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Title: Analysis of the relationship between pluripotency and DNA damage tolerance recombination in mammalian stem cells
Author: Abduljaleel, Rafat Hamdi
ISNI:       0000 0004 5365 5158
Awarding Body: Prifysgol Bangor University
Current Institution: Bangor University
Date of Award: 2014
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Embryonic stem cells {ESCs} are isolated from the inner cell mass of the blastocyst. These cells are responsible for the development of the embryo proper. Accumulation of DNA damage at this stage of development would affect multiple / all lineages of the embryo which may poses danger to the health or survival of the organism and could be passed on through the germ line. One of the most threatening DNA lesions to the cell is DNA doublestrand break {DSB}. The repair of these DNA lesions in mouse ESCs {mESCs} is carried out mainly by homologous recombination {HR}. When using the correct DNA sequence as a template for DNA DSB repair, HR is of a high fidelity, otherwise it may lead to chromosomal aberrations. The low level of mutation rate measured in mESCs coupled with high rate of efficient HR during gene replacement / genome modulation experiments showed by these cells suggested that mESCs might use inter-sister recombination for repairing DNA DSBs in favour of the inter-homologue mitotic recombination to prevent loss of heterozygosity {LOH}. To test this hypothesis we designed a reporter cassette for measuring inert-sister chromatid exchange events in mESCs. Treated mESCs that carries a reporter cassette with the DNA replication perturbing agent aphidicolin and the differentiation of mESCs into neural progenitor cells revealed clonal heterogeneity among these cells. The measurement of GFP positive cells was hampered by the low intensity level of GFP. To overcome the problems of non-specific integration and the weak GFP signal, a system consisting of an improved version of the reporter cassette for stronger GFP signal, combined with the use of TALENs for better targeting has been designed and constructed. The repair of DNA DSBs or stalled DNA replication fork is often mediated by the formation of Holliday junctions {HJs}. The Bloom's syndrome protein {BLM} is a key player in choosing HR, in restarting stalled DNA replication forks and in dissolving HJs. The impacts of DNA replication perturbing drugs and differentiation on BLM levels were tested in mESCs and human NTERA2 cells - teratocarcinoma cells that have pluripotent properties and considered as the malignant counter parts of hESCs. Differentiated mESCs displayed a decrease in BLM levels compared to undifferentiated mESCs. Here we present evidence to suggest that BLM levels varies according to the lineage which the cells are differentiating and that BLM levels might be related to the survival of stem-like cells after DNA damage.
Supervisor: Wakeman, Jane ; Mcfarlane, Ramsay Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available