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Title: Identification of genes regulating glucocorticoid-induced and antigen-mediated thymocyte apoptosis
Author: Woodward, Martin James
ISNI:       0000 0004 2670 3804
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2008
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The default pathway for developing thymocytes is apoptosis they must be actively instructed to survive and proliferate. Approximately 95% of developing thymocytes undergo apoptosis only 5% survive to enter the periphery. Thymocytes commit to apoptosis either because of a lack of survival signalling (death by neglect), or by encountering high avidity T cell receptor signalling that might predicate potential autoimmunity (negative selection). We have performed two Affymetrix DNA microarray screens to attempt to identify candidate regulatory genes for thymocytes undergoing apoptosis in response to the synthetic glucocorticoid dexamethasone and in the F5 TCR Rag-1"7" Tap-1"" transgenic model for antigen-mediated negative selection. The up-regulation and expression of genes identified in these screens has been confirmed by qPCR. The action of these genes was assayed in both foetal thymic organ culture (FTOC) and OP9-Delta-Like 1 (OP9-DL1) thymocyte co-culture systems. We have used modified pMSCV (murine stem cell virus) over-expression and knockdown retroviral vectors to infect haematopoietic progenitor cells (HPCs). These cells were then used to reconstitute depleted FTOCs or placed into culture on OP9-DL1 stromal cell layers. Transduced HPCs then differentiate into thymocytes, allowing us to assay the effect of target genes upon apoptosis and the T cell development program. We assayed target genes from both screens for their effect on thymocyte apoptosis in the presence and absence of apoptosis inducing agents, and have identified several that may be critical in the context of thymocyte apoptosis. One gene, Tnfaip8, has been shown to markedly enhance apoptosis in response to dexamethasone, and to inhibit apoptosis when knocked down using RNA interference. This gene is both up-regulated at the double negative (DN) to double positive (DP) transition and highly up-regulated by glucocorticoids. We have also demonstrated that Krox24, a highly induced gene in our F5 TCR7" Rag- lv" Tap-1A antigen screen, appears to have a role in enhancing apoptosis induced by negative selection in over-expression studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available