http://bura.brunel.ac.uk/handle/2438/166 2013-05-23T19:13:36Z 2013-05-23T19:13:36Z Ojani, Maryam http://bura.brunel.ac.uk/handle/2438/7441 2013-05-20T15:11:26Z 2012-01-01T00:00:00Z

Title: Relationship between DNA damage response and telomere maintenance Authors: Ojani, Maryam Abstract: Telomeres are regions of repetitive DNA bound with a set of specialized proteins required to protect chromosomes from fusing with each other and from eliciting DNA damage response. Dysfunctional telomere maintenance can lead to premature cellular senescence, premature organismal aging and cancer predisposition. In the last few years the evidence has emerged indicating a link between dysfunctional maintenance of telomeres and defective DNA damage response. The objective of this project was to explore further this link by examining effects of some DNA damage response proteins on telomeres that have not been examined before and examining DNA damage response in cells in which telomeres are dysfunctional as a result of alterations in genes not directly involved in DNA damage response. We have developed a method, termed IQ-FISH, for accurate identification of average telomere length in interphase cells from individuals with defective DNA damage response. By applying IQ-FISH we could successfully measure telomere lengths in cell lines from patients that are heterozygous (+/-) and cell lines from patients or animals that are homozygous (-/-) with respect to mutations in these genes. We then analysed telomere length and function, as well as DNA damage response, in lymphoblastoid cell lines originating from BRCA1 and BRCA2 carriers (+/-) and also a single fibroblast cell line from a patient with bi-allelic mutations in BRCA2 (-/-). In addition we have analysed a mouse embryonic stem cell line in which Brca1 was deleted (Brca1-/-) by gene targeting. Our results show lack of correlation between DNA damage response and telomere maintenance in heterozygous cell lines (with the exception of one BRCA1+/- cell line) but a clear positive correlation in the case of cell lines with homozygous mutations. Finally, as a model for telomere dysfunction we have chosen cell lines from Dyskeratosis Congenita (DC) patients. DC is a rare progressive congenital disorder which results in premature aging. DC is primarily a disorder of dysfunctional telomere maintenance and we used cell lines from patients with mutations in DKC1, a gene encoding a protein termed Dyskerin which forms a part of the telomerase enzyme complex. Our results indicate that DC cells with dysfunctional DKC1 may have a dysfunctional DNA damage response. Description: This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.

2012-01-01T00:00:00Z Alshehri, Areej http://bura.brunel.ac.uk/handle/2438/7433 2013-05-10T14:26:41Z 2013-01-01T00:00:00Z

Title: The role of homeobox gene in leukaemia Authors: Alshehri, Areej Abstract: Homeobox genes are known to be active during development and they are turned off after the early stages of developmental life. The HLXB9/MNX1 gene is a homeobox gene localized on human chromosome 7 and is involved in the development of pancreas and the nervous system. However, some leukaemia research groups have reported an over-expression of HLXB9 in leukaemia patients who carry the t(7;12) and in the GDM-1 cell line that carries the t(6;7). The mechanisms of leukaemogenesis in t(7;12) patients are still unclear. The t(7;12) is one of the recurrent cytogenetic abnormalities that is associated with infant acute myeloid leukaemia (AML) patients and has been linked to poor prognosis. The aim of this study was (i) to determine the involvement of HLXB9 in cell lines known to express this gene at the transcript level and (ii) to investigate the position on HLXB9 in AML patients with abnormalities of chromosome 7. This aim was achieved through a series of experiments involving the use of both conventional and molecular cytogenetics. In the first place, the chromosomal abnormalities in leukaemia and lymphoma cell lines (GDM-1, K562 and Pfeiffer) have been analysed using G-banding and Multiplex FISH (M-FISH) techniques. Furthermore, FISH using whole chromosome painting technique was performed on 7 AML patients to investigate chromosome 7 rearrangements. Thirdly, the involvement of the homeobox gene HLXB9 has been investigated in the acute myeloid leukaemia (AML) derived cell line GDM-1 and in 4 AML patients. Fluorescence in situ hybridization (FISH) analysis was carried out using a specific probe for the HLXB9 gene on the AML patients in single and dual colour FISH in combination with an additional probe distal to HLXB9 on the GDM-1 cell line. FISH analysis showed no involvement of the HLXB9 gene in any rearrangement or breaks at chromosomal level on the AML cell line (GDM-1) and AML patients. Nevertheless, a breakpoint either proximal or distal to HLXB9 has been identified. In particular, the breakpoint in the GDM-1 cell line has been confirmed on between the two probes used. This thesis poses the basis for further studies to investigate the mechanisms of oncogenesis in leukaemias with over-expression of HLXB9 in relation to possible breakage of chromosome 7 in the vicinity of the gene Description: This thesis was submitted for the degree of Master of Philosophy and was awarded by Brunel University

2013-01-01T00:00:00Z Sandi, C Al-Mahdawi, S Pook, MA http://bura.brunel.ac.uk/handle/2438/7332 2013-04-03T12:39:50Z 2013-01-01T00:00:00Z

Title: Epigenetics in Friedreich's ataxia: Challenges and opportunities for therapy Authors: Sandi, C; Al-Mahdawi, S; Pook, MA Abstract: Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by homozygous expansion of a GAA·TTC trinucleotide repeat within the first intron of the FXN gene, leading to reduced FXN transcription and decreased levels of frataxin protein. Recent advances in FRDA research have revealed the presence of several epigenetic modifications that are either directly or indirectly involved in this FXN gene silencing. Although epigenetic marks may be inherited from one generation to the next, modifications of DNA and histones can be reversed, indicating that they are suitable targets for epigenetic-based therapy. Unlike other trinucleotide repeat disorders, such as Huntington disease, the large expansions of GAA·TTC repeats in FRDA do not produce a change in the frataxin amino acid sequence, but they produce reduced levels of normal frataxin. Therefore, transcriptional reactivation of the FXN gene provides a good therapeutic option. The present paper will initially focus on the epigenetic changes seen in FRDA patients and their role in the silencing of FXN gene and will be concluded by considering the potential epigenetic therapies. Description: Copyright © 2013 Chiranjeevi Sandi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2013-01-01T00:00:00Z Ulus-Senguloglu, G Arlett, CF Plowman, PN Parnell, J Patel, N Bourton, EC Parris, CN http://bura.brunel.ac.uk/handle/2438/7233 2013-02-27T11:07:06Z 2012-01-01T00:00:00Z

Title: Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis Authors: Ulus-Senguloglu, G; Arlett, CF; Plowman, PN; Parnell, J; Patel, N; Bourton, EC; Parris, CN Abstract: Background: The objective of this study was to determine the molecular mechanism(s) responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double strand break (DSB) repair in cells. Quantitative-PCR (Q-PCR) established the expression levels of key DNA DSB repair proteins. Imaging flow cytometry using Annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Over-expression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity. Description: Copyright @ 2012 Nature Publishing Group

2012-01-01T00:00:00Z