Apoptosis is known for its physiological role in development, sculpting of tissues, and regulation of immune cells. Although much of the signalling pathway has been dissected, the mechanism of many apoptotic events remains undefined. Furthermore, the lack of knowledge of how cells commit to apoptosis has impeded our understanding of how this process is altered in disease states. This study aimed to determine the sequence of key apoptotic events using novel combinations of flow cytometric assays, and to investigate the irrevocable point-of-no-return. To induce apoptosis, Jurkat T-cells were treated with camptothecin, anti-CD95- antibody or cycloheximide, in the presence/absence of inhibitors. Various apoptotic events were detected by flow cytometry. Secondly, cells were treated with apoptotic inducers in the presence of caspase inhibitor, and the differential gene expression pattern was studied using microarray. Four hours after treatment with stimuli, apoptosis were detected in approximately 50% of the cells. This thesis presents evidence that cell cycle may play a decisive role. Transcriptional regulation of p21 and GADD45B were found to control cell-cycle arrest and cell death. Multicolour flow cytometric assays also demonstrated that a proportion of cells in the AnnV7AAD- population (conventionally defined as live cells) underwent mitochondrial depolarisation and caspase activation after camptothecin or anti-CD95 treatment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617004 |
| Date | January 2014 |
| Creators | Pal, Rubina |
| Publisher | St George's, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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