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Moye, Aaron Lavel. "Understanding the relationship between telomeres, telomerase, and DNA G-quadruplexes". Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17713.
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Cancer cells elongate their telomeres - G-rich repetitive sequences found at the end of linear chromosomes, allowing limitless replicative potential in these cells. Approximately 85% of cancers use telomerase to extend telomeres, making it an attractive potential anti-cancer target. The G-rich nature of telomeres allows the formation of DNA G-quadruplex secondary structures. Previous data had demonstrated that telomeric G-quadruplex substrates could not be extended by ciliate telomerase (Zahler et al., 1991). However, while the above observation is true for anti-parallel G-quadruplexes, parallel G-quadruplexes were shown to be substrates for ciliate telomerase (Oganesian et al., 2006). Whether human telomerase could extend parallel G-quadruplexes was unknown. In this thesis, I confirmed that human telomerase, like ciliate telomerase, can extend parallel, intermolecular G-quadruplexes in vitro. The ability of telomerase to extend G-quadruplexes is also true for parallel, intramolecular G-quadruplexes, indicating that the parallel nature of the structure allows telomerase extension. Extension of parallel G-quadruplexes using both biochemical and single-molecule FRET microscopy revealed that parallel G-quadruplexes are bound by telomerase as a distinct substrate and partially unfolded, allowing hybridisation of the RNA template. This partially unwound G-quadruplex is extended by human telomerase to the hTR template boundary, followed by translocation and complete G-quadruplex unfolding. Stabilisation of the parallel G-quadruplex using a parallel-G-quadruplex-specific ligand NMM did not inhibit telomerase activity demonstrating that chemically-stabilised parallel G-quadruplexes can be extended by human telomerase. Using a G-quadruplex specific antibody I showed that G-quadruplexes at telomeres increased after NMM treatment, indicating that parallel G-quadruplexes exist at human telomeres in vivo, and that telomeres with G-quadruplexes are a site of localisation for human telomerase. A potential protective effect of Gquadruplexes at uncapped telomeres was also investigated. In Saccharomyces cerevisiae lacking cdc13, equivalent in function to mammalian POT1, the DNA damage response could be suppressed by stabilising Gquadruplexes, showing that G-quadruplexes can have a protective effect at uncapped telomeres, but whether this is true at mammalian telomeres was unknown. In chapter 3 of this thesis I demonstrated that the DNA damage response at uncapped telomeres was suppressed by G-quadruplex stabilising ligands in G1 cells. I showed that G-quadruplex-telomere colocalisation increase in the absence of POT1, consistent with in vitro FRET experiments (Hwang et al., 2012). Treatment of POT1-deficient telomeres in G1 with G-quadruplex stabilising ligands reduced G-quadruplex-telomeres colocalisation. I provide preliminary data indicating that the nucleotide excision repair pathway is responsible for this phenotype, and that loss of stabilised telomeric G-quadruplexes is linked to the DNA damage response suppression phenotype. This thesis provides a body of work that improves our understanding of the role of G-quadruplexes at telomeres.
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Kamnert, Iréne. "Classes of DNA associated with telomeres in the chironomids C. pallidivittatus and C. tentans". Lund : Dept. of Genetics, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39009480.html.
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Xing, Xuekun. "DNA replication and telomere resolution in vaccinia virus". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq23557.pdf.
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Ojani, Maryam. "Relationship between DNA damage response and telomere maintenance". Thesis, Brunel University, 2012. http://bura.brunel.ac.uk/handle/2438/7441.
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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.
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Carlos, A. R. "DNA damage responses to loss of telomere integrity". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:27bcf3b6-edb9-47e2-af7c-c7ba9b431572.
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Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.
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Cabuy, Erik. "Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours". Thesis, Brunel University, 2005. http://bura.brunel.ac.uk/handle/2438/5157.
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Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.
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Xu, Mengyuan. "The Role of Shelterin Proteins in Telomere DNA Protection and Regulation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1585760345643995.
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Brown, Karen E. "Telomere-directed breakage of the human Y chromosome". Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260731.
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Denham, Elizabeth. "The Effects of Relocating the Ku-binding Stem-loop of Telomerase RNA on Telomere Healing Events". Thesis, Boston College, 2008. http://hdl.handle.net/2345/528.
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Thesis advisor: Anne E. StellwagenThesis advisor: Clare O'Connor
In most eukaryotes, the enzyme telomerase adds telomeric DNA repeats to the 3' ends of chromosomes in order to stabilize them and protect them from degradation. In the budding yeast Saccharomyces cerevisiae, telomerase is a ribonucleoprotein complex consisting of multiple protein subunits and an approximately 1.3 kb RNA component termed TLC1. Among the various proteins involved in telomerase, Ku is a heterodimer that binds both to double-stranded DNA and to a 48 nucleotide stem loop on the TLC1 RNA. Beyond its function of extending telomeres at the ends of chromosomes, telomerase can also be instrumental in repairing double-stranded DNA breaks (DSBs) by adding telomeric repeats at the site of the break. This stabilizes the damaged chromosome, but also silences genes proximal to the break. Ku is an important factor in the recruitment of telomerase to these double stranded breaks, so this investigation explored whether TLC1 structural variants with relocated Ku-binding sites are still capable of healing chromosomes via the addition of telomeres. It was determined that the TLC1 RNA is flexible and can retain its function with relocated and additional Ku-binding stem loops
Thesis (BS) — Boston College, 2008
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program
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Tuntiwechapikul, Wirote. "Studies of a G-quadruplex-specific cleaving reagent, expansion of long repetitive DNA sequences, and a cytosine-specific alkylating aza-enediyne /". Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3055255.
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Yasaei, Hemad. "Analysis of telomere maintenance in artemis defective human cell lines". Thesis, Brunel University, 2009. http://bura.brunel.ac.uk/handle/2438/4406.
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Telomeres are physical ends of chromosomes consisting of (TTAGGG)n DNA sequence and a specialized set of proteins that protect chromosomal ends from degradation and from eliciting DNA damage response. These specialized set of proteins, known as shelterin, directly bind to telomeric DNA. In addition, some DNA double-strand break (DSB) repair proteins such as, DNA-PKcs and KU70/80, play active roles in telomere maintenance. Mouse knock-out experiments have revealed that deletion of either DNA-PKcs or Ku70/80 resulted in elevated levels of telomeric fusion, indicative of dysfunctional telomeres. Artemis protein is involved in DNA DSB repair through non-homologous end joining (NHEJ) and it is phosphorylated by DNAPKcs. Human cells defective in Artemis have been identified and shown to be radiosensitive and patients with an Artemis defective gene suffer from radiosensitive severe-combined immune deficiency syndrome (RS-SCID). Mouse cells defective in Artemis have elevated levels of telomeric fusion. We have demonstrated in this thesis that Artemis defective human cell lines show a mild telomeric dysfunction phenotype detectable at the cytological level. The nature of telomere dysfunction phenotype appears to be similar to that observed in DNAPKcs defective cells as exemplified by the presence of IR induced chromatid telomeric fusions. We have also shown that (a) DNA damage occurring within the telomeric DNA is difficult to repair or irreparable in older cells and that (b) Artemis defective older cells show higher proportion of DNA damage at telomeres than their normal counterparts. Finally, we have demonstrated that inhibition of DNA-PKcs causes (a) an increase in telomeric fusions in Artemis defective cell lines relative to both normal cell lines after inhibition and Artemis cell lines before inhibition and (b)elevated levels of DNA damage at telomeres following exposure of cells to radiation relative to both irradiated normal cells exposed to a DNA-PKcs inhibitor and irradiated Artemis defective cells but not exposed to the DNA-PKcs inhibitor. These results suggest that the effects of Artemis and DNA-PKcs on telomeres are cumulative. We have also performed (a) experiments to examine telomere function in Artemis defective cell lines after knocking down DNA-PKcs levels by RNAi and b) preliminary experiments to knock-down Artemis in DNA-PKcs defective cells. Taken together, our results suggest that the Artemis defect causes mild telomere dysfunction phenotype in human cells.
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Ebrahimi, Hani. "The effect of DNA replication on telomere positioning in S. cerevisiae". Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=26454.
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Castillejo-López, Casimiro. "Repetitive DNA in search of a function a study of telomeric and centromeric sequences in Chironomus /". Lund : Lund University, 1998. http://catalog.hathitrust.org/api/volumes/oclc/68945096.html.
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Ballal, Rahul. "BRCA1 localization to the telomere and its loss upon DNA damage". Connect to Electronic Thesis (CONTENTdm), 2010. http://worldcat.org/oclc/642695417/viewonline.
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Denham, Joshua. "Telomere, DNA Methylation and Gene Expression changes caused by exercise training". Thesis, Federation University Australia, 2016. http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/99858.
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Exercise training is one of the few therapeutic interventions that improves health span by delaying the onset of age-related diseases and preventing early death. Despite the clear benefits to health conferred by exercise training, our understanding of the underlying molecular mechanisms remain crude. The primary purpose of this thesis is to determine and analyse the molecular biology changes that occur with strenuous aerobic exercise. Specifically, the main objectives were to investigate the impact of strenuous aerobic exercise training on structural DNA modifications, measured in context with cardiovascular health and fitness adaptations. In the first part of this thesis I investigated the influence of endurance exercise training on leukocyte telomere length and cardiovascular health. Leukocyte telomere length reflects biological age. Indeed, excessively short leukocyte telomeres are associated with age-related chronic diseases. Epidemiological studies indicate endurance athletes live longer than people from the general public who do not engage in extensive aerobic exercise training. In Chapter 2, my literature review on the subject of exercise and telomere biology suggested that, at the time of this study, the impact of exercise training on leukocyte telomere length was equivocal. Therefore, to determine whether strenuous aerobic exercise training influences biological ageing (assessed by leukocyte telomere length), I conducted two cross-sectional studies on leukocyte telomere length differences between endurance athletes and healthy controls. The first study (Chapter 3) was a cross-sectional analysis of leukocyte telomere length between athletes and controls, determined by quantitative polymerase chain reaction (qPCR). This is a relative measurement of telomere length expressed as a telomere (T) to single copy gene (S) ratio. Relative to the healthy controls (n = 56), the ultra-marathon runners (n = 67) possessed 11% longer leukocyte telomeres in age-adjusted analysis (ultra-marathon runners vs controls; average T/S ratio: 3.56 vs 3.16, p = 1.4 × 10-4) and the difference was not explained by the favourable cardiovascular health profile exhibited by the athletes (p = 2.2 × 10-4). The difference in leukocyte telomere length indicated the athletes had reduced their biological age by 16.2 years. To elucidate the potential mechanism for the longer leukocyte telomeres observed in endurance athletes, I recruited another cohort of athletes and controls and measured leukocyte telomere length and gene expression of genes involved in telomere length regulation. In the second study (Chapter 4), I describe data replicating the finding that endurance athletes possess longer leukocyte telomeres compared to healthy controls (athletes v controls mean T/S ratio ± SE: 3.64 ± 0.06 vs 3.38 ± 0.06, p = 0.002). This difference was associated with a concomitant increased activity of two important telomere regulating genes, telomerase reverse transcriptase (TERT) and adrenocortical dysplasia homolog (TPP1) (2- fold and 1.3-fold, respectively, both p < 0.05). The difference in leukocyte telomere length and leukocyte telomere-regulating gene (TERT and TPP1 mRNA) expression was ameliorated after adjusting for maximal oxygen uptake and resting heart rate (all p > 0.05). This finding indicates that cardiorespiratory fitness is an important determinant of telomere biology. Together, these two cross-sectional studies suggest that regular endurance exercise training is associated with longer leukocytes telomeres and that this is likely achieved through higher TPP1 and TERT mRNA expression gained through improved cardiorespiratory fitness. The findings in Chapters 3 and 4 provide evidence for extensive endurance exercise training as an effective lifestyle strategy to attenuate biological ageing. In parallel to telomere length changes, epigenetic modifications (e.g. DNA methylation) caused by environmental factors alter the transcriptomic milieu of cells. My thorough literature review (Chapter 5) revealed that exercise training seems to rearrange chromatin by modifying the DNA methylome in a variety of cells and that the extent is dictated by exercise duration and intensity. Therefore, in the second part of my thesis, I investigated the DNA methylation changes in leukocytes (which are somatic cells) and sperm (male germ cells) from healthy men before and after sprint interval training (SIT). Unlike traditional, long duration training at moderate intensity training, SIT involves short, intense (>85% VO2max to supra-maximal) efforts followed by periods of rest (3–4 min), typically repeated 3–8 times. It is an effective type of training that improves cardiorespiratory fitness quicker than traditional long slow distance training. Thus, to establish the DNA methylome changes associated with SIT, I conducted two training studies and analysed the leukocyte and sperm methylomes using the Infinium HumanMethylation450 BeadChip (Illumina). My third study (Chapter 6) provides the first evidence showing an association between DNA methylation changes paralleled with improvements to lipid profile and cardiorespiratory fitness in humans. Twelve young men (18–24 years) undertook SIT (thrice weekly) for four weeks. Resting blood samples were obtained and whole-blood leukocytes were isolated by red blood cell lysis. Genome-wide DNA methylation was assessed using the 450K BeadChip (Illumina). Cardiorespiratory fitness, determined by maximal oxygen uptake, was improved by 2.1 ml.kg-1.min-1 and low-density lipo-protein cholesterol was decreased by 3.9% after SIT (p < 0.05). Notably, the leukocyte methylome was significantly affected by SIT, in regions throughout the genome in relation to CpG islands – CpG islands, North shores, N shelves, South shores and South shelve – and the nearest genes – 3’ untranslated region (UTR), 5’ UTR, exonic, intergenic, intronic, non-coding and promoter regions (all p < 0.001). Genes with differentially methylated CpG sites (q < 0.005) after SIT were enriched for cardiovascular gene ontology (GO) terms that included metabolic activity, biological adhesion and antioxidant activity. Similarly, pathway analysis revealed genes involved in focal adhesion, calcium signaling and mitogen activated protein kinase were modulated by SIT-induced DNA methylation changes. Amongst the 205,987 probes relating 32,445 transcripts differentially methylated after SIT (q < 0.05), with methylation changes between 0.1 – 62.8%, the largest and most statistically significant demethylated site was in the epidermal growth factor (EGF) gene, causing decreased mRNA expression. As with EGF, the microRNA-21 and microRNA-210 genes (MIR21 and MIR210, respectively), known for their roles in cardiovascular disease (ischemic heart disease and coronary atherosclerosis), had modest but consistently statistically significant DNA methylation changes at numerous CpG sites, which altered mature microRNA abundance. Together, these data suggest that genome-wide DNA methylation changes occur after short-term intense exercise training concurrently with improvements to blood cholesterol profile and cardiorespiratory fitness. The data presented in this thesis provided evidence that the epigenome of somatic cells is malleable to exercise. There is mounting evidence supporting the premise that environmental perturbations cause DNA methylation changes and these are subsequently transgenerationally inherited, altering phenotypes of future generations. In the current study I also asked the question; can exercise training reconfigure the DNA methylome of male germ cells (sperm)? Therefore, my next study (Chapter 7) entails an analysis of the impact that three months of SIT has on genome-wide DNA methylation of sperm in healthy men. Thirteen subjects undertook twice-weekly SIT for three months, while the controls were asked not to change their current physical activity habits (if any). Sperm samples were donated before and after the three-month intervention. Mature sperm were isolated using density gradient centrifugation and DNA was extracted using the Purelink Genomic DNA Mini Kit (Life Technologies). Global and genome-wide DNA methylation was assessed using an enzyme-linked immunosorbent assay-based kit and the 450K BeadChip (Illumina), respectively. Relative to controls, the cases decreased their resting heart rate and had a higher maximal treadmill speed during exercise testing (both p < 0.05). Cases had decreased global DNA methylation after SIT compared to controls (p < 0.05). Genome-wide DNA methylation analysis revealed numerous modest (0.3 – 6%) methylation changes to 7509 CpG sites, relating to 4602 transcripts (q ≤ 0.1). Differentially methylated CpG sites were in genes associated with developmental biology, which included GO terms, such as developmental process, anatomical structure, embryonic morphogenesis and organ development, together with known pathways regulated by exercise training (MAPK, ErbB and PI3K-Akt signalling). Genes with increased methylation were associated with numerous human diseases, with most overrepresented being psychiatric disorders (schizophrenia, Parkinson’s disease and autism). Notably, paternally imprinted genes associated with other diseases were also differentially methylated after SIT. Therefore, exercise training is associated with the modifications to genome-wide DNA methylation of both somatic and germ cells. In conclusion, the studies presented as a series of peer-reviewed publications, outlines investigations that describe an influence of strenuous exercise training on leukocyte telomere length regulation and the DNA methylome of both leukocytes and germ cells. Both of these molecular changes in leukocytes and sperm provide evidence for novel molecular mechanisms by which exercise improves cardiovascular health and fitness. Future investigations should focus on longitudinal studies determining whether these changes are required for improved health and fitness, and should establish whether exercise-induced DNA methylation changes are transgenerationally inherited, and if so, what impact this has to future generations. Such discoveries could change national physical activity guidelines and policies, by emphasising the benefit of regular exercise both in the present and to future offspring.Doctor of Philosophy
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Oikemus, Sarah R. "Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways: A Dissertation". eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/229.
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Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion.
Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion.
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Pedroso, Ilene Marie. "The Outer Limits: Telomere Maintenance by TRF2 and G-Quadruplex DNA Structures". Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/24.
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Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3' assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. We show that K+ and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells. The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, a protein essential for telomere maintenance, is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3'-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5'-d(TTAGGG)4-3', to 5'-dTTAGGG(TTAGAG)2TTAGGG-3', reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexes. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2 G-quadruplex interactions, suggesting a novel mechanism for TRF2 recognition of the ds/ss junction of telomeres.
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Martinez, Alaina R. "Variant requirements for DNA repair proteins in cancer cell lines that use alternative lengthening of telomere mechanisms of elongation". The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1479924417740462.
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Méndez-Bermúdez, Aarón. "Telomere stability in cancer cells and the role of DNA mismatch repair". Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/30378.
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Human telomeres are essential for maintaining chromosome stability and are composed of TTAGGG repeat arrays with interspersed variant repeats at the proximal end. This work has focused on understanding the role of DNA mismatch repair on telomere stability by mapping the interspersion pattern of variant telomere repeats in search of mutations. Telomere instability was analysed in clones of mismatch repair (MMR) defective cell lines and colorectal carcinomas with MSH2 mutations. The combined mutation frequency found in four MMR-defective cell lines (3.6% per telomere per genome) and two colorectal tumours lacking the MSH2 gene (1.2% per telomere per genome) was significantly different compared to MMR proficient cell lines. The mutations resulted in losses, gains and telomere repeat-type changes suggesting that mutations originated by intra-allelic mechanisms. In addition, downregulation of the MSH2 gene was carried out in a normal fibroblast cell line using RNA interference. Downregulation of MSH2 caused microsatellite and telomere instability with a possible effect on telomere length. These results indicate that the MMR pathway is involved in maintaining the stability of telomeres. One variant telomere repeat found in humans is the CTAGGG repeat. When multiple tandem copies of CTAGGG are present in a telomere, it is highly unstable in the male germ-line with a mutation rate as high as 0.83 per sperm. In this work, the analysis of instability of CTAGGG-containing telomeres was extended in the germ-line and conducted in somatic tissue. The results indicated that the presence of more than five CTAGGG repeats cause instability in that telomere, with a mutation frequency of 3.8% per telomere per genome in somatic cells. Finally, the basis of the telomere maintenance mechanism (TMM) was investigated in a panel of liposarcomas where around 50% are reported to lack a TMM. By mapping telomeres from liposarcomas with unknown TMM, it was possible to identify complex mutations that arose, most probably, by recombination-like processes similar to that seen in the alternative lengthening of telomeres pathway (ALT). However, these tumours lack some of the markers for ALT such as ALT-associated PML bodies.
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Roberts, Jennifer Nicole. "Investigating the DNA binding properties of the Telomere End-Binding Protein Cdc13". Connect to online resource, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3337145.
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Smiraldo, Phillip G. "The Rad51d DNA Repair Gene is Required for Chromosome and Telomore Stability in Mammalian Cells". University of Toledo Health Science Campus / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=mco1146675938.
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Langston, Rachel Elizabeth, i Rachel Elizabeth Langston. "DNA Replication Defects in the Telomere Induce Chromosome Instability in a Single Cell Cycle". Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/622910.
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Errors in DNA replication can cause chromosome instability and gross chromosomal rearrangements (GCRs). For my thesis work I investigate how chromosome instability can originate in the telomere. Here I report how defects in Cdc13, a telomere specific protein, lead to chromosome instability and GCRs in Saccharomyces cerevisiae. Using a temperature sensitive mutant of Cdc13, I find that cdc13-induced instability can be induced in a single cell cycle and synergizes with replication stress (dNTP depletion via hydroxyurea). Additionally, I find that Cdc13 has to be functional during the cell’s S phase to suppress chromosome instability. Further genetic analysis suggests that that cdc13-induced chromosome instability depends on the generation of single stranded (ss)DNA, but not on the activity of canonical double strand break (DSB) repair pathways such as homologous recombination or non-homologous end joining. Finally, I demonstrate that telomeric unstable chromosomes can later progress and trigger rearrangements at centromeric loci. This system, using the conditional nature of the cdc13 mutation, promises a more complex analysis of the ontogeny of chromosome instability: in this case from errors semi-conservative DNA replication through the telomere to the formation and resolution of unstable chromosomes.
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Santos, Gabriel Arantes Galvão Dias dos. "Caracterização molecular da atividade de interação da proteína RPA-1 com os telômeros de Leishmania spp". Universidade Estadual Paulista (UNESP), 2018. http://hdl.handle.net/11449/153962.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Entre as espécies do gênero Leishmania estão os protozoários que causam leishmaniose, uma doença tropical negligenciada endêmica em muitos países, incluindo o Brasil. Métodos de controle e tratamento ainda são ineficientes e a resistência a drogas é um desafio. Por isso, pesquisas para entender melhor a biologia molecular desses parasitos são encorajadas. Uma possível estratégia para isso, é o estudo dos telômeros, estrutura fundamental para a homeostase do genoma. Os telômeros são estruturalmente diferentes do resto do cromossomo, e contam com proteínas específicas que realizam sua manutenção. A Replication Protein A subunit 1 (RPA-1) é uma proteína que interage de DNA de simples fita que tem diversas funções relacionadas com o metabolismo do DNA eucarioto, incluindo os telômeros. A RPA-1 é parte de um complexo heterotrimérico conservado nos eucariotos, incluindo Leishmania spp.. Recentemente nós mostramos por modelagem molecular que a estrutura terciária da LaRPA-1 difere dos seus ortólogos em humanos e leveduras, além de mostrar interações específicas nos telômeros dos parasitos, que na ausência de homólogos canônicos para telomere-end binding protein (TEP) elegem a LaRPA-1 como um potencial candidato para essa função. Neste trabalho, avaliamos a capacidade da LaRPA-1 como uma TEP, cujo papel principal é proteger a extremidade 3' dos telômeros de ataques por exonucleases. Uma busca estrutural por proteínas que compartilham com as TEP domínios de interação proteína-DNA, mostrou que no genoma de Leishmania spp. não existem homólogos estruturais para as mesmas. Aqui mostramos por diferentes abordagens que a LaRPA-1 tem a capacidade de interagir com no mínimo uma repetição telomérica e também é capaz de proteger in vitro a simples fita telomérica rica em G (5’ TTAGGG 3’) da digestão por Exonuclease I bacteriana cuja atividade é no sentido 3’-5’. Somando esses dados, com dados anteriores que mostram que a LaRPA-1 tem preferência pela fita telomérica rica em G e o fato dela ter sido co-purificada com a atividade de telomerase sugerem fortemente que ela está diretamente relacionada com a manutenção da maquinaria telomérica, podendo inclusive ser considerada a principal ligante de simples fita telomérica rica em G (3’ G-overhang) em Leishmania spp.
Among the protozoa parasites of the Leishmania genus are the causative agents of leishmaniasis, a neglected tropical disease endemic in many countries, including Brazil. Disease control and treatment are still inefficient and parasite drug resistance is a challenge. Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged. One possible strategy is to study parasite telomeres, a vital chromosome structure important to maintain genome homeostasis. Telomeres are significantly different from the rest of the chromosome and are associated with proteins involved in their maintenance. Replication Protein A subunit 1 (RPA1), a single-stranded DNA-binding protein that plays multiple roles in eukaryotic DNA metabolism, including telomeres, is part of a conserved heterotrimeric complex which is present in most eukaryotes including Leishmania spp. Recently, using molecular dynamics simulations we have shown that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1 and that it also shows parasitespecific interactions with telomeric DNA. In the absence of real homologues to telomere-end binding proteins, LaRPA-1 could be considered a potential candidate. If LaRPA-1 is a telomere-end binding protein, one of its main role would be to protect the telomeric 3`-end termini from nuclease attack. A structural search for proteins that share with the TEP domains of protein-DNA interaction, showed that in the genome of Leishmania spp. there are no structural homologues for them. In this work, we show by different methods, that in vitro LaRPA-1 can bind at least one telomeric repeat and it can also protect the telomeric G-rich sequence (5’ TTAGGG 3’) from the bacterial 3’-5’Exonuclease I digestion. These data compiled to previous data showing that LaRPA-1 preferentially binds the G-rich telomeric DNA and that it co-purifies with telomerase activity strongly suggest that LaRPA-1 is directly involved with parasite telomere maintenance and, possibly, is the main G-rich single-stranded (3’ G-overhang) telomere-binding protein in Leishmania spp.
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AGUADO, PEREZ JULIO. "THE ROLE OF TELOMERIC RNA AT DYSFUNCTIONAL TELOMERES AND ITS IMPACT ON SENESCENCE AND AGING". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/556299.
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A novel class of small non-coding RNAs discovered in our laboratory, termed DNA damage response RNAs (DDRNAs), has been demonstrated to be generated upon DNA double strand break (DSB) induction, and to be necessary for full DNA Damage Response (DDR) activation. DDRNAs are generated following DSB induction upon transcription of the damaged locus and the synthesis of an RNA precursor further processed by the endoribonucleases DICER and DROSHA.
The aim of this PhD dissertation was to investigate the mechanism underlying DDRNA-dependent DDR activation specifically at telomeres, important chromosomal regions required for genomic stability that, if disrupted, are associated with aging-related diseases. In this dissertation, I show that telomere dysfunction, like DSBs, induces the transcription of telomeric DDRNAs and their precursors from both DNA strands of the telomere. Such transcripts are necessary for DDR activation and maintenance at dysfunctional telomeres. Most importantly, the use of sequence-specific antisense oligonucleotides (ASOs) allows the inhibition of telomere transcripts’ functions, thereby specifically inhibiting telomeric DDR.
Telomere dysfunction is rising as a key feature in Hutchinson–Gilford Progeria Syndrome (HGPS) and other premature aging syndromes. Here I show that progerin, the protein whose expression causes HGPS, induces the transcription of telomere transcripts, both in vitro and in vivo. Furthermore, signaling inhibition of progerin-driven telomere dysfunction improves the growth potential of progerin-expressing cells. Finally, this inhibition also increases the lifespan of an HGPS mouse model, opening the possibility for the use of this approach as a viable therapy to treat HGPS.
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CESENA, DANIELE. "The RNA processing proteins Xrn1 and Rrp6 regulate DNA damage checkpoint activation and telomere metabolism". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158272.
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L’instabilità genomica è una delle caratteristiche principali delle cellule tumorali e può essere causata da difetti nella riparazione del DNA, dal mancato arresto del ciclo cellulare e dalla perdita della protezione telomerica all’estremità dei cromosomi, che porta alla degradazione e alla fusione delle estremità. Tra i vari tipi di danno al DNA, le rotture della doppia elica del DNA (Double-Strand Break o DSB) rappresentano una delle lesioni più pericolose, poiché possono causare mutazioni o riarrangiamenti cromosomici. In presenza di DSBs, le cellule eucariotiche attivano un checkpoint, dipendente dalle protein chinasi Tel1/ATM e Mec1/ATR, che arresta il ciclo cellulare finché il danno non è stato riparato. Mec1/ATR è attivata dal DNA a singolo filamento (ssDNA) ricoperto da RPA che si forma dopo il processamento nucleolitico (resection) delle estremità del DSB.
Una simile risposta è attivata anche quando le estremità naturali dei cromosomi eucariotici perdono la loro protezione, generando delle estremità simili ad un DSB che vengono riconosciute dal checkpoint e dai meccanismi di riparazione. Questa protezione è fornita da complessi nucleoproteici specializzati, chiamati telomeri. Il DNA telomerico è costituito da sequenze ripetute ricche in G che terminano con una coda a singolo filamento sporgente in 3’ (detta coda G), la quale è importante per l’estensione dei telomeri ad opera della telomerasi. Diverse proteine, tra cui il complesso CST, sono necessarie al mantenimento della struttura e della lunghezza dei telomeri sia in lievito che nei mammiferi.
Recenti dati sperimentali indicano che i fattori che processano l’RNA hanno un ruolo fondamentale nella stabilità del genoma e nel metabolismo telomerico, anche se il meccanismo è ancora poco compreso. In questa tesi abbiamo dimostrato che in Saccharomyces cerevisiae le proteine che degradano l’RNA Xrn1, Rrp6 e Trf4 promuovono l’attivazione di Mec1/ATR facilitando la formazione di DNA a singolo filamento ricoperto da RPA ai DSB. Inoltre, Xrn1 e Rrp6 sono necessarie per attivare il checkpoint anche ai telomeri deprotetti a causa del malfunzionamento di Cdc13, una delle subunità del complesso CST coinvolto nella protezione dei telomeri. Xrn1 facilita la formazione di DNA a singolo filamento ai DSBs promuovendo il caricamento del complesso MRX, mentre come svolga questa funzione ai telomeri deprotetti rimane ancora da chiarire. Al contrario, la generazione di ssDNA ai DSBs non è influenzata dalla mancanza di Rrp6 o Trf4, ma la loro assenza ostacola il reclutamento di RPA e quindi di Mec1 al sito di danno. L’inattivazione di Rrp6 e Trf4 non influenza né l’associazione di Rad51/Rad52 ai DSB né la riparazione della rottura attraverso la ricombinazione omologa (Homologous Recombination o HR), suggerendo che la piena attivazione di Mec1 richieda più DNA a singolo filamento ricoperto da RPA di quanto ne sia richiesto per la riparazione attraverso la ricombinazione omologa. Infine, Xrn1, regolando negativamente il trascritto di RIF1, è coinvolto nel mantenimento della lunghezza dei telomeri promuovendo l’associazione di Cdc13 indipendentemente dalla formazione di DNA a singolo filamento.
In conclusione, i nostri risultati forniscono un nuovo collegamento tra il processamento dell’RNA e il mantenimento della stabilità del genoma.Genome instability is one of the most pervasive characteristics of cancer cells. It can be due to DNA repair defects, failure to arrest the cell cycle and loss of telomere-end protection that lead to end-to-end fusion and degradation. Among the many types of DNA damage, the DNA Double Strand Break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Eukaryotic cells respond to DSBs by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR, in order to arrest the cell cycle until DSBs are repaired. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA) that arises upon nucleolytic degradation (resection) of the DSB. A similar checkpoint response is triggered when the natural ends of eukaryotic chromosomes lose their protection, resembling and being recognized as DSBs. This protection is provided by specialized nucleoprotein complexes called telomeres. Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3’-ended single-stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidences indicate that RNA processing proteins play critical, yet poorly understood, roles in genomic stability and telomere metabolism. We provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate activation of Mec1/ATR by promoting the generation of RPA-coated ssDNA at intrachromosomal DSBs. Xrn1 and Rrp6 are also required to activate a Mec1/ATR-dependent checkpoint at uncapped telomeres due to loss of the CST component Cdc13. Xrn1 promotes checkpoint activation by facilitating the generation of ssDNA at both DSBs and uncapped telomeres. Xrn1 exerts this function at DSBs by promoting the loading of the MRX complex, whereas how it does at uncapped telomeres remains to be determined. By contrast, DSB resection is not affected by the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Finally, we demonstrate that Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the RIF1 transcript. Our results provide novel links between RNA processing and genome stability.
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Gozaly, Chianea Yaghoub. "Investigation of telomere maintenance in BRCA2 defective mammalian cell lines". Thesis, Brunel University, 2014. http://bura.brunel.ac.uk/handle/2438/8724.
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BRCA2 is a highly penetrant breast cancer predisposing gene. The protein product of the BRCA2 gene mediates repair of breaks in DNA, through Homologous Recombination (HR). Understanding the mechanism(s) behind BRCA2 involvement in HR will help clarify its clinical importance and may pave the way for possible therapy. In this work we show that BRCA2 affects telomere maintenance in mammalian cells. Telomeres are physical ends of chromosomes implicated in cell senescence and carcinogenesis. In particular, the enzyme telomerase that synthesizes telomeric DNA is highly active in ~90% cancers and it is considered one of the cancer markers. The remaining 10% of cancers do not show telomerase activity and they maintain their telomeres by an alternative pathway known as Alternative Lengthening of Telomeres (ALT). We observed telomere shortening, loss of telomere function in the form of end chromosome fusions and increased incidence of Telomere Sister Chromatid Exchanges (T-SCE), one of the recognized markers of ALT, in 3 sets of Chinese hamster and human BRCA2 defective cell lines, all of which maintained telomeres by conventional mechanisms. We have also inhibited BRCA2 expression in ALT positive cells by transfecting them with si (short interfering) RNA oligonucleotides specific for BRCA2 and monitored its expression by Real Time-PCR and Western blot. Results indicate that BRCA2 knock-down in ALT positive human cells that causes reduction in T-SCE frequencies, thus suggesting that ALT cells and those that maintain telomeres by conventional mechanisms differ in this respect. One interesting scenario that emerges from these results is that BRCA2 deficiency could potentially suppress the ALT pathway. We wanted to explore this possibility further by creating a permanent BRCA2 knock-down. Our preliminary results suggest that our method for the permanent BRCA2 knock-down based on the SMARTvector 2.0 system and sh (short hairpin) iv RNA approach is still not working effectively. We identified hyper-methylation of the promoter within the vector as a possible cause. Finally, we examined repair kinetics of interstitial telomeric sites (ITSs) in BRCA2 deficient Chinese hamster cells in order to test the hypothesis that defective DNA double strand break repair may be responsible for their increased sensitivity to DNA damaging agents. Our results indicate that DNA damage within ITSs is repaired effectively thus disproving the above hypothesis. In conclusion, this work demonstrates the involvement of BRCA2 in telomere maintenance.
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Wainwright, Linda Jane. "Studies of mean telomere length in human skin : changes with age and in malignancy". Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260056.
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Khan, Sheik Jamaludin. "Functions of TRF2: From Telomere Protection to DNA Damage Signaling and Vascular Remodeling". Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/123.
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TTAGGG repeat factor 2 (TRF2) is a protein that plays an important role in capping telomere ends from DNA damage responses. Telomere DNA consists of double strand repeats of the TTAGGG sequence ending with a 3'single-stranded overhang of the guanine strand (the G-strand overhang). TRF2 protects telomeres from being recognized as double-stranded breaks. It is thought that this protection is performed through the formation of T-loop structures and recruitment of proteins into a complex called shelterin. The exact mechanism of T-loop formation is unknown. I show with in vitro biochemical studies that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the G-strand overhang and double-stranded DNA sequence at the junction. Binding assays with TRF2 truncation mutants suggest that TRF2 interacts with both the double-stranded DNA through the C-terminal DNA binding domain and the G-strand overhang through the N-terminus. Mobility shifts and atomic force microscopy with truncation mutants bound to telomeric DNA also show that a previously uncharacterized "linker" region within TRF2 is involved in DNA-specific TRF2 oligomerization. From these observations, I suggest that TRF2 forms protective loops by oligomerizing through both a previously characterized dimerization domain and the linker region. I propose that loop formation involving the telomere ends is accomplished through direct interactions between TRF2 and the G-strand overhang. In addition to DNA protection, a new role has emerged for TRF2 in sensing DNA damage. TRF2 can be phosphorylated within its dimerization domain by ATM and recruited to DNA damage foci in cells. The inhibition of TRF2 function alone has been shown to induce senescence and apoptosis in vascular endothelial cells. Since the common stimuli for a senescence phenotype is activation of a DNA damage response, I studied the relationship between DNA damage and TRF2 phosphorylation. Ex-vivo characterization of DNA damage-induced changes in vascular smooth muscle cells (VSMC) was undertaken. VSMC treated with H202 induced an increase in reactive oxygen species (ROS), and 8-oxo-guanine accumulation resulting in cell cycle arrest, chromatin condensation and a senescent phenotype. Interestingly phosphorylated TRF2 and ATM were also up regulated. Balloon injury was used to test the connection between phosphorylated TRF2 and senescence during vascular remodeling in rat arteries. Vascular remodeling as judged by neointima formation was associated with accumulation of 8-oxo-guanine, DNA damage signaling, including phosphorylated TRF2, an increase in cell cycle inhibitors and senescence. These events were exaggerated in aged animals and are consistent with a role in telomere dysfunction, and age related diseases.
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Hills, Mark. "The Xp/Yp telomere and its adjacent DNA : structure, sequence organisation and mutation". Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/30355.
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My research has focused on developing an understanding of processes operating in and around the Xp/Yp telomere, combined with detailed analysis of the unusual properties seen immediately adjacent to it. To this end, 7 kb of telomere adjacent-sequence was analysed, revealing a SNP density, pi, of 1.73 x 10-3 across the first 3 kb, 2-fold above average genome level. Furthermore, the sequence analysis revealed a 1.9 kb tandem duplication in 86% of chromosomes. This included the minisatellite DXYS14, and both copies were found to be hypervariable, with pedigree analysis revealing mutation rates of 0.94% and 0.62% for DXYS14a and DXYS14b respectively, with double mutations often occurring.;Haplotype analysis revealed strong SNP associations across the entire 7 kb region. High-resolution crossover analysis was carried out using allele-specific PCR methods to selectively amplify recombinant sperm molecules, which indicated that the Xp/Yp telomere-adjacent DNA is recombinationally inert, with a rate of 2.4 x 10-6 cM Mb-1 calculated from 1.250,000 amplifiable molecules.;It was thought secondary structure may be implicated in both the high SNP density and low recombination rate seen, so analysis of t-loops and nucleosome phasing was carried out at the Xp/Yp telomere. While the efficiency of t-loop visualisation prevented specific targeting of the Xp/Yp telomere, preliminary nucleosome data suggest that nucleosomes are present, but not phased in this region.;Finally, analysis was conducted on the presence of the CTAGGG variant repeat at the Xp/Yp telomere. This variant repeat is presented at ∼7% of Xp/Yp telomeres, and in CEPH families, two telomeres containing arrays of CTAGGG were shown to display a ∼46% germline mutation rate, higher than the 0.6% rate estimated in telomeres not harbouring this repeat. It is possible that secondary structure may be responsible for the instability associated with this repeat type.
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Ji, Yingjie, Xindi Dang, Lam Ngoc Thao Nguyen, Lam Nhat Nguyen, Jaun Zhao, Dechao Cao, Sushant Khanal i in. "Topological DNA Damage, Telomere Attrition and T Cell Senescence During Chronic Viral Infections". Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/6522.
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Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions.
Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival.
Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.
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Bonnell, Erin. "The role of Tbf1 in telomere homeostasis in Saccharomyces cerevisiae". Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/11077.
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Abstract: By differentiating chromosomal ends from internal breaks, telomeres prevent DNA damage checkpoint activation and provide protection from inappropriate DNA repair activity that could create genomic instability. In Saccharomyces cerevisiae, a large number of genes have been identified that are implicated in telomerase and telomere structure and/or function. However, a comprehension of the mechanism of action of these genes and how they relate to other genes is lacking. The function of end protection is based on the telomeric repeats and associated proteins, but evidence is accumulating that the subtelomeric region also plays a role. This region contains binding sites for various proteins, notably Tbf1. TBF1 is an essential gene and the protein has been implicated in telomere homeostasis, chromatin remodelling, and the DNA damage response. My master’s project is based on the observation that cells harbouring a thermosensitive (tbf1-ts) allele have abnormally short telomeres. However, all four known mutant tbf1 alleles have multiple point mutations, which renders their analyses difficult. In order to be able to more precisely determine the origin of the phenotypic variations, we used site-directed mutagenesis to create single point mutation tbf1 alleles. These experiments yielded two particular mutations, tbf1-82 and tbf1-453, which were found to have growth defects at various temperatures as well as increased sensitivity to DNA damaging drugs. Although the alleles had only minor telomere length phenotypes, it was discovered that Tbf1 could have a direct role in telomere stability in special situations. For example, in the absence of telomerase, which normally maintains telomeres, cells enter replicative senescence after about 60 population doublings and stop dividing. A small subset of the cellular population is able to evade this growth arrest by maintaining telomeres via a recombination-dependent process. An introduction of the tbf1-82 or tbf1-453 mutation into strains that also lacked telomerase caused a dramatic advance in time of onset of senescence. Thus this work uncovered that Tbf1 is a previously unknown regulator of senescence. Various genetic assays with homologous recombination genes and chromatin regulators were performed to help further characterize TBF1 and its interactors. Characterization of these novel tbf1 alleles has given new insights into the multiple roles of Tbf1.En différenciant les extrémités chromosomiques des cassures d’ADN internes, les télomères empêchent l'activation de la signalisation d’un dommage à l'ADN et fournissent une protection contre des activités inappropriées qui sont associées à une réparation de l'ADN. Une telle réparation pourrait en fait créer une instabilité génomique. Chez Saccharomyces cerevisiae, un nombre de protéines sont impliquées dans la structure du télomère et / ou la fonction de la élomérase. On pense que la protection des télomères est gérée par les répétitions télomériques et les protéines associées, mais il y a de plus en plus d’indices que la région sous-télomérique joue également un rôle. Cette région contient des sites de liaison pour plusieures protéines, notamment pour Tbf1. TBF1 est un gène essentiel et la protéine est impliquée dans l'homéostasie des télomères et dans la réponse aux dommages de l’ADN. Toutefois, les mécanismes moléculaires restent à être précisés. Mon projet de Maîtrise est basé sur l’observation que dans les cellules qui ont un allèle thermosensible (tbf1-ts), les télomères sont anormalement courts. Malheureusement, les 4 allèles mutants de tbf1 connus présentent tous des mutations ponctuelles multiples ce qui rend leur analyse difficile. Pour clarifier l'origine des variations phénotypiques de ces mutations, la mutagenèse dirigée a été utilisée pour créer des allèles tbf1 avec une seule mutation. Mes résultats montrent que deux mutations spécifiques, tbf1-82 et tbf1-453, causent des défauts de croissance cellulaires, ainsi qu'une sensibilité aux drogues qui endommageant l'ADN. Une analyse détaillée de ces nouveaux allèles de tbf1 a montré que la protéine pourrait avoir un rôle direct dans le maintien de la stabilité des télomères. Par exemple, en absence de la télomérase qui est responsable du maintien des télomères, les cellules entrent en sénescence réplicative après environ 60 générations et arrêtent de se diviser. Par contre, une petite fraction de la population est capable de contourner cet arrêt de croissance car ces cellules maintiennent les télomères par un processus dépendant de la recombinaison homologue. L'introduction de mutations tbf1 dans des souches sans télomérase provoque une accélération d’entrée en sénescence; donc Tbf1 est un régulateur précédemment inconnu de la sénescence. Divers tests génétiques avec des gènes de recombinaison homologue et des régulateurs de chromatine ont été effectués pour aider à caractériser TBF1 et ses interactions. La caractérisation de ces nouveaux allèles a permis de mieux comprendre les multiples rôles de Tbf1.
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Zhou, Jia. "Dna Glycosylases Remove Oxidized Base Damages From G-Quadruplex Dna Structures". ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/529.
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The G-quadruplex DNA is a four-stranded DNA structure that is highly susceptible to oxidation due to its G-rich sequence and its structure. Oxidative DNA base damages can be mutagenic or lethal to cells if they are left unrepaired. The base excision repair (BER) pathway is the predominant pathway for repair of oxidized DNA bases. DNA glycosylases are the first enzymes in BER and are responsible for removing base lesions from DNA. How DNA glycosylases remove base lesions from duplex and single-stranded DNA has been intensively studied, while how they act on G-quadruplex DNA remains to be explored.
In Chapter II of this dissertation, we studied the glycosylase activity of the five mammalian DNA glycosylases (OGG1, NTH1, NEIL1, NEIL2 and mouse Neil3) on G-quadruplex DNA formed by telomere sequences that contain a single base lesion. We found that telomeric sequences that contain thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) or spiroiminodihydantoin (Sp) all formed the basket form of an antiparallel G-quadruplex DNA structure in Na+ solution. We also showed that no glycosylase was able to remove 8-oxoG from quadruplex DNA, while its further oxidation products, Sp and Gh, were good substrates for mNeil3 and NEIL1 in quadruplex DNA. In addition, mNeil3 is the only enzyme that removes Tg from quadruplex DNA and the glycosylase strongly prefers Tg in the telomere sequence context in both single-stranded and double-stranded DNA.
In Chapter III, we extended our study to telomeric G-quadruplex DNA in K+ solution and we also studied quadruplex DNA formed by promoter sequences. We found that 8-oxoG, Gh and Sp reduce the thermostability and alter the folding of telomeric quadruplex DNA in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases are able to remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplex DNA in K+ solution. Interestingly, NEIL1 or NEIL3 do not efficiently remove hydantoin lesions at the site that is most prone to oxidation in quadruplex DNA. However, hydantoin lesions at the same site in quadruplex DNA are removed much more rapidly by NEIL1, NEIL2 and NEIL3, when an extra telomere TTAGGG repeat is added to the commonly studied four-repeat quadruplex DNA to make it a five-repeat telomere quadruplex DNA. We also show that APE1 cleaves furan in selected positions in Na+-coordinated telomeric quadruplex DNA structures. We use promoter sequences of the VEGF and c-MYC genes as models to study promoter G-quadruplex DNA structures, and show that the NEIL glycosylases primarily remove Gh from Na+-coordinated antiparallel quadruplex DNA but not from K+-coordinated parallel quadruplex DNA containing VEGF or c-MYC promoter sequences.
Taken together, our data show that the NEIL DNA glycosylases may be involved in both telomere maintenance and gene regulation.
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Kargaran, Kobra. "The role of BRCA1 in telomere maintenance". Thesis, Brunel University, 2015. http://bura.brunel.ac.uk/handle/2438/13671.
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Telomeres are fundamental structures found at the end of all eukaryotic chromosomes that function to protect the end of chromosomes from end-to-end fusion, erosion and subsequent telomere dysfunction. Telomerase and alternative lengthening of telomere (ALT) mechanisms maintain the telomeres by compensating natural telomeric loss. ALT is found to be present in 15% of human tumours lines and it may be expressed at low levels in the normal mouse tissues. However, the exact mechanism behind ALT depression and/or activation in the mammalian cells is not fully understood. Previous studies have highlighted the role of BRCA1 in telomere dysfunction. Also, it has recently been shown that BRCA1 co-localises at telomeres in the ALT + human cells through BLM and Rad50. However, it is still unclear whether BRCA1 plays a direct role on telomere length maintenance and integrity. The aim of this project was to examine the role of BRCA1 in telomere maintenance associate with ALT in BRCA1 defective mammalian cells. Therefore to achieve this, we have set up series of experiments to look at, (a) hallmarks of ALT activity at the cytological level, (b) measuring of ALT activity using biochemical and immunocytochemistry techniques and (c) understanding the role of BRCA1 in DNA damage response mechanism and telomere dysfunction. Firstly, we found elevated levels of recombination at telomeres in the two human BRCA1 carrier cell lines and mouse embryonic stem cell with deficiency in Brca1-/-. Secondly, our data showed that human and mouse BRCA1 defective cells are significantly more sensitive to ionizing radiation in line with the DNA repair function of BRCA1. Moreover, we found persistent DNA damage at telomeres in the BRCA1 defective environment when after exposure of cells to ionizing radiation. Thirdly, we found evidence of ALT activity in some mouse cell lines, and elevated ALT in mouse cells defective in Brca1. Finally, we examined some other ALT markers using immunofluorescence. Our data indicate differences between human and mouse cells in regulating ALT. Taken together data presented in this thesis revealed that (i) BRCA1 plays a major role in telomere maintenance and defective BRCA1 mammalian cells show evidence of telomere dysfunction and telomere length shortening in line with previous publish data, (ii) BRCA1 defective mouse cells have elevated levels of ALT, (iii) the mouse lymphoblastoid LY-S cells have complete absence of ALT.
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Bourns, Brenda. "Development and characterization of a new assay to examine telomere-protein interactions in vivo /". Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6336.
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Lannan, Ford. "Folding of the human telomere sequence DNA in non-aqueous and otherwise viscous solvents". Thesis, Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/47598.
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G-quadruplex forming human telomere sequence (HTS) DNA, has been widely studied due to the telomere's implied role in biological processes, including cellular ageing and cancer physiology. The goal of these previous efforts has been to characterize the physiologically relevant structures and their stability and dynamics in order to develop therapeutic applications. Unfortunately, understanding the biologically relevant form of the human telomere DNA is complicated by the fact that HTS-derived sequences are highly polymorphic. To further complicate the issue, recent investigations have demonstrated the ability of "cell-like" co-solvents to alter the preferred G-quadruplex fold of HTS DNA. However, the origins of G-quadruplex structure selection, the relative contributions of crowding versus dehydration, and the possible effects of co-solvents on kinetically determined folding pathways remain unresolved. Towards answering these questions, I investigated HTS DNA G-quadruplex in extreme anhydrous and high viscosity conditions utilizing a deep eutectic solvent (DES) consisting of choline chloride and urea. Herein I report that the water-free DES supports an extremely stable parallel stranded structure, consistent with observations that diminished water activity is the main cause of structural transitions to the "parallel-propeller" form. Furthermore, my research shows that the highly viscous nature of the solvent enables significant diffusion based control over HTS g-quadruplex folding rates and topology, fully consistent with Kramers rate theory. To the best of my knowledge, this is the first example of the kinetic exploration of G-quadruplex folding utilizing high friction solvent; the results of which display a decreased intramolecular folding rate of HTS DNA to a never before encountered time scale on the order of days at physiological temperature. Moreover, I have demonstrated that the folding pathway of a G-quadruplex can be altered with increased solvent friction. These discoveries are important because they highlight the need to consider the viscosity when exploring the dynamics of human telomeres specifically drug binding and folding of G-quadruplexes in vivo where cellular viscosity has been reported to be as high as 140cP. Lastly, it appears that tuning solvent viscosity could prove useful to the continued study of G-quadruplex dynamics.
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Liew, Li Phing. "Characterization of the TLH1-4+ telomere-linked recq DNA helicase genes in schizosaccharomyces pombe". Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510984.
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Beyer, Tracey Elaine, i Tracey Elaine Beyer. "Ontogeny of Unstable Chromosomes Formed by Telomere Replication Error". Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621103.
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The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.
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Borssén, Magnus. "DNA methylation as a prognostic marker i acute lymphoblastic leukemia". Doctoral thesis, Umeå universitet, Patologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-127225.
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Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Most ALL cases originate from immature B-cells (BCP-ALL) and are characterized by reoccurring structural genetic aberrations. These aberrations hold information of the pathogenesis of ALL and are used for risk stratification in treatment. Despite increased knowledge of genetic aberrations in pediatric T-cell ALL (T-ALL), no reliable molecular genetic markers exist for identifying patients with higher risk of relapse. The lack of molecular prognostic markers is also evident in patients with relapsed ALL. During the last decades, aberrant epigenetic mechanisms including DNA methylation have emerged as important components in cancer development. Telomere maintenance is another important factor in malignant transformation and is crucial for long-term cell survival. Like DNA methylation, telomere length maintenance has also been implicated to reflect outcomes for patients with leukemia. In this thesis, the prognostic relevance of DNA methylation and telomere length was investigated in pediatric ALL at diagnosis and relapse. The telomere length (TL) was significantly shorter in diagnostic ALL samples compared to normal bone marrow samples collected at cessation of therapy, reflecting the proliferation associated telomere length shortening. Prognostic relevance of TL was shown in low-risk BCP-ALL patients where longer telomeres at diagnosis were associated with higher risk of relapse. Genome-wide methylation characterization by arrays in diagnostic T-ALL samples identified two distinct methylation subgroups denoted CIMP+ (CpG Island Methylator Phenotype high) and CIMP- (low). CIMP- T-ALL patients had significantly worse outcome compared to CIMP+ cases. These results were confirmed in a Nordic cohort treated according to the current NOPHO-ALL2008 protocol. By combining minimal residual disease (MRD) status at treatment day 29 and CIMP status at diagnosis we could further separate T-ALL patients into risk groups. Likewise, the CIMP profile could separate relapsed BCP-ALL patients into risk groups, where the CIMP- cases had a significantly worse outcome compared to CIMP+ cases. From these data we conclude that DNA methylation subgrouping is a promising prognostic marker in T-ALL, as well as in relapsed BCP-ALL two groups where reliable prognostic markers are currently missing. By elucidating the biology behind the different CIMP profiles, the pathogenesis of ALL will be further understood and may contribute to new treatment strategies.
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Conway, Colin. "The role of Ku in antigenic variation, DNA repair and telomere maintenance in African trypanosomes". Thesis, University of Glasgow, 2002. http://theses.gla.ac.uk/30956/.
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The process of antigenic variation in African trypanosomes allows the survival of the parasite by constantly switching the variant surface glycoprotein (VSG) expressed in their surface. There are believed to be several hundred copies of these silent VSG genes in the parasite's genome and they are expressed differentially. The majority of these genes are not capable of being transcribed in situ and must therefore be expressed from specialised transcriptional units known as bloodstream expression sites (BESs). Only one such site is active at any one time, ensuring that a single VSG is expressed in the trypanosome's surface coat. Switching the expressed VSG involves replacing the VSG in the active BES, or activating a new BES in conjunction with silencing the previously active. Differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres. All BESs are telomeric and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with yeast telomeres that is directly involved in DNA recombination and gene silencing. The main aim of this thesis was to test the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. In order to compare trypanosome Ku homologues with those from other organisms, it was necessary to compile homology alignments with other Ku homologues using Clustal W analysis. Subsequent experiments looked at the fate of exogenously introduced restriction enzyme target sites after transient transformation with cassettes encoding the restriction enzyme. A final analysis looked for the presence of NHEJ in homologous recombination- deficient trypanosomes. Disrupting this element of DNA repair would hopefully lead to other forms of repair becoming detectable, and even up-regulated. Rad51, in yeast a member of the Rad52 epistasis group (integral in yeast homologous recombination), had previously been demonstrated to be involved in DNA repair in trypanosomes (McCulloch & Barry, 1999). rad51 mutants were electroporated with cassettes containing noncompatible ends that would prevent their integration into the endogenous genome via conventional homologous recombination. This cassette also contained promoter DNA sequence to allow selection in the event of integration into non-transcribed regions of the genome. Study of the junctions encompassing the integration sites of the cassette allowed investigation into how the cassettes were integrated, and revealed to us the extent of the sequence homology required to catalyse integration. The method of repair detection observed indicated that classical homologous recombination is not the only pathway utilised by African trypanosomes to metabolise DNA double-strand breaks.
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Boerckel, Julie Ann Ahmed Shawn. "Isolation and characterization of genes involved in DNA damage response and telomere maintenance in Caenorhabditis elegans". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,843.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of PhD in the Department of Biology." Discipline: Biology; Department/School: Biology.
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Eisenstatt, Jessica R. "Histone H4 Acetylation in the DNA Damage Response and Telomere Formation of Schizosaccharomyces pombe". Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1440417554.
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Lee-Bellantoni, Margaret S. "Antioxidant defense and redox responses to telomere homolog oligonucleotides in human dermal fibroblasts: a model for investigating redox signaling responses to DNA damage". Thesis, Boston University, 2005. https://hdl.handle.net/2144/37162.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
It has been demonstrated that oligonucleotides homologous to the 3' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H202 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H20 2 showed no H20 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated β-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of β-galactosidase positive cells by 72 hours (<30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms.
2031-01-01
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Jasti, Madhuri. "Identification and characterization of tac5, a telomerase activation mutant, characterization of DNA damage responses and assessment of interactions between telomere-related proteins in Arabidopsis thaliana". [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1238.
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Da, Silva Marcelo Santos 1982. "Estresse oxidativo em Leishmania amazonensis = do encurtamento dos telômeros ao deslocamento de LaRPA-1 do complexo telomérico = Oxidative stress in Leishmania amazonensis : from telomere shortening to displacement of LaRPA-1 from telomeric complex". [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317093.
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Orientador: Maria Isabel Nogueira CanoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-26T06:26:57Z (GMT). No. of bitstreams: 1 DaSilva_MarceloSantos_D.pdf: 9189361 bytes, checksum: 79ecad917f6be7c8b200b549dafc493e (MD5) Previous issue date: 2014
Resumo: A leishmaniose é um espectro de doenças causadas por parasitos do gênero Leishmania, que afeta milhões de pessoas em todo o mundo. Durante a infecção, os parasitos usam diferentes estratégias para sobreviver as defesas do hospedeiro, incluindo superar a exposição intensa a espécies reativas de oxigênio (ROS), principais responsáveis por causar danos no DNA, sobretudo nos telômeros, induzindo instabilidade genômica, senescência e morte celular. Telômeros são estruturas nos terminais dos cromossomos compostos por sequências de DNA repetitivas e proteínas, cuja função é proteger as extremidades dos cromossomos, evitando fusões terminais e degradação nucleolítica. Neste trabalho nós induzimos estresse oxidativo agudo em formas promastigotas de L. amazonensis através do tratamento com 2 mM de peróxido de hidrogênio (H2O2) por 1h, o qual foi capaz de aumentar os níveis de ROS intracelular, como demonstrado pela reação utilizando CM-H2DCFDA. Além disso, o estresse oxidativo induziu danos no DNA, como mostrado por análise quantitativa de 8-oxodG e núcleos positivos para o ensaio TUNEL. Observamos também, através de parâmetros qualitativos e quantitativos (Southern blot, telomere-PCR e flow-FISH), que o estresse oxidativo, assim como em mamíferos, induziu encurtamento dos telômeros. Analisando a co-localização e interação proteína:DNA por FISH-IIF e ensaios ChIP, foi possível demostrar que o estresse oxidativo causou erosão da extremidade 3¿G overhang, fazendo com que a proteína LaRPA-1 perdesse seu sítio de interação nos telômeros. Além disso, pudemos observar uma maior afinidade de LaRPA-1 para com a fita telomérica rica em C, nesse caso uma região de simples-fita gerada dentro da dupla fita telomérica, provavelmente como consequência do reparo de DNA, sugerindo a participação de LaRPA-1 na resposta a danos oxidativos. Por análise de curvas de crescimento e incorporação de EdU, foi possível observar que o estresse oxidativo induziu diminuição acentuada no número de parasitos em cultura, enquanto os sobreviventes continuaram proliferando e replicando DNA. Observamos também que o estresse oxidativo agudo provocou arrest de ciclo celular na fase G2/M em parte da população em crescimento exponencial. Em conjunto, esses resultados sugerem a presença de um sistema muito eficiente de resposta a danos oxidativos no DNA telomérico, que permite que os parasitos sobrevivam e repliquem DNA mesmo após um estresse agudo
Abstract: Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects million people around the world. During infection, parasites use different strategies to survive host defenses including overcoming exposure to Reactive Oxygen Species (ROS), mainly responsible for causing DNA damage, especially at telomeres which frequently results in genome instability, senescence and cell death. Telomeres are chromosomes end termini structures composed by repetitive DNA coupled with proteins whose function is to protect chromosome ends and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H2O2) for 1 hour, which was able to increase intracellular ROS levels, as demonstrated by CM-H2DCFDA reaction. In addition, oxidative stress induced DNA damage, as confirmed by quantitative analysis of 8-oxodG and TUNEL-positive nuclei. We have also observed using qualitative and quantitative parameters (Southern blot, telomere-PCR and flow-FISH) that oxidative stress, as in mammals, induced telomere shortening. Analysing the protein:DNA co-localization and interaction by FISH-IIF and ChIP assays, it was possible to show that oxidative stress is able to induce erosion of the 3¿G overhang, inducing a displacement of LaRPA-1 from its telomeric interaction site. In addition, we observed an increase in the affinity between LaRPA-1 and the telomeric C-rich strand, in this case, a single-strand region inside the double-strand telomeric DNA generated probably as a consequence of DNA repair, suggesting the participation of LaRPA-1 in oxidative DNA damage response. Analysis of growth curves and EdU incorporation showed that oxidative stress induced a decrease in the number of parasites in culture, while the survivors continued proliferating and replicating DNA. Moreover, as result of acute oxidative stress, part of the parasites in exponential growth shows a G2/M cell cycle arrest. Taken together, these results suggest the presence of a very efficient oxidative damage response in the telomeres that allows parasites to survive and to replicate DNA even after acute stress
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular
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Madalena, Christiane Rodriguez Gutierrez. "DNA repetitivo e seu papel na estrutura cromossômica terminal em Rhynchosciara americana (Diptera: Sciaridae)". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-02102008-144225/.
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A localização cromossômica do DNA ribossômico (rDNA) foi estudada em cromossomos politênicos e em tecidos diplóides de quatro espécies de sciarídeos: Trichosia pubescens; Rhynchosciara americana; R. milleri e Schwenkfeldina sp.. Resultados de hibridação em cromossomos mitóticos mostraram a existência de um único locus de rDNA; entretanto, sondas ribossomais hibridaram em mais de uma região dos cromossomos politênicos em todas as espécies analisadas devido à adesão de micronucléolos em regiões específicas dos cromossomos. Os micronucléolos são estruturas arredondadas que contêm, provavelmente, DNA extracromossômico transcricionalmente ativo. Em T. pubescens, o rDNA está predominantemente localizado nas secções cromossômicas X-10 e X-8. Em R. americana o rDNA está freqüentemente associado à heterocromatina centromérica dos cromossomos X, C, B e A, e também às secções X-1 e B-13. Sondas ribossômicas em R. milleri hibridaram, em alta freqüência, em regiões teloméricas e pericêntricas de cromossomos politênicos. Schwenkfeldina sp. apresenta uma distribuição incomum do rDNA em núcleos politênicos, caracterizada pela adesão de micronucléolos em muitas regiões cromossômicas. Os resultados mostraram que os micronucléolos estão preferencialmente associados à heterocromatina intercalar ou terminal de todos os sciarídeos analisados e, dependendo da espécie, estão aderidos a um número pequeno (Trichosia), moderado (Rhynchosciara) e grande (Schwenkfeldina sp.) de sítios em cromossomos politênicos. Este trabalho também descreve a caracterização de seqüências presentes nas extremidades cromossômicas de R. americana, que se iniciou através da triagem de uma microbiblioteca plasmidial, feita a partir de uma extremidade microdissecada B-1. Uma repetição do tipo satélite foi identificada e sua composição de bases, estrutura genômica e localização cromossômica são semelhantes às repetições teloméricas complexas de Nematocera que já foram descritas. Contudo, dados obtidos em outras espécies de Rhynchosciara, assim como a localização desse satélite e da transcriptase reversa, sugerem que o elemento repetitivo caracterizado neste trabalho não atinge as extremidades dos cromossomos. A caracterização de seqüências terminais e subterminais presentes nos cromossomos de R. americana foi continuada através da triagem de uma biblioteca de DNA desse díptero clonada em fagos Dash. Escolhemos como sonda para a triagem o clone pRaM47.33, representativo do elemento repetitivo M22, caracterizado em R. americana. Foram analisados cerca de 12kb de um único inserto de fago, que continha, alem das repetições M22, uma nova repetição de 16pb, organizada em tandem e que denominamos de M16. Resultados de hibridações in situ revelaram a presença da repetição M16 nas 5 extremidades cromossômicas não-telocêntricas de R. americana. Essa repetição também foi utilizada como sonda em uma outra triagem da mesma biblioteca genômica, o que permitiu a seleção e análise de aproximadamente 50kb de DNA cromossômico terminal de R. americana. Encontramos também, ao longo dessas 50kb de DNA analisado, repetições de 414pb anteriormente caracterizadas em R. americana; parte de seqüências do transposon Ramar1 e do retrotransposon RaTART . Além disso, foram observadas também seqüências que não apresentam semelhança significativa com seqüências depositadas no banco de dados GenBank, e que tampouco apresentam motivos repetitivos. Os resultados obtidos apontam para a possibilidade de que a região telomérica de R. americana seja composta por mais de um tipo de elemento repetitivo.The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites. This work also describes the characterization of chromosome end sequences of Rhynchosciara americana, initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome end. We report the identification and sequencing of an R. americana satellite displaying base composition, genomic structure and chromosomal localization similar to the complex telomeric repeats of Nematocera that have previously been characterized. However, data obtained in other Rhynchosciara species, as well as distinct chromosomal localization of satellite and reverse transcriptase loci in R. americana, suggest that the repetitive element characterized does not reach the very end of the chromosome. The characterization of chromosome end sequences of Rhynchosciara americana continued with the screening of a phage library made with its genomic DNA. We choose pRaM47.33, a clone whose insert is a repetitive microsatellite characterized in the subtelomeric region of R. americana chromosomes, as a probe for the screening. We analyzed 12kb of a single phage insert, composed of M22 tandem arrays and a new microsatellite which was 16pb long, arranged in tandem (named M16). In situ hybridization showed the presence of M16 repeats in the five telomeric termini of R. americana chromosomes. The M16 repeat was used as a probe in another screen of the same phage library, which allowed us to analyze approximately 50kb of terminal DNA. We find that repetitive sequences, such as the 414pb repeat previously characterized in R. americana and stretches of Ramar1 and RaTART mobile elements, also characterized in R. americana, compose the subtelomeric region of R. americana chromosomes. Additionally, we find sequences that do not match sequences in the GenBank database and do not present repetitive motifs. Our results suggest that the telomeric regions of R. americana chromosomes are composed of more than one type of repetitive sequence.
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dos, Santos Soares Martins de Castro Alicia Maria. "A mechanistic investigation into candidate markers of telomere-induced senescence in normal human epidermal keratinocytes". Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8034.
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Telomere dysfunction is one mechanism of cellular and tissue ageing. Dysfunctional telomeres in fibroblasts are recognised as DNA double-strand breaks (DSBs) and trigger the DNA damage pathway of senescence. However, telomere uncapping in normal human epidermal keratinocytes, via expression of the dominant negative mutant of the telomere repeat-binding factor 2 (TRF2!B!M), resulted in a senescent-like arrest without a significant DNA damage response (DDR). This suggests that either keratinocytes are unusually sensitive to telomere uncapping and the low DDR is sufficient to induce senescence or that dysfunctional telomeres may also be signalled through an alternative pathway. Subsequent analysis revealed genes HIST2H2BE, ICEBERG, S100A7 and HOPX as potential markers for telomere dysfunction-induced senescence (TDIS) since they were induced by telomere uncapping and seemed to be regulated by telomerase. The aim of this project was to assess the specificity of these candidate markers for TDIS and to select the most promising for use as a biomarker. To this end, keratinocytes were exposed to doses of ionising radiation, capable of generating transient or permanent damage to the DNA, or transduced with retroviral constructs expressing p14ARF, p16INK4a, p53 or TRF2!B!M and the gene expression levels of the candidates assessed after a recovery period or at the early stages of senescence. Whilst S100A7, HOPX or ICEBERG were not induced by a transient or persistent DDR or by p16INK4a, ICEBERG and HOPX were induced by p53 and p14ARF when these were ectopically expressed at higher levels. Thus, S100A7 seems to be the most specific early marker for telomere dysfunction in keratinocytes since it was selectively induced by telomere uncapping via expression of TRF2!B!M and not by DSBs or by over expression of p14ARF, p53 or p16INK4a. S100A7 may have the potential to identify cells with telomere dysfunction in human epithelia and body fluids.
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Smith, Zoe Elizabeth. "Analysis of the 16p telomere to examine the relationship between DNA replication, chromosome structure and gene expression". Thesis, University of Oxford, 1998. https://ora.ox.ac.uk/objects/uuid:fefcbb7b-9829-4a48-8edd-9608a613922d.
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The overall aim of the laboratory is to understand how the human α globin genes are expressed in a tissue-specific and developmental-stage specific manner from their natural chromosomal environment. This gene cluster has been studied in great detail from this point of view, and it now represents an important general model to address how eukaryotic genes are regulated. Ultimately, this information may be important for the further development of strategies for effective gene transfer, particularly for patients who suffer from inherited anaemias which result from abnormal globin gene expression (thalassemias). The human α globin gene cluster is located close to the telomere, in the terminal band of the short arm of chromosome 16 (16p13.3). Many aspects of α gene regulation have been studied in detail, including the identification of the structural genes, their proximal regulatory elements (promoters) and distal regulatory element (HS-40). Erythroidspecific trans-acting factors which bind these elements in vitro and in vivo have been identified. In an attempt to define the regulatory domain containing the α genes, the surrounding chromosomal environment (~400kb) has also been extensively studied. The region is fully sequenced, and the chromatin structure, methylation patterns and scaffold attachment sites have been established. Over 100 natural mutants that perturb α globin expression have been characterised and numerous fragments from this region have been analysed in transgenic mice. One potentially important aspect of α globin gene expression that has not been previously examined is DNA replication. Study of replication throughout this particular region provides an excellent opportunity to integrate this information with what is already known about the regulation of the α cluster. Initially, studies were undertaken to analyse the pattern and timing of DNA replication of the human α globin gene cluster and the surrounding chromosomal region in erythroid and non-erythroid cell-types. Replication timing of this region has also been compared in the individual paternal and maternal alleles of chromosome 16. Once the in vivo pattern of replication was established, the role of chromosomal position upon replication timing was assessed by analysing various constructs of the a cluster in transgenic mice. The role of the distal regulatory element HS-40 in influencing the pattern and timing of replication of the extended region has been examined. Naturally-occurring deletions within this region have facilitated the study of the importance of the telomere and subtelomeric region in determining the replication characteristics of this region. Replication has also been studied in another natural mutant, which contains a deletion which may perturb α gene expression by a novel mechanism.
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Nguyen, Lam Nhat, Juan Zhao, Dechao Cao, Xindi Dang, Ling Wang, Jianqi Lian, Ying Zhang i in. "Inhibition of TRF2 Accelerates Telomere Attrition and DNA Damage in Naïve CD4 T Cells During HCV Infection". Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etsu-works/6523.
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T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.
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Musetti, Caterina Livia. "Heterocyclic Cations as Potential Anticancer Agents: An Approach that Targets G-quadruplex with Different Binding Modes". Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/chemistry_theses/26.
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G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.
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BUSNELLI, ANDREA. "MITOCHONDRIAL DNA COPY NUMBER, TELOMERE LENGTH AND DNA METHYLATION IN PERIPHERAL BLOOD OF WOMEN UNDERGOING IN VITRO FERTILIZATION CYCLES AS NEW PREDICTORS OF LIVE BIRTH". Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/891706.
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Background: The peripheral biomarker of female reproductive biological age proposed so far (i.e., mitochondrial DNA copy number (mt-DNAcn), telomere length (TL) and DNA methylation (DNAm) in peripheral blood are promising. Unfortunately, the results of studies aimed at investigating their predictive capacity are conflicting. The conduction of a prospective study including women at the beginning of their natural pregnancy seeking, at the present state of knowledge, goes beyond the possibilities offered by even the most favorable settings. In this context, assisted reproductive technology (ART) has emerged as the most reliable study model. Importantly, considering the absence of non-invasive predictors of ART success, the identification of a reliable biomarker would also have positive implications for the determination of the ART risk-benefit ratio and, in a public health perspective, for the rational allocation of economic resources.
Objective: To evaluate whether mt-DNAcn, TL or epigenetic age estimators based on DNAm pattern (biological age, epigenetic age acceleration, LINE-1 methylation rate), could be considered reliable predictors of in vitro fertilization (IVF) success in terms of live birth rate (LBR).
Design: Prospective cohort study
Setting: Infertility Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Italy.
Patients: 181 women aged 37-39 years who underwent IVF at a single center between January 2017 and December 2018.
Interventions: On the day of recruitment, blood samples were collected, and genomic DNA was isolated from white blood cells. TL, mt-DNAcn and DNAm assessment was performed using quantitative real-time polymerase chain reaction (qPCR). Biological age (DNAm age) was computed as the algorithm based on methylation pattern of five genes. Epigenetic age acceleration was estimated from the residuals of the linear model of epigenetic age regressed on chronological age. Long Interspersed Nuclear Elements-1 (LINE-1) methylation pattern was used as a surrogate for global DNA methylation.
Main outcome Measures: This study investigated whether peripheral TL, mt-DNAcn and DNAm could predict live birth in IVF cycles.
Results: TL, mt-DNAcn and LINE-1 methylation were not associated with IVF success. Conversely, DNAm age resulted significantly lower in women who had a live birth compared to women who did not (36.1 ± 4.2 and 37.3 ± 3.3 years, respectively, p=0.04). For DNAm age, odds ratio (OR) for live birth per year of age was 0.90 (95%CI: 0.82-0.99, p=0.036) after adjusting for FSH and antral follicle count (AFC) and 0.90 (95%CI: 0.82-0.99, p=0.028) after adjusting also for number of oocytes retrieved. A significant association also emerged for epigenetic age acceleration after adjustments (OR=0.91, 95%CI: 0.83-1.00, p=0.048).
Conclusion: DNAm age is associated with IVF success but the magnitude of this association is insufficient to claim a clinical use. However, our findings are promising and warrant further investigation. Assessment of biological age using different epigenetic clocks or focusing on different tissues may reveal new predictors of IVF success.