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Title: A system level approach to identify novel cell size regulators
Author: Dul, Zoltan
ISNI:       0000 0004 7963 9260
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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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. A system level approach to identify novel cell size regulators A Thesis presented for the degree of Doctor of Philosophy by Zoltán Dul 5 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.
Supervisor: Csikasz-Nagy, Attila Istvan ; Thomas, Nicholas Shaun Bevan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available