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Gene Expression and Cell Cycle Regulation in Human Pancreas Development and Congenital Hyperinsulinism

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Gene Expression and Cell Cycle Regulation in Human Pancreas Development and Congenital Hyperinsulinism Gene expression and cell cycle regulation in human pancreas development and congenital hyperinsulinism A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2014 Rachel Jayne Salisbury School of Medicine Table of contents List of figures ..................................................................................................................... 7 List of tables ...................................................................................................................... 9 Abstract ........................................................................................................................... 10 Declaration and copyright statement ................................................................................ 11 Acknowledgements .......................................................................................................... 12 List of abbreviations ......................................................................................................... 13 Publications arising from this thesis ................................................................................. 15 Chapter 1- Introduction .................................................................................................... 16 1.1. Introduction ......................................................................................................... 16 1.2. Role of the pancreas ............................................................................................. 17 1.2.1. Anatomy of the pancreas ................................................................................ 17 1.2.2. Role of the exocrine cells ................................................................................ 18 1.2.3. Role of the ductal cells .................................................................................... 18 1.2.4. Role of the endocrine cells .............................................................................. 19 1.2.4.2. Beta cells ..................................................................................................... 20 1.2.5. Dysfunction of the beta cell ............................................................................. 23 1.2.5.1 Diabetes ....................................................................................................... 23 1.2.5.2 Hyperinsulinism of Infancy ............................................................................ 24 1.3. Lessons learned from development ....................................................................... 25 1.3.1. Morphological development ............................................................................ 25 1.3.2. Endocrine differentiation ................................................................................. 26 1.3.2.1. Neurogenin factors ....................................................................................... 30 1.4. Maintenance of islets postnatally ........................................................................... 32 1.4.1 Postnatal islet cell “replication” vs “differentiation” vs “trans-differentiation” ...... 32 1.4.2. Cells that have potential as progenitors .......................................................... 35 1.5. Lessons learned from hyperinsulinism ................................................................... 37 1.5.1. Overview of hyperinsulinism of infancy ........................................................... 37 1.5.2. Mutations in the KATP channel ......................................................................... 39 1.5.3. Evidence for a progenitor population ............................................................... 41 1.6. Aims of the project ................................................................................................. 44 Chapter 2- Materials and methods ................................................................................... 45 2.1. Preparation of tissue sections................................................................................ 45 2.1.1. Tissue collection ............................................................................................. 45 2.1.2. Tissue fixing and embedding .......................................................................... 46 2.1.3. Microtome sectioning and mounting ................................................................ 47 2.1.4. H&E staining ................................................................................................... 47 2 2.2. Immunostaining ..................................................................................................... 48 2.2.1. Immunohistochemistry .................................................................................... 48 2.2.2. Dual immunofluorescence ............................................................................... 49 2.2.3. TUNEL ............................................................................................................ 52 2.2.4. Image analysis ................................................................................................ 54 2.2.5. Cell counting ................................................................................................... 54 2.3. Laser capture microdissection ............................................................................... 55 2.3.1. Preparation of slides for LCM .......................................................................... 56 2.3.2. Laser capture microdissection ........................................................................ 57 2.4. cDNA synthesis from RNA .................................................................................... 57 2.4.1. RNA extraction (Qiagen RNeasy Kit) .............................................................. 57 2.4.2. RNA quantification .......................................................................................... 58 2.4.3. First strand cDNA synthesis (ABI Method) ...................................................... 58 2.5. RNA amplification .................................................................................................. 59 2.5.1. Ovation® first strand cDNA synthesis ............................................................. 59 2.5.2. Ovation® generation of DNA/RNA heteroduplex double-stranded cDNA ........ 59 2.5.3. Ovation® purification of cDNA ........................................................................ 59 2.5.4. Ovation® SPIA® amplification ........................................................................ 60 2.5.5. Ovation® cDNA purification ............................................................................ 60 2.6. RNA sequencing ................................................................................................... 61 2.6.1. Library preparation and QC ............................................................................. 62 2.6.2. Illumina® RNA-seq ......................................................................................... 62 2.6.3. RNA-Seq mapping, quantification and analysis ............................................... 63 2.7. Polymerase chain reaction .................................................................................... 64 2.7.1. Primer design .................................................................................................. 64 2.7.2. qRT-PCR ........................................................................................................ 66 2.7.3. Primer Efficiencies .......................................................................................... 67 2.8. Statistical analysis ................................................................................................. 68 Chapter 3- Characterising histology and key developmental transcription factor expression in CHI-D pancreas ........................................................................................................... 69 3.1. Introduction ........................................................................................................... 69 3.1.1. Nesidioblastosis in diffuse-CHI........................................................................ 69 3.1.2. Progenitor population within ductal epithelium ................................................ 70 3.1.2. Endocrine differentiation during human fetal development .............................. 71 3.1.3. Classification of CHI ........................................................................................ 73 3.1.4. Mutations in KATP ............................................................................................. 74 3.2. Aims ...................................................................................................................... 75 3 3.2.1. To characterise hormone expression and islet architecture in CHI-D samples; A comparison with age-matched controls ..................................................................... 75 3.2.2. To Investigate a process of islet neogenesis in CHI-D; A comparison with normal human fetal development .............................................................................. 75 3.2.3. To determine whether CHI-D is consistent with a prolonged immaturity of islets ................................................................................................................................. 75 3.3. Results .................................................................................................................
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