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Title: Dissection of signalling pathways involved in the induction of apoptosis in a murine thymocyte cell line
Author: Windsor, Richard Hector Stanley
ISNI:       0000 0001 3570 7493
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1996
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Apoptosis is the process of programmed cell death by which cells commit suicide. Apoptosis occurs in order to dispose of unwanted cells without cell lysis or release of intracellular components which could result in inflammation or autoimmunity. During development much cell death occurs by apoptosis and this is important not only for morphological sculpting, such as limb development, but also in the generation of a functional antigen-reactive repertoire in the development of the immune system. During differentiation of T lymphocytes in the thymus, many cells die by apoptosis in the cortex, either because they fail to receive survival signals or they have self-reactive antigen receptors. Therefore cortical thymocytes are a good model for cells which are sensitive to the induction of the suicide pathway. In order to investigate the signalling pathways involved in the induction of apoptosis, I have studied a cortical thymocyte cell line, F57T3.2 (T3.2), which undergoes apoptosis in response to a range of stimuli which induce cell death in normal thymocytes. The occurrence of apoptosis in T3.2 was examined in detail by comparing the effects of different stimuli: Ionomycin, Phorbol 12,13-Dibutyrate (PdBu), anti-CD3, Concanavalin-A (Con-A), Hydrocortisone, Thapsigargin and Staurosporine. The effects of these stimuli were analysed with a variety of different assays including, growth arrest, DNA fragmentation. Transmission Electron Microscopy, [Ca2+]i flux and tyrosine phosphorylation. The onset and extent of apoptosis in T3.2 was found to be similar to that reported for freshly isolated thymocytes indicating that T3.2 is a good model for thymocyte apoptosis. Mutants of T3.2 were generated which were resistant to apoptosis induced by the different stimuli, in order to try and identify apoptosis specific gene products. All mutant cell lines were found to be resistant only to the stimulus with which they were selected which suggests that the mutagenesis protocol targeted only early events in the apoptosis induction pathway. To remove sibling mutations, complementation assays were performed on mutants which exhibited similar phenotypes. Two separate and distinct mutations were found in the Ionomycin and Thapsigargin pathways and one each in the Hydrocortisone and Con-A pathways. Additionally, I examined whether the expression of any genes known to be involved in apoptosis had been altered in the mutants by Western blotting and probing for, c-Myc, Bcl-2, Bax, ICE and glucocorticoid receptor (GR). The differences between wild type and mutant cells are analysed and discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genetics