This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ A system level approach to identify novel cell size regulators Dul, Zoltan Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 03. Oct. 2021 A system level approach to identify novel cell size regulators ZOLTAN DUL A THESIS PRESENTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2019 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul Declaration I declare that this thesis has been composed solely by myself and that it has not been submitted, in whole or in part, in any previous application for a degree. Except where stated otherwise by reference or acknowledgement, the work presented is entirely my own. Zoltán Dul January 2019 2 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul Acknowledgements Thank you to my primary supervisor Dr Attila Csikász-Nagy for giving me the opportunity to work in his computational biology group in London and for guiding me so well throughout my PhD studies. His sound ideas and plans immeasurably helped my way to complete my PhD. I’m also exceptionally grateful to my second supervisor Professor Shaun Thomas for helping and introducing myself to wet lab experiments with T-Cells. His academic experience and inspirational ideas have been invaluable to me. I also would like to thank you, my Italian supervisor, Dr Azeddine Si-Ammour for guiding my path in Arabidopsis research and giving me research ideas. I’m very grateful to him introducing me to a new research area in science. I would like to acknowledge the help of the Leukaemia Cell Cycle Lab members in the Rayne Institute, King’s College London especially to Dr Stephen Orr who introduced me the various techniques of T-Cell experimentation and guided my path until I mastered the technique. Also, I’m thankful to Dr Nermina Lamadema and Dr Sun Sook Chung without whom this work would not have been possible to complete. Moreover, I’m indebted to Dr Loretta Brown and others at King’s College Hospital for helping me in some areas of research. I would like you to thank the current and former members and Csikasz-Nagy group: Dr Federico Vaggi, Rosa Hernansaiz and Kirsten Jenkins for their help in the Bioinformatic field. Moreover, I’m very grateful to Dr Asquini Elisa, Dr Mirko Moser and Dr Valentina Cappelletti and all other people from Fondazione Edmund Mach, San Michele all’ Adige, Italy for guiding me in the endless field of Arabidopsis research. Finally, I would like to thank the Research and Innovation Centre in Fondazione Edmund Mach, San Michele all’ Adige, Italy for funding my PhD studies. Last but not least to my family and friends, thank you for all the support and encouragement. 3 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul Abstract It is known that evolutionarily conserved pathways regulate several essential biological processes, such as the cell cycle, DNA replication and protein synthesis. I hypothesise that there exists a conserved regulatory mechanism which controls cell size as well. To test this proposition, I carried out two types of experiment: Bioinformatics. Development of a bioinformatics tool to predict cell size regulators, based on information of evolutionarily-conserved proteins which have been described as cell size regulators in genome-wide studies. I collected existing large-scale data from the literature from five evolutionarily distant organisms (A. thaliana, D. melanogaster, H. sapiens, S. cerevisiae and S. pombe), then looked for conserved orthologous proteins with conserved cell size regulatory functions. I used the eggNOG orthology database as the primary source for orthology information, in addition to one manually curated list by PomBase curators. I added biological pathway information from KEGG and functional data from Gene Ontology. This approach allowed me to identify a core conserved cell size regulatory network and based on data on three of these species I created a list of predicted novel cell size regulators for the remaining two organisms. I focussed on those orthologous groups of proteins, which have only one ortholog in all five organisms and further reduced the list by concentrating on those which lack pathway annotations. 20 conserved orthologous groups matched these criteria. Some of these were tested in wet- lab experiments. Additionally, I have extended this tool with the capability to find the conserved core of any biological function, listed in the Gene Ontology SLIM database and extended the list of the investigated model species to seven (A. thaliana, C. elegans, D. melanogaster, D. rerio, H. sapiens, S. cerevisiae and S. pombe). This bioinformatics tool is also able to predict novel functional annotations to Gene Ontology, based on orthologous group involvement in eggNOG. Wet-lab experiments. Analysis of some of the predicted cell size regulators by carrying out experiments on Arabidopsis thaliana and human T-Cells. 4 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul T-Cells. I isolated human peripheral blood T-Cells that are in a quiescent state, which increase in size and enter the cell cycle when stimulated via CD3/CD28. The quiescent T-Cells were transfected with siRNA to reduce the induction of each of the predicted proteins, which normally occurs as the cells respond to CD3/CD28. I analysed the size distribution by Flow Cytometry, measuring FSC-A values. My data show that reducing the induction of TCTP, a protein that plays a role in microtubule stabilisation, apoptosis and calcium binding, affects T-Cell size. I also show that reducing the expression of the MCM7 protein, a member of the mini-chromosome maintenance protein complex, reduced the size of T-Cells in G1 and increased their size in the S-phase of the cell cycle. Arabidopsis thaliana. I confirmed previously published data that knockout mutants of pfd6, a member of the prefoldin complex, produce small plants. I show that knock-out pfd3 mutants have a significantly smaller cell size compared with the control. The knock-out pfd3 mutants also have significantly different seed and silique sizes. Furthermore, I found that the knockout of mos14, encoding a nuclear import receptor, results in significantly smaller leaves. In summary, I have created a flexible bioinformatics tool that can predict novel cell size regulators, some of which are verified by experiments on human cells and Arabidopsis. 5 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul Table of Contents 2 3 4 6 13 16 17 20 21 22 22 22 24 25 26 28 28 30 31 31 33 35 36 37 38 39 6 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 41 42 43 44 45 46 46 47 47 48 48 49 51 51 52 53 53 54 55 55 55 55 55 56 57 57 58 58 59 60 60 60 61 61 61 62 62 64 7 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 64 64 64 65 65 66 66 67 67 69 73 74 74 75 75 76 77 78 78 79 81 81 83 86 88 88 91 91 91 93 94 94 95 96 96 97 98 98 99 99 100 101 103 105 8 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 105 106 108 112 112 113 114 114 114 115 115 117 118 119 119 119 120 120 120 121 121 121 123 123 124 124 125 125 126 126 126 126 127 128 129 130 130 132 132 135 136 137 138 139 139 139 140 141 9 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 142 143 144 146 147 147 148 148 149 149 150 153 156 158 158 159 159 162 164 164 167 167 170 170 172 172 173 178 182 186 186 187 188 188 189 190 190 192 10 A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 193 194 194 194 196 196 197 200 200 200 200 201 201 201 202 202 202 203 204 204 204 208 209 209 210 211 212 213 213 214 215 216 217 217 220 220 223 #1 225