The Functional Analysis of Granulins 2014 By Kate Young School of Medicine A thesis submitted to the University of Manchester for the degree of Master of Philosophy (MPhil) in the Faculty of Medical and Human Sciences -1- Table of Contents Declaration ........................................................................................................................ 6 Copyright Statement ......................................................................................................... 6 Acknowledgements ........................................................................................................... 7 Abstract ............................................................................................................................. 8 Abbreviations ........................................................................................................................ 9 Chapter 1 Introduction ........................................................................................................ 10 1.1 Frontotemporal Lobar Degeneration. ................................................................... 11 1.2 Clinical Syndromes. ................................................................................................ 11 1.3 Neuropathology. .................................................................................................... 13 1.4 FTLD Proteinopathies. ............................................................................................ 14 1.4.1 FTLD Tau/FTLD-17 ........................................................................................... 14 1.4.2 FTLD-TDP43 ..................................................................................................... 15 1.4.3 Less Common types of FTLD ........................................................................... 16 1.5 Gene Mutations Associated with FTLD. ................................................................. 16 1.5.1 MAPT linked FTLD ........................................................................................... 16 1.5.2 GRN Linked FTLD ............................................................................................. 17 1.5.3 Chromosome 9 linked FTLD ............................................................................ 18 1.5.4 Rare Mutations................................................................................................ 19 1.6 Progranulin............................................................................................................. 21 1.6.1 Progranulin Function ....................................................................................... 22 1.6.2 Progranulin Receptors..................................................................................... 23 1.6.3 Inflammation and Wound Healing .................................................................. 23 1.6.4 Cell Signalling .................................................................................................. 24 1.6.5 Tumorigenesis ................................................................................................. 24 1.6.6 Progranulin and Neurodegeneration .............................................................. 24 1.7 The Functional Analysis of Granulins. .................................................................... 25 1.8 Aims and Objectives. ............................................................................................. 25 Chapter 2 Progranulin and Granulin Cloning ...................................................................... 26 2.1 Introduction ........................................................................................................... 27 2.2 Construct DNA Amplification ................................................................................. 29 -2- 2.2.1 Transformation and Clonal Selection .............................................................. 30 2.2.2 Restriction Digestion ....................................................................................... 30 2.2.3 Agarose Gel Electrophoresis ........................................................................... 30 2.2.4 Ligation ............................................................................................................ 31 2.2.3 Transient Transfection .................................................................................... 32 2.2.4 Gel Electrophoresis ......................................................................................... 32 2.2.5 Western Blotting ............................................................................................. 33 2.3.1 PCR Protocol .................................................................................................... 35 2.3.2 Sequencing. ..................................................................................................... 36 Chapter 3 Protein Expression and Purification ................................................................... 40 3.1 Introduction ........................................................................................................... 41 3.2 Stable Transfection ................................................................................................ 41 3.3 Protein Purification. ............................................................................................... 42 3.3.1 Cell harvesting ................................................................................................. 42 3.3.2 Halotag resin purification ................................................................................ 42 3.3.3 Coomassie Staining ......................................................................................... 44 3.3.4 High-Performance Liquid Chromatography (HPLC) ........................................ 45 3.4 Gel Electrophoresis ................................................................................................ 46 3.5 Silver Staining. ........................................................................................................ 46 Chapter 4 Cell Treatment and Next Generation Sequencing ............................................. 49 4.1 Introduction. .......................................................................................................... 50 4.2 Differentiating SH-SY5Y cells.................................................................................. 50 4.3 Cell Treatment ....................................................................................................... 50 4.4 RNA Extraction and Homogenisation. ................................................................... 51 4.5 Library Preparation for Next Generation Sequencing ........................................... 52 4.5.1 Ribosomal RNA Depletion ............................................................................... 53 4.5.2 RNA Fragmentation ......................................................................................... 54 4.5.3 Library Preparation ......................................................................................... 56 4.5.4 Library Amplification ....................................................................................... 57 4.5.5 Library Sequencing .......................................................................................... 59 4.6 Next Generation Sequencing Data Analysis .......................................................... 60 -3- 4.6.1 Alignment to the Reference Genome. ............................................................ 60 4.7 Validation by Real Time PCR .................................................................................. 62 4.7.1 Introduction .................................................................................................... 62 4.7.2 RNA Extraction and cDNA Synthesis ............................................................... 63 4.7.3 Real Time PCR ................................................................................................. 64 4.8 Gene Expression Results ........................................................................................ 66 Chapter 5 Discussion ........................................................................................................... 74 RNA Seq Discussion ...................................................................................................... 77 Possible Future Work. .................................................................................................. 82 Supplementary information ................................................................................................ 84 Materials ...................................................................................................................... 84 References ................................................................................................................... 86 Tables Table 1.1 Clinical, diagnostic and pathological features associated with the different sub- types of FTLD. ...................................................................................................................... 13 Table 4.1 Cell Treatments were performed as shown in the table below. ........................ 51 Table 4.2 Overview of sequencing statistics. The ERRC Spike in control counts show between 60 and 70 transcripts detected out of a possible 92. .......................................... 62 Table 4.3 Genes selected using the P=0.01 cut off by class ...............................................