One of the causes of ageing is thought to be the accumulation of senescent cells. Since normal ageing is very complex, diseases with single gene mutations (progeroid syndromes) which show many features of normal ageing have been used as models in ageing research. Werner's syndrome is the progeroid syndrome which mimics most of the features of normal ageing. It is caused by a mutation of the WRN gene that encodes a RecQ helicase/exonuclease involved in DNA fork stabilization and repair during synthesis. Werner's syndrome fibroblasts have been shown to exit their replicative lifespan three to five times more rapidly than normal fibroblasts. The mechanisms of Werner's syndrome senescence have been extensively studied in fibroblasts. However, it is clear that accelerated replicative senescence is not a universal feature of the disease in all tissues. Thus, it is important to study the relationship between Werner's syndrome senescence in different tissue lineages. Donor to donor differences in replicative potential are also known to occur, thus it would be advantageous if replicative lifespan could be studied in cells with isogenic backgrounds. Accordingly, this thesis describes an attempt to knockdown the WRN gene III normal cell lines to produce WRN-1- isogenic cell lines. RNAi knockdown using shRNA and hammerhead ribozymes was carried out previously but did not demonstrate a reduction in protein levels. However, an increased sensitivity to camptothecin was demonstrated by Comet assay. Therefore, we expressed both the shRNA and the ribozyme in a single cell line in order to improve the knockdown. The work was initially carried out on cell lines to test whether the RNAi method was effective prior to applying it to irreplaceable primary cell strains. This would have helped elucidate whether premature senescence in Werner's syndrome fibroblasts is due to DNA replication fork stalling or due to telomere erosion. In normal individuals, keratinocyte senescence has been shown to occur by either telomere dependent or independent senescence depending on culture conditions. Thus, we have used keratinocytes from Werner's syndrome to explore the relationship between loss of WRN and replicative senescence in vitro. This was carried out using standard cytokinetic tests in the presence and absence of SB203580, a selective inhibitor of p38 MAP kinase which plays an important role in Werner's syndrome fibroblasts.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:590022 |
| Date | January 2012 |
| Creators | Ibrahim, Badr |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://research.brighton.ac.uk/en/studentTheses/e18d673c-7638-4686-b9cb-ed8f47f13605 |
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