ebook img

eg PhD, MPhil, DClinPsychol PDF

186 Pages·2016·5.96 MB·English
by  
Save to my drive
Quick download
Download
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

Preview eg PhD, MPhil, DClinPsychol

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. Effects of Glucocorticoids in Macrophages Alasdair Jubb Presented for the degree Doctor of Philosophy University of Edinburgh 2015 PhD – The University of Edinburgh – 2015 PhD – The University of Edinburgh – 2015 Declaration Unless explicitly stated otherwise the work presented here is my own. The work has not been submitted for any other degree or qualification. ……………………………………….. Date ………………… Alasdair Jubb PhD – The University of Edinburgh – 2015 Contents Acknowledgements ..................................................................................................... 9! Abstract ..................................................................................................................... 11! Figures ....................................................................................................................... 13! Tables ......................................................................................................................... 15! Abbreviations .............................................................................................................. ii! Published, presented and submitted work from this thesis ..................................... i! Chapter 1: Introduction ............................................................................................. 1! 1.1! Glucocorticoids ................................................................................................ 1! 1.1.1! Discovery, use and problems .................................................................... 1! 1.1.2! Towards a mechanism for glucocorticoid action ...................................... 6! 1.1.3! The role of GR dimerization and the ‘dissociated’ glucocorticoid ........ 10! 1.2! Chromatin and the control of gene expression .............................................. 11! 1.2.1! Chromatin ............................................................................................... 11! 1.2.2! Long range control of gene regulation ................................................... 12! 1.2.3! GC effects on chromatin ......................................................................... 25! 1.3! Macrophages .................................................................................................. 26! 1.3.1! Overview and relevance ......................................................................... 26! 1.3.2! Macrophage response to GC .................................................................. 27! 1.4! Models, conservation and divergence ........................................................... 29! 1.5! Summary ........................................................................................................ 31! 1.6! Thesis aims .................................................................................................... 31! Chapter 2: Methods .................................................................................................. 33! 2.1! Laboratory procedures ................................................................................... 33! 2.1.1! Ethics ...................................................................................................... 33! 2.1.2! Cell culture ............................................................................................. 33! 2.1.3! RNA extraction and processing .............................................................. 34! 2.1.4! Chromatin Immunoprecipitation ............................................................ 34! 2.1.5! Single cell RT-qPCR .............................................................................. 37! 2.1.6! 3D DNA FISH ........................................................................................ 37! 2.2! Data Analysis ................................................................................................. 40! 2.2.1! Expression data ....................................................................................... 41! 2.2.2! Single cell PCR ....................................................................................... 41! 2.2.3! Promoter Analysis .................................................................................. 41! 2.2.4! Comparison to GWAS results and inflammatory genes ......................... 42! 2.2.5! Functional Annotation ............................................................................ 42! 2.2.6! ChIP-sequencing ..................................................................................... 42! 2.2.7! Interspecies and evolutionary analysis ................................................... 43! 2.2.8! Analysis of 3D FISH data ....................................................................... 44! Chapter 3: The transcriptional response of macrophages to glucocorticoids .... 45! 3.1! Introduction ................................................................................................... 45! 3.2! The response of mouse bone marrow derived macrophages to dexamethasone ....................................................................................................... 46! 3.3! The response of human monocyte derived macrophages to dexamethasone 51! 3.4! The expression response is different and is not linked to promoter variation 54! 3.5! Targets of glucocorticoids in human macrophages enrich for risk variants for inflammatory disease .............................................................................................. 61! 3.6! Discussion ...................................................................................................... 63! 3.7! Summary ........................................................................................................ 65! Chapter 4: Glucocorticoid receptor binding in macrophages .............................. 67! 4.1! Introduction ................................................................................................... 67! 4.2! GR binding in Mouse Bone Marrow Derived Macrophages ......................... 68! 4.3! GR binding in Human Monocyte Derived Macrophages .............................. 76! 4.4! Glucocorticoid receptor binding is associated with induced, not repressed genes ....................................................................................................................... 80! 4.5! Inter-species differences of glucocorticoid receptor binding are associated with sequence changes that lead to motif loss ........................................................ 82! 4.6! Discussion ...................................................................................................... 91! 4.7! Summary ........................................................................................................ 95! Chapter 5: Dynamic regulation of chromatin structure in macrophages by glucocorticoids .......................................................................................................... 97! 5.1! Introduction ................................................................................................... 97! 5.2! GR binding is associated with rapid and prolonged chromatin decompaction 98! 5.3! Rapid chromatin decompaction of Fkbp5 locus does not depend on transcription .......................................................................................................... 105! 5.4! GR binds rapidly at enhancers in the Fkbp5 locus ...................................... 108! 5.5! Rapid decompaction may be a feature of GR bound loci ............................ 112! 5.6! Discussion .................................................................................................... 115! 5.7! Summary ...................................................................................................... 117! Chapter 6: Conclusions and future work ............................................................. 119! 6.1! Conclusions ................................................................................................. 119! 6.1.1! The genome wide response to GC in macrophages .............................. 119! 6.1.2! GC effects on chromatin organisation in macrophages ........................ 119! 6.2! Future work ................................................................................................. 120! 6.2.1! Enhancer turnover and variability ........................................................ 120! 6.2.2! Kinetics of the macrophage response to GC and mechanisms of repression .......................................................................................................... 123! 6.2.3! GC driven chromatin dynamics ............................................................ 123! 6.3! Summary ...................................................................................................... 127! References ............................................................................................................... 129! Appendix 1 .............................................................................................................. 149! Appendix 2 .............................................................................................................. 155! Acknowledgements Thanks must first go to the directors of the Edinburgh Clinical Academic Track Brian Walker and John Iredale and the Wellcome Trust/NES for giving me the post in the first place. The flexibility of the programme and advice they have given me, more recently also Andrew Jackson, has enabled me to identify and take on a much more ambitious project than I could have done otherwise. Thanks to my supervisors David Hume and Wendy Bickmore for taking me on without any track record to speak of and helping me put together a worthwhile project. Both were and continue to be incredibly supportive of my attempts to scale what remains at times a pretty precipitous learning curve. There is no way I could have done any of this without the teaching and support provided by the Bickmore and Hume and wider Maclab group members. Thankfully there are plenty of them to spread the load, some are named below but thanks are due to all. From the Bickmoreans Rob Illingworth has to get first mention due to the sheer amount of time I spent bending his ear about ChIP. Rob Young added welcome and necessary computational and enhancer expertise and Shelagh Boyle enabled all things FISH related. Thanks are due to all : Hazel, Pradeep ‘should be fine’ MM, Liz, Gillian (GD), Sehrish, Iain, Nezha, Pierre, Celine, Leisha, Jess and Andrew as well as the wider E3 community for the unfailingly entertaining environment. Paul and Matt for helping me out in imaging at HGU. At Roslin thanks must go to the core of the lab Clare, Kristen, Lindsay, Anna, and Gemma; to those who helped me get going Kyle Upton and Ronan Kapetanovic; to Tom Freeman for introducing me to the world of expression analysis and his lab for providing good chat and gender balance; to Malcom Fisher and latterly Sara Clohisey and Gemma Davies for blood days Maggie for the fish, Bob in imaging. Specific mention to my ECAT forerunner at Roslin, Kenny Baillie, for help, advice, and being consistently distractible by the promise of a haggis roll. And finally, of course, thanks to Francesca and Saoirse.

Description:
PhD – The University of Edinburgh – 2015. Effects of Glucocorticoids in Macrophages. Alasdair Jubb. Presented for the degree Doctor of Philosophy. University of Edinburgh. 2015
See more

The list of books you might like

Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.