To see the other types of publications on this topic, follow the link: Telomere.
Dissertations / Theses on the topic 'Telomere'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Telomere.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Johnston, Jeffrey Scott. "Combination therapy targeting telomere and telomerase /." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486462067841929.
Full text
2
Hirsch, Erica. "Telomerase activity and telomere lengths in fibroblast cells treated with ependymin peptide mimetics." Link to electronic thesis, 2005. http://www.wpi.edu/Pubs/ETD/Available/etd-050505-134911/.
Full text
3
Shakirov, Yevgeniy Vitalievich. "Telomeres and telomere binding proteins in Arabidopsis thaliana." Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/422.
Full textAbstract:
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.
4
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.
Full text
5
Gonzàlez, Busqué Núria 1980. "Snail1 controls telomere integrity and transcription and telomerase expression." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/663193.
Full textAbstract:
Snail1 transcription factor is the key inducer of epithelial-to-mesenchymal transition (EMT). Here, we describe a novel role for Snail1 on the regulation of telomere integrity and transcription as well as telomerase expression. FISH assays indicate an increase of telomere alterations in Snail1-depleted mouse mesenchymal stem cells (MSCs) and shorter telomeres. However, these cells present higher levels of TERT since Snail1 represses its expression, meaning that other mechanisms in telomere homeostasis are involved. In fact, telomeres are transcribed into a long non-coding RNA called telomeric repeat-containing RNA (TERRA). Here we report that Snail1 regulates TERRA transcription by repressing TERRA 2q, 11q and 18q. TERRA and TERT are transiently down-regulated during EMT in NMuMG cells. Ectopic expression of TERRA affects the transcription of some genes induced during EMT such as fibronectin whereas TERT does not modify those genes. We propose that Snail1 control of TERRA besides being required for telomere maintenance is also necessary for the expression of a subset of mesenchymal genes.El factor de transcripció Snail1 és el principal inductor de la transició epiteli mesènquima (EMT). Aquí describim un nou paper de Snail1 en la regulació de la integritat i transcripció telomèrica i també en l’expressió de la telomerasa. El FISH mostra un augment de les alteracions telomèriques en les MSCs deficients per Snail1 així com telòmers més curts. Malgrat això, aquestes cèl·lules presenten nivells més alts de telomerasa degut a que Snail1 en reprimeix la seva expressió, la qual cosa significa que hi ha altres mecanismes involucrats en l’homeòstasi telomèrica. De fet, els telòmers es transcriuen en uns llargs RNA no codificants anomenats TERRA. Aquí mostrem que Snail1 regula la transcripció de TERRA reprimint TERRA 2q, 11q I 18q. L’expressió de TERRA i TERT disminueix de forma transitòria durant l’EMT en les cèl·lules NMuMG. L’expressió ectòpica de TERRA afecta la transcripció d’alguns gens induïts durant l’EMT com la fibronectina, mentres que TERT no modifica aquests gens. Proposem que el control de TERRA per part de Snail1 no només és necessari pel manteniment telomèric sinó també per l’expressió d’un subconjunt de gens mesenquimals.
6
Drummond, Mark William. "Telomere dynamics and telomerase expression in chronic myeloid leukaemia." Thesis, University of Glasgow, 2003. http://theses.gla.ac.uk/41113/.
Full textAbstract:
INTRODUCTION: Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell (HSC), with a variable clinical course. Chronic phase (CP) disease, typically of 4-5 years duration, progresses to accelerated (AP) and then blastic phase (BP), with the latter behaving as a particularly aggressive acute leukaemia of either myeloid (MBP) or lymphoid (LBP) lineage. Treatment is most successful when delivered in CP, and accurate prognostic indices are required to individualise treatment. Increased telomere shortening has been described during progression of CML, and may be of prognostic relevance. Paradoxically, telomerase activity (TA, as determined by the TRAP assay) has been shown to be elevated in the CP stem/progenitor cell (CD34+) compartment: however this may not accurately reflect telomere maintenance in-vivo. We sought to further define the prognostic significance of telomere shortening at diagnosis of CML, monitor the rate of telomere loss during the disease and characterise expression of the major telomerase components (hTR and hTERT) in CD34+ selected cells at diagnosis and during disease progression. METHODS: Peripheral blood leucocyte (PBL) telomere length measurement was performed by flow-FISH on cohorts of normal individuals, patients at diagnosis and all stages of CML. To define the degree of telomere shortening in individual patients at diagnosis ex-vivo expanded (BCR-ABL') T-cells were used as an internal control for 'normal' somatic cell telomere length. Expression of hTERT and hTR was quantified by Q RT-PCR and hTERT mRNA splice variants detected by RT-PCR. CD34+ selected cells from CML patients were confirmed as BCR-ABL+ by FISH. TA was determined by TRAP assay. RESULTS: Telomere shortening in CP and AP CIVIL patients progressed at 10-20 times the rate of age-related shortening observed in the normal control group. Furthermore, high-risk prognostic score patients at diagnosis had significantly shorter telomeres than low-risk patients. High purity CD34+ selected cells from CML, as compared to normal, demonstrated increased TRAP activity which correlated with the proportion of cycling cells. However, hTERT mRNA expression was not significantly elevated. Unexpectedly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the CML samples, raising the possibility that telomere homeostasis is disrupted in CML. In BP samples, hTERT expression was significantly lower in MBP than LBP and this was mirrored by a corresponding shift in hTERT splicing patterns. MBP hTERT expression correlated inversely with telomere length. CONCLUSIONS; In summary, increased TRAP activity is not synonymous with telomere maintenance in CML, and dysregulated expression of hTR may contribute to the telomere loss observed in these patients. Indeed TRAP activity appeared largely dependent on the proportion of cycling cells. In the context of progressive (i.e. BP) disease, hTERT expression is lineage and telomere length dependent, thus explaining inconsistent reports of TA levels in BP samples. We have also demonstrated that subtle shifts in splicing of hTERT mRNA is likely to have a regulatory role in primary HSC. In prognostic terms telomere shortening in CML is greatest in high-risk score patients at diagnosis, and occurs rapidly during disease progression. These data further emphasise the potential clinical utility of telomere length measurement for prognostic modelling and monitoring of disease progression.
7
Karpov, Victor. "A study on telomere protection and telomerase-and-cap-independent mechanisms of telomere maintenance in yeast Saccharomyces cerevisiae." Mémoire, Université de Sherbrooke, 2008. http://savoirs.usherbrooke.ca/handle/11143/3940.
Full textAbstract:
An SGA approach to discover cdc13-1ts supressors. Telomeres, the DNA-protein complexes at the end of eukaryotic chromosomes, are essential for chromosomal stability. In yeast, the telomeric single-strand binding protein Cdc13p has multiple important roles related to telomere maintenance: (1) telomeric"capping"--protection of telomeres by forming complexes with yKu70/80 and with Stn1p/Ten1p; (2) positive regulation of telomere replication via interaction with Est1p, which is a part of telomerase; (3) negative regulation of telomerase by the recruitment of telomere elongation suppressors Stn1p and Ten1p. In an attempt to identify genes that are involved in the deleterious outcome of an absence of Cdc13p, we screened the yeast gene knock-out library for genes that could suppress the growth defect of cdc13-1 cells at 33ê C. For this purpose, we performed an SGA array experiment. We scored for the ability of double mutant haploids to grow at 33ê C. Eventually, we hoped to find the elusive genes involved in telomere 5'-end processing (exonucleases). Based on the comparative analysis of growth properties of the strains (23ê C vs 33ê C), the initial screen identified up to 111 genes that displayed an apparent growth at 33ê C. In order to verify these results, diploids were regenerated, sporulated, microdissected, and haploid double mutants cdc13-1 yfg[deletion] were isolated from 38 potential cdc13-1 suppressors. Unfortunately, this verification failed to reproduce a suppression of the growth defect by any of the selected genes at any temperature. While disappointing, the results reemphasize that careful re-examination of large scale SGA approaches are indispensable before going on to more involved experimentation. Similarities and differences between adaptation to DNA double-strand break and to telomere uncapping in yeast Saccharomyces cerevisiae. It was previously shown that a certain proportion of telomerase negative survivor cells (both type I and type II cells) is able to survive in the absence of the telomere capping protein Cdc13p. These strains (named [deletion]13s) were characterized in great detail and one of their discovered features was a striking ability to continuously inactivate DNA-damage checkpoints. Based on structural similarities between DNA double strand breaks (DSB) and unprotected telomeres, we attempted to verify if the molecular mechanisms regulating adaptation to a single irreparable DSB also regulate adaptation to a loss of Cdc13p. For this purpose we created three tlc1[deletion] cdc13[deletion] strains also harboring DSB adaptation related mutations tid1[deletion], ptc2[deletion] and rfa1-t11. After deprotection of their telomeres, mutant survivor cells showed similar cell cycle progression patterns as compared to the cells where a single irreparable DSB was introduced. Adaptation defective mutants tid1[deletion] and ptc2[deletion] demonstrated an inability to adapt to telomere uncapping and to resume cell cycle. Interestingly, cells harboring the rfa1-t11 allele, which was reported to suppress adaptation defects of other mutations, did not show any distinguishable phenotype in terms of initial adaptation to telomere deprotection; i.e. rfa1-t11 mutant survivors do escape the G2/M arrest and re-enter the cell cycle. However, all three mutant survivor strains failed to produce viable [deletion]13 capping independent cells, which is consistent with the hypothesis that adaptation to loss of Cdc13p depends on the same pathway as the previously reported adaptation phenomenon. Finally, we report the surprising finding that if cells had once experienced an adapted [deletion]13 state, they will re-produce capping negative survivors much more readily. Thus, while a culture of type II survivor cells generates [deletion]13s at a rate of about 1×10 -5 events per division, cells that had been [deletion]13s and re-transformed with a Cdc13p carrying plasmid will produce capping independent cells at about 1×10-2 events per division. We are currently examining why these cells re-generate [deletion]13 cell lines more readily and suspect structural differences in telomere terminal sequence arrangements.
8
Fakhoury, Johans. "Conserved and divergent mouse and human telomerase and telomere regulation: implications for the development and validation of telomerase and telomere-specific anticancer strategies." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=94905.
Full textAbstract:
Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) (TERT) and RNA (TR). We reconstituted heterologous mouse and human TERT-TR and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity, and telomere function map not only to TR, but also to the TERT component. hTERT-hTR, but not heterologous TERT-TR complexes, nor chimeric mTERT-hTERT-hTR complexes, significantly reduced the percentage of chromosomes without telomeric signals in ALT cells. Moreover, heterologous and chimeric complexes were defective in recruitment to telomeres. Our results suggest a requirement for several hTERT domains and interaction with multiple proteins for proper recruitment of telomerase to the shortest telomeres in human ALT cells. The ability of hTERT to elongate short mouse telomeres, and the inability of mTERT to elongate short human telomeres suggest that mechanisms regulating recruitment and activity of hTERT at short telomeres may be less stringently regulated than mechanisms regulating mTERT recruitment and activity at short telomeres. Results such as these may lead to the design of better strategies for inhibiting telomerase and validation using rodent models. For example, TERT domains that confer similar functions in human and mouse cells may be better targets than domains with species-restricted functions. We also tested the specificity of a novel class of platinum(II) G-quadruplex stabilizers at inhibiting telomerase activity. We showed that these ligands efficiently stabilize telomeres and inhibit telomerase activity with comparable potency to telomestatin (a potent telomerase inhibitor). Additionally, these ligands may present a potent dual action strategy to not only inhibit telomerase function, but also disrupt telomere function and assembly. Accordingly, targeting telomere functionLa télomérase synthétise les séquences télomériques et se compose minimalement d'une sous-unité transcriptase inverse (TERT) et d'un fragment d'ARN (TR). Nous avons reconstitué des complexes hétérologues TERT-TR humains et murins ainsi que des complexes chimériques mTERT-hTERT-hTR in vitro et dans des cellules immortalisées utilisant un mécanisme alternatif d'élongation des télomères (cellules ALT). Nos résultats suggèrent que les déterminants espèce-spécifiques de l'activité, la processivité et la fonction télomérique sont attribués non seulement au composant TR mais aussi au composant TERT de la télomérase. Les complexes hTERT-hTR, mais non les complexes hétérologues TERT-TR ou mTERT-hTERT-hTR ont diminué le pourcentage de chromosomes sans signal télomérique de façon significative lorsqu'exprimés dans des cellules ALT. De plus, il a été démontré que les complexes hétérologues et chimériques sont déficients quant à leur recrutement aux télomères. Nos résultats suggèrent que plusieurs domaines de TERT et la présence d'interactions entre plusieurs protéines sont requis pour le recrutement de la télomérase aux télomères les plus courts dans les cellules ALT. La capacité de hTERT à allonger les télomères murins les plus courts, et l'incapacité de mTERT à allonger les télomères humains les plus courts suggèrent que les mécanismes régulant le recrutement et l'activité de hTERT aux télomères les plus courts seraient régulés de façon moins rigoureuse que les mécanismes régulant ceux de mTERT. De tels résultats pourraient mener à la création de meilleures stratégies visant à inhiber la télomérase et la validation de celles-ci dans des modèles murins. Par exemple, les domaines de TERT qui confèrent des fonctions similaires dans les cellules humaines et murines risquent de représenter de meilleurs cibles thérapeutiques que les domaines de TERT possédant des fonctions espèce-spécifiques.$
9
Choi, Eugene Ho Yan. "Investigating spontaneous activation of two telomere maintenance mechanisms in the same cancer cells." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20766.
Full textAbstract:
Telomeres are biological constructs that protect the genomic information from DNA damage repair pathways and chromosomal fusions. Telomeres shorten every cell division and when the telomeres become critically short the cells become senescent. Cancer cells overcome this hindrance and proliferate infinitely via the activation of a telomere maintenance mechanism (TMM). Current literature suggests most cancers maintain their telomeres via two known mechanisms. The first involves telomerase, a ribonucleoprotein, and the other TMM, Alternative Lengthening of Telomeres (ALT), is independent of this ribonucleoprotein. Normal somatic cells do not utilise a TMM, TMMs are ideal targets for the generation of selective anti-cancer therapeutics. There is a widespread assumption in this field of research that ALT and telomerase are mutually exclusive. A prior study in the Reddel laboratory investigated the possibility that spontaneous activation of both TMMs within a single cell can occur. A melanoma cell line, LOX IMVI, was characterised to have telomerase activity in addition to the phenotypic characteristics of ALT. To identify whether both TMMs actually contributed to telomere length maintenance in LOX IMVI cells, telomerase activity was abrogated. My main contribution to this study was to examine telomere lengths in these telomerase-null cells over the course of 200 population doublings. Telomere lengths continued to decrease throughout that entire time. Therefore, no evidence was obtained that ALT contributes to telomere length maintenance in LOX IMVI cells. I then found that another human cell line, 1301, which is derived from a paediatric acute lymphoblastoid leukaemia, has telomerase activity and features of ALT activity in every subclone. To determine whether ALT and telomerase are contributing to telomere length maintenance in these cells, I used CRISPR/Cas9 to knock out the TERC gene, which encodes the RNA subunit of telomerase. Knockout was confirmed in eight subclones and were passaged long-term with seven controls. In seven of the treated clones telomere length declined detectably, but in one clone there was a negligible rate of decline. To my knowledge, this is the first evidence indicating that functional levels of telomerase and ALT activity may be spontaneously activated in the same cancer cells.
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.
Full textAbstract:
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.
11
Sfeir, Agnel J. "Telomere dynamics and end processing in mammalian cells." Access to abstract only; dissertation is embargoed until after 5/15/2007, 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=166.
Full text
12
Lemos, Carla Alexandra Ribeiro Cerqueira de. "Aspetos estruturais e funcionais do complexo telómero/telomerase." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5320.
Full textAbstract:
Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasOs telómeros são estruturas nucleoproteicas heterocromáticas de comprimento variável presentes nas extremidades dos cromossomas. Os telómeros, conjuntamente com a telomerase, permitem ultrapassar a limitação replicativa dos segmentos terminais de DNA que se verifica na maioria das células humanas somáticas e que está associada à diminuição do comprimento do DNA telomérico em cada divisão celular. As estruturas cromossómicas teloméricas são constituídas por longas extensões de repetições hexaméricas 5’-TTAGGG-3’ de cadeia dupla e por um complexo proteico específico designado por shelterin. As sua principais funções incluem a proteção das extremidades dos cromossomas contra uma eventual fusão ou degradação prematura da molécula de DNA, regulação da síntese de DNA telomérico e regulação/manutenção do comprimento do telómero. Os telómeros de mamíferos são transcritos por ação da RNA polimerase II a partir de vários loci subteloméricos e as moléculas de RNA produzidas são constituídas por um número variável de repetições da sequência 5’-UUAGGG-3’. A sua associação ao telómero ocorre através da formação do segmento híbrido RNA-DNA ou da interação de ribonucleoproteínas específicas. Apesar da função deste transcripto ainda não estar completamente estabelecida, várias evidências sugerem a sua participação no processo de regulação do comprimento dos telómeros através de vários mecanismos: inibição da atividade da telomerase, ativação de exonucleases, regulação do nível de eucromatina e/ou atuação como fator de proteção. A telomerase é um complexo ribonucleoproteico constituído por uma parte central, que inclui a subunidade catalítica com atividade de transcriptase reversa, e por RNA, que representa a sequência molde para a síntese do DNA telomérico. A sua biossíntese, maturação e atividade catalítica estão relativamente bem caracterizadas. A base molecular para a regulação da atividade da telomerase é complexa e envolve vários níveis de controlo, principalmente transcripcional. No entanto, existem diversas evidências que sugerem que a enzima é também submetida a processos de controlo pós-trancripcional e pós-traducional. Dada a relação observada entre o comprimento do DNA telomérico e processos celulares aparentemente antagónicos tais como o envelhecimento e a tumorigénese, pretende-se com o presente trabalho de revisão bibliográfica caracterizar os principais aspetos estruturais e funcionais do complexo telómero/telomerase e compreender o seu papel na manutenção do comprimento dos telómeros. Adicionalmente, é explorada a importância do conhecimento sobre a biologia molecular e celular deste complexo para a identificação e o desenvolvimento de novas abordagens para a prevenção do envelhecimento ou do cancro.
Telomeres are heterochromatic nucleoproteicas structures of varying length present at the ends of chromosomes. Telomeres, together with the telomerase, allow to overcome the limitation imposed by terminal DNA segments of chromosome to the semi-conservative DNA replication of most human somatic cells, which is associated with the decreased length of telomeric DNA in each cell division. The telomeric cromosomal structures are composed of long extensions of double stranded hexameric repetitions 5’-TTAGGG-3’ and a specific proteinaceous complex termed as shelterin. The main functions of this nucleoprotein complex include the protection of the chromosomal endings against an eventual fusion or premature DNA degradation, regulation of the telomeric DNA synthesis and regulation/maintenance of the telomere length. The telomeres of mammals are transcribed through the action of RNA polymerase II from several subtelomeric loci. The produced RNA molecules are composed of a variable number of 5’-UUAGGG-3’ sequence repetitions. The association of this RNA molecule with telomeres occurs through the formation of a RNA-DNA hybrid or interation of ribonucleoproteins. Although the function of this transcript molecule is not completely established, yet, several evidences suggest its participation in the process of regulation of telomere lenght through several mechanisms: telomerase activity inhibition, exonucleases activation, regulation of the chromatin level and/or protection factor. The telomerase is a ribonucleoprotein complex formed by a central core including the catalytic subunit with activity of reverse transcriptase and RNA, which represents the template sequence used in the telomeric DNA elongation. The biosynthesis, maturation and catalytic activity of telomerase are relatively well characterised. The molecular basis of telomerase activity regulation is complex and involve several levels of control, mainly the transcriptional level. Nonetheless, several evidences suggest that enzyme is also subjected to pos-transcriptional and pos-tradutional regulation. Given the relation observed between the length of telomeric DNA and the apparently antagonic processes of aging or tumorigenesis, the present work of bibliographic review aims to characterise the main structural and functional aspects of the complex telomere/telomerase and to understand its role in the maintenance of telomere length. Additionally, it also explores the importance of the knowledge on the molecular and cell biology of this complex to the identification and development of novel approaches to prevent aging or cancer.
13
Bestilny, Leslie James. "Regulation of telomere length and telomerase activity during aging and immortality." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0014/NQ38456.pdf.
Full text
14
Betts, Dean Harvey. "Analysis of telomerase activity and telomere length during early bovine development." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ61967.pdf.
Full text
15
Marzec, Paulina. "NR2C/F telomeric association drives telomere-genome rearrangements in ALT cells." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20179.
Full textAbstract:
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'ADNCellular 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
16
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.
Full textAbstract:
Thesis advisor: Clare O'ConnorTelomeres 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
17
Foote, Christopher Graham. "Avian telomere dynamics." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/539/.
Full textAbstract:
Telomeres, the repetitive DNA sequences that cap eukaryotic chromosomes, are thought to play an important role in linking life conditions and senescence. In vertebrate somatic cells, telomeres shorten at each cell division, and the rate at which they do so has been linked to cellular and organismal senescence. Although telomeres generally shorten with age in vertebrates, in most species studied there is considerable variation between same age individuals. In this thesis, I examined the telomere dynamics of various avian species, investigating both the causes of variation in telomere length among individuals and what effect this variation has on attributes such as survival rates. Previous studies have shown that most telomere loss occurs in young individuals and it thus makes sense that early life conditions are responsible for much of the inter-individual variation in telomere length. I investigated this idea by studying chick telomere dynamics in a wild population of lesser black-backed gulls Larus fuscus. There was considerable variation in hatching telomere length among individuals and much of this variation was related to circumstances during embryonic growth. Larger hatchlings had shorter telomere lengths, suggesting that embryonic growth rate could have affected telomere attrition. Independent of this trend, males had longer telomeres at hatching than females. Although telomere length did decrease with age post-hatching, these initial variations remained consistent during the initial post-hatching period. The relationship between early life conditions and telomere length was investigated further with a longitudinal study of telomere length in chicks of the European shag Phalacrocorax aritotelis. A previous study on this population of birds had shown that telomere length declines with age within individuals over a period of several years. However no change in telomere length was detected over a period of 11-13 days during the chick period. Body size had no effect on telomere length, but males did have longer telomere than females. These initial chapters investigate telomere length in chicks; however there are very few studies that investigate telomere length over the entire lifespan of long-lived species. I thus next examined the telomere dynamics of two species of long-lived seabird, the northern and southern giant petrels (Macronectes spp.). In both giant petrel species, telomeres were shorter in adults than chicks, but there was no trend for adult telomere length to decrease with age. In southern giant petrels, there was a significant relationship (independent of age and sex) between an individuals telomere length and whether it was still alive 8 years after it was initially sampled. This relationship was not present in northern giant petrels, possibly due to a smaller sample size. The results thus support both the idea that most telomere loss occurs in young individuals and that telomere length may be an indicator of life expectancy. Various methods exist to measure telomeres. As the number of taxa whose telomere dynamics are being studied increases, it becomes increasingly important to know which methods are the best to use and to what extent these methods are applicable across species. These questions were investigated in relation to work conducted on the telomere dynamics of the blue-footed booby Sula nebouxxi. Both the TRF and qPCR techniques were used to measure booby telomeres, but problems arose with both methods. It is possible that these problems occurred because blue-footed boobies have a particularly large amount of interstitial telomeric DNA, although a more detailed analysis of booby telomeres would be necessary to determine this. These findings suggest that standardised methods to measure telomeres cannot necessarily be applied to every new species whose telomere dynamics are studied. The evidence presented here suggests that the study of telomere dynamics can be a very powerful tool for behavioural ecologists. It now seems possible that telomeres might provide both a way of measuring the long-term costs of early life-conditions and a way to measure the quality of an individual. However, further research is still needed to fill in the considerable gaps in our knowledge and fully exploit the potential telomeres have for behavioural ecology.
18
Chomal, Manish R. "Analysis of telomerase activity and telomere lengths in human umbilical cord cell populations during ex vivo amplification of hematopoietic stem cells." Digital WPI, 2002. http://www.wpi.edu/Pubs/ETD/Available/etd-1205102-085840.
Full text
19
Gocha, April Renee Sandy. "Mechanisms of alternative telomere elongation in human cancer cells." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1351190051.
Full text
20
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.
Full textAbstract:
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.
21
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.
Full textAbstract:
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.
22
Shawi, May. "Telomerase and telomere regulation by associated proteins and in primary malignant lymphocytes." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107575.
Full textAbstract:
Telomeres are repeated sequences of DNA at the ends of chromosomes that protect the cell from death. Telomerase is a ribonucleoprotein (RNP), consisting of a catalytic subunit, TERT, and an RNA component, TR, that synthesizes telomeres. Telomerase is active in 85% of cancers, including leukemias. Telomerase is thus a potential pharmacological drug target for cancer therapy, and it is important to understand this enzyme and its regulation by associated proteins. One goal of this thesis was to identify novel telomerase interacting partners that specifically regulate telomerase function and that could serve as potential anticancer targets. Additionally, it is important to understand how telomerase inhibition works in a cancer model. Telomere length is a prognostic factor in chronic lymphocytic leukemia (CLL). Previous reports have assessed Imetelstat, a telomerase inhibitor which targets the human telomerase RNA component hTR, in a phase I-II clinical trial in CLL patients. We further investigated Imetelstat in lymphocytes from CLL patients. We found that telomerase activity was present in 47% of lymphocyte samples from CLL patients and that treatment with Imetelstat alone did not affect the survival of primary CLL lymphocytes in vitro. Nonetheless, Imetelstat increased the sensitivity of CLL lymphocytes to fludarabine, independent of basal telomerase activity. Imetelstat inhibited fludarabine-induced DNA-PK autophosphorylation, a surrogate of DNA-PK activity in CLL lymphocytes, to the same extent as the DNA-PK inhibitor NU7026. The effect of Imetelstat on fludarabine sensitivity was associated with a lower basal expression of the DNA-PK subunit protein Ku80. Our results suggest that Imetelstat can inhibit fludarabine-induced DNA-PK activity in primary CLL lymphocytes. We conclude that there may be a functional interaction between hTR and DNA-PKcs in primary CLL lymphocytes and that Imetelstat in combination with fludarabine may be useful to decrease the tumour burden in CLL. To investigate other telomerase targets, we identified telomerase associated proteins via mass spectrometry. One potential target is NOP17, which could be involved in the early assembly of telomerase. We created a human cell line stably expressing NOP17 fused to a FLAG tag. Telomerase activity was coimmunoprecipitated using an antibody against FLAG. Moreover, downregulating the expression of this Box C/D SnoRNP protein resulted in decreased telomerase activity. Characterization of telomerase-associated proteins is vital to our understanding of how this enzyme contributes to tumorigenesis. Understanding the regulation of telomere length and telomerase function by associated proteins will be important for the discovery of targeted therapeutics in cancers.La télomérase synthétise les séquences télomériques et se compose minimalement d'une sous-unité transcriptase inverse (TERT) et d'un fragment d'ARN (TR). La télomérase est active dans 85% des cancers, y compris la leucémie, elle représente donc une cible pour les thérapies du cancer. Afin de découvrir de meilleures cibles thérapeutiques, il est important de comprendre cette enzyme et sa régulation par des protéines associées. Un des buts de cette thèse est d'identifier de nouvelles protéines interagissant avec la télomérase, pouvant réguler ses fonctions de façon spécifique et ainsi potentiellement devenir des cibles anticancéreuses. De plus, il est important de bien comprendre l'effet de l'inhibition de la télomérase dans un modèle de cancer. Nous avons choisi la leucémie car la taille des télomères est un facteur pronostique de la leucémie lymphoïde chronique (LLC). De précédentes études ont conduit à l'évaluation d'Imetelstat, un inhibiteur de la télomérase qui cible la composant ARN de l'enzyme, présentement en essais cliniques de phases I-II. Nos résultats montrent qu'il y a activité de la télomérase dans 47% des échantillons de lymphocytes de patients atteints de LLC et que le traitement seul d'Imetelstat n'a aucun effet sur la survie des lymphocytes primaires de LLC in vitro. Cependant, Imetelstat augmente la sensibilisation des lymphocytes à la Fludarabine, et ce indépendamment de l'activité basale de la télomérase. Imetelstat inhibe l'autophosphorylation de DNA-PK (l'autophosphorylation de DNA-PK est corrélée à son activité enzymatique dans les lymphocytes de LLC) induite par la fludarabine au même niveau que l'inhibiteur de DNA-PK NU7026. L'effet d'Imetelstat sur la sensibilité à la fludarabine est associé à l'expression basale de la protéine Ku80. Nos résultats suggèrent que l'Imetelstat inhibe l'activité DNA-PK dans les lymphocytes de LLC primaire. Nous pouvons conclure qu'il peut s'agir d'une interaction fonctionnelle entre hTR et DNA-PKcs dans les lymphocytes de LLC et que la combinaison de la fludarabine et d'Imetelstat pourrait être utile afin de diminuer la masse tumorale dans la LLC. Afin de découvrir de nouvelles cibles contre la télomérase, nous avons purifié le complexe de la télomérase par spectrométrie de masse. Cela nous a permis d'identifié une nouvelle protéine associée à la télomerase, NOP17, qui pourrait être impliquée dans l'assemblage de la télomérase. Nous avons créé une lignée cellulaire humaine exprimant de façon stable la protéine NOP17 fusionnée à une étiquette FLAG. L'Immunoprecipitation de NOP17 montre une activité de la télomérase indiquant qu'elle est associée à un complexe actif. Par ailleurs, la régulation négative de l'expression de la boîte C/D SnoRNP de la protéine réduit l'expression et l'activité de la télomérase. La caractérisation des protéines associées à la télomérase est essentielle afin de mieux comprendre le fonctionnement de cette enzyme ainsi que sont implication dans la formation de cancers. De plus, ces protéines associées à la télomérase pourraient également servir de cibles potentielles dans les thérapies contre le cancer.
23
McKevitt, Tom Patrick. "A study of telomere and telomerase biology in the dog and cat." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443374.
Full text
24
Perera, Yatawarage Omesha Nalindri. "A non-canonical function of human telomerase reverse transcriptase in telomere protection." Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14963.
Full textAbstract:
Telomerase is a ribonucleoprotein complex with a well-established role in telomere maintenance. There is growing evidence of non-canonical functions of telomerase that promote tumorigenesis. Here we have demonstrated that hTERT can mediate telomere protection independent of its canonical function in telomere maintenance, potentially enhancing positive feedback pathways that may facilitate oncogenesis. hTERT mediated telomere protection was found to be independent of catalytic activity and telomere recruitment, required nuclear export of hTERT, and required an intact N-terminal portion of hTERT. We have further shown that hTERT appears to regulate two seemingly independent pathways, with mutual requirement of both NFκB and Hsp70 to exert telomere protection. hTERT over-expression resulted in an increased localization of Hsp70 at the telomeric protein TRF2. This increase was dependent on the presence of the shelterin accessory protein Apollo, suggesting that Apollo acts as the bridge between Hsp70 and TRF2, facilitating the stabilization of TRF2 to form a fully capped telomere. Conversely, depletion of hTERT down-regulated NFκB signaling, with a concomitant increase in telomeric DNA damage and consequential G1 arrest. These two pathways may intersect physiologically through Hsp70 regulation of the NFκB pathway, further enforcing the feed-forward mechanism between hTERT and its transcriptional targets to promote oncogenesis.
25
Perumal, Kuppusamy Senthilkumar. "Telomerase and telomere dysregulation in Polychlorinated Biphenyl (PCB) exposed human skin keratinocytes." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2957.
Full textAbstract:
Polychlorniated Biphenyls (PCBs), a group of 209 individual congeners, are ubiquitous environmental pollutants and classified as probable human carcinogens. Hallmarks of aging and carcinogenesis are changes in telomerase activity and telomere length. I hypothesize that PCBs modulate telomerase activity and telomeres via interference in gene regulation and generation of reactive oxygen species (ROS) resulting in the dysregulation of cell growth. To explore this possibility, I exposed human skin keratinocytes (HaCaT) to a synthetic airborne PCB mixture (CAM) and individual congeners, i.e. PCB28, PCB52, PCB126 and PCB153. To mimic the chronic human exposure to PCBs and the slow process of carcinogenesis, a long term exposure period of 48 days and beyond was employed. All PCB congeners and CAM reduced telomerase activity, telomere length and cell growth. Among all PCBs, PCB126 had the most pronounced effect with reduction in telomerase activity, telomere length, hTERT and hTR gene expression and cell growth, while increasing TRF1 & TRF2 gene expression. PCB126 elicited an increase in CYP1A1 mRNA, CYP1A1 activity, DHE and DCFH oxidation levels from days 6 to 48, suggesting that increased ROS might be a causative factor for the reduction in telomerase activity and telomere length. However, transduction with hTERT and hTR subunits partly rescued telomerase activity, while treatment with PEG-catalase did not rescue telomerase activity suggesting that telomerase subunits play an important role on PCB126 induced effects on telomerase activity and telomere length. Since cells with shortened telomeres may escape crisis through telomerase reactivation, PCB126 treatment was continued until day 90. A change in growth behavior was observed from day 54 to 90, with cells recovering the proliferation rate, and increasing c-Myc, hTERT, and hTR gene expression level, re-activating telomerase activity and re-elongating telomere length. TRF1 & 2 gene expression started to decrease after day 66. From day 78, no increase in CYP1A1mRNA and its activity as well as CYP1B1, ALDH3A1, UGT1A1 and AhRRmRNA was observed suggesting that the AhR response pathway may have been altered. This study shows for the first time that PCBs initially reduce telomerase activity, telomere length, and cell growth, and can later lead to telomerase re-activation, telomere lengthening and increased cell growth with modulation of the AhR receptor pathway. This observation has broad implications for chronic PCB exposure scenarios.
26
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.
Full textAbstract:
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.
27
Silva, Filipa Isabel Serra e. "Mistranslation and telomere stability." Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7648.
Full textAbstract:
Mestrado em Biologia AplicadaThe regulation of a stable proteome is crucial for the cell homeostasis. The translation process from the nucleotide sequence of a gene into the aminoacid sequence of a protein is associated with a basal error of 10-4 which the cell deals with through quality control mechanisms. The misincorporation of aminoacids into de novo synthesized proteins tends to rise when the cell is exposed to stressful conditions. The increase of dysfunctional proteins produced by mistranslation may induce expression of genes related to stress response and genome destabilization. In this work we used yeast as a model to study the impact of high mistranslation rates in telomere stability, since the telomeric and adjacent sub-telomeric regions are key elements on the preservation of genome integrity. Results shown that some types of induced mistranslation may in fact have an impact on telomere length, and also that some proteins’ activity, such as Pnc1 and Sir2, is important regarding telomeric DNA stability. These results shed a new light over the importance of controlling mRNA mistranslation rates in eukaryotic cells.
A manuten¸c˜ao de um proteoma est´avel ´e crucial para a homeostase celular. Ao processo de tradu¸c˜ao da sequˆencia de nucle´otidos de um gene para a sequˆencia de amino´acidos de uma prote´ına est´a associada uma taxa de erro basal de cerca de 10 −4, com a qual a c´elula lida atrav´es de mecanismos de controlo de qualidade das prote´ınas. A incorpora¸c˜ao incorrecta de amino´acidos nas prote´ınas sintetizadas de novo tende a aumentar quando as c´elulas est˜ao expostas a condi¸c˜oes de stress. Por sua vez, o aumento de prote´ınas disfuncionais provocado por erros de tradu¸c˜ao pode induzir a express˜ao de genes de resposta ao stress e destabiliza¸c˜ao do genoma. Neste trabalho, utilizou-se a levedura como modelo para o estudo do impacto de uma elevada taxa de erros de tradu¸c˜ao ao n´ıvel da estabilidade dos tel´omeros, uma vez que esta regi˜ao, juntamente com a regi˜ao subtelom´erica, representa um elemento-chave na preserva¸c˜ao da integridade do genoma. Metodologias de engenheria de tRNA foram utilizadas para induzir erros de tradu¸c˜ao, sendo a t´ecnica de Southern blot escolhida para an´alise do padr˜ao de migra¸c˜ao electrofor´etica de fragmentos de ADN correspondentes `a regi˜ao subtelom´erica. Os resultados obtidos demonstraram que os erros de tradu¸c˜ao podem, de facto, ter impacto a n´ıvel do comprimento dos tel´omeros, dependendo do tipo de erro de tradu¸c˜ao induzido, permitindo ainda confirmar a importˆancia da actividade de prote´ınas como Pnc1 e Sir2 no controlo da estabilidade do ADN telom´erico, lan¸cando uma nova luz sobre a importˆancia do controlo da taxa de erros de tradu¸c˜ao nas c´elulas eucari´oticas.
28
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.
Full textAbstract:
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.
29
Henson, Jeremy D. "The role of Alternative Lengthening of Telomeres in human cancer." University of Sydney, 2006. http://hdl.handle.net/2123/1533.
Full textAbstract:
Doctor of PhilosophyActivation 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.
30
D'Souza, Yasmin. "Processivity domains within human telomerase reverse transcriptase that regulate telomere length and immortalization." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116879.
Full textAbstract:
Short, repetitive G-rich DNA sequences present at telomeres are synthesized by telomerase, a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase, TERT, and an integrally associated RNA, TR. Human TERT (hTERT) can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. We investigated if threshold levels of telomerase activity and processivity are required to maintain telomere length and/or function and immortalize human cells with limited lifespan. Specifically, we assessed hTERT variants with mutations in several motifs implicated in processivity, namely the N terminus (E79A, E90K), RT motif 1 (I624M), the 'Insertion in Fingers' domain (V791Y), the C 'catalytic center' motif (L866Y), the E 'primer grip' motif (W930F) and the C terminus (∆1047-1056 and ∆1107-1118). The N-terminal and motif 1 hTERT mutants did not reveal any interesting phenotypes in vitro or in cells. On the other hand, the remaining variants, except L866Y, displayed a substantial decrease in processivity. Despite the presence of short telomeres in cells expressing these low processivity telomerase variants, only W930F could immortalize limited lifespan human cells. We demonstrate that limiting levels of DNA synthesis on the order of 20% of wild-type, and extension of as few as three telomeric repeats displayed by W930F are sufficient to maintain functional telomeres and immortalize limited lifespan human cells. The hTERT-C-terminal mutants likely could not immortalize cells due to synthesis of only 2 or less telomeric repeats. V791Y could not maintain telomere function due to a failure to localize to telomeres. On the other hand, L866Y displayed a 2-3 fold increase in proccessivity compared to wild-type telomerase. Cells expressing this mutant displayed telomere elongation followed by heterogenous telomere lengths and an increase in short telomeres, and fragile sites at telomeres accompanied by telomere trimming, indicating that processivity levels above that displayed by wild-type telomerase lead to telomere replication stress. These results suggest that telomere function and length, and immortalization in human cells are regulated by telomerase enzyme processivity.Des courtes séquences répétées et G-riches d'ADN présentes aux télomères sont synthétisées par télomérase, une ribonucléoprotéine constituée d'une sous-unité catalytique, 'telomerase reverse transcriptase' ou 'TERT', et un ARN associé nommé 'TR'. TERT humain (hTERT) peut diriger de façon répétitive la transcription inverse de son ARN, agissant processivement en ajouteant de multiples répétitions télomériques sur le substrat d'ADN. Nous avons étudié si des niveaux limites d'activité ou de processivité de télomérase sont nécessaires pour maintenir la taille ou la fonction des télomères et pour immortaliser des cellules humaines possédant une durée de vie limitée. Plus précisément, nous avons évalué plusieurs variants de hTERT avec des mutations dans des motifs impliqués dans la processivité, incluant l'extrémité N-terminale (E79A, E90K), le motif 1 du Reverse Transcriptase (RT) (I624M), le domaine 'Insertion in Fingers' (V791Y), le motif C (L866Y), le motif E (W930F) et l'extrémité C-terminale (Δ1047-1056 et Δ1107-1118). Les mutations dans le terminus N et le motif 1 de hTERT n'ont pas révélées de phénotypes intéressants. Les autres variants, sauf L866Y, ont demontré une diminution substantielle des niveaux de processivité. Malgré la présence de télomères courts dans les cellules exprimant ces variantes de processivité faibles, seul W930F pouvait immortaliser les cellules. Nous démontrons que le niveau de synthèse d'ADN de l'ordre de 20% de hTERT sauvage, et l'extension de seulement trois répétitions télomériques par W930F sont suffisants pour maintenir des télomères fonctionnels et immortaliser les cellules. Les variants avec des mutations dans le terminus C ne pouvaient pas immortalizer les cellules dues à la synthèse de seulement 2 ou moins de répétitions télomériques. V791Y ne pouvait pas maintenir la fonction des télomères en raison d'une incapacité à se localiser aux télomères. D'autre part, L866Y a demontré une augmentation des niveaux de proccessivité de 2-3 fois par rapport à la télomérase sauvage. Les cellules exprimant ce mutant ont presenté un rallongement des télomères, suivi de télomères de tailles hétérogènes et une augmentation du nombre de télomères courts, accompagné d'une augmentation de sites fragiles aux télomères et de télomères tronqués, tout ce qui indique que des niveaux de processivité plus élevés que ceux du type sauvage mènent à des difficultés réplicatives aux télomères. Ces résultats suggèrent que la fonction et la taille des télomères, et l'immortalisation des cellules humaines sont réguléss par la processivité de l'enzyme télomérase.
31
Thomson, Philippa. "Isolation and characterisation of telomere and telomere-related sequences in the chicken genome." Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/29768.
Full textAbstract:
Telomeres are the physical ends of eukaryotic chromosomes, and are essential for chromosome stability and complete replication. Chicken telomere and telomere-related sequences were characterised using a variety of approaches including: cosmid libraries, anchored PCR strategies, and fluorescence in situ hybridisation (FISH). The results suggested that chicken chromosomes terminate in 6.7-14.0 kb tandem arrays of the vertebrate telomere repeat, (TTAGGG)n. The proximal boundaries of these arrays are degraded, and include many variant repeat types which differ from the telomere repeat by one base change. Southern analysis suggested that the degraded repeats can be digested with restriction enzymes, increasing the probability of locating telomere-related sequences in standard cosmid libraries. Complex repeats associated with the terminal arrays and the degraded repeats were isolated, and shown to be located proterminally on metaphase chromosomes. Hybridisation of a telomere repeat probe to genomic DNA separated by pulse field gel electrophoresis (PFGE) suggested that complex subtelomeric repeats may form large satellite blocks at the ends of a subset of chromosomes, resulting in terminal MboI restriction fragments of up to 620kb. Fluorescence in situ hybridisation (FISH) experiments indicated that the distribution of subterminal and terminal sequences within the genome were correlated with distance from the chromosome end. Sequences located towards the ends of chromosomes (i.e. interspersed telomere repeats) were found on more chromosomes than those from more proximal locations (i.e. complex repeats). Screening of a large insert cosmid library (Stratagene) with a telomere repeat probe resulted in the isolation of 59 clones. Single-locus restriction fragment length polymorphisms (RFLPs) were identified from ten of these clones, and the inserts were localised by FISH on metaphase chromosomes. The cosmid probes showed no bias towards location on macrochromosomes. The results suggested that some of the clones may contain DNA from subtelomeric locations, but that others clearly identify interstitial loci.
32
Wen, Victoria Wei-Yu Women's & Children's Health Faculty of Medicine UNSW. "Molecular alterations during immortalisation of human endothelial cells." Awarded by:University of New South Wales. Women's & Children's Health, 2009. http://handle.unsw.edu.au/1959.4/44743.
Full textAbstract:
Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.
33
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/.
Full text
34
Revie, John. "Identification and characterisation of telomere regulatory and signalling pathways after induction of telomere dysfunction." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7358/.
Full textAbstract:
Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.
35
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.
Full text
36
Nanavaty, Vishal P. "Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009.
Full text
37
Wiley, Emily A. "Yeast telomere structure : genetic analysis implicating a novel terminus-specific factor in telomeric silencing /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/6359.
Full text
38
Gerasimopoulos, Efthalia. "Topoisomerase-mediated poxviral telomere resolution." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0021/MQ47325.pdf.
Full text
39
Qi, Qi. "Mathematical modelling of telomere dynamics." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12258/.
Full textAbstract:
Telomeres are repetitive elements of DNA which are located at the ends of chromosomes. During cell division, telomeres on daughter chromomeres shorten until the telomere length falls below a critical level. This shortening restricts the number of cell divisions. In this thesis, we use mathematical modelling to study dynamics of telomere length in a cell in order to understand normal ageing (telomere shortening),Werner’s syndrome (a disease of accelerated ageing) and the immortality of cells caused by telomerase (telomere constant length maintenance). In the mathematical models we compared four possible mechanisms for telomere shortening. The simplest model assumes that a fixed amount of telomere is lost on each replication; the second supposes that telomere loss depends on telomere length; for the third case the amount of telomeres loss per division is fixed but the probability of dividing depends on telomere length; the fourth cases has both telomere loss and the probability of division dependent on telomere length. We start by developing Monte Carlo simulations of normal ageing using these four cases. Then we generalize the Monte Carlo simulations to consider Werner’s syndrome, where the extra telomeres are lost during replication accelerate the ageing process. In order to investigate how the distribution of telomere length varies with time, we derive, from the discrete model, continuum models for the four different cases. Results from the Monte Carlo simulations and the deterministic models are shown to be in good agreement. In addition to telomere loss, we also consider increases in telomere length caused by the enzyme telomerase, by appropriately extending the earlier Monte Carlo simulations and continuum models. Results from the Monte Carlo simulations and the deterministic models are shown to be in good agreement. We also show that the concentration of telomerase in cells can control their proliferative potential.
40
Richter, Torsten. "Mechanisms of telomere-dependent senescence." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435572.
Full text
41
Kedziora, Sylwia Maria. "How Rif1 controls telomere length." Thesis, University of Aberdeen, 2017. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=235993.
Full textAbstract:
The activation of replication origins is temporally regulated in S phase, with some origins activating early and some late. The molecular events controlling the temporal programme are not well understood, but in S. cerevisiae there is a close relationship between telomere length and nearby origin activation time. In the first part of this thesis I explore how the initiation time of origins near to telomeres is regulated by telomere length in a manner dependent on the Tel1 kinase. I demonstrate that an induced short telomere drives early activation of a nearby origin, but that in the absence of Tel1 the same origin activates late. In the second, major part of this thesis I focus on how the Rif1 protein negatively regulates length of the terminal TG1-3 repeats. While Rif1 has long been known to control telomere length, the mechanism through which Rif1 prevents telomere over-extension has remained unclear. Recently Rif1 was discovered to act in DNA replication control as a Protein Phosphatase 1-targeting subunit, directing Protein Phosphatase 1 (PP1) to dephosphorylate the MCM replicative helicase complex. I therefore investigated whether Rif1 also controls telomere length through PP1 interaction. I examine the effects of a mutant Rif1 with its PP1 interaction sites mutated to ablate PP1 binding. I found the mutant Rif1 binds normally to telomeres but causes a long telomere phenotype, similar to that in ∆rif1 cells, implicating Rif1-PP1 interaction in telomere length control. In further experiments I show that tethered PP1 can partially substitute for Rif1 in telomere length control. I also establish that the effect of Rif1-PP1 on telomere length does not operate indirectly through replication timing control, but rather appears to act through a direct pathway controlling telomerase recruitment. I discuss potential dephosphorylation targets, and the mechanism through which Rif1 and PP1 may control telomere length homeostasis. To summarise, my PhD research demonstrates that S. cerevisiae Rif1 acts with PP1 to repress telomerase-mediated TG1-3 repeat extension.
42
Pooley, Karen Anne. "Genetic factors in telomere length." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609670.
Full text
43
Zhao, Y. "Pot1 phosphorylation regulates telomere function." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1380712/.
Full textAbstract:
The telomere is a conserved nucleoprotein structure at the ends of eukaryotic chromosomes. It is essential for maintenance of genomic stability: on the one hand, it suppresses DNA damage response and protects the natural chromosome ends from repair activities; on the other hand, it recruits telomerase, the specialized reverse transcriptase, to counteract the end-replication problem. The telomeric G-strand ssDNA-binding protein Pot1 plays a crucial role in both of these functions. In fission yeast S. pombe, inhibition of Pot1 induces rampant 5’ resection and loss of telomere signal in a single cell cycle. It was recently shown that spPot1 interacts with, and is phosphorylated by, the master cell cycle regulator DDK. Alleles of a V5-tagged version of pot1+ were constructed with mutations at the putative phosphorylation sites, which reside in the N-terminal OB-fold DNA binding domain of Pot1 {Kuznetsov, 2008 #6380}. The goal of this study was to determine the molecular mechanism by which phosphorylation of Pot1 regulates telomere function. We found that the phospho-deficient mutants of Pot1 induce telomere elongation, checkpoint activation, and deregulation of ssDNA generation, suggesting reduced association with the ssDNA. Our data point to a model in which cell cycle-regulated Pot1 phosphorylation coordinates telomere replication and telomerase activity in different cell cycle phases. Furthermore, we showed that the C-terminal V5-tagging of Pot1 also affects its functions, suggesting an additional layer of complexity governing Pot1 function.
44
Kamnert, Iréne. "Classes of DNA associated with telomeres in the chironomids C. pallidivittatus and C. tentans." Lund : Dept. of Genetics, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39009480.html.
Full text
45
Bernal, Martínez Aina. "Telomere deprotection and the maintenance of genome integrity: discrepancy between telomere shortening and shelterin dysfunction." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/666888.
Full textAbstract:
Els telòmers són estructures nucleoprotèiques que segellen l’extrem cromosòmic i el protegeixen de la reparació il·legítima mitjançant la formació d’un llaç telomèric anomenat t-loop que està modulat per la proteïna TRF2. En les últimes dècades, s’ha demostrat que la disfunció telomèrica és un mecanisme capaç d’originar inestabilitat cromosòmica (CIN) en cèl·lules humanes i de ratolí i promoure la tumorigènesis en models murins.
L’objectiu d’aquesta tesi és immortalitzar cèl·lules telomèricament inestables i avaluar el seu potencial tumorigènic mitjançant la desprotecció telomèrica a través de l’escurçament telomèric o de la disfunció de la proteïna TRF2. En el Treball I, cèl·lules epitelials mamàries humanes deficients per p16INK4a (vHMECs) i deficients o no per p53 (p53+/+) foren cariotipades a diferents temps de doblatge per avaluar la presència d’anomalies cromosòmiques i l’evolució del cariotip. En l’absència de la telomerasa, els telòmers s’escurçaren de forma progressiva fins esdevenir desprotegits, i la subseqüent reparació il·legítma dels extrems afavorí l’entrada en cicles de ruptura-fusió-pont (BFB) i disparà la CIN. Tanmateix, les cèl·lules inestables finalment moren independentment de la funcionalitat de p53. Per contra, la sobreexpressió d’hTERT en les cèl·lules vHMEC-p53+/+ afavoreix la proliferació indefinida amb un cariotip gairebé estable, mentre que la immortalització de les cèl·lules vHMEC-shp53 amb signes de CIN és permissiva amb la proliferació cel·lular i l’evolució del cariotip.
Al Treball II i al Treball III, la desprotecció telomèrica aguda va ser induïda mitjançant l’expressió transitòria d’un dominant negatiu de TRF2 (TRF2BM). El dany telomèric agut fou avaluat en la línia cel·lular epitelial mamària MCF-10A (Treball II) i en cèl·lules epitelials mamàries (HMECs) immortalitzades derivades de pacients sanes (Treball III). Al Treball II, la desprotecció telomèrica mitjançant TRF2BM generà un increment de TIFs, fusions telomèriques i ponts anafàsics. Els ponts anafàsics es consideren els inductors dels cicles (BFB) i de la posterior reorganització cariotípica. No obstant, no s’observaren reorganitzacions cromosòmiques derivades dels cicles BFB després de l’expressió transitòria del TRF2BM, independentment de la funcionalitat de les proteïnes p53 i pRb. Després de successius cicles de desprotecció telomèrica, s’observà un increment en el nombre de cèl·lules diploides, tot suggerint que l’excessiu dany telomèric evitaria la proliferació d’aquelles cèl·lules que podrien esdevenir altament reorganitzades. Al Treball III s’expressà TRF2BM en HMECs immortalitzades mitjançant hTERT i l’antigen SV40LT (HMEC-TO). L’expressió transitòria del TRF2BM induí l’increment de ponts anafàsics, però les cèl·lules tampoc presentaren reorganitzacions pròpies dels cicles BFB en marxa. A diferència del Treball II, les cèl·lules poliploides incrementaren arrel del procés d’immortalització. Independentment de la causa d’aquest increment en les cèl·lules poliploides, les cèl·lules exposades als cicles d’expressió de TRF2BM no presentaven un fenotip associat a la disfunció telomèrica, ni tampoc un potencial tumorigènic, tot suggerint que l’expressió del mutant TRF2BM provoca un efecte deleteri sobre la viabilitat cel·lular i l’inici de la CIN.
En resum, la present tesi evidencia que el grau de dany telomèric és una eina de doble fil en el manteniment de la integritat genòmica. Per una banda, l’escurçament telomèric indueix un dany cel·lular progressiu i lleu, compatible amb la viabilitat cel·lular. Quan el dany telomèric assoleix un cert llindar, la cèl·lula activa mecanismes dependents i independents de p53 que indueixen la seva mort. Pel contrari, la disfunció de TRF2 afecta a un nombre molt elevat de telòmers i indueix una exagerada resposta cel·lular que la fa incompatible amb la viabilitat cel·lular i la reorganització del cariotip. Aquesta tesi demostra que la desprotecció simultània d’un nombre elevat de telòmers pot ser una eina útil per generar un dany al DNA molt elevat i mantenir la integritat genòmica.Telomeres are nucleoprotein structures that cap the end of chromosomes and protect them from illegitimate recombination through a lariat conformation or t-loop that is mainly promoted by TRF2 protein. Dysfunctional telomeres have been proved to be a mechanism capable of originating chromosome instability (CIN) in mouse and human cells, and promote tumorigenesis in mouse models. This dissertation thesis aims to generate immortalised but unstable cells due to telomere deprotection through progressive telomere shortening and by TRF2 depletion, and to evaluate their tumorigenic potential. In Work I, p16INK4a-deficient human mammary epithelial cells (vHMECs) lacking or not for p53 function through specific short-hairpin RNA inactivation, were karyotyped at different population doublings to evaluate chromosomal abnormalities and their evolution. In the absence of telomerase, vHMECs progressively shortened their telomeres and subsequent end-to-end fusions initiated breakage-fusion-bridge (BFB) cycles and promoted CIN. However, these unstable cells finally succumbed to cell cycle arrest, independently of their p53 checkpoint status. In contrast, hTERT overexpression in p53-proficient vHMECs resulted in cells able to proliferate indefinitely with a nearly stable karyotype, while immortalised p53-deficient cells showed signs of CIN that could be permissive with an evolving karyotype. In Work II and Work III, acute telomere deprotection was induced by t-loop disassembly through transient expression of the dominant negative form of TRF2 (TRF2BM) in the mammary cell line MCF-10A and in immortalised HMEC derived from cosmetic reductions of four healthy donors, respectively. In Work II, acute telomere deprotection phenotype was reflected by the presence of TIFs and by an increase of end-to-end fusions and anaphase bridges after TRF2BM induction. Anaphase bridges are considered the prelude to breakage-fusion-bridge cycles and subsequent karyotype reorganisations. However, no scars of BFB cycles or highly reorganised cells have been observed after transient expression of TRF2BM, independently of the status of p53 and pRb proteins. Instead, diploid cells were enriched after successive cycles of telomere deprotection induction, thus suggesting that excessive telomere deprotection could be detrimental for the origin of cells with highly reorganised karyotypes. According with these results, in Work III, immortalised HMEC through hTERT and SV40LT overexpression transiently expressing TRF2BM (HMEC-TO) exhibited an increase of anaphase bridges. But after a minimum of five cycles of telomere protection and deprotection, TRF2BM expressing cells did not display scars of telomere deprotection and ongoing BFB cycles. In contrast to MCF-10A derived cell lines, the HMEC-TO cell lines exhibited a progressive increase of polyploid cells as a consequence of SV40LT immortalisation process. Independently of the cause of polyploidy increase, cells exposed to TRF2BM expression cycles did not exhibit a telomere dysfunction phenotype or either a tumorigenic potential, thus suggesting that TRF2BM expression provoked a deleterious effect over TRF2BM expressing cells and prevented CIN emergence. In conclusion, the present dissertation provides evidence that telomere dysfunction acts as a double sword mechanism for genome integrity. On the one hand, telomere shortening induces a mild and progressive DNA damage that firstly is compatible with cell viability, until damage is high enough to induce cell death. On the contrary, shelterin dysfunction affects widely to all chromosomes inducing an exacerbated cell response that is deleterious for cell viability and karyotype reorganisation. This Thesis illustrate that acute telomere deprotection through shelterin dysfunction could be a useful tool to impinge an exacerbated DNA damage and maintain genome integrity in human mammary cells.
46
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.
Full textAbstract:
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.
47
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.
Full textAbstract:
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.
48
Cabuy, Erik. "Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours." Thesis, Brunel University, 2005. http://bura.brunel.ac.uk/handle/2438/5157.
Full textAbstract:
Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.
49
Barbaro, Pasquale. "Telomere dynamics in children undergoing cancer treatment and the relationship between telomere length and treatment outcome." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/21162.
Full textAbstract:
Telomeres are specialised DNA structures that protect the ends of linear chromosomes, and their length is important for cell viability. Telomere length declines with age, and there is a large variability seen within the general population. Patients with short telomere disorders (STD) suffer increased toxicity from chemotherapy given for haematopoietic stem cell transplant (HSCT), however little is known of the relationship between telomere length, outcome and toxicity in the general population undergoing chemotherapy. This thesis shows that patients with STDs have poor short- and long-term outcomes following HSCT with mortality mostly due to pulmonary disease or infections. To define the relationship between telomere length and outcome in patients receiving therapy for cancer, we undertook retrospective analysis of outcome and telomere dynamics in children who had undergone HSCT, treatment for childhood acute lymphoblastic leukaemia (ALL) and who were long term survivors of childhood cancer. Children undergoing cancer treatment have an acute decline in telomere length at the time of cancer therapy, however there is no evidence that this telomere attrition continues in the long term, with the exception of patients with some cancer types, and who received certain specific chemotherapy agents. Patients undergoing HSCT or treatment for ALL with shorter telomere length have higher rates of specific organ toxicity and certain infections. However there does not appear to be any relationship between telomere length and significant late effects from childhood cancer, aside from avascular necrosis. These findings highlight the complex relationship between telomere length and cancer therapy. They require further confirmation in prospective clinical trials, and in a wider population with differing initial malignancies, and undergoing different chemotherapy regimes in order to better define the complex relationship between telomere length, chemotherapy and outcome.
50
Surovtseva, Yulia V. "Telomere-associated proteins in Arabidopsis thaliana." [College Station, Tex. : Texas A&M University, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-2656.
Full text