Abstract
The focus of my thesis is to apply systems biology approaches to obtain a better understanding of complex cellular systems. In particular, my work concentrates on the function of haematological cells. The first part of the thesis applies a novel, predictive strategy to identify new regulators of a signalling pathway in human immune cells. Cdc42 is a membrane associated GTPase that is an important regulator of cytoskeleton rearrangements and is required for natural killer (NK) cell activation. Previous work had shown that Cdc42 activity oscillates during NK immune surveillance after an initial increase, suggesting that Cdc42 activity is regulated in NK cells by a number of signalling molecules. The aim of the project is to predict proteins required for Cdc42 activity in NK cells and test the predictions. Using a bioinformatics methodology, 13 different proteins were predicted to interact with Cdc42 and form feedback loops. To determine whether any of the predicted proteins were required for Cdc42 activity, NK cells were transfected with a Cdc42 biosensor, each of the predicted targets was downregulated with siRNA and Cdc42 activity was quantified by FRET/FLIM microscopy in the presence of target cells. The screen identified AKT1 and the p85a subunit of PI3K as novel regulators of Cdc42 activity. Depletion of each of these targets also results in an impaired cellular cytotoxic response. This proof of principle study demonstrates the power of a predictive approach in the NK immune surveillance context to identify novel regulators of Cdc42. The second part of my thesis is based on using a predictive methodology, called the ’Phenolog approach’ to predict novel proteins involved in cancer. To determine whether the predicated proteins are required to maintain genome stability, two of the predicted targets were tested using a human primary T cell system, in which cellular mechanisms are normal.Quiescent T cells were transfected with siRNA, the cells were stimulated to enter the cell cycle and chromosome integrity was analysed by interphase FISH. The two predicted targets tested were AND1, a DNA replication protein, and SEC13, a component of the nuclear pore complex. Depletion of each of the targets led to a number of chromosomal abnormalities,indicating that their normal expression during the G0—>G-i transition is required to maintain genome stability. The third part of my thesis focuses on the systematic analyses of the chromatin proteome of T cells during cell cycle progression and identifying changes in the proteome caused by depleting the DMA replication protein Mcm7. Reducing the induction of Mcm7 causes DMA damage, premature chromatid separation and genome instability (291). Initially, the chromatin proteome obtained by a native, non-crosslinked chromatin extraction method was compared with that obtained after formaldehyde crosslinking. In addition, the methodology was compared with the proteome obtained previously using the CSK extraction method (290). To analyse the effects of depleting Mcm7, quiescent primary T cells were transfected with Mcm7 siRNA and the cells were stimulated to enter the cell cycle. The proteins were crosslinked with formaldehyde, chromatin was isolated and the chromatin-bound proteome in Mcm7-depleted and control cells were analysed by LC-MS/MS. Changes in the nuclear proteome caused by depleting Mcm7 were quantified by a label-free spectral counting method. Network analyses (HumanNet) were used to identify protein interaction sub-networks. The analyses identified that downregulation of Mcm7 affects a number of processes, including DNA replication, DNA damage, transcription and ribosome biogenesis.
| Date of Award | 1 Apr 2013 |
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| Original language | English |
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| Supervisor | Shaun Thomas (Supervisor) & Farzin Farzaneh (Supervisor) |