Copyright 2008 By Nathan G. Salomonis ii Acknowledgments Publication Reprints The text in chapter 2 of this dissertation contains a reprint of materials as it appears in: Salomonis N, Hanspers K, Zambon AC, Vranizan K, Lawlor SC, Dahlquist KD, Doniger SW, Stuart J, Conklin BR, Pico AR. GenMAPP 2: new features and resources for pathway analysis. BMC Bioinformatics. 2007 Jun 24;8:218. The co-authors listed in this publication co-wrote the manuscript (AP and KH) and provided critical feedback (see detailed contributions at the end of chapter 2). The text in chapter 3 of this dissertation contains a reprint of materials as it appears in: Salomonis N, Cotte N, Zambon AC, Pollard KS, Vranizan K, Doniger SW, Dolganov G, Conklin BR. Identifying genetic networks underlying myometrial transition to labor. Genome Biol. 2005;6(2):R12. Epub 2005 Jan 28. The co-authors listed in this publication developed the hierarchical clustering method (KP), co-designed the study (NC, AZ, BC), provided statistical guidance (KV), co- contributed to GenMAPP 2.0 (SD) and performed quantitative mRNA analyses (GD). The text of this dissertation contains a reproduction of a figure from: Yeo G, Holste D, Kreiman G, Burge CB. Variation in alternative splicing across human tissues. Genome Biol. 2004;5(10):R74. Epub 2004 Sep 13. The reproduction was taken without permission (chapter 1), figure 1.3. iii Personal Acknowledgments The achievements of this doctoral degree are to a large degree possible due to the contribution, feedback and support of many individuals. To all of you that helped, I am extremely grateful for your support. This dissertation would not have been possible without the guidance of my advisor, Dr. Bruce Conklin. In addition to contributing to the majority of the intellectual work provided herein, Bruce has been an extremely supportive mentor who has fostered the creativity of his lab members. Bruce has always remained confident in my abilities and has encouraged me to think outside of the box. I am grateful for his generous time and intellectual contribution to my projects. In addition to Bruce, I would like to thank the support of the Gladstone Institutes and in particular Gladstone president Dr. Robert Mahley for making Gladstone one of the best academic environments around to work in the world. Much of the work presented herein was through the personal and professional support of my lab mates who I would like to specifically acknowledge. Dr. Alex pico, a Conklin lab Postdoctoral fellow, is an extremely talented computational biologist who has provided ongoing support, help and encouragement with the bioinformatics of nearly all of the projects I’ve worked on. Alex has always been available to provide feedback, assistance and new insights into problems that I often iv thought were too daunting to achieve. Specifically, Alex developed several programs required to generate domain -evel predictions from splicing data to visualize splicing data at the level exons and introns in Cytoscape (SubgeneViewer). He developed tools to identify primers for validation of alternative splicing events in a high-throughput manner (AltPrimer) (Chapters 5-6). Kristina Hanspers, a senior research associate in the lab has been a long-term colleague without her assistance, much of the work provided in this thesis would not be possible. Kristina is a multi-talented genomics researcher who is the heart and soul of the GenMAPP project. Kristina is always able to approach difficult problems and tackle complex projects with a level head and achieve great successes as a result. Kristina has been one of the main developers of the GenMAPP applications (chapters 2, 3, 4, 5 and 6) and contributed to the development of SubgeneViewer and BubbleRouter applications (Chapters 2, 5). Alex Zambon, a former postdoctoral fellow in the Conklin lab, has been a long-term collaborator who has also contributed heavily to the work discussed in this thesis. Alex and I have written several papers together, working very successfully as a team. Alex has an excellent ability to see unique problems and design elegant solutions, both at the bench and at the computer. In particular, Alex did considerable experimental and analysis work for Chapters 2, 3, 4 and 6. v Karen Vranizan, a bioinformatics specialist and statistician in the Conklin lab, has provided wise advice and consul for all nearly all bioinformatics projects conducted during the course of this thesis and before. Karen has directed many of the specific analyses discussed in this dissertation including assistance with clustering (Chapter 3), algorithms for permutation based tests (Chapter 5-7), over-representation analysis (Chapters 2 and 4), splicing statistics (Chapter 5-7) and study design (Chapter 3). Dr. David Williamson is a recent graduate of the bio-medical Informatics (BMI) program at UCSF who has provided valuable input into my projects. David is an excellent bioinformatics programmer with the ability to approach seemly impossible problems and identify quick and effective solutions. Stan Gaj in the Chris Evelo group, a part of the BiGCAT bioinformatics consortium an early adopters of GO-Elite and provided considerable input and design suggestions that were incorporated into the final software (Chapter 4). I would like to especially thank the members of my thesis and orals committee. Drs. Ru- Fang Yeh and Susan Fisher have served on both committees and provided excellent support, input and advice throughout this process. Drs. Patsy Babbitt (chair) and Andrej Sali, who served on my orals examination committee, made significant sacrifices to be there (Andrej’s first child was delivered the night before) and made critical contributions to the direction this work has taken. vi Although most of my work has been done at the computer, I have relied heavily on the assistance and support of both academic and industrial collaborators on what have become fairly extensive and involved research projects. Chris Schlieve, a talented cell biologist, partnered up with me for the Tcf3 embryonic stem cell analyses (Chapter 6) and conducted a large portion of that work, resulting in the production of data that I would not have been able to produce on my own. Laura Pereira in the Brad Merrill lab, has significantly contributed to the Tcf3 isoform project and along with Brad has contributed greatly to the scientific content and descriptions in this analysis. In the Mark Mercola lab, the contributions by both Brandon Nelson and Mark have provided valuable reagents which have facilitated the large scale human exon array analyses conducted in this thesis (Chapter 5). Furthermore, the excellent work of the Gladstone Genomic’s core, lead by Dr. Chris Barker and conducted by Linda Ta have been vital to obtaining high quality data. In addition, industrial collaborators at Affymetrix and Agilent were also instrumental in this work. Without the assistance from John Blume, Melissa Cline, Hui Wang, and Allan Williams at Affymetrix, the technology and support needed to perform the mouse exon- exon junction array analyses would not be possible. At Agilent, work by Allan Kuchinsky, an expert Java programmer who is one of the primary contributors to Cytoscape development, worked closely with Kristina Hanspers and Alex Pico to develop the SubgeneViewer and BubbleRouter software, used extensively in these analyses. vii To re-implement published algorithms (ASPIRE, linear regression analysis of splicing, perfect match DABG p-value calculation), heavily used in these projects, the authors of these algorithms graciously provided their assistance (Jernej Ule and Chuck Sugnet). I am especially grateful to my friends, family and fellow students who have provided invaluable support and understanding throughout this process. To my wife Heather, thank you so much for your love and support from day one of this process, I don’t know what I’d do with out you. You have patiently stood with me and helped throughout this process, from isolating EBs with me on your days off and editing the drafts of this thesis, to helping me cope with the ups and downs of graduate student life. Finally, I would like to thank Dr. Frank Szoka and Debbie Acoba for their impressive administrative accomplishments in the Pharmaceutical Sciences and Pharmacogenomics graduate program. They have both been extremely helpful and supportive during my graduate studies, from day-to-day needs to funding and academic advice. I would like to further acknowledge the funding opportunities that allowed to me to accomplish this work, including the Achievement Rewards for College Scientists (ARCS) Scholarship 2005 – 2006, National Institutes of Health Training Grant (T32 GM07175) 2003-2004, and the kind awards provided by the PSPG graduate program (Ira Herskowitz award) and the Gladstone Institutes. viii Abstract Nathan G. Salomonis The development of an organism from conception to adulthood requires the specification of cell types to distinct fates. In an adult organism, tissues can similarly undergo dramatic transformation, altering their structure, physiology, and overall biochemical properties. In both of these paradigms, the study of gene transcription and its contribution to protein content in the cells has been the primary focus. While clearly important, more recently, alternative splicing and microRNA regulation have been recognized as significant processes that can have crucial regulatory influences on the diversity of mRNAs produced and their over-all expression in the cell. In this dissertation, I have