The Role of Nuclear Factor-B in -Cell Survival and Function David Liuwantara A submission to the University of New South Wales in candidature for the degree of Doctor of Philosophy Gene Therapy and Autoimmunity Research Program Department of Inflammation and Immunology Garvan Institute of Medical Research Darlinghurst, Sydney, Australia November 2007 Statement of Originality ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowleged.’ Signed …………………………………………………………… In Christ alone will I glory Though I could pride myself in battles won For I've been blessed beyond measure And by His strength alone I overcome Oh I could stop and count successes Like diamonds in my hands But those trophies could not equal To the grace by which I stand In Christ alone will I glory For only by His grace I am redeemed And only His tender mercy Could reach beyond my weakness to my need And now I seek no greater honor Than just to know Him more And to count my gains But losses to the glory of my Lord In Christ alone I place my trust And find my glory in the power of the cross In every victory let it be said of me My source of strength, my source of hope is Christ alone In Christ Alone by: Don Koch and Shawn Craig Paragon Music Corp © 1990 BMG Music Pty. Ltd. The fear of the LORD is the beginning of knowledge; fools despise wisdom and instruction. Proverbs 1: 7 (ESV) For the word of the cross is folly to those who are perishing, but to us who are being saved it is the power of God. For it is written, "I will destroy the wisdom of the wise, and the discernment of the discerning I will thwart." Where is the one who is wise? Where is the scribe? Where is the debater of this age? Has not God made foolish the wisdom of the world? For since, in the wisdom of God, the world did not know God through wisdom, it pleased God through the folly of what we preach to save those who believe. For Jews demand signs and Greeks seek wisdom, but we preach Christ crucified, a stumbling block to Jews and folly to Gentiles, but to those who are called, both Jews and Greeks, Christ the power of God and the wisdom of God. For the foolishness of God is wiser than men, and the weakness of God is stronger than men. 1 Corinthian 1:18-25 (ESV) i Acknowledgement The past four years have been the toughest in my life, but an incredible experience I would not have missed for the world. Along the way I’ve met and worked with some of the most incredible people, and unfortunately I cant mention all of you here (otherwise printing cost would be expensive for twice the volume of this thesis). Firstly, I would like to thank my primary supervisor Dr. Shane Grey, who has been to me a great mentor and friend. Your continued effort and encouragement has been the driving force in the completion of this project. I admire your knowledge in the field and passion for science. I am privileged to have the opportunity to work with you in the duration of this thesis. Over the past four years, you have taught me a great deal of knowledge and skills, from islet biology, diabetes immunology, western blotting, islet isolation, islet transplantation, beer drinking and hunting for good steak. I’ve enjoyed the time working with you and I look forward to future engagements that we may have. I would also like to thank a number of secondary supervisors that I’ve had over the past four years. To Prof. Charles Mackay, thank you for allowing me to join the department, and helping my transfer from the Bone Department. To Dr. Cecile King, thank you for your guidance and encouragement in my second PhD year. To Dr. Jenny Gunton, thank you for all the help and skills you have taught me over the past two years, I look forward also to any future collaboration that we may have. A special thanks to Stacey Walters, our honorary Lab Manager, for your patience and generosity in bestowing upon me your skills in islet isolation and transplantation. You have been a great friend and tutor. I know I promise to employ you someday but I doubt I could ever match the last offer you got. I think it is unfair to give Shane all the glory for my increased tolerance to alcohol, I don’t think I would ever had the skill to have a second round if not for your persistent encouragement. I hope that you continue to run into successful projects and I look forward to working with you again someday. Eliana Marino (The Princess), many thanks for your help, advice and most importantly friendship, throughout the course of this thesis. Andrew Yam, many thanks for your help with the bioinformatics, and a whole lot of primers. Thank you also for Civ III, Ricochet and especially the AirPort Extreme (you’re the first person who gave me a wireless experience). Thankyou also for inventing “Fatty Friday”, though it’s not the same without you. Fred and David Zhara, thank you for your help and friendship over the past years. To Bennett, Marry, Zoe, Tim, Trina and Sue Liu, thank you for welcoming me to ‘Chinatown’, Fatty Friday would not happen without your participation, but most importantly for all the help, advise and encouragements. To Bennet and Sue especially for welcoming me into their home in Boston. To Sue-Mei, Rebecca, Ken, Kim, Chris and Sue-Lyn, many thanks for your help and friendship over the past years, you guys have made lab 3 a livelier place to work. To Carrie, Hyun, Julie, Joanna, Heidi and Kendal, many thanks for your help and advise as well as friendship over the past years. To Carrie especially, thank you for welcoming me into your home in London; I had a great time there. i I would like to also thank Louis Tsai, Lewis Cox, Jess, Cindy, Helen, Santi, Gerard, Peta, Anita, Alex and all the friends on level 10. Thank you all for your encouragement and friendship, you have made level 10 an enjoyable place to work. To all the staff and students of Inflammation and Immunology department, many thanks for the great conversations we’ve had scientific or social… more social. I would like to also thank the BTF staff for your help in maintaining the animal facilities. To the IT staff past and present, especially George, Ryan and John, thank you all for your help. To all the friends from class 2000 UNSW, Mel, Shaz, Will, Leon, Danny, Sam, Iris, Quynh, thanks for the fun time we had over the past years. To the brothers and sisters in Christ at IPC: to Rev. Joni Stephen and Rev. Joe Mock, Hatta, Mariana, Darwin and Deb, thank you for your constant prayers and support; to James, Natalie, Kenneth, Jasmine, Victor, Adi, Hanvy, Muriani, Deviani, Ryan and Monnie, thank you for your prayers; and all the members of Pemuda thank you for your prayers and encouragements. To my fiancée Inggrid, thank you for your patience with me throughout the course of this thesis. Thank you for your encouragements and unconditional love over the past years. To my extended families in Sydney and beyond, thank you for your help throughout the different stages of my life, which altogether made it possible for me to complete this thesis. To mum and dad, thank you for sacrificing so much to get me here. A great lesson I learnt from you both over the past years is the unending love of a parent should give to their children. You have reflected to me the love Christ has taught us all to give. Thank you mum and dad, for everything. ii Abstract In Type 1 diabetes, -cells is subjected to an autoimmune-mediated apoptotic and inflammatory attack. Whilst lymphocytes are the primary contributor of -cell death, exposure of -cells to stress signals such as cytokines, transform these cells into an inflammatory state activating transcripts for toxic agents. Demonstrating a significant role for -cells in participating in their own destruction through the elaboration of toxins and chemotactic molecules that could contribute to increased cellular infiltration. The Nuclear Factor-B (NF-B) is a transcription factor that provides for early immediate stress responses governing inflammation and cell survival. In islets, NF-B is thought to have an important role in -cell inflammation and apoptosis. Few studies however, have explored the role of NF-B in -cell protection. Indeed, we found that the expression NF-B is responsible for the islet-intrinsic immediate-early pro-inflammatory gene expression. Importantly however, we also found that in islets, NF-B is responsible for the expression and regulation of anti-apoptotic genes.