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Depletion of the Phosphatase Inhibitor, PPP1R1A, May Contribute to Β-Cell Loss in Type 1 Diabetes Jessica Rose Chaffey Doctor O

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Depletion of the Phosphatase Inhibitor, PPP1R1A, May Contribute to Β-Cell Loss in Type 1 Diabetes Jessica Rose Chaffey Doctor O Depletion of the phosphatase inhibitor, PPP1R1A, may contribute to β-cell loss in Type 1 diabetes Jessica Rose Chaffey Doctor of Philosophy May 2020 1 Depletion of the phosphatase inhibitor, PPP1R1A, may contribute to β-cell loss in Type 1 diabetes Submitted by Jessica Rose Chaffey, to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Medical Studies, May 2020. This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that any material that has previously been submitted and approved for the award of a degree by this or any other University has been acknowledged. (Signature) ………… …………… 2 Acknowledgements Firstly, I would like to thank my outstanding supervisors: Professor Noel Morgan and Professor Sarah Richardson whom have supported me non-stop throughout my PhD studies. Thank you for your continuous inspiration, direction and encouragement. I would also like to thank all members of the IBEx team, past and present. In particular, thank you to: Dr Mark Russell for giving me the opportunity to do a summer placement during my undergraduate degree, and guiding me throughout the past few years, Dr Pia Leete for not only sharing her expert skills in immunostaining and microscopy, but for emotional support (and the roast beef dinner!), and Dr Shalinee Dhayal for good experimental advice and even better Indian food! I could not have undertaken my PhD in the company of a kinder, more helpful group, and with that would like to thank the whole of the IBEx team. Finally, I would like to thank my friends and my family, particularly my parents; Brian and Caroline, for always being there and guiding me down the science path; and my sister and grandparents for all their love and support over the years. Lastly, but most definitely not least, to my fiancé, Steven, for keeping me going when I didn’t think I could, and for immense patience and kindness every day – Thank you. 3 Abstract Increasing evidence implicates a persistent enteroviral infection of β-cells as a potential trigger for the development of Type 1 diabetes (T1D). In support of this, findings presented in this thesis demonstrate that interferon-stimulated genes are upregulated in pancreas samples from T1D donors, but absent from donors without T1D, despite evidence of viral protein in their islets. The reasons for this exaggerated response are unclear but may be related to altered regulation of the viral recognition protein, MDA5. Protein phosphatase 1, regulatory, inhibitory, subunit 1A (PPP1R1A) is a largely unstudied molecule and has a restricted tissue distribution, but is highly expressed in β-cells. PPP1R1A specifically regulates protein phosphatase 1 (PP1) which has a central role in coordinating MDA5 activity. Findings presented in this thesis demonstrate that PPP1R1A is depleted from β-cells in T1D. To explore the impact of PPP1R1A on β-cell function, clonal lines of tetracycline inducible β-cells were developed using the PPP1R1A-deficient 1.1B4 cells as a host line for the Flp-In T-REx system. Two cell lines were generated which express either wild-type (WT) PPP1R1A or a phosphorylation-null (T35A) mutant form, upon addition of tetracycline. During the development of these Flp- In T-REx lines, I made the discovery that the parental 1.1B4 line was contaminated with an unidentified rat cell line. Data are presented on how this contamination was discovered and the steps taken to re-derive and characterise new human 1.1B4 cells lines. These findings have resulted in the withdrawal of 1.1B4 (and other related cell lines) from the European Collection of Authenticated Cell Cultures (ECACC) and a change in international practice for the authentication of cell lines. 4 Despite these difficulties, the Flp-In T-REx PPP1R1A cell lines and other human cell lines available, were used to explore (1) the role of PPP1R1A in cell cycle progression and (2) the role of PPP1R1A in regulation of secretion from β-cells. Cell cycle progression was found to be reliant upon the timing of sequential PPP1R1A phosphorylation and dephosphorylation. Phosphorylation of PPP1R1A is critical for successful completion of the cell cycle and sustained phosphorylation of PPP1R1A resulted in apoptosis. Previous studies had identified PPP1R1A as a critical component necessary for insulin secretion. The studies reported in this thesis demonstrate that PPP1R1A could also play a previously unrecognised role in regulating constitutive secretion of molecules from cells. 5 6 Table of Contents Acknowledgements ............................................................................................ 3 Abstract .............................................................................................................. 4 List of Figures ................................................................................................... 15 List of Tables .................................................................................................... 24 Abbreviations .................................................................................................... 26 1 Introduction ............................................................................................... 32 1.1 Diabetes Mellitus ................................................................................. 32 1.1.1 Gestational diabetes mellitus ........................................................ 33 1.1.2 Monogenic diabetes ...................................................................... 33 1.2 Type 2 diabetes ................................................................................... 35 1.3 Type 1 diabetes ................................................................................... 36 1.3.1 A brief overview of genetic links and Type 1 diabetes .................. 38 1.3.2 A brief overview of islet autoantibodies ......................................... 39 1.3.3 Role of a virus infection? .............................................................. 40 1.3.4 Other potential triggers of T1D...................................................... 44 1.4 Pancreas in health............................................................................... 48 1.4.1 Structure and function ................................................................... 48 1.4.2 Cellular secretory pathways .......................................................... 52 1.5 How can we study the pancreas? ....................................................... 60 1.5.1 Animal models .............................................................................. 60 7 1.5.2 Tissue biobanks ............................................................................ 60 1.5.3 In-vitro β-cell models ..................................................................... 62 1.6 Protein phosphatase 1, regulatory (inhibitory) subunit, 1A (PPP1R1A) 66 1.6.1 Regulation of PPP1R1A ............................................................... 67 1.6.2 Expression of PPP1R1A ............................................................... 69 1.6.3 PPP1R1A isoforms ....................................................................... 72 1.6.4 PPP1R1A and regulation of antiviral responses ........................... 74 1.7 Project aims ........................................................................................ 76 2 Methods .................................................................................................... 81 2.1 Cell culture .......................................................................................... 81 2.1.1 Freezing of cells ............................................................................ 88 2.1.2 Transfections ................................................................................ 88 2.1.3 Pseudoislet preparation ................................................................ 88 2.2 Methods for investigation into gene expression .................................. 91 2.2.1 RNA extraction.............................................................................. 91 2.2.2 DNA Extraction ............................................................................. 91 2.2.3 RNA / DNA estimation .................................................................. 92 2.2.4 cDNA synthesis ............................................................................ 92 2.2.5 Primer Design ............................................................................... 93 2.3 PCR ..................................................................................................... 94 2.3.1 Gel extraction................................................................................ 95 8 2.4 Sub-cloning of DNA ............................................................................. 97 2.4.1 Transformation.............................................................................. 97 2.4.2 Glycerol stocks ............................................................................. 98 2.4.3 Restriction digest .......................................................................... 98 2.4.4 DNA ligation .................................................................................. 98 2.5 Southern Blotting ............................................................................... 100 2.5.1 DNA digestion ............................................................................
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