIDUALS.......................12 Introduction.................................................................................................................12 Materials and Methods ...............................................................................................13 Participants ..........................................................................................................13 RNA Isolation......................................................................................................14 cDNA Library Construction................................................................................14 PCR Amplification of cDNA Clones ..................................................................15 Generation of Microarray Slides .........................................................................16 Data Acquisition..................................................................................................18 Data Flagging ......................................................................................................19 Data Normalization .............................................................................................19 Univariate Analysis .............................................................................................19 Graphic Illustration..............................................................................................19 Sequencing Analysis and Clone Identification....................................................20 Results.........................................................................................................................21 Gene Expression Profiling Distinguishes T1D Phenotypes ................................21 vi Identification of Genes Involved in Distinguishing T1D Phenotypes.................22 Discussion...................................................................................................................24 3 NOVEL TYPE 1 DIABETES BIOMARKERS BASED ON MULTIGENIC MODELS OF GENE EXPRESSION.........................................................................43 Introduction.................................................................................................................43 Materials and Methods ...............................................................................................44 Participants ..........................................................................................................44 Microarray Analysis ............................................................................................45 Multivariate Analysis ..........................................................................................45 Results.........................................................................................................................46 Multi-Gene Models for Prediction ......................................................................46 Identification of AbP Subsets..............................................................................47 Discussion...................................................................................................................49 4 EXAMINATION OF INFLAMMATORY GENES IDENTIFIED BY MICROARRAY ANALYSIS ....................................................................................56 Introduction.................................................................................................................56 Materials and Methods ...............................................................................................58 Subjects................................................................................................................58 S100A8 and S100A9 ELISA...............................................................................59 S100A8/A9 ELISA..............................................................................................60 Soluble L-Selectin ELISA...................................................................................60 Statistical Analysis ..............................................................................................61 PCR Confirmation...............................................................................................61 Results.........................................................................................................................62 Serum Levels of S100A8, S100A9, and S100A8/A9..........................................62 Serum Levels of L-Selectin.................................................................................63 Confirmation of Expression Differences via Real-Time PCR ............................64 Discussion...................................................................................................................64 5 GENE EXPRESSION PROFILING OF CD3+ T CELLS.........................................90 Introduction.................................................................................................................90 Materials and Methods ...............................................................................................91 Subjects................................................................................................................91 Samples................................................................................................................91 Cell Culture .........................................................................................................92 RNA Isolation......................................................................................................93 Flow Cytometry...................................................................................................94 Microarray Analysis ............................................................................................94