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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. The effects of mutations in the eEF1A2 gene in mouse gene expression profiles and identification of potential markers for motor neuron degeneration. Vesa Qarkaxhija MSc Genetics and Molecular Medicine. University of Edinburgh 2017 Declaration page I declare that this thesis is my own work unless stated otherwise and any inclusions of contributions or publications from others have been clearly acknowledged. This thesis has not been submitted for any other degree or professional qualification. Vesa Qarkaxhija 1 Acknowledgments I would like to thank Cathy Abbott, whose guidance and support has made this year both incredibly enlightening and enjoyable. I have learnt an inexpressible amount from her and could not have asked for a more wonderful supervisor. I would also like to thank the Abbott lab group, who have also taught me so much, in the lab and out, whom I had many a titillating conversations and debates with that made my time here most entertaining. My family and friends both old and new who had enriched this experience and accompanied me on many Scottish adventures during the course of my masters. 2 Contents: Declaration page ......................................................................................................................... 1 Acknowledgments ....................................................................................................................... 2 Abstract: ........................................................................................................................................ 6 Lay Summary: .............................................................................................................................. 7 Chapter 1: Introduction ............................................................................................................. 10 1.1 Translation and Eukaryotic Elongation factor Alpha (eEF1A). ........................... 10 1.2 eEF1A isoforms. ........................................................................................................ 11 1.3 Translation factors and EEF1A in disease. ................................................................ 19 1.4 eEF1A isoforms in Neurological disease. ................................................................... 20 1.5 Motor neuron disease and eEF1A2. ............................................................................ 23 1.6 Mouse Models: ................................................................................................................ 25 1.7 Biomarkers for Motor neuron degeneration. ............................................................... 27 1.8 Project Aims: ................................................................................................................... 29 Chapter 2: Materials and methods. ........................................................................................ 30 2.1: Clustal Alignments and Phylogenetic trees. .............................................................. 30 2.2: Genotyping. .................................................................................................................... 30 2.2.1: Genomic DNA extraction. ..................................................................................... 30 2.2.2: Nested PCR. ........................................................................................................... 30 2.2.3: TOPO Cloning. ....................................................................................................... 31 2.3: Protein expression. ........................................................................................................ 31 2.3.1: Sample preparation................................................................................................ 31 2.3.2: Protein separation. ................................................................................................. 31 2.3.3: Protein transfer, immunoblotting and quantification. ........................................ 31 2.4: Immunohistochemistry. ................................................................................................. 32 2.4.1: Sample preparation................................................................................................ 32 2.4.1: Section staining. ..................................................................................................... 32 2.5: Proteomics. ..................................................................................................................... 33 2.5.1: Sample extraction and preparation. .................................................................... 33 2.5.2: Mass spectrometry. ................................................................................................ 33 3 2.5.3: Statistical tests. ....................................................................................................... 34 2.6: Microarray. ...................................................................................................................... 34 2.6.1: Sample extraction and preparation. .................................................................... 34 2.6.2: Statistical tests. ....................................................................................................... 34 2.7: Gene Ontology analysis. .............................................................................................. 34 Chapter 3: Characterisation of the D252H mutation in eEF1A2 in mice. ......................... 35 3.1 Development of mouse lines. ....................................................................................... 35 3.2 Genotyping. ..................................................................................................................... 36 3.3 Protein Expression. ........................................................................................................ 40 3.4 Discussion. ...................................................................................................................... 44 Chapter 4: Analysis of eEF1A2 null and Wildtype spinal cord quantitative proteome and neuronal damage. ..................................................................................................................... 47 4.1 Biological specimens. .................................................................................................... 47 4.2 Establishing the extent of neurodegeneration in the new homozygous mice. ...... 48 4.3 Mass Spectrometry. ....................................................................................................... 52 4.3.1 Quantitative Label-free Mass spectrometry. ........................................................... 52 4.3.2 Peptide identification. .............................................................................................. 53 4.3.3 Determination of protein abundances. ................................................................. 54 4.4 Data Analysis. ................................................................................................................. 56 4.4.1 Expression profiles: ................................................................................................. 58 4.5 Gene Ontology analysis: ............................................................................................... 64 4.6 Discussion........................................................................................................................ 69 Chapter 5: RNA expression profiles of wasted mice and wild type mice. ........................ 75 5.1 Biological specimens. .................................................................................................... 76 5.2 Expression profiles. ........................................................................................................ 77 5.3 Gene Ontology analysis. ............................................................................................... 81 5.4 Discussion........................................................................................................................ 86 Chapter 6: Proteins and genes in the context of MND. ....................................................... 88 6.1 Fibroblast growth factor 12 (Fgf12).............................................................................. 88 6.1 Cytoplasmic dynein 1 intermediate chain 2 (Dync1i2). ............................................. 89 6.2 DnaJ homolog subfamily C member 5 (Dnajc5). ....................................................... 92 6.3 Kinesin heavy chain isoform 5C (Kif5C). .................................................................... 95 4 6.4 Discussion. .....................................................................................................................
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