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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. Investigating the roles of Translation Elongation Factor 1B in mammalian cells Yuan Cao Thesis submitted for the degree of Doctor of Philosophy The University of Edinburgh 2012 Declaration I here declare that this thesis has been composed by me and that all the work presented within is my own, unless clearly indicated otherwise. This work has not been submitted for any other degree or professional qualification. Yuan Cao i Acknowledgements First of all I wish to express my deepest gratitude to my supervisor Professor Cathy Abbott who has been so patient and understanding in the last four years. I appreciate her scientific guidance throughout my research, her encouragement when I was being negative, as well as her support and help in both my work and life. I would also like to thank my second supervisor Rebecca Barnetson, and Ian Jackson and Elaine Nimmo of my thesis committee for their helpful advices for my project. I am also very grateful for the scholarship offered by the Chinese Scholarship Counsel and the College of Medicine and Veterinary Medicine and the opportunity to be a student of the University of Edinburgh. Thanks to all those who have kindly offered technical support for my lab work. I thank Abby, Barbie for their help with microscope training, cell culture and lending me the humidity chamber for the PLA, Liang for his advice of the GelQuant software, and Shaun for the antibody. Thanks to Paul Perry in the MRC for his tutorial on the multi-purpose microscope and data analysis using the IPLab software. I also thank David and Chris at the BRF of the Western General Hospital for helping out with the mice. A big thank you to all the past and present members of the Abbott group: to Helen for her guidance with lab techniques especially when I first started my PhD, to Jennifer for her help especially with the transgenic mice, and to Miriam from whom I took over this project. Thanks also to Justyna, Lowri and Mariam for all the experimental tips and the time we spent together in the lab as well as in the cinema and restaurants. I wish Yan and Faith, the current PhD students in the Abbott group, further success with their PhDs. I would also like to thank all other fellow PhD students in the MMC for organizing activities and wish them a bright future ahead. I could not forget to thank my friends outside the lab, particularly Ling for her love and kindness, Lucy for being an inspiring, positive and trustworthy friend, and Yewen for all the fun (and silly) times as well as her encouragement and comfort in sad times. I also wish to thank Dr Linda Watson and David Purves for their patient ii proofreading of the manuscript of this thesis, and Nelly for her help with the diagram drawing in this thesis and my other reports. Last but by no means least, I would like to thank the brothers and sisters in the Bristo Baptist Church, in the Mandarin Fellowship of the Chinese Evangelical Church in Edinburgh, as well as those who are from other churches: Eleanor, Lindsey, Paul and Pete. They have been a great blessing and, in many ways, my family in Edinburgh. I appreciate so much their prayers, love, friendship and encouragement that supported me through difficult times. iii Table of contents Declaration ................................................................................................................... i Acknowledgements ..................................................................................................... ii Table of contents ....................................................................................................... iv List of figures ........................................................................................................... viii List of tables ................................................................................................................ x Abbreviations and symbols ...................................................................................... xi Abstract .................................................................................................................... xvi Chapter 1 Introduction .............................................................................................. 1 1.1 Protein translation ............................................................................................ 1 1.1.1 Initiation ....................................................................................................... 1 1.1.2 Elongation .................................................................................................... 2 1.1.3 Termination ................................................................................................. 3 1.1.4 Regulation of protein translation .............................................................. 4 1.2 Eukaryotic translation elongation factor 1A ................................................. 7 1.3 Eukaryotic translation elongation factor 1B .................................................. 9 1.3.1 eEF1Bα ........................................................................................................ 9 1.3.2 eEF1Bδ ...................................................................................................... 10 1.3.3 eEF1Bγ ...................................................................................................... 11 1.3.4 The eEF1B complex .................................................................................. 12 1.3.5 Regulation of eEF1B by phosphorylation ................................................. 15 1.4 The functions of eEF1B .................................................................................. 18 1.4.1 eEF1B and the cell cycle control ............................................................... 18 1.4.2 eEF1B and the cytoskeleton ...................................................................... 20 1.4.3 eEF1B and the stress response ................................................................... 21 1.4.4 eEF1B and diseases ................................................................................... 24 1.5 The wasted mouse, a model for MND ........................................................... 30 1.6 Proximity ligation assay ................................................................................. 32 iv 1.7 Project aims ..................................................................................................... 34 Chapter 2 Materials and Methods .......................................................................... 35 2.1 Materials .......................................................................................................... 35 2.1.1 Solutions and buffers ................................................................................. 35 2.1.2 Cell lines .................................................................................................... 37 2.1.3 Animals ...................................................................................................... 38 2.1.4 Antibodies .................................................................................................. 38 2.1.5 Slides ......................................................................................................... 38 2.1.6 DNA primers ............................................................................................. 42 2.1.7 siRNAs ....................................................................................................... 42 2.2 Cell culture ...................................................................................................... 44 2.2.1 Cell culture and maintenance..................................................................... 44 2.2.2 Cell counting .............................................................................................. 44 2.2.3 Cryopreservation of cells ........................................................................... 45 2.2.4 RNAi .......................................................................................................... 45 2.2.5 MTT assay ................................................................................................. 46 2.3 Protein-related methods ................................................................................. 48 2.3.1 Protein extraction from cells ...................................................................... 48 2.3.2 Protein extraction from tissues .................................................................. 48 2.3.3 Western blotting......................................................................................... 48 2.3.4 Immunohistochemistry .............................................................................
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