Dissertations / Theses: 'Telomeres'

1

Shakirov, Yevgeniy Vitalievich. "Telomeres and telomere binding proteins in Arabidopsis thaliana." Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/422.

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Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary to prevent chromosome fusions and exonuclease attack. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2-9kb. Unexpectedly, telomeres in WS plants displayed a bimodal size distribution with some individuals exhibiting 4-8 kb telomeres and others 2-5 kb telomeres. We also examined the dynamics of telomere tracts on individual chromosome ends. Following the fate of telomeres in plants through successive generations, we found that the shortest telomeres were typically elongated in the subsequent generation, while the longest telomeres were usually shortened. Thus, telomere length homoeostasis is achieved through intermittent telomerase action on shorter telomeres to attain an optimal size.Single-strand telomere binding proteins were also analyzed. Four major telomere binding protein complexes from cauliflower were identified and their DNA-binding properties characterized. The DNA-binding component of one of the complexes was purified and analyzed by mass-spectrometry. Peptide mass data was used to search for putative protein candidates from the Arabidopsis thaliana database. Additionally, two Arabidopsis genes, AtPot1 and AtPot2, were identified and characterized. The genes encode two single-strand telomeric DNA binding proteins. AtPot1 and AtPot2 proteins can homo- and heterodimerize in vitro. Pot1 protein predominantly localizes to the nucleolus, whereas Pot2 is exclusively nuclear. Plants over-expressing full-length Pot1 and Pot2 proteins had no obvious phenotype, while over-expression of P2DBD and P1∆DBD caused moderate telomere shortening. Plants over-expressing P2DBD had severe morphological and reproductive defects, multiple chromosome abnormalities and aneuploidy. Over-expression of a chimeric protein DBD-P1∆DBD led to rapid telomere shortening, confirming the involvement of Arabidopsis Pot proteins in telomere length maintenance. Intriguingly, telomerase in DBD-P1∆DBD-EYFP plants is inactivated, suggesting that Pot proteins are also involved in regulation of telomerase activity. The analysis of Arabidopsis telomeres and telomere binding proteins will provide additional information towards understanding the role of the telomeric nucleoprotein complex in eukaryotic chromosome biology.

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2

Maddison, Rachelle Louise. "Telomeres in the absence of telomerase in Saccharomyces cerevisiae." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/30365.

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The aim of this study was to screen a list of proteins which were suggested to have roles in telomerase-independent telomere maintenance. Of the proteins screened some had no effect on post-senescent survival (Nup53p and Crp1p). Other proteins are suggested to function in post-senescent survival (M1p1p, M1p2p, Dhh1p, Rrm3p and Stm1p).;The importance of the affect of the chromatin context on post-senescent survival was identified with proteins involved in chromatin remodelling (Chd1p, Isw1p and Isw2p) and modifying (Rdp3p) identified as also affecting survival. Of particular interest was the strain dependency of their effects, with opposing roles in post-senescent survival in the YP1 and Y55 strains. More work is needed to fully characterise these roles. Their effects on post-senescent survival could be by them directly affecting the telomeric and subtelomeric chromatin or by indirectly affecting the chromatin of the rest of the genome, and hence the transcription of other genes involved in post-senescent survival.;In agreement with previous studies a role for Exo1p in post-senescent survival was identified. In addition the other RAD2 family members (Rad27p, Rad2p, Din7p and Yen1p) were also found to be involved in post-senescent survival. The RAD2 family members are involved in either inhibiting Type I or promoting Type II survival mechanisms immediately following senescence. The data is also suggestive of a role for the family in maintaining post-senescent survival.

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3

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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4

Lee, Joyce Hiu Yan. "Detection of Alternative Lengthening of Telomeres in Telomerase-Positive Cancers." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17252.

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Immortalisation is a hallmark of cancer and requires activation of a telomere lengthening mechanism (TLM) to counteract natural telomere shortening. There are two TLMs: telomerase, reported in 85% of cancers, and Alternative Lengthening of Telomeres (ALT), reported in 13% of cancers. Because most somatic tissues do not have TLM activity, TLMs are widely regarded as targets for the development of anti-cancer therapies. Preliminary data from the Reddel laboratory indicated that in non-small cell lung carcinoma (NSCLC), ALT was more common than previously reported but mostly at very low levels, and together with telomerase activity. A panel of NSCLCs were examined for ALT and telomerase activity by the C-Circle Assay (CCA) and TRAP assay, respectively. ALT (at least a low level) and telomerase coexisted in 18% of the panel, suggesting that ALT is a more important therapeutic target in NSCLC than previously appreciated. ALT+/telomerase+ tumours could result from intratumoral heterogeneity, or from individual cancer cells being dual-positive. It was hypothesised that ALT and telomerase may be activated spontaneously in the same cancer cell. As functional studies are not possible in human tumours, a panel of 384 cancer cell lines was examined to determine whether some telomerase+ lines have ALT markers. Three cell lines positive for CCA and TRAP activity were subcloned, each of which were positive for both TLMs, indicating that both TLMs may occur spontaneously in a cancer cell. One of the dual-positive lines (LOX IMVI) was used for functional studies. A DNA tag inserted into one of its telomeres was copied on to other telomeres; this is regarded as the most definitive empirical evidence for the presence of a functional ALT mechanism. Therefore, telomere lengthening is occurring via ALT activity. The functional significance of telomerase activity was also addressed by CRISPR/Cas9-mediated knockout of the TERC gene. A blood-based diagnostic for ALT could be important for patient management and could be provided by the CCA because C-Circles have been reported in the blood of patients with ALT+ osteosarcomas. It was demonstrated that C-Circles are secreted by cancer cells in vesicles, which may be lost during plasma isolation. This knowledge may allow correct implementation of the CCA as a blood-based diagnostic for ALT activity.

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5

Lee, Michael. "Next Generation Sequencing Strategies to Investigate Telomeres in Cancer." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21844.

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Telomeres are regions of repetitive DNA at the ends of human chromosomes that function to maintan the integrity of the genome. Telomere attrition is associated with celluar ageing, whilst telomere maintenance is a prerequisite for replicative immortality in cancer. There are two telomere maintenance mechanisms (TMM) that cancer cells can utilize, the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These two mechanisms synthesise telomeres in very distinct ways leading to differences in their telomere sequence composition and length. The molecular pathways involved with the selective activation of one TMM over the other remain unclear. In the last decade, whole genome sequencing (WGS) has proven to be an invaluable tool for the study of cancer, leading to the discovery of novel gene mutations that either drive the disease or confer an increased risk of developing it. The utility of this technique has led to the creation of vast cancer WGS data resources, in particular The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), that are available for cancer researchers worldwide to use. This provides an excellent resource from which we can better understand and associate genetic markers and telomere sequence content across cancers, as well as between tumours that utilise the telomere maintenance mechanisms telomerase and ALT. In order to utilise these available datasets, we require a WGS‐based approach to determine the TMM status of a tumour, as experimental validation requires obtaining cellular material. We propose that differences exist in telomere sequence composition and length between ALT and telomerase cancers that can be used to determine the TMM status of a tumour from WGS data. In this thesis, we first compared a range of WGS‐based telomere content measurement tools against the lab‐based technique q‐PCR, in order to assess their accuracy in quantitating telomere content, whilst simultaneously enriching for variant telomeric sequences. We then applied the best of these tools to two experimentally validated tumour datasets, pancreatic neuroendocrine tumours and melanomas, in order to directly analyse and compare the telomere sequence content between tumours that utilise ALT and those that do not. Finally, we exploited the differences in telomere sequence content in order to develop a classifier capable of determining the ALT status of a tumour from WGS data, and applied it to WGS data from 821 TCGA tumours, to identify the molecular pathways associated with the activation of ALT. We were able to demonstate that WGS‐based telomere content measurement tools perform well, producing comparable results to q‐PCR, with R2 = 0.9516. We have developed a methodology for the accuracte quantification of variant repeats within telomere sequences, identifying a number of differences in telomere sequence composition between ALT positive (+ve) and ALT negative (‐ve) tumours. We have demonstated the utility of this methodology to develop a WGS‐based classifier capable of predicting the ALT status of a tumour with 91.6% accuracy. Analysis of pathway mutations that were under‐represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways were involved in the survival of ALT tumours. Overall, we have demonstrated the capability and utility of WGS to investigate telomere sequence content, shown how telomere sequence content can be used to stratify cancers by TMM, and applied this to cancer WGS datasets to elucidate the genetic changes that associate with each TMM. This thesis provides a useful resource for future studies seeking to investigate the role of telomere sequence content in disease and overall health.

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6

Sherwood, Rebecca. "The Effect of the Copy Number of the Telomerase RNA Gene on the Elongation of Telomeres in Saccharomyces cerevisiae." Thesis, Boston College, 2008. http://hdl.handle.net/2345/532.

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Thesis advisor: Clare O'Connor
Telomeres are repeated sequences at the ends of chromosomes, which promote chromosome stability by preventing the loss of necessary nucleotides from the DNA with successive rounds of replication. Telomeres are elongated by the enzyme telomerase, which has both a protein component and an RNA component. In the yeast Saccharomyces cerevisiae, the TLC1 gene encodes the RNA component of the enzyme. Telomerase RNA interacts with several proteins to perform its function, including the Ku protein, which binds to the end of the DNA and helps to recruit telomerase to the chromosome thereby facilitating the lengthening of chromosome ends. Ku interacts with telomerase RNA at the site of a 48-nucleotide stem-loop on the RNA's structure. Previous experiments have shown that yeast strains engineered to carry two copies of the TLCI gene exhibit higher levels of telomerase RNA than those that have only one copy of the gene. Also, a yeast strain carrying a copy of the mutant tlc1Δ48 gene, which contains a deletion of the 48-nucleotide stem-loop, contains lower levels of telomerase RNA than a strain with the wild type TLC1 gene. This series of experiments is investigating whether the copy number of the telomerase RNA gene affects the elongation of telomeres in S. cerevisiae. In order to determine this effect, the de novo telomere addition of four strains was examined, as were the native telomere lengths of these strains. The assay indicated that the efficiency of telomere elongation was unchanged by increasing the copy number of the wild type gene but was increased upon increasing the copy number of the mutant gene. Analysis of the native telomere lengths showed that increasing the copy number of either the wild type or the mutant gene allowed the cells to maintain their telomeres at a longer length
Thesis (BS) — Boston College, 2008
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program

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7

Schuller, Christine Children's Cancer Institute Australia for Medical Research Faculty of Medicine UNSW. "Telomeres and telomerase in haematopoietic progenitors and bone marrow endothelial cells." Publisher:University of New South Wales. Children's Cancer Institute Australia for Medical Research, 2008. http://handle.unsw.edu.au/1959.4/41098.

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In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

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8

Henson, Jeremy D. "The role of Alternative Lengthening of Telomeres in human cancer." Thesis, The University of Sydney, 2006. http://hdl.handle.net/2123/1533.

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Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

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9

Henson, Jeremy D. "The role of Alternative Lengthening of Telomeres in human cancer." University of Sydney, 2006. http://hdl.handle.net/2123/1533.

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Abstract:

Doctor of Philosophy
Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

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10

Dagg, Rebecca Ann. "The extensive proliferation of human cancer cells with ever-shorter telomeres." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17341.

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Cellular immortalisation is currently regarded as an essential step in malignant transformation and is consequently considered a hallmark of cancer. Acquisition of replicative immortality is achieved by activation of a telomere lengthening mechanism (TLM), either telomerase or the alternative lengthening of telomeres (ALT), to counter normal telomere attrition. However, a proportion of malignancies are reported to be TLM-negative. The lack of serial untreated malignant human tumour samples over time has made it impossible to examine telomere length over time and hence determine whether they are truly TLM-deficient, or whether this is the result of false-negative assays. Here we describe a subset (11%) of high-risk neuroblastomas (NB) that lack evidence of any significant TLM activity despite a 51% 5-year mortality rate. Two NB cell lines derived from such tumours proliferated for 500 population doublings (PDs) with ever-shorter telomeres (EST). The EST cells had exceptionally long and heterogeneous telomere lengths as measured by terminal restriction fragment analysis and telomere fluorescence in situ hybridisation. Both cell lines were telomerase negative during culturing and did not have elevated markers of ALT or associated gene mutations. The telomeres of these cells shortened by 80 and 55 bases/PD, consistent with telomere attrition due to normal cell division, but did not reach senescence after 500 PDs in culture. This is conclusive evidence that cells from highly malignant, lethal tumours are able to undergo continuous proliferation in spite of an EST phenotype. The EST phenotype was rescued by activation of telomerase (via transduction with hTERT expression constructs) or ALT (spontaneous occurrence of a nonsense TP53 mutation, followed by spontaneous activation of ALT after 100 PDs). We also found that NB EST cells are very sensitive to topoisomerase I inhibitors indicating the potential to target the EST phenotype with topoisomerase I inhibitors in high-risk NB.

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Kartawinata, Maria Melissa. "Regulation of the recruitment of telomerase to telomeres in human cancer cells." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17128.

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Telomerase activity allows normal cells to achieve immortality, which is one of the hallmarks of cancer. It is required to maintain telomere length to protect chromosome ends from being recognised as damaged DNA. The protein TCAB1 is necessary for telomerase trafficking to telomeres, while telomeric protein TPP1 is essential for its interaction with telomeres. However, the mechanisms regulating these proteins are poorly understood. This thesis identifies TPP1 as a novel disease-causing gene in humans. A mutation in the ‘TEL patch’ (the surface area of TPP1 that interacts with telomerase) is likely to have caused short telomeres in a family with inherited bone marrow failure. We demonstrated that other amino acids within the TEL patch, F209 and S210, are also vital for the recruitment of telomerase to telomeres. Mutational analysis demonstrated that phosphorylation of S255 of TPP1, possibly by ATM, regulates the function of TPP1 in recruiting telomerase to telomeres. Phosphorylation of S64 of TCAB1 was hypothesised to regulate its function in telomerase localisation to telomeres; while the involvement of phosphorylation of this residue remains undetermined, we demonstrated that this region of the protein is important. Furthermore, S491 of TCAB1 emerged as a residue that is potentially involved in regulating telomerase recruitment to telomeres. Additionally, this thesis also provided the first evidence for an association between nuclear actin polymerisation and telomerase recruitment to telomeres. Inhibition of actin polymerisation or nuclear import disrupted telomerase recruitment, suggesting a novel role for nuclear structural integrity in this process. Collectively, these data increase our understanding of the regulation of telomerase recruitment to telomeres by the proteins TCAB1, TPP1, and actin. This understanding will inform new strategies for development of cancer therapeutic drugs and for developing better diagnostics and treatments for telomere biology disorders.

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12

Schulze, Franziska. "Die Telomerlänge als Prognosefaktor in MYCN nicht-amplifizierten Neuroblastomen." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-200943.

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Eines der charakteristischen Merkmale des Neuroblastoms stellt seine einzigartige biologische Heterogenität dar, die eine genaue Ausage des weiteren klinischen Verlaufes stark erschwert. Bestimmte prognostisch wirksame klinische, molekularbiologische und genetische Faktoren, wie zum Beispiel Alter bei Erstdiagnose, Tumorstadium, MYCN-Amplifikation und 1p Deletion, werden seit längerem zur Risikostratifizierung genutzt. Bereits in anderen Tumorerkrankungen konnte nun der Einfluß einer Telomerlängenveränderung auf das Gesamtüberleben von Patienten nachgewiesen werden. Telomere sichern die genomische Integrität und bestimmen maßgeblich die proliferative Kapazität jeder somatischen Zelle. Aktuelle Forschungsergebnisse legen die Vermutung nahe, dass Veränderungen der Telomerlänge auch in Neuroblastomen einen prognostischen Effekt auf das Gesamtüberleben haben. In diesem Kontext untersucht die vorliegende Arbeit den Zusammenhang zwischen Telomerlänge und Gesamtüberleben in 420 MYCN nicht-amplifizierten primären Neuroblastomen mit Erstdiagnosen von 1983-2001. Hierfür wurden die relativen Telomerlängen mithilfe einer neu etablierten monochromen multiplex q-RT-PCR ermittelt. Anschließend wurden diese sowohl mit ausgesuchten klinischen Variablen (Alter bei Erstdiagnose, Tumorstadium, Primärlokalisation des Tumors, Histologie, Geschlecht und Rezidivauftreten) korreliert als auch auf ihren Einfluß auf das Gesamt- und ereignisfreie Überleben untersucht. In Korrelation mit den klinischen Parametern konnte zwischen Alter bei Erstdiagnose und Telomerlänge ein eindeutiger Zusammenhang nachgewiesen werden. Je älter die Patienten bei Erstdiagnose, desto höher war sowohl der Anteil verlängerter Telomere als auch der extremer Telomerlängenveränderungen. Neuroblastome mit verlängerten Telomeren zeigten in der gleichen Altersgruppe ein verringertes Gesamtüberleben der betroffenen Patienten verglichen mit Neuroblastomen mit verkürzten Telomeren. Somit könnte eine Telomerlängenveränderung, insbesondere verlängerte Telomere, im klinischen Alltag als Hinweis auf einen prognostisch ungünstigen Verlauf genutzt werden.

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13

Hidalgo, Bravo Alberto. "Human telomeres and recombination." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/27809.

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Telomeres are DNA-protein complexes that help protecting the end of linear chromosomes. They consist of repetitive DNA, in mammals the repeat unit is the hexanucleotide TTAGGG, these repeats span 5-20 kb. Under normal conditions in somatic cells, telomeres get shorter with every population doubling until they reach a critical length and then, the cell enters a checkpoint called senescence or M1 where it stops dividing. If the cell escapes senescence and continues dividing with further telomere shortening, it reaches a second checkpoint called crisis or M2. Crisis is characterized by telomere dysfunction leading to genomic instability that can end with cell death. However, some cells achieve to maintain telomere length by activating a telomere maintenance mechanism (TMM). The presence of a TMM is a hallmark of cancer cells. Two TMM have been described in human cells, one is the through the enzyme telomerase, which is active in 85% of cancers, and the second is a homologous recombination (HR) based mechanism called Alternative Lengthening of Telomeres (ALT) active in 15% of cancers. The evidence that the ALT pathway relies in HR was the observation that sequences can be copied from one telomere to another in ALT+ but not in telomerase+ cells and that several genes involved in HR are necessary for ALT progression. The ALT pathway is not the only event involving HR at telomeres. It has been shown that the human herpesvirus 6 (HHV-6) can integrate into human telomeres. Interestingly, HHV-6 possesses perfect telomeric repeats within its genome. The proposed mechanism for integration if through HR between the telomeric repeats present in the virus with the human telomere repeats. The aim of this work is to unravel the molecular mechanism underlying the ALT pathway and HHV-6 integration. The data obtained will contribute to the understanding of HR in human telomeres.

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14

Bruneau, Julie. "Hemopathies spontanément regressives : exemples de la matocytose et de la papulose lymphomatoide." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00980884.

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En hématologie, du fait de leur évolution favorable, les tumeurs spontanément regressives comme lesmodèles myéloïde de la mastocytose, et lymphoïde de la papulose lymphomatoïde sont peu étudiés. Lamastocytose est une hémopathie myéloproliférative clonale dont les lésions cutanées peuvent régresserspontanément chez l'enfant alors que la maladie est chronique chez l'adulte. Les mutations chezl'enfant sont en partie différentes de celles retrouvées chez l'adulte. Nous avons montré in vivo dansles mastocytoses pédiatriques une expression diminuée de la télomérase, associée à des télomèrescourts. In vitro, grâce à deux modèles cellulaires comportant différents mutants de KIT de " typepédiatriques " ou de " type adulte ", nous avons montré une augmentation de la longueur destélomères dans le mutant adulte, associée à une moindre sénescence comparées aux mutantspédiatriques sans pour autant mettre en évidence de différence dans l'expression et l'activité de latélomérase. Ces observations permettent en partie d'expliquer la régression des formes pédiatriques.La papulose lymphomatoïde est une lymphoprolifération cutanée T CD30+ dont les lésions régressentspontanément sans traitement. Cependant 10 à 20% des cas sont associés à un lymphome. Nous avonsétudié la physiopathologie d'expression du PDGFRβ dans les cellules tumorales via l'activation deNotch1. L'étude des télomères et de la télomérase in vivo et in vitro est préliminaire, et montrenotamment des télomères courts dans les cellules tumorales. En conclusion, nous montrons d'une partque la longueur des télomères dans les mastocytoses et la papulose lymphomatoïde est corrélée àl'évolution de la maladie, d'autre part, nous identifions un type de mutation potentiellement agressivedans les mastocytoses. Nous recommandons le génotypage systématique de cette pathologie dans lebut d'un suivi clinique attentif lorsque les lésions sont persistantes ou évolutives.

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15

Alotaibi, Mohammad Kdaimes H. "Genes required to maintain telomeres in the absence of telomerase in Saccharomyces cerevisiae." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12589/.

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In the absence of telomerase, Saccharomyces cerevisiae telomeres erode leading to senescence. Rare cells can survive after this stage as they can elongate their telomeres utilizing homologous recombination. Two different types of survivors can be easily distinguished by Southern blot. Type I survivor cells, elongate the telomere by amplifying Y elements and require RAD51, RAD54, RAD55 and RAD57 for establishment. Type II survivors elongate their telomere by amplifying TG1-3 repeats, however, they require the following genes to be established: RAD50, MRE11 and XRS2, RAD59, SGS1 and KU80 in some cases. Both types require the gene RAD52. In this study several candidate genes were deleted individually in diploid type II survivor strains. The main aim of this work was to see if these genes were required for type II telomere maintenance. Most of these genes are not required for type II telomere maintenance at least until ~150 generations after deleting these genes. The exceptions were KU80 and RPB9. Ku80Δ strains switched to a new survivor type that is similar to type I and continued for the long term. RPB9 was required for two independent type II survivor strains to survive, whereas the third type II strain did not require this gene at ~150 generations after deleting the gene. After many generations (~ 350), this strain switched to type I. At long term propagation (~500 generations) after deletion of the candidate genes, all type II strains displayed telomere shortening until the propagation was stopped. However, Rad50Δ strains switched to type I after long term. Finally, the absence of the candidate genes did not affect the sensitivity of type II survivor strains to temperature. On the other hand, type II survivor strains with some genes deleted displayed sensitivity to UV.

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16

Elizalde, Violeta Serra. "Modulation of telomere length by oxidative stress in vitro and in vivo." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366579.

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17

Baldassarri, Enrico Junior. "Effetto dei controioni sul processo di autoassemblaggio di molecole di guanosina: uno studio strutturale." Doctoral thesis, Università Politecnica delle Marche, 2014. http://hdl.handle.net/11566/242740.

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Il progetto di dottorato presentato in questa tesi riguarda lo studio del self-assembling di molecole di Guanosina. Il progetto nasce dalla pi ambiziosa idea di studiare il comportamento, la stabilit´a e l’ aggregazione sopramolecolare di DNA a singolo filamento che porta alla formazione di strutture non convenzionali chiamate G-quadruplex. I G-quadruplex sono forme non canoniche del DNA, e l’interesse per queste strutture ´e iniziato quando comparve, nel 1962, un articolo di Gellert et.al., nel quale veniva dimostrata, per la prima volta, l’esistenza di queste inusuali strutture ad elica, formate dall’acido guanilico. I G-quadruplex sono correlati con le regioni terminali dei cromosomi chiamate telomeri, ricche in Guanosina. I telomeri sono strettamente legati all’attivit´a della Telomerasi, un enzima che replica le sequenze terminali, ripetute in tandem - TRs, nel cromosoma. Tale enzima ´e espresso a livelli molto bassi nelle cellule somatiche di diversi organismi, ma esente in elevate quantit´a in cellule cancerose (permettendo loro di replicarsi indefinitamente e di portarle ad una condizione di immortalizzazione che ´e alla base del processo di cancerogenesi). L’interesse per i G-quadruplex ´e legato ad una loro possibile attivit´a anticancro. In questa tesi viene presentato un esteso studio strutturale sul processo di autoassemblaggio della Guanosina 5’-monofosfato in diverse condizioni fisiche. Vengono introdotti e discussi di↵erenti esperimenti e↵ettuati presso i laboratori Di.S.V.A. dell’ Universit´a Politecnica delle Marche e presso le Large Scale Facilities utilizzando prevalentemente tecniche di di↵razione (XRD) di di↵usione a piccolo angolo dei raggi X (SAXS), ottenendo informazioni sui parametri strutturali, stabilit´a ed e↵etto dei controioni ed interpretando quindi l’interazione tra i telomeri utilizzando un semplice modello basato sulla formazione di G-quadruplex da singole molecole di Guanosina.
My PhD project described in this thesis concerns the study of supramolecular aggregation of guanosine molecules. The project starts from the more ambitious idea of studying the behavior, stability and supramolecular aggregation of single strands DNA that lead to the formation of unusual structures called G-Quadruplexes. G-Quadruplexes are noncanonical forms of DNA and the interest for these structures origins from an article of Gellert et.al. in 1962, when these helical structures formed by guanylic acid were identified for the first time. G-Quadruplexes were then correlated with the Telomeres rich in guanosine at the end of chromosomes. Telomeres are tightly bound to Telomerase, the enzyme that replicates the tandem repeated sequences (TRs) in chromosome, and is expressed at very low levels in somatic cells of di↵erent organisms but is present in high amounts in cancer cells (allowing them to replicate indefinitely and bringing to an immortalization condition that due the carcinogenesis process). Therefore, the interest in G-Quadruplexes is linked with the several hypotheses on possible anti-cancer activities. In this thesis I present an extended study on the self-assembly process and behavior of Guanosine 5’-monophosphate under di↵erent physical conditions. I performed several experiments at Di.S.V.A. Laboratories and at di↵erent European Large Scale Facilities (LSF) using various techniques such as Small Angle Scattering (SAS), X-ray Di↵raction (XRD), obtaining information on the structural parameters, stability, counter-ion e↵ects and interactions between telomeres using a simple model based on G-quadruplex formation by Guanosine molecules.

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18

Brouilette, Scott Wayne. "Telomeres and coronary heart disease." Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/29899.

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Using mean telomere length as a marker of biological age, I show that: 1. Subjects with premature myocardial infarction (MI) have significantly shorter telomeres than age-sex matched, healthy, controls. The mean telomere length in MI subjects was similar to controls almost 11 years older. 2. Healthy young adult children of families with a strong history of premature MI have shorter telomeres than age matched children of families without such a history. 3. Shorter telomere lengths are associated with increase risk of subsequent CHD events in a prospective study. This analysis was carried out on samples collected in the West of Scotland Coronary Prevention Study (WOSCOPS). This randomised blinded trial was designated to examine the benefits of statin treatment on preventing CHD and showed a 30% reduction of events in those treated with pravastatin. Interestingly, my analysis showed that this benefit of statin is only seen in those subjects at higher risk of CHD based on their telomere length.;As the final part of the thesis I carried out a quantitative linkage trait (QTL) analysis in sib-pairs in an attempt to identify genetic loci regulating telomere length. I report the mapping of a major QTL on chromosome 12 that determines almost 50% of the inter-individual variation in mean telomere length.;These findings support a novel "telomere" hypothesis of CHD. They indicate that telomere biology is intimately linked to the genetic aetiology and pathogenesis of CHD. Specifically, the findings suggest that (i) those individuals born with shorter telomeres may be at increased risk of CHD (ii) rather than individual genes, a more global structural property of the genetic material may explain the familial basis of CHD (iii) variation in telomere length may explain, in part, the variable age of onset of CHD. The findings provide several new avenues for future research.

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Arora, Amit, Mark A. Beilstein, and Dorothy E. Shippen. "Evolution of Arabidopsis protection of telomeres 1 alters nucleic acid recognition and telomerase regulation." OXFORD UNIV PRESS, 2016. http://hdl.handle.net/10150/622915.

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Protection of telomeres (POT1) binds chromosome ends, recognizing single-strand telomeric DNA via two oligonucleotide/oligosaccharide binding folds (OB-folds). The Arabidopsis thaliana POT1a and POT1b paralogs are atypical: they do not exhibit telomeric DNA binding, and they have opposing roles in regulating telomerase activity. AtPOT1a stimulates repeat addition processivity of the canonical telomerase enzyme, while AtPOT1b interacts with a regulatory lncRNA that represses telomerase activity. Here, we show that OB1 of POT1a, but not POT1b, has an intrinsic affinity for telomeric DNA. DNA binding was dependent upon a highly conserved Phe residue (F65) that in human POT1 directly contacts telomeric DNA. F65Amutation of POT1aOB1 abolished DNA binding and diminished telomerase repeat addition processivity. Conversely, AtPOT1b and other POT1b homologs from Brassicaceae and its sister family, Cleomaceae, naturally bear a non-aromatic amino acid at this position. By swapping Val (V63) with Phe, AtPOT1bOB1 gained the capacity to bind telomeric DNA and to stimulate telomerase repeat addition processivity. We conclude that, in the context of DNA binding, variation at a single amino acid position promotes divergence of the AtPOT1b paralog from the ancestral POT1 protein.

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20

Xin, Xing. "Effects of polychlorinated biphenyls (PCBs) on telomere maintenance in hematopoietic stem cells and progenitor cells." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/2026.

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Polychlorinated biphenyls (PCBs) are synthetic persistent organic compounds that are known to be carcinogenic to humans. Changes in telomerase activity and telomere length are hallmarks of aging and carcinogenesis. Retention of telomerase activity and long telomeres are key characteristics of stem cells and progenitor cells. I hypothesize that PCBs modulate telomerase activity and telomeres of hematopoietic stem cells and progenitor cells via interference of gene regulation and potentially disrupt cell differentiation. To investigate this possibility, I used progenitor-like cells, human promyelocytic leukemia cells (HL-60), and stem cells from rat bone marrow. I show that PCB126 and PCB153 display toxic effects on telomerase activity, telomere length and their related gene expression in progenitor-like HL-60 cells, but they did not exert much effect on differentiation. Further, an in vivo/in vitro study using rat bone marrow cells shows that PCB126-induced hematotoxicity, evidenced by reduction in telomerase activity and TERT gene expression, an increase of the differentiation and a change in the differentiation direction towards granulocytes, which indicate an effect on stem cell function. I also show that the most potent dioxin-like congener, PCB126, regulates hTERT gene expression by activation of the AhR pathway. Both AhR and ARNT work together as a repressor of hTERT transcription. This research improves our understanding of mechanisms of PCB126 and PCB153 toxicity on hematopoietic stem cells and progenitor cells, which will ultimately have significant implications for human health.

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21

Westin, Erik R. "Characterization of telomeric defects and signal transduction pathways in Dyskeratosis Congenita cells." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/1276.

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Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

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Garg, Aggarwal Mansi. "Characterization of the role of SUMO in telomere length homeostasis and overhang processing at yeast telomeres." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/68661/.

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Lu, Robert. "The FANCM-BLM-TOP3A-RMI1/2 complex suppresses telomere replication stress and Alternative Lengthening of Telomeres." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23414.

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In humans, telomeres consist of ′TTAGGG′ repeats, which form a nucleoprotein complex that caps the ends of chromosomes. Telomeres are constitutively bound by a six-member protein complex called shelterin. The telomere nucleoprotein structure is essential for preventing the recognition of chromosome ends as DNA double-strand breaks, and for suppressing aberrant repair processes, including end-joining and recombination. When telomeres become critically short, they can initiate a DNA damage response, thereby triggering replicative senescence. Cancer cells are able to overcome this proliferative barrier and immortalise through the reactivation of a telomere maintenance mechanism. The majority (85-90%) of cancers utilise a ribonucleoprotein reverse-transcriptase complex called telomerase to extend telomere repeats, while the remaining 10-15%, especially several paediatric cancers, utilise a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT). ALT cancers tend to have poor prognosis and ALT activity is enriched in several paediatric cancers. ALT activity can also be activated as an adaptive response to treatment therapies that inhibit telomerase. Consequently, there is a need to develop therapies that inhibit or exploit the mechanistic weaknesses of ALT. We sought to study the role of FANCM, a DNA fork translocase that remodels and protects stalled replication forks, at ALT telomeres. FANCM canonically functions as the key sensor and fork remodeller in the Fanconi Anaemia (FA) pathway of inter-strand crosslink (ICL) repair. Its canonical function is to recruit the FA core complex and facilitate ID2 complex (FANCI/FANCD2) mono-ubiquitination at ICL-stalled forks. Yet FANCM has broader roles in preventing replication defects induced by non-ICL sources. We found that FANCM depletion dramatically induces ALT phenotypes, including extrachromosomal telomeric DNA species such as C-circles, large APBs, and break-induced replication events resulting from damaged telomeres. This induction was dependent on the key break-induced replication proteins POLD3 and BLM, the telomeric G-strand-binding shelterin component POT1, and the replication fork translocase SMARCAL1. We then determined which functional domains of FANCM were required to suppress ALT activity, and whether known domains required for ICL repair were necessary. We found that the K117R mutant and, to a lesser extent, the MID and ERCC4 domain mutants were defective at suppressing several ALT phenotypes and telomere dysfunction. Overall, the MM2 domain mutant produced the most striking results, indicating that the MM2 domain was the most important domain required for the suppression of ALT activity. This suggests that the role of FANCM in suppressing ALT requires its interaction with the BLM/TOP3A/RMI1/2 (BTR) complex. We then demonstrated the therapeutic potential of inhibiting the MM2 domain interaction with the BTR complex using a small molecule inhibitor and an inducible fusion peptide construct. While the drug was moderately selective towards ALT cells, the fusion peptide displayed remarkable inhibition specifically of ALT-cells. Overall, we demonstrate that ALT telomeres are particularly dependent on FANCM to prevent excessive ALT activity, which is ultimately detrimental for viability. Therefore, we present a means to selectively kill ALT cells via disruption of the MM2 domain of FANCM.

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Brault, Marie Eve. "Telomeres and telomerase: role in human cancer, the premature aging syndrome dyskeratosis congenita and frailty." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117043.

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Telomeres and telomerase stand at a junction of cellular processes that govern aging, cancer and disease. Premature aging syndromes and age-related diseases are characterized by short telomeres which compromise cell function and viability, whereas cancer cells are able to reactivate telomerase or alternative lengthening of telomeres (ALT) mechanisms to maintain their telomeres and become immortal.Telomeres and telomerase represent very attractive targets for the development of anticancer therapies. However, there is concern that these therapies may lead to cell resistance, including the reactivation of telomerase or ALT in telomerase-positive cells. Here we show that telomeric recombination can be promoted by telomere-induced dysfunction, an anticancer strategy currently in development, despite the presence of an active telomerase. Our results highlight an important potential mechanism of cancer cell resistance, but might also help to understand the interplay between telomerase and the ALT pathway in the cell. Defective telomere maintenance is associated with premature or accelerated-aging disease. Mutations in almost every component of the telomerase holoenzyme have been found to be implicated in Dyskeratosis congenita (DC), revealing the importance of a functional telomerase for stem cell maintenance and cell proliferative potential. We found that the telomerase component dyskerin is sumoylated on highly conserved lysines and that lysine-to-arginine dyskerin mutants reproduce the phenotype observed in DC. Our findings identify that impaired post-translational modifications can lead to DC, and importantly point to new possibilities in the treatment of DC.Finally, we investigated the complex relationship between telomere maintenance, frailty and cardiovascular disease. We examined the feasability of measuring telomere length as a predictor of morbidity in elderly patients undergoing cardiac surgery. Our preliminary data do not identify telomere length as a predictor of surgery outcomes. Our findings also suggest that telomere length measurement in an epidemiological/clinical context must be interpreted with great caution.
Les télomeres et la télomérase se rencontrent à la jonction de processus cellulaires qui régulent le vieillissement, le cancer et certaines maladies. Plusieurs maladies de vieillissement précoce ou maladies associées au vieillissement sont caractérisées par la présence de courts télomères, qui compromettent la viabilité et la fonction de la cellule, alors que les cellules cancéreuses sont capables de réactiver la télomérase ou un mécanisme alternatif d'élongation des télomères (ALT) pour maintenir leurs télomères et devenir immortelles. Les télomères et la télomérase représentent des cibles très attrayantes pour le développement de thérapies anti-cancer. Il existe toutefois certaines possibilités que ces thérapies conduisent au développement d'une résistance chez la cellule. Ces mécanismes de résistance peuvent inclure la réactivation de l'enzyme télomérase ou réactivation du mécanisme ALT dans les cellules télomérase-positives. Nous démontrons que la recombination des télomères peut être induite par une dysfonction des télomères, une stratégie anti-cancer présentement en développement, et cela malgré la présence de télomérase dans la cellule. Nos résultats mettent l'emphase sur un mécanisme potentiel de résistance, et pourraient aussi permettre de mieux comprendre comment le mécanisme ALT et la télomérase sont régulés dans la cellule. Un maintien déficient des télomères est associé aux maladies de vieillissement précoce ou accéléré. Des mutations dans la majorité des composants de l'enzyme télomérase ont été retrouvées chez des patients atteints de Dyskératose congénitale (DC), révélant l'importance d'une télomérase fonctionnelle pour la fonction des cellules souches et le potentiel réplicatif des cellules. Nous avons identifié que la protéine dyskérine est sumoylée sur des lysines hautement conservées et que la présence de protéines mutantes dans la cellule imite les phénotypes associés à la maladie DC. Nos résultats démontrent qu'un défaut de modifications post-traductionnelles peut conduire à la maladie DC mais surtout, identifient de nouvelles possibilités pour le traitement des patients atteints de DC. Finalement, nous avons investigué la relation complexe qui existe entre le maintien des télomères, la fragilité et les maladies cardiovasculaires. Nous avons examiné le potentiel de mesurer les télomères pour prédire la mortalité et morbidité chez des patients âgés sur le point de subir une chirurgie cardiaque. Nos résultats préliminaires indiquent que la taille des télomères ne peut servir à prédire l'issue d'une chirurgie cardiaque. Nos résultats suggèrent que l'utilisation de la taille des télomères comme marqueur dans un contexte épidémiologique ou clinique doit être étudiée et interprétée avec précaution.

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25

DUCRAY, CAROLINE. "Dynamique de la taille des telomeres, activite telomerase et instabilite chromosomique dans des cellules humaines." Paris 6, 1999. http://www.theses.fr/1999PA066166.

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La plupart des cellules somatiques presentent des capacites proliferatives limitees. La duree de vie des cellules serait programmee et en partie gouvernee par la taille des telomeres. Les sequences telomeriques, structures specialisees localisees a l'extremite des chromosomes, raccourcissent a chaque division mitotique. Un raccourcissement trop important des telomeres serait un signal de sortie du cycle cellulaire. Les cellules tumorales, caracterisees par une proliferation illimitee et anarchique, peuvent contourner ces arrets obligatoires. Un des mecanismes d'immortalisation des cellules passerait par l'activation de la telomerase. Le travail presente ici est axe sur une etude approfondie de l'evolution de la taille des telomeres dans differents contextes telomerasiques et de proliferation cellulaire. Une premiere etude consiste a suivre l'evolution de la taille des telomeres dans des cellules en proliferation intense. Un raccourcissement telomerique progressif est observe dans les lymphocytes humains stimules a se renouveller rapidement par le virus du sida. Cette hyperproliferation des cellules du systeme immunitaire se traduit par une disparition des lymphocytes t4. Les lymphocytes des patients severement immunodeprimes, contenant moins de 200 lymphocytes t4 par mm 3, presentent des telomeres de taille diminuee par rapport a ceux des patients en cours d'infection. Le raccourcissement telomerique serait correle a terme a l'epuisement des capacites de renouvellement des cellules immunitaires. Les cellules immortelles sont egalement poussees a se diviser activement. Un deuxieme modele base sur la transformation de fibroblastes humains par l'agt de sv40 a permis une etude approfondie du lien entre la regulation de la taille des telomeres, l'etat d'activation de la telomerase et leur relation avec l'evolution de l'instabilite chromosomique au cours de l'immortalisation. L'absence de telomerase, avant crise, est associee a une diminution de la taille des telomeres et une augmentation de l'instabilite chromosomique dans les quatre lignees etudiees. Apres crise, l'activation de la telomerase ne previendrait pas systematiquement l'erosion telomerique mais serait reliee a une stabilisation de l'instabilite chromosomique. De plus, l'heterogeneite intracellulaire de la taille des telomeres observee dans la lignee tp15. 5 tend vers une evolution autour d'une taille optimale en presence de telomerase avec diminution des telomeres longs en parallele avec une elongation des telomeres courts. Cela suggere que la telomerase aurait une action differentielle sur les telomeres suivant leur taille. La telomerase permettrait l'allongement et/ou la protection des extremites chromosomiques. Cette enzyme, marqueur de la majorite des cancers, serait une cible anticancereuse de choix. Le traitement a des doses croissantes de cisplatine de lignees, derivees de cellules tumorales ovariennes, sensibles et resistantes a cette drogue a montre un effet transitoire du cisplatine sur l'activite telomerase et la taille des telomeres. Cette drogue agirait indirectement sur la telomerase en perturbant la proliferation cellulaire. La telomerase serait une enzyme regulee ayant des roles multiples au niveau des telomeres, leur permettant d'osciller autour d'une taille optimale quelle que soit la lignee etudiee.

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26

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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27

Marzec, Paulina. "NR2C/F telomeric association drives telomere-genome rearrangements in ALT cells." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20179.

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L'immortalité cellulaire est toujours accompagnée par l'activation du mécanisme de maintien des télomères. Dans la plupart des cancers humains, ce rôle est assuré par l'enzyme télomérase. Cependant, dans 15 % des tumeurs, la télomérase n'est pas activée et les télomères sont maintenus par l'allongement alternatif des télomères (ALT), voie qui implique la recombinaison des télomères. ALT est plus fréquent dans les tumeurs provenant de tissus mésenchymateux (sarcomes), representant 40-60 % des cas, que dans les tumeurs épithéliales. Comprendre le mécanisme ALT est primordial dans les thérapies anti-cancéreuses puisque certaines drogues inhibant la télomérase conduisent souvent à l'activation de l'ALT.La voie ALT est définie par de caractéristiques typiques des télomères. Dans les cellules ALT, les recombinaisons aberrantes d'ADN ne se limitent pas aux télomères puisque les génomes sont souvent fortement réarrangés. Les liens de ces caractéristiques génomiques anormales et la maintenance des télomères atypique ne sont pas connues, mais l'instabilité du génome contribue certainement à la transformation. Notre équipe a montré que les récepteurs orphelins appartenant aux familles NR2C/F ont été trouvés enrichies dans les télomères des lignées cellulaires ALT. Nous avons proposé que ces facteurs puissent être recrutés aux télomères par liaison directe à la séquence répétée GGGTCA, un site de liaison à haute affinité pour ces protéines. Mon projet vise à comprendre (i) leur mécanisme de liaison et (ii) leur rôle, dans le processus d'ALT.Dans cette étude nous montrons que dans les sarcomes primaires humains, les télomères d'ALT sont souvent liés par des récepteurs nucléaires orphelins des sous-familles NR2C/F, en particulier dans les tumeurs au stade avancé. Ceci suggère un rôle actif de ces facteurs dans la progression tumorale ALT. En utilisant la technique de ChIP-sequencing, nous avons montré que les protéines NR2C/F se lient à une répétition directe amplifiée (DR0) aux télomères, et pas de manière significative à toute autre combinaison de motif GGGTCA. Nous avons également analysé la distribution sur tout le génome de NR2C2/F2 et TRF2, une protéine de liaison des télomères, dans des cellules ALT (-) et ALT (+). Bien qu'il n'y ait que peu de sites génomiques liés par TRF2 dans les cellules ALT (-), nous avons été surpris d'identifier plusieurs centaines de régions liées par TRF2 dans les cellules ALT (+). Plus surprenant, la grande majorité de ces régions spécifiques TRF2 ALT chevauche des sites endogènes de NR2C2/F2. Étant donné que ces sites ne contiennent généralement pas les répétitions des télomères, TRF2 est probablement recruté de façon indirecte. Conformément à cette interprétation, nous montrons que les facteurs NR2C/F entrainent un rapprochement des loci et sont responsables du regroupement atypique des télomeres dans ALT. De plus, un sous-ensemble de ces régions génomiques uniques a des additions hétérogènes des séquences télomeriques ALT, suggérant un rôle dans le recrutement des télomères par des protéines NR2C/F mais aussi une fonction de ciblage de recombinaison génomique. Systématiquement, nous trouvons que ces réarrangements des télomères/génome sont situés à proximité des motifs GGGTCA endogènes. Le caryotype spectral des lignées cellulaires ATL montre que les sites télomériques interstitielles sont fréquemment localisés aux niveaux des sites de translocations/réarrangements entre deux ou plusieurs chromosomes, ce qui est également observé dans les données de ChIPseq. Ces résultats suggèrent que les réarrangements entres les télomères et le génome pourraient participer à la formation d'un caryotype complexe ce qui caractérise environ 50% des sarcomes. De plus, l'addition de sites télomériques interstitielles dans le génome est spécifique des cellules ALT et est favorisée par les dommages de l'ADN
Cellular immortality is always accompanied by the activation of telomere maintenance mechanism. In most human cancers this role is fulfilled by the telomerase enzyme. However in 15% of tumors, telomerase is not activated and telomeres are maintained by an Alternative Lengthening of Telomeres (ALT) pathway that involves telomere-telomere recombination. Interestingly ALT is more prevalent in tumors originating from mesenchymal tissues (sarcomas), where it is present in 40-60% of cases, than in epithelial tumors. Understanding ALT maintenance is critical since inhibiting telomerase in tumors leads to the activation of ALT. The ALT pathway is operationally defined by typical telomere hallmarks. In ALT cells, aberrant DNA transactions are not restricted to telomeres since genomes are often highly rearranged. Whether these abnormal genomic features are linked to atypical telomere maintenance is not known, but genome instability is certainly contributing to transformation. We have previously shown that orphan receptors of the NR2C/F families were enriched at telomeres in ALT cell lines. We proposed that these factors could be recruited to telomeres through direct binding to the GGGTCA variant repeat, a high affinity binding site for these proteins. My project is aimed at understanding (i) their mechanism of binding and (ii) their role, if any, in the ALT process.We show that in human primary sarcomas, ALT telomeres are often bound by orphan nuclear receptors of the NR2C/F subfamilies, particularly in more advanced-stage tumors. This suggests an active role for these factors in ALT tumor progression. Using ChIP-sequencing, we show that NR2C/F proteins bind to an amplified direct repeat (DR0) at telomeres, and not significantly to any other GGGTCA motif combination. We also analyzed the genome wide distribution of NR2C2/F2 and TRF2, a telomere binding protein, in ALT(-) and in ALT(+) cells. While there are only few genomic sites bound by TRF2 in ALT(-) cells, we were surprised to identify several hundred regions bound by TRF2 in ALT(+) cells. More surprisingly, the great majority of these ALT specific TRF2 regions overlap with endogenous NR2C2/F2 sites. Since these sites usually do not contain telomere repeats, TRF2 is likely indirectly recruited. Consistent with this interpretation, we show that NR2C/F factors drive locus proximity. Moreover, a subset of these unique genomic regions harbor heterogeneous ALT telomere sequence additions, not only suggesting a telomere recruitment role for NR2C/F proteins but also a recombination targeting function in the genome. Consistently, we find these telomere/genome rearrangements are located close to endogenous GGGTCA motifs. Next, we wanted to evaluate a role of these rearrangements in formation of complex karyotype which characterize approximately 50% of sarcomas. We found by spectral karyotyping that interstitial telomeric sites are frequently located at translocation/ rearrangements sites between two or more chromosomes, which we could also observe in our ChIPseq data. Furthermore, we demonstrate that addition of interstitial telomeric sites to the genome is enhanced by DNA damage and specific for ALT genome. Therefore we conclude that NR2C/F factors target telomere proximity to defined NR2C/F regions which enables telomere-genome rearrangements under DNA damage condition. This contributes not only to efficient telomere recombination, but also it drives further genomic instability at selected NR2C/F sites.We believe we identified a new mechanism of telomere dysfunction potentially driving targeted genome instability and mediated by NR2C/F proteins in ALT cells which probably underlie complexity of sarcomas genome. Understanding the ALT mechanism allows designing NR2C/F-targeted therapies in treatment of ALT tumors and therapies for patients treated with anti-telomerase drugs to prevent ALT appearance

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28

Sousa, Rute Inês Silva e. 1983. "The Epigenetic regulation of Drosophila telomeres." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/96908.

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Drosophila telomere maintenance depends on the transposition of three specialized retrotransposons – HeT-A, TART and TAHRE (HTT). Controlling the activation and silencing of these elements is crucial to maintain telomere length homeostasis without compromising genomic instability. In this thesis, I have identified the role of different chromosomal proteins involved in creating the correct chromatin environment to achieve telomere length homeostasis and stability. JIL-1, together with HP1a and Z4, act as a boundary at the telomere-subtelomere frontier. The interplay of these proteins leads to an equilibrium in the activation/repression state of the telomere retrotransposons. Additionally, I have contributed to the finding that the HeT-A Gag protein is a key component targeting different protein complexes to the telomeres and guaranteeing genome stability. I have also been able to demonstrate that the Z4 partners DREF, TRF2 and KEN are also involved in the silencing of HTT, probably by mediating chromatin remodeling. Finally, I have identified a special subtelomere domain at the 4R telomere with different chromatin characteristics and demonstrated that SETDB1, HP1a and POF are involved in the regulation of the telomeric retrotransposons in the 4th chromosome. These results provide important insights to better understand how in Drosophila the telomere retrotransposons are orchestrated to achieve a telomere function analogous to telomerase telomeres in other eukaryotes.
El manteniment dels telòmers de Drosophila depèn de la transposició especialitzada de tres retrotransposons, HeT-A, TART i TAHRE (HTT). El control de l’activació i la repressió d’aquests elements és crucial a l’hora de mantenir la llargada telomèrica sense comprometre l’estabilitat genòmica. En aquesta tesi jo he pogut identificar el paper de diferents proteïnes cromosòmiques involucrades en crear un estat de la cromatina adient per mantenir la longitud i l’estabilitat telomèrica. JIL-1 juntament amb HP1a i Z4 ajuden a crear el llindar entre la frontera dels domini telomèric i subtelomèric. L’actuació conjunta d’aquestes proteïnes aconsegueix un estat d’equilibri activació/repressió dels retrotransposons telomèrics. A més a més, he contribuït a la descoberta de la implicació de la proteïna HeT-A Gag en el reclutament de diferents complexes proteics als telomèrs de Drosophila per poder garantir l’estabilitat telomèrica. També he pogut demostrar que altres membres dels complexes on participa Z4, com ara: DREF, TRF2 i KEN, estan també implicats en el silenciament dels retrotransposons telomèrics segurament per mitjà de la remodelació de la cromatina. Finalment he pogut demostrar que el domini subtelomèric del telòmer 4R, té una estructura cromatínica diferent a la resta dels dominis subtelomèrics dels altres cromosomes i he pogut demostrar que les proteïnes SETDB1, HP1a i POF estan implicades en la regulació de l’HTT del cromosoma 4. Els resultats d’aquesta tesi ajuden de manera substancial a comprendre com els retrotransposons telomèrics estan orquestrats per tal de poder fer una funció anàloga als telòmers de telomerasa en altres eucariotes.

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29

Wang, Zhuo. "Extracellular Inflammatory Signaling from Dysfunctional Telomeres." Thesis, University of the Sciences in Philadelphia, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10692989.

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Telomere dysfunction describes the catastrophic damage at telomeres, which often leads to genomic instability at the cellular level. There is rising evidence showing that telomere dysfunction also influences the extracellular environment with the inflammatory response. However, little is known about the molecular mechanism of this dysfunctional telomere-associated inflammation. In this dissertation, we identified extracellular forms of Telomeric repeat-containing RNA (TERRA), and demonstrated it might play a role in mediating the crosstalk of telomere dysfunction and inflammation. We found this cell-free TERRA (cfTERRA) is present in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. By characterizing extracellular fractions of the human lymphoblastoid cell line (LCL) culture media, cfTERRA is shown as a shorter form (∼200 nt) of cellular TERRA and co-purifies with CD63- and CD81-positive exosome vesicles that could be visualized by cryo-electron microscopy. Mass spectrometry and extracellular chromatin immunoprecipitation (ChIP) assays revealed that regular cfTERRA was physically interacting with histones and telomeric DNA. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells (PBMCs) stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10). Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. The levels of cfTERRA and DNA damage marker γH2AX were increasingly incorporated into the exosomes during telomere dysfunction. These dysfunctional telomere-derived exosomes activated a more robust transcription of inflammatory cytokines in PBMCs. These findings imply a previously unknown extrinsic function of TERRA and a potentially molecular mechanism of communication between telomeres and innate immune signaling in tissue and tumor microenvironments.

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30

Cross, Sally H. "Isolation and characterisation of human telomeres." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/13500.

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31

ORRU', FEDERICA. "Role of Telomeres in Onco-Hematology." Doctoral thesis, Università degli Studi di Cagliari, 2017. http://hdl.handle.net/11584/249705.

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Telomeres are protective structure located on the endings of human chromosomes, that prevent translocation and progressive shortening of DNA coding sequences. Several studies showed that telomeres are involved in regulation of cell lifespan and proliferative potential. The role of telomeres in oncogenesis is still a matter of active investigation. Recently, some papers identified telomere length alterations in patients affected by Chronic Myeloid Leukemia and Myelofibrosis. My research activity focused on the relation between telomere length and the outcome of patients with Chronic Myeloid Leukemia and Myelofibrosis. My aim was to investigate new therapeutic targets and outcome predictive factors. In particular, I pursued two research lines: first, I tried to evaluate the role of telomeres as biomarker to discriminate patients with Chronic Myeloid Leukemia that could safely stop tyrosine kinase therapy. Next, I investigated the relation between telomere length and response to Ruxolitinib, a recently approved JAK-STAT inhibitor, who proved to be highly beneficial to high-risk patients with Myelofibrosis. I enrolled a population of 32 patients affected by Chronic Myeloid Leukemia who discontinued imatinib after achieving complete molecular remission and 32 age-sex-matched controls, for whom was possible to evaluate telomere length. I performed the clinical management of these patients and the data analysis. The relative telomere length (RTL) was determined by q-PCR as the telomere to single copy gene (36B4) ratio normalized to a reference sample (K-562 DNA). Age-corrected RTL (acRTL) was also obtained. The 36-month probability of treatment-free remission (TFR) was 59.4 %. TFR patients showed shorter acRTL compared to relapsed (mean ± SD = 0.01 ± 0.14 vs 0.20 ± 0.21; p = 0.01). TFR was significantly higher in CML patients with acRTL ≤0.09 (78.9 vs 30.8 %, p = 0.002). These findings indicate that telomere length seems to be related to treatment free remission in CML patients, and may be a useful biomarker to select patients candidate to treatment interruption. Next, I enrolled a population of 11 patients affected by Myelofibrosis, eligible to Ruxolitinib treatment, for whom was possible to evaluate telomere length before and after a median of 1000 days of Ruxolitinib therapy. I performed the clinical management of this cohort, and the data analysis. The RTL was determined as stated before. Related samples Wilcoxon signed-rank test performed before treatment with Ruxolitinib showed that the mean RTL was shorter in patients compared with age-and sex-matched healthy controls (1.08 vs 1.26, respectively; P = 0.09). The most interesting finding was that Mann-Whitney Utest showed shorter acRTL in MF patients with high IPSS compared to patients with intermediate-2 IPSS (mean ± SD = 1.016 ± 0.22 vs 1.34 ± 0.14; p= 0.03); furthermore, median RTL increased significantly (1.30 vs 1.08; p = 0.018), showing overlapping values with the healthy controls. Median RTL elongation from baseline was 15%. These findings seem to indicate that telomeres are involved in the dynamics of Myelofibrosis evolution and treatment response, and may be a possible therapeutic target in these patients. In conclusion, this research highlights some interesting points of the complex relation between telomere length and outcome of patients treated for Chronic Myeloid Leukemia and Myelofibrosis. If confirmed, these findings will be a useful advance in understanding response to treatment in these populations.

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Elchinova, Elena Georgieva. "Analysis of the levels of monocyte subsets in patients with heart failure." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667760.

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La insuficiencia cardíaca es un síndrome, caracterizado por diferentes signos y síntomas clínicos debidos a una anomalía estructural o funcional del corazón. Es una de las cardiopatías más predominantes en los países desarrollados, tanto desde el punto de vista epidemiológico como de sus manifestaciones clínicas. La insuficiencia cardíaca es un problema médico creciente relacionado con una alta tasa de hospitalización e importante mortalidad y un pronóstico desfavorable con coste socioeconómico muy elevado en todo el mundo. Los monocitos son una población heterogénea de células efectoras con funciones clave en el mantenimiento y la restauración de la integridad del tejido y del sistema inmunológico. Mediante citometria de flujo se pueden separar tres subpoblaciones de monocitos humanos distintos: clásicos (CD14 ++ / CD16–), intermedios (CD14 ++ / CD16 +) y no clásicos (CD14– / CD16 +). Poco se sabe acerca de la importancia, la relación entre los niveles de los monocitos circulantes en sangre periférica y su distribución en la insuficiencia cardíaca, incluso menos se conoce si estos parámetros podrían usarse como marcadores predictores de la progresión de la enfermedad. El objetivo principal del proyecto actual fue evaluar la relación entre los niveles y la distribución de las diferentes subpoblaciones monocitarias y la longitud de sus telómeros en pacientes con insuficiencia cardíaca y los eventos adversos, comomortalidad y hospitalización por insuficiencia cardíaca. La tesis doctoral actual describe tres estudios, respectivamente de 28, 400 y 101 pacientes ambulatorios, tratados consecutivamente en una unidad multidisciplinaria de insuficiencia cardíaca desde diciembre de 2013 hasta mayo de 2015. Todos los procedimientos del estudio se realizaron de acuerdo con todos los estándares éticos y todos los participantes proporcionaron un consentimiento informado por escrito. Se extrajeron muestras de sangre periférica de todos los pacientes para su posterior análisis mediante citometría de flujo. Las muestras se incubaron directamente con anticuerpos monoclonales con fluorocromos contra antígenos de superficie específicos de monocitos, tipo CD86 (o HLA -DR), CD14 y CD 16, y en paralelo (en 101 muestras) se analizaron marcadores genéticos (telómeros) mediante un citómetro de flujo. (BD LSRFortessa) en el Departamento de Citolatría de la IGTP. Se analizó la distribución porcentual de cada subconjunto de monocitos y también se determinó cuantitativamente su recuento de células absoluto (U/mL). Durante nuestro proyecto pudimos establecer un nuevo método de análisis conjunta de subpoblaciones de monocitos y con determinación de la longitud relativa de los telómeros, que resulto rápido, preciso y mucho más barato que otros métodos utilizados previamente. En nuestro estudio, la subpoblación intermedia se asoció de forma independiente en el análisis multivariable con mortalidad por todas las causas y con la variable compuesta (mortalidad por todas las causas o ingreso por insuficiencia cardiaca). La determinación cuantitativa del recuento de células absoluto de cada subpoblación de monocitos expresado en U/mL fue superior desde el punto de vista pronóstico al porcentaje de estas subpoblaciones. Se observó una reducción de aproximadamente el 22% en la longitud de los telómeros durante un año en los monocitos de nuestros pacientes, aunque la longitud relativa y el cambio en la longitud de los telómeros no se asociaron significativamente con los resultados. Por lo tanto, no es probable que el cambio en la longitud de los telómeros sea un biomarcador útil de la progresión de la insuficiencia cardíaca. Los monocitos y las subpoblaciones de monocitos podrían usarse en el futuro no solo como un factor predictor, sino que también podrían tomarse en consideración como parte de una terapia de inmunomodulación para los pacientes con insuficiencia cardíaca.
Heart failure is a disorder characterized by different clinical signs and symptoms due to a structural or functional anomaly of the heart. It is the most predominant heart disease in developed countries, both from epidemiological point of view and clinical implications. Indeed, it is a growing medical problem related to major hospitalization needs and high mortality, with significant economic and population burden worldwide. Established prognostic factors, such as age, sex, aetiology, comorbidities, New York Heart Association functional class, left ventricle ejection fraction, and routine laboratory markers might fail to completely and individually predict disease progression and mortality. A good risk stratification strategy is crucial as risk might be refined using several biological biomarkers of different pathophysiological processes that the former mortality risk factors do not necessarily directly reflect. That is why efficient and reliable new prognostic predictor markers are of upmost importance and relevance for the future management of the disease. Monocytes are a heterogeneous population of effector cells with key roles in the maintenance and restoration of tissue integrity. Three distinct human monocyte subsets can be identified by flow cytometry: classical (CD14++/CD16–), intermediate (CD14++/CD16+) and non-classical (CD14–/CD16+). Little is known about the importance, relationship between the levels of the circulating monocytes and their distribution in heart failure, even less if these parameters could be used as a predictor markers for the progression of the disease. The main objective of the current project was to assess the relationship between the levels and distribution of the different circulating monocyte subsets and the length of its telomeres in outpatients with heart failure with adverse events, namely mortality and heart failure hospitalizations. Three cohorts of respectively 28, 400 and 101 ambulatory patients, consecutively treated at a multidisciplinary heart failure Clinic from December 2013 to May 2015 were included in the studies described in this doctoral thesis, independently of the data of their entry into the heart failure Clinic program. All study procedures were performed in accordance with all ethical standards and all participants provided written informed consent. Peripheral blood samples of all patients were extracted for subsequent analysis by flow cytometry. The samples were incubated directly by means of monoclonal antibodies with fluorocromes against monocyte specific surface antigens, type CD86 (or HLA -DR), CD14 and CD 16 and in parallel (in 100 samples) genetic markers (telomeres) were subsequently analyzed by flow cytometer (BD LSRFortessa) in the Department of Citolatry of the IGTP. The percentage distribution of each monocyte subset was analyzed and their absolute cell count (U/mL) was also determined quantitatively. We were able to establish an innovating, accurate and much less expensive method than established ones for simultaneously measuring the different monocyte subsets and the its relative telomere length. In our study, the intermediate subset was independently associated with all-cause death and the composite end-point of all-cause death or heart failure hospitalization, in multivariable analyses. The quantitative determination of the absolute cell count of each monocyte subset expressed by U/mL was superior from the prognostic point of view than the percentage of these monocyte subsets in outpatients with Heart failure. We observed about 22% reduction in telomere length over 1 year in the monocytes of our patients, being the baseline telomere length and change in telomere length not significantly associated with outcomes. Therefore, the change in telomere length is not likely to be a useful biomarker of heart failure progression. The monocytes and monocyte subsets could be used not only as a predictor factor but also might be taken into consideration as part of an immuno-modulation therapy in the future for the heart failure patients.

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33

Roberts, Amity Rondalyne. "Studies on the Control of Telomere Length and the Effects of Shorter Telomeres on Gene Expression in the Mouse." Thesis, Griffith University, 2013. http://hdl.handle.net/10072/366153.

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Telomeres are structures located at the ends of our chromosomes that protect against DNA damage and chromosome end-to-end fusions. The length of a telomere is important, as telomeres that are too short can elicit a DNA damage response that can lead to replicative cell senescence. Many studies have been carried out looking at the factors that affect telomere length. Two examples of such factors include ageing and epigenetic state. Telomere shortening has been observed with ageing in human and mouse tissue. However, studies in the human population have been confounded by extensive genetic variation. In inbred mice, whether telomere length changes in somatic and germline tissue with age remains unclear. In this thesis, an ageing study was performed on inbred C57BL/6J mice and telomere length was measured using terminal restriction fragment analysis. Surprisingly, little change in telomere length was found in somatic and germline tissue from these mice. The pattern of telomere lengths in one individual is complex and differs among inbred mice. However, a similar pattern was observed in somatic and germline tissue from the same mouse, confirming previous reports that telomere length is already set early in development and is either occurring in the gametes of the parents or early in the zygote.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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34

Sridhar, Akila. "The role of Tel1 at short telomeres." Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203802.

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DNA replication is initiated at replication origins, which are temporally regulated within the S phase of the cell cycle so that some origins initiate early, while others initiate late. S. cerevisiae telomeres of normal length replicate late during the S phase. However, shortened telomeres replicate early – coupling the regulation of length to the replication timing. The mechanism through which telomere length regulates the activation time of nearby replication origins is unknown. In this thesis, I find that Tel1, a homolog of human ATM kinase, is required for early replication of short telomeres. In the absence of Tel1, short telomeres of the yku70Δ mutant no longer replicate early. I tested the role of Histone H2A(X) phosphorylation at Serine-129, as the target of Tel1 kinase, in regulation of telomeric replication times. However, preventing this phosphorylation had only a minor effect, so H2A(X) is unlikely to be the sole Tel1 target in telomeric replication regulation. On the other hand, deletion of Rif1 – a telomeric length regulator – showed complete epistasis to tel1Δ in telomeric replication regulation, implying that Rif1 acts downstream of Tel1 in the regulation of telomeric replication. Proteomic analysis revealed Tel1-dependent phosphorylation of Rif1 upon telomere shortening, implicating Rif1 as a direct downstream target of Tel1. However, mutation of the Tel1-phosphorylation sites on Rif1 had only a small effect on telomere replication times, indicating that phosphorylation of Rif1 by Tel1 is not the sole mechanism governing replication timing of short telomeres.

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Markiewicz-Potoczny, Marta. "Responding to different types of damaged telomeres." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3745.

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Telomeres are bound by multiple ‘capping’ proteins, which protect chromosome ends from fusion and degradation. Mutations of telomeric proteins of Saccharomyces cerevisiae, cdc13-1 and yku70Δ, induce a telomere uncapping, generation of ssDNA by nucleases, cell cycle arrest and the DNA Damage Response (DDR). In other context the Mec1 checkpoint kinase is activated by Ddc1, Dpb11 and Dna2 in response to DNA damage. I tested how Ddc1, Dpb11 and Dna2 affect cdc13-1 and yku70Δ fitness. DPB11 and DNA2 are essential genes, therefore I analysed checkpoint defective alleles of DPB11, DNA2 and DDC1. I showed that Dpb11 and Dna2 have no effect on yku70Δ fitness in contrast to Ddc1, which slightly affects yku70Δ fitness and might have a role in cell cycle arrest of yku70Δ mutants. I confirmed that Ddc1 strongly, whereas Dpb11 slightly, affects cdc13-1 fitness. Dpb11 may contribute to the checkpoint function of Ddc1. I also found that the cdc13- 1 defect was suppressed by the dna2-W128A,Y130A allele when on a plasmid, but enhanced when integrated into the genome, and these effects might be due to variable dna2-W128A,Y130A copy number (in the genome versus on a plasmid). Surprisingly, in the course of my experiments, I found that deletion of DDC1 suppressed dna2Δ lethality. I confirmed that deletion of RAD9 suppresses dna2Δ lethality, and I found that deletion of RAD17, CHK1, MEC1 and POL32 also allows dna2Δ viability. I observed that elimination of Tel1, Rad53, Exo1, Mre11 or Rad27 do not suppress dna2Δ lethality. Based on the observation that checkpoint gene deletions suppress dna2Δ, whereas they exacerbate defects in other core DNA replication proteins, I propose that the essential function of Dna2 is in the telomeric Okazaki fragment processing. I also found that deletions of HCM1, XBP1 and ULS1, genes selected from a genome-wide screen, suppress growth defect of cdc13-1 and yku70Δ mutants, and ULS1 suppresses stn1-13 defect. Finally, I showed that thermo-sensitive cdc13-1 and cdc15-2 strains adapt to chronic low-dose stress. Adapted cells are fitter if more stress is applied, but are less fit in the absence of stress. I found the consequences of adaptation to be reversible.

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36

Walsh, Monica Eve. "The role of SUV methyltransferases at telomeres." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16620.

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Telomeres are nucleoprotein structures at the ends of linear chromosomes composed of 5′-TTAGGG-3′ tandem repeat arrays, bound by histone proteins and the telomere-specific binding protein complex shelterin. In somatic cells, telomeres undergo gradual attrition with each cell division, accompanied by a loss of proliferative capacity and eventual cell death. Cancer cells are required to activate a telomere maintenance mechanism in order to circumnavigate this telomere attrition, and thereby gain unlimited proliferative capacity. This can be accomplished through the activation of the ribonucleoprotein telomerase, or through the activation of a homologous recombination (HR) mediated mechanism known as Alternative Lengthening of Telomeres (ALT). Telomere specific proteins, along with telomeric nucleosomes, cap chromosome ends to prevent them from being recognised as DNA double strand breaks. Cancers that use ALT display elevated levels of telomere-specific DNA damage response (DDR) and aberrant telomeric chromatin, implicating a role for telomere chromatin in maintaining structural integrity. Tandem repeat sequences are characterised as being highly recombinogenic. In order to prevent aberrant telomere recombination events, telomeres are maintained in a heterochromatin-like state, being enriched in the H3K9me3 and H4K20me3 heterochromatin marks. The post-translational methylation of H3K9 is achieved by the histone methyl transferase SUV39 family of proteins, which function to increase chromatin compaction. Alteration of these heterochromatin marks in mice results in the induction of ALT phenotypes. As ALT telomeres rely on a HR mechanism of telomere extension, aberrations in telomeric heterochromatin are believed to mediate HR, and therefore facilitate the growth of ALT cancers. The aim of this thesis was to explore the role of SUV39 proteins, and their heterochromatic mark H3K9me3, in maintaining telomere structural integrity. By modulating the expression of SUV39 proteins in a panel of human cancer cell lines, we were able to investigate whether telomeric chromatin state affected telomere recombination, telomere protection and telomere length maintenance. Through SUV39 depletion studies, we demonstrated that loss of H3K9me3 at telomeres results in exacerbated telomere dysfunction, detected by the association of DDR proteins at telomeres. This result revealed for the first time that heterochromatin state is important to the maintenance of telomere structure. One caveat was that this increase was only observed in cells with an already high basal level of DNA damage, implying that loss of H3K9me3 was only able to perturb telomeres with underlying structural defects. This result was independent of the employed TMM of the cell. Through SUV39 overexpression studies, we demonstrated that increased compaction resulted in a decrease in DDR at telomeres only in ALT cells, suggesting that the structural defects that underlie ALT telomeres can be supressed through increased heterochromatin. Not all ALT cell lines were able to be protected from DDR, suggesting that factors other than heterochromatin mediate the extent of telomere dysfunction at these cancer cell lines. Through depletion of the telomere capping protein, telomere repeat binding protein 2 (TRF2), we revealed that SUV39 overexpression was able to confer a protective effect in certain ALT cells. This reduction in DDR, however, could not be maintained following further telomere deprotection by the depletion of both telomere repeat binding protein 1 (TRF1) and TRF2. These results suggest that while telomeric heterochromatin may have a role in telomere DDR suppression, it is not able to confer a protective role upon exhaustive telomere insult. Overall in this thesis we demonstrated that both the depletion and overexpression of SUV39 proteins did not alter telomere length in cells which utilise either ALT or telomerase-mediated telomere extension. These findings are in direct contrast to heterochromatin-based telomere length studies carried out in both mouse and swine cell lines. Furthermore, we were able to show through chromatin immunoprecipitation studies that the SUV39 family of proteins directly associate with telomeric chromatin, albeit at a very low abundance. This result provides evidence demonstrating that there is a direct interaction between telomeres and SUV39 in human cells, but its association does not regulate telomere length.

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Östlund-Lagerström, Lina. "Effect of long-term ultra-endurance training on telomere length and telomere regulatory protein expressions in vastus lateralis of healthy humans." Thesis, Örebro universitet, Hälsoakademin, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-15859.

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Viviescas, Maldonado Maria Alejandra. "Análise Estrutural do Componente Telomerase Transcriptase Reversa de Leihmania major." Botucatu, 2018. http://hdl.handle.net/11449/154850.

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Orientador: Maria Isabel Nogueira Cano
Resumo: Os parasitos do gênero Leishmania são protozoários primitivos entre os quais estão os causadores de um espectro de doenças conhecidas como leishmaniose, as quais afetam milhões de pessoas no mundo inteiro, sendo o Brasil um dos países que apresenta maior número de casos por ano. Os tratamentos e vacinas disponíveis para a leishmaniose apresentam problemas como toxicidade, alto custo e baixa eficiência, tornando importante a busca por novos alvos terapêuticos. Dada sua importância para a estabilidade genômica e proliferação celular, os telômeros têm sido considerados como alvos terapêuticos potenciais no tratamento da leishmaniose. Os telômeros são as extremidades físicas dos cromossomos lineares compostos por complexos ribonucleoproteicos que envolvem a interação entre proteínas, DNA e RNA. A maioria dos eucariotos mantêm o tamanho dos telômeros pela ação do complexo telomerase, que é minimamente composto por uma proteína (TERT, Telomerase Reverse Transcriptase) e um RNA longo não codificador (TER, Telomerase RNA). Os dois componentes do complexo telomerase em Leishmania sp. foram identificados, porém não há informação disponível sobre suas estruturas. O objetivo principal deste estudo foi a caracterização estrutural do componente TERT de Leishmania major. Utilizando alinhamentos múltiplos de sequências, foi possível verificar que os quatro domínios estruturais das telomerases canônicas estão presentes no componente TERT em Leishmania sp. Três destes domínios foram estudados ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Parasites of the Leishmania genus are primitive protozoa and among them are the causative agents of a spectrum of diseases called leishmaniasis, which affect millions of people in tropical countries worldwide, being Brazil one of the countries with higher number of cases each year. The drugs or vaccines available to treat leishmaniasis present problems such as toxicity, excessive cost a low efficiency, so it is important to search for new potential therapeutic targets. Due to their importance in genome stability and cell proliferation, telomeres have been considered a potential therapeutic target against leishmaniasis. Telomeres are the ends of linear chromosomes composed by ribonucleoproteic complexes that involve the interaction between proteins, DNA and RNA. Most eukaryotes maintain their telomere length by the action of the telomerase complex, minimally composed by a protein (TERT, Telomerase Reverse Transcriptase) and a long non-coding RNA (TER, Telomerase RNA). Both components of the Leishmania sp. telomerase complex have already been identified, however, little is known about their structure. This study had the aim to characterize the structure of the Leishmania major TERT component. Using multiple sequence alignments, we were able to verify that the four structural domains of canonical telomerases are present in Leishmania sp. Using different bioinformatic approaches three of these domains were independently studied. The TEN (Telomerase essential N-terminal) domain is... (Complete abstract click electronic access below)
Doutor

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39

Giardini, Miriam Aparecida. "Caracterização bioquimica e molecular do componente transcriptase reversa da telomerase de Leishmania spp." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317096.

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Orientador: Maria Isabel Nogueira Cano
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-10T06:46:56Z (GMT). No. of bitstreams: 1 Giardini_MiriamAparecida_D.pdf: 16478216 bytes, checksum: 881eff76c52bbb67a22537e86deba896 (MD5) Previous issue date: 2007
Resumo: Telômeros são complexos DNA-proteínas que protegem os cromossomos eucarióticos da degradação, garantindo estabilidade genômica. As seqüências teloméricas são ricas em G e apresentam uma protrusão 3¿ simples-fita que se estende em direção ao terminal cromossômico. Em Leishmania, os telômeros são compostos pela seqüência repetida 5¿-TTAGGG-3¿ e são replicados pela telomerase, a principal responsável pela manutenção dos terminais cromossômicos em eucariotos. Além de replicar os telômeros, o complexo holoenzimático da telomerase, composto pela transcriptase reversa da telomerase (TERT), pelo RNA da telomerase (TER) e por proteínas associadas, também atua como parte de um complexo de ordem maior que protege os terminais teloméricos. O entendimento do mecanismo de regulação da manutenção telomérica será de grande valor científico e poderá levar ao descobrimento de algum alvo potencial para o desenvolvimento de novas drogas anti-leishmania. Com esse objetivo, identificamos, clonamos e caracterizamos o gene que codifica o componente TERT em Leishmania spp.. O alinhamento múltiplo das seqüências através do programa ClustalW demonstrou que as telomerases de Leishmania apresentam muito mais homologia entre si do que com as proteínas de outros kinetoplastídeos e eucariotos. Experimentos de caracterização indicaram que a seqüência putativa do gene da telomerase de Leishmania localiza-se provavelmente em cópia única nos maiores cromossomos. Um único transcrito de RNA mensageiro foi encontrado nos promastigotas. Análises filogenéticas sugeriram que a telomerase de Leishmania pode representar uma ligação entre os mais antigos e os mais novos ramos das telomerases. Além disso, proteínas recombinantes foram expressas em sistema bacteriano, tornando possível a produção de anticorpos policlonais em coelhos. Experimentos de ¿Western blotting¿ e imunoprecipitação de cromatina indicaram que o anticorpo foi capaz de reconhecer a proteína nativa e que a telomerase de L. amazonensis interage in vivo com a seqüência telomérica rica em G. A atividade de telomerase de L. amazonensis foi purificada utilizando-se uma combinação de colunas cromatográficas. Testou-se a atividade enzimática em cada passo da purificação utilizando-se o ensaio ¿Two-tube TRAP¿. Os resultados mostraram que a atividade enzimática é encontrada nas frações purificadas pelas cromatografias de troca iônica, de afinidade por Heparina e de gel filtração. A atividade foi altamente enriquecida após a purificação por afinidade utilizando um oligonucleotídeo de DNA telomérico rico em G. Quando foi utilizado um oligorribonucleotídeo 2¿O-metil complementar à putativa seqüência molde do TER de Leishmania como ligante na cromatografia de afinidade, pouca ou nenhuma atividade enzimática foi eluída da resina, sugerindo que a interação entre a telomerase de L. amazonensis e este oligorribonucleotídeo é tão forte que não permite sua dissociação nas condições de eluição gentis necessárias para manter a atividade enzimática. Formas procíclicas de Trypanosoma brucei foram utilizadas para a construção do sistema ¿PTP-tagging¿, no intuito de futuramente purificar o complexo holoenzimático da telomerase. Em paralelo, ensaios de ¿primer extension¿ foram padronizados e identificou-se uma seqüência candidata ao gene do RNA da telomerase de T. brucei. Também foi identificada e clonada em L. amazonensis, uma seqüência homóloga à PinX1 humana, descrita como uma proteína que interage diretamente com a TERT humana e considerada um inibidor natural da atividade de telomerase
Abstract: Telomeres are protein-DNA complexes that protect linear chromosomes from degradation, providing genomic stability. The telomeric sequences are G-rich and contain a 3¿ single-stranded region that protrudes toward the chromosome end. In Leishmania, the telomeric DNA is composed by the conserved 5¿-TTAGGG-3¿ repeated sequence and it is replicated by telomerase. Telomerase is responsible for maintaining chromosome ends in most eucaryotes by adding new telomeric sequences to the G-rich strand. Besides replicating telomeres, the telomerase holoenzyme complex, composed by the reverse transcriptase component (TERT), the telomerase RNA (TER) and associated proteins, also works as part of the higher order complex that protects telomeric ends. Understanding the regulation of the telomeric maintainance mechanism may allow the discovery of potential targets to the development of new antileishmania drugs. Therefore, we identified, cloned and characterized the TERT gene in Leishmania spp.. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania TERT gene was probably located in single copy at the largest chromosomes. A single messenger RNA transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases. Besides that, recombinant proteins were expressed in bacterial system, allowing production of anti-LaTERT polyclonal serum in rabbits. Western blotting and chromatin immunoprecipitation assays indicated that the anti-LaTERT serum was able to recognize a native protein in nuclear and total extracts of the parasite and that L. amazonensis telomerase interacts in vivo with the G-richtelomeric sequence. We have also purified the L. amazonensis telomerase activity in order to better understand its biochemical features. Protein extracts of L. amazonensis containing telomerase activity were purified using combined chromatographic columns. Enzyme activity was tested in each purification step using the ¿Two-tube TRAP¿ assay. The results showed that enzyme activity is found in fractions purified by ion exchange (DEAE), Heparin affinity and gel filtration chromatographic methods. The activity was greatly enriched after affinity purification using a G rich telomeric DNA oligonucleotide as the ligand. When a 2¿O-methyl oligoribonucleotide complementary to the putative L. amazonensis TER template was used as a ligand in the affinity purification, little or no enzyme activity was eluted from resin, suggesting that the interaction between L. amazonensis telomerase and this oligoribonucleotide is too strong that disables its dissociation under the gentle elution conditions necessary to maintain enzyme activity. In order to identify the telomerase holoenzyme components, procyclic forms of Trypanosoma brucei were used to construct the PTP-tagging system. ¿Primer extension¿ reactions were also done in order to isolate and sequence an RNA candidate for the telomerase RNA gene in T. brucei. In addition, we have cloned a L. amazonensis homologue of the human PinX1 protein, previously known as a hTERT-interacting factor and as a potent telomerase inhibitor
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

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40

Lee, Hyemin [Verfasser]. "mEst1A (mouse ever shorter telomeres 1A) regulates telomere length and RNA quality control in murine stem cells / Hyemin Lee." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2012. http://d-nb.info/1024534235/34.

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41

Perrem, Kilian Thomas. "Molecular Studies of an alternative lengthening of telomeres (ALT) mechanism." Thesis, The University of Sydney, 2001. http://hdl.handle.net/2123/793.

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Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the 'end replication problem'. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the 'Telomere Hypothesis of Senescence'. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

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42

Perrem, Kilian Thomas. "Molecular Studies of an alternative lengthening of telomeres (ALT) mechanism." University of Sydney. Children's Medical Research Institute, 2001. http://hdl.handle.net/2123/793.

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Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

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43

Idol, Rachel A. "Telomeres and their associated factors in Arabidopsis thaliana." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4141.

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Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary for genome integrity. Telomeres are maintained by intermittent action of telomerase. I explored the kinetics of telomere length homeostasis in the model plant Arabidopsis thaliana by crossing wild type plants to different generations of telomerase deficient plants, and then analyzing telomere length in the resulting progeny. Unexpectedly, I found plants lacking telomerase for seven generations can lengthen telomeres when telomerase is reintroduced, but one generation is not sufficient to reestablish the telomere set point. Est1 is a non-catalytic component of the Saccharomyces cerevisiae telomerase holoenzyme. To investigate the role of Est1 in higher eukaryotes, I identified two putative Est1 homologues in Arabidopsis, AtEST1a and AtEST1b. Plants deficient in AtEST1a displayed no vegetative or reproductive defects. However, plants deficient for AtEST1b were sterile and had severe vegetative and reproductive irregularities. Surprisingly, no defects in telomere maintenance were observed in any single or double mutant line. This suggests that the Est1- like proteins in plants have evolved new functions outside of telomere length maintenance and end protection.One consequence of telomere dysfunction is end-to-end chromosome fusion. In mammals, telomere fusion is mediated through NHEJ and requires DNA Ligase IV (Lig4). Lig4 is an essential component of the NHEJ pathway along with the Ku70/Ku80 heterodimer and DNA-PKcs. To address the mechanism of chromosome fusion in Arabidopsis, we investigated the role of Lig4 in mutant combinations lacking TERT, the catalytic subunit of telomerase, and Ku70. Surprisingly, telomere end-to-end fusions were observed in ku70 tert lig4 triple mutants, suggesting that neither Lig4 nor Ku70 are required for the fusion of critically shortened telomeres in Arabidopsis. To investigate the origin of genome instability, terminal restriction fragment analysis was performed on triple mutants. Strikingly, telomeres diminished five to six-fold faster than in a tert single mutant. Moreover, in the triple mutants, telomere tracts were extremely heterogeneous, suggesting that the telomeres were exposed to catastophic nucleolytic attack. These data provide the first evidence that Lig4 contributes to telomere maintenance and chromosome end protection.

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44

Coleman, Joanna. "The analysis of variation at human autosomal telomeres." Thesis, University of Leicester, 1998. http://hdl.handle.net/2381/30306.

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The terminal regions of 7q and 12q show a high level of similarity (96 %) for at least 2 kb of DNA adjacent to the telomere. In addition, the start of the telomere repeat arrays coincide with respect to the flanking DNA at these two chromosome ends. This suggests that a duplication has occurred and that the proximal region of the telomere was involved. The 7q and 12q telomere-adjacent regions contain a diverged subterminal repeat that suggests multiple rearrangements have been involved in the formation of the modern 7q and 12q telomeres. A polymorphic telomere (the 'Nitru' telomere) has been isolated, and is located at 16p and 16q. The telomere-adjacent sequence from the Nitu telomere is present on 18 autosomes, but it is only adjacent to a telomere repeat array at these two chromosome ends. The Nitu telomere is present in approximately 8 % of individuals from various populations. Analysis of the interspersion patterns of TTAGGG and variant repeats, at the proximal end of these three autosomal telomeres, indicates that there is no similarity between them. The 7q and 12q telomeres can be distinguished easily despite the similarity in the flanking DNA. The Nitu telomere repeat arrays are different again, although it is not possible to determine the location (16p or 16q) by the telomere interspersion pattern or the 200 bp of DNA immediately adjacent. This may suggest that the Nitu telomere duplication is relatively recent. Analysis of single alleles at the 12q and Nitu telomeres demonstrates a high level of variation, suggesting a high level of turnover. The allelic variation within these autosomal telomeres indicates that the major mechanism involved in the turnover of repeats is intra-allelic.

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45

Vaziri, Homayoun. "Telomeres, DNA damage signaling molecules and cellular aging." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0020/NQ45836.pdf.

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46

Tsolou, Avgi. "Cellular responses to uncapped telomeres in eukaryotic cells." Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442349.

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47

White, Elizabeth W. "Selective Recognition of Quadruplex DNA by Small Molecules." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/chemistry_diss/10.

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Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

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48

Abbou, Scarlette. "Exploration préliminaire du rôle de la jonction à trois branches de la sous-unité ARN de la télomérase chez Saccharomyces cerevisiae dans le maintien de la longueur des télomères et dans la viabilité des cellules." Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/10647.

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La télomérase est essentielle pour le maintien des télomères. Elle compense le problème de réplication de l’ADN télomérique par l’ajout de séquences d’ADN aux extrémités des chromosomes. Chez l’humain, elle est très active dans les premiers stades du développement (embryon, fœtus). Puis, son activité est réprimée pour devenir indétectable dans la plupart des cellules. Ceci conduit au raccourcissement de l’ADN télomérique, à la déprotection de l’ADN et à un arrêt de division cellulaire, appelé senescence. Par contre, dans 90% des cellules de type cancéreux, elle est suractivée. Elle contribue donc à une capacité continue de la prolifération de ces cellules et à leur immortalisation. Notre organisme d’étude est la levure bourgeonnante, Saccharomyces cerevisiae. En plus de ses nombreux avantages d’utilisation, chez cette levure, la télomérase est exprimée constitutivement, ce qui signifie qu’elle nous rapproche le plus du contexte de cellule cancéreuse. Mon projet de maîtrise vise à étudier un des composants de la télomérase chez la levure S. cerevisiae, c’est-à-dire la sous-unité ARN, appelée Tlc1, et plus particulièrement une sous-partie de cet ARN, formant une jonction à trois branches (« Three Way Junction »). Jusqu’à présent, cette structure a été considérée comme étant non essentielle. Pourtant, cette structure très conservée a été démontrée comme étant essentielle à l’assemblage de la télomérase et à son activité, chez une grande variété d’espèces. Avec ce projet, j’ai tenté de déterminer si cette structure à trois branches a un quelconque rôle à jouer que ce soit dans l’assemblage ou dans l’activité de la télomérase. J’ai exploré cette structure en y réalisant des mutations et en analysant leurs effets sur la croissance cellulaire et sur la longueur des télomères. Parmi tous les mutants, la simple substitution d’un nucléotide spécifique, l’adénine 119, conduit à des télomères plus courts qui demeurent stables au fil des générations et les levures sont viables. De plus, ce raccourcissement est de l’ordre de la centaine de paires de bases lorsque la délétion d’une partie ou de la structure au complet est réalisée. C’est donc un raccourcissement significatif, représentant près d’un tiers de la longueur normale des télomères. Par ailleurs, sur des cellules présentant des télomères anormalement courts, l’ajout de ces mutations de la TWJ de TLC1 crée un phénotype létal.
Abstract : Telomerase is essential for telomere maintenance. It compensates for the End-replication problem by adding DNA sequences to the ends of chromosomes. In humans, telomerase is very active in the early stages of development (embryos, foetus). Later, its activity is repressed, and in most cells its activity becomes undetectable. This leads to telomere shortening, a deprotection of chromosome ends and to an arrest of cellular divisions, a highly regulated process also called cellular senescence. However, in cancer cells of 90% of all subtypes, telomerase is up-regulated. Hence, this enzyme promotes the proliferative capacity of cancer cells and their immortalization. The budding yeast Saccharomyces cerevisiae is our organism of study. In addition to its ease of access, telomerase is constitutively expressed in this yeast, which makes it a useful and inexpensive model of cancer cells. My master’s project aims at studying one of the telomerase components in S. cerevisiae, namely the RNA subunit Tlc1, and more specifically a part of this RNA, forming a Three-Way Junction (TWJ). So far, this structure was considered as non-essential for cell viability. However, this structure is highly conserved among species, and in diverse species it was shown to be crucial for telomerase assembly and activity. My project hence consisted in trying to determine whether or not this structure plays a role in telomerase assembly or activity. The requirements on this structure were explored by creating mutations and by analyzing their effects on cell growth and telomere length. Of all the mutants, a specific nucleotide substitution, Adenine 119 in the TWJ, leads to shortened telomeres, and this shortening is stable during further outgrowth. Furthermore, a telomere shortening of up to 100 base pairs is observed when a part or the complete TWJ structure is deleted. This shortening is quite significant as it represents about one third of the normal length of telomeres. Moreover, expressing these mutants of the TWJ in cells with short telomeres creates a synthetic lethal effect.

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49

VENTURINI, LORENZA. "TELOMERE MAINTENANCE MECHANISMS IN TUMOR OF MESENCHYMAL ORIGIN: EVALUATION OF PROGNOSTIC SIGNIFICANCE AND CHARACTERIZATION OF RELEVANT MOLECULAR PATHWAYS." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/171334.

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A limitless proliferative potential is one of the hallmarks of tumour cells and can be achieved through the activation of telomere maintenance mechanisms (TMM), which rely on telomerase reactivation (TA) or, alternatively, on recombination-based processes known as alternative lengthening of telomeres (ALT). Since a substantial fraction of tumours of mesenchymal origin utilizes ALT mechanisms, they represent an interesting model to study the molecular pathways involved in the activation of TMM. With the present work, we extended our knowledge about the prevalence and the prognostic significance of the two known TMMs in different soft-tissue sarcoma histotypes (Malignant peripheral nerve sheath tumors –MPNST-, leiomyosarcoma, liposarcoma) and mixed origin tumours (Wilms’ tumour), showing the relatively low frequency of telomerase activity in soft tissue sarcomas if compared to tumors of epithelial origin, and the important role of ALT in these malignancies. Controversial results have been obtained about the clinical relevance of TMMs, whose prognostic role seems to be dependent on tumor histotypes. Specifically, ALT is a strong determinant of an unfavorable outcome in liposarcoma and leiomyosarcoma patients, whereas it failed to significantly affect the outcome of patients with MPNST (for whom TA proved to be an important predictor of poor survival). This scenario could be due, at least in part, to the lack of standardized methods to properly classify tumours with respect to their TMM status; in this context, we comparatively analysed the prognostic relevance of ALT in a series of liposarcoma as a function of the characteristic used to classify the tumour, with the final aim to identify the most suitable ALT marker. Moreover, we also proposed to identify microRNAs expressed as a function of the different TMM operating in the tumour, which could be either involved in the regulation of such mechanisms or represent surrogate markers of TA-positive or ALT-positive phenotypes. The knowledge of the specific factors/pathways by which the two known TMMs are differentially regulated in distinct tumour histotypes of mesenchymal origin might be important for a better understanding of the pathogenesis of these malignancies and for the identification of new therapeutic targets.

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50

Kvaloey, Kirsti. "The long arm telomeres of the human sex chromosomes." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358686.

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Dissertations / Theses on the topic 'Telomeres'

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