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Tgfβ Superfamily Signalling in Pluripotency and Differentiation Of TGFβ SUPERFAMILY SIGNALLING IN PLURIPOTENCY AND DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS by VARVARA CHALMANTZI A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham January 2019 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. To my daughter Iouliani for teaching me that human beings can learn, evolve and thrive in every breath they take. Summary The discovery of human embryonic stem cells (hESCs) 20 years ago (Thomson et al., 1998), has significantly benefited the field of stem cell biology (Ilic & Ogilvie, 2017). Despite ethical (and legal in some cases) obstacles, therapies utilising hESCs and their derivatives are attempting to tackle several diseases (Ilic & Ogilvie, 2017). The TGFβ superfamily is known to be implicated in crucial decisions from early development of the embryo to the adult life (Mullen & Wrana, 2017). ACTIVIN A and BMP4 are two members of this superfamily and their activation status has an important impact on hESCs (James et al., 2005). On the one hand, ACTIVIN A is associated with the maintenance of the undifferentiated state (James et al., 2005), (Beattie et al., 2005), while on the other BMP4 is considered a differentiation stimulus (R. Xu et al., 2002), (P. Yu, Pan, Yu, & Thomson, 2011), (Richter et al., 2014). The aim of this Thesis is to study in depth several key cellular responses of hESCs to BMP4 and investigate the role of SARA, a TGFβ signalling mediator (Tsukazaki et al., 1998) in pluripotency and differentiation. In particular, Chapter 3 establishes the culture system of H1 cells and shows the effect of BMP4 on the expression of several genes associated with differentiation. Moreover, it introduces a new culture system based on a modified version of a commercial medium which is later used for phosphoproteomic studies in Chapter 4. Chapter 4 describes the experimental procedure followed in order to metabolically label H1 cells and use Mass Spectrometry (MS) to elucidate changes in protein phosphorylation status upon 60min of BMP4 induction. The regulated phosphosites were further categorised depending on three parameters (biological process, molecular function and cellular component). Some of the upregulated phosphosites were selected and further tested as putative BMP4 targets using phos-tag acrylamide gels. Chapter 5 focuses on SARA; it investigates the presence of one or more isoforms in human cell lines and the possibility that SARA shuttles to the nucleus. Moreover, it studies the expression levels of SARA, both at the mRNA and at the protein level in differentiating H1 cells and the effect it has in SMAD2 phosphorylation. Additionally, it compares the protein levels between fibroblasts and human induced pluripotent stem cells (hiPSCs) and addresses the notion that SARA is degraded by the lysosome. Last, this chapter attempts to generate GFP-SARA stable H1 cell lines, by transfection, and SARA knockout H1 cell lines, using a genome editing technique (CRISPR). Acknowledgments «All men by nature desire knowledge» (Aristotle, Metaphysics) and this powerful quote is the best way I found in order to summarise the reasons behind my decision to start a PhD. This endeavour would have never started if it wasn’t for my husband Dr. Anastatios Batilas who encouraged me to pursue it. I would like to thank Prof. Theodore Fotsis and Dr. Carol Murphy for giving me the space and time to adapt in an, unknown for me, laboratory environment a couple of years ago and express my scientific curiosity under their supervision. I also want to thank Prof. John Heath for helping me throughout my studies in the University of Birmingham and for his willingness to assist me and supervise me during my last, and most crucial, year of studies. «When setting out upon your way to Ithaca, wish always that your course be long, full of adventure, full of lore» (Konstantine P. Kavafy, Ithaca) and indeed this PhD has been a long adventurous journey. I would like to thank all the friends and people with whom I have worked all these years, for teaching me new skills and making this journey less daunting. I also want to express my gratitude to my family for standing next to me and supporting me morally when I was facing several adversities. TABLE OF CONTENTS Summary ..................................................................................................................................... 3 Table of Contents ........................................................................................................................ i List of Figures ............................................................................................................................ iv List of Tables .............................................................................................................................. v i Abbreviations .............................................................................................................................. 1 1. Introduction ....................................................................................................................... 5 1.1 Introduction in Stem Cells (SC) ....................................................................................... 5 1.1.1 Stem Cells ........................................................................................................................................ 5 1.1.2 Early Embryogenesis ....................................................................................................................... 6 1.1.3 Human embryonic stem cells, origins and properties .................................................................... 13 1.1.4 Human embryonic stem cells, therapeutic potential ...................................................................... 14 1.1.5 Human embryonic stem cells, culture systems .............................................................................. 16 1.1.6 Generation of induced pluripotent stem cells (iPSCs) ................................................................... 18 1.2 Dissecting Pluripotency ................................................................................................. 19 1.2.1 Key pluripotency transcription factors .......................................................................................... 19 1.2.2 Naïve and Primed Pluripotency ..................................................................................................... 22 1.3 TGFβ superfamily signalling .......................................................................................... 24 1.3.1 Ligands and antagonists ................................................................................................................. 24 1.3.2 Receptors and SMADs .................................................................................................................. 26 1.3.3 Non SMAD signalling ................................................................................................................... 29 1.3.4 Endocytosis overview .................................................................................................................... 30 1.3.5 TGFβ superfamily trafficking ........................................................................................................ 34 1.4 Key Signalling pathways regulating pluripotency and differentiation ............................. 39 1.4.1 TGFβ/ACTIVIN/NODAL signalling ............................................................................................ 39 1.4.2 BMP signalling .............................................................................................................................. 43 1.4.3 WNT signalling ............................................................................................................................. 46 1.4.4 FGF signalling ............................................................................................................................... 49 1.4.5 IGF, NOTCH and HIPPO signalling ............................................................................................. 51 1.5 SARA (ZFYVE9) protein ................................................................................................ 53 1.5.1 Introduction in SARA .................................................................................................................... 53 1.5.2 SARA in TGFβ/ACTIVIN signalling ............................................................................................ 54 1.5.3 SARA in trafficking ....................................................................................................................... 57 1.5.4 SARA in Epithelial to Mesenchymal Transition ........................................................................... 58 1.5.5 SARA in Asymmetric Cell Division ............................................................................................
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