Cells exposed to DNA damaging agents activate a network of mechanisms called DNA damage response, including telomere length regulation. Telomeres are specialized structures that protect chromosome ends from degrading and being fused together. Mouse-knockout experiments revealed that cell lines deficient of DNA-PKcs or Ku70/80 resulted in high amount of telomere end-to-end fusion. Numerous other studies have shown a functional interplay between DNA damage response and telomere maintenance. The aim of this project is to examine this interplay further by investigating mechanisms of DNA damage response, using cell lines from X-linked homozygous recessive form of Dyskeratosis Congenita (DC) patients, which have dysfunctional telomere maintenance. DC is a multi-system disorder characterised by abnormalities of the bone marrow, immune deficiency and a predisposition to cancer. In this work we have shown that cells with defective DKC1 (the gene implicated in the X- linked homozygous recessive form of DC) exhibit a defective DNA damage response by examining two types of cells: fibroblast and lymphoblastoid cell lines. By using various biomarkers (H2AX, TIF assay etc) we analysed the DNA damage response by exposing DC cell lines to ionizing radiation. Our results demonstrated that DC cell lines have an abnormal DNA damage response and as a result show radiosensitivity. We have also knocked down the DKC1 gene in normal cell lines using siRNA oligonucleotides and demonstrated that this knock-down causes radiosensitivity. Therefore our results conclusively show an abnormal DNA damage response in cells derived from DC patients. Finally we used TA-65, a novel telomerase activator derived from the plant Astragalus membranaceus and showed radioprotective effects of this compound in normal lymphoblastoid cell lines. Taken together our results potentiate further the link between telomere maintenance and DNA damage response.

Telomeres are repetitive DNA sequences found at the ends of eukaryotic chromosomes that help maintain genome stability. Telomeres shorten every time a cell divides, eventually inducing replicative senescence. To gain replicative immortality cancer cells establish mechanisms to maintain telomere length over many cell divisions. Around 10% of cancers do this using a recombination-based pathway called the Alternative Lengthening of Telomeres (ALT). ALT resembles a specific type of homology-directed repair called break-induced replication (BIR). Through this body of work, we aimed to better understand both the genetics underlying ALT positive cancers and the mechanistic basis of ALT. ALT positive cancers frequently carry loss of function mutations in the genes for ATRX/DAXX, which function to regulate heterochromatin. Recently, we identified a novel chromosomal fusion event in ALT positive osteosarcoma causing defects in DAXX function. Additionally, we identified several osteosarcoma tumors with wild-type ATRX/DAXX that had abnormalities in SLX4IP or SMARCAL1, proteins recently shown to regulate the ALT pathway. These data suggest that a more thorough understanding of the ALT mechanism may reveal additional factors that are defective in ALT positive tumors. Building on this, we aimed to further define the mechanism of ALT by investigating the DNA translocase RAD54 in the ALT pathway. During BIR, a broken DNA strand invades a homologous template, forming a structure called a displacement loop (D-loop) where a strand of template DNA is displaced to allow base pairing between the broken DNA strand and the homologous template. The D-loop recruits DNA polymerases, leading to extension and repair of the broken DNA strand. RAD54 is known to regulate both the formation and resolution of D-loops. In this work, we found that RAD54 promotes elongation at ALT telomeres by mediating branch migration and dissolution of the D-loop. D-loops formed at ALT telomeres must be resolved before mitosis to prevent the formation of ultra-fine anaphase bridges. These data demonstrate that by mediating D-loop migration RAD54 plays an important role in both promoting telomere elongation and maintaining genome stability in ALT cells. Together this body of work represents advances in defining both the genetic and mechanistic basis of ALT. / 2021-01-30T00:00:00Z

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Background: Telomeres are repetitive, gene-poor regions that cap the ends of DNA and help to maintain chromosomal integrity. Their shortening is caused by inflammation and oxidative stress within the cellular environment and ultimately leads to cellular senescence. Shortened leukocyte telomere length (LTL) is hypothesized to be a novel biomarker for age-related diseases and may therefore be useful in the prediction of cardiometabolic outcomes above conventional risk factors. Methods: A systematic review and meta-analysis was undertaken to summarize existing literature on the association between LTL and myocardial infarction (MI), stroke, and type 2 diabetes (T2D). MEDLINE (1966–present), and EMBASE (1980-present) were last searched on September 9th 2013. Studies were combined using the generic inverse variance method and both fixed and random effects models. Additionally, LTL was measured in 3972 MI patients and 4321 controls from an international study on risk factors for MI (INTERHEART), and 8635 participants from an epidemiological study on dysglycemia and T2D (EpiDREAM) prospectively followed (approximately 3.5 years) for incident cardiometabolic events. Results: Based on current literature, a 1-standard deviation decrease in LTL was significantly associated with stroke (OR=1.21, 95% CI=1.06-1.37; I2=61%), myocardial infarction (OR=1.24, 95% CI=1.04-1.47; I2=68%), and type 2 diabetes (OR=1.37, 95% CI=1.10-1.72; I2=91%). Stratification by measurement technique, study design, study size, and ethnicity explained heterogeneity in certain cardiometabolic outcomes. Within INTERHEART participants, a 1 unit decrease in LTL was associated with an increased risk of MI (OR=2.17, 95% CI=1.74-2.72). Effect estimates were consistent across all ethnic groups (p=0.19). In EpiDREAM a significant association between LTL and T2D or incident cardiometabolic outcomes was not observed. Conclusion: Telomere length appears to be a marker for MI above conventional risk factors. Further research is needed to explain existing heterogeneity in the literature with respect to LTL and T2D. / Thesis / Master of Science (MSc)

"Human umbilical cord blood (CB) hematopoietic stem cells (HSCs) have well established applications for cellular therapy. Current protocols for isolating HSCs from bone marrow or cord select for CD34 + cells, however some CD34 - populations have recently been shown to also contain strong HSC activity. Thus the positive selection of HSCs based on cell surface markers remains controversial. However, it is clear from the literature that differentiated hematopoietic cells (lineage positive, Lin + ), representing the vast majority (>90%) of most blood populations, contain no long-term reconstitution potential. Thus Viacell Inc. (Worcester, MA) expands and enriches its populations of cells containing HSCs by removing only those Lin + cells known not to contain HSCs. This is accomplished on two separation columns (post-sep-1, and post-sep-2) (separated by 7 days of cell growth) that contain a variety of antibodies to known differentiation surface markers. Although this process strongly enriches functional HCSs, these primitive cell populations remain biochemically uncharacterized. Because HSC populations containing long chromosomal telomeres and high telomerase activity (which helps maintain telomeres) have been shown to display the strongest long-term reconstitution potential, the purpose of this thesis was to investigate these two parameters in selected samples of Viacell’s ex vivo amplification procedure. Two specific hypotheses were tested: 1. the removal of Lin + cells will appear to increase the telomerase activity and telomere lengths in the remaining cell population, and 2. these two parameters will decrease upon hematopoietic cell differentiation and proliferation. Telomerase activity was assayed using a telomeric repeat amplication protocol (TRAP), and normalized relative to a cancer cell line positive control. Relative to fresh cord blood, telomerase activity was found to increase significantly in post-sep-1 (from 8.5 ± 1.5% to 76.2 ± 4.9%, p = 0.0001, n = 5) and post-sep-2 (8.5 ± 1.5% to 111.3 ± 4.9%, p = 0.0001, n = 5) fractions following the removal of Lin + cells. This increase was found to be highly reproducible, showing very low intra-cord and inter-cord variability. Telomere lengths were assayed using a telomere length assay (TLA). Relative to fresh cord blood, telomere lengths increased significantly in post-sep-1 (from 10 to 12 kb, n = 2) and post-sep- 2 (from 10 to 14 kb, p = 0.001, n = 2) fractions. These apparent increases likely result from the direct removal of cells low in telomerase activity with short telomeres since the Lin + cells from the post-sep-1 column were found to contain relatively low telomerase activity (32.1 ± 15%, p = 0.001, n = 2) and short telomeres (7.5 kb, p = 0.001), which supports our first hypothesis. Finally, we show that telomerase activity and telomere lengths decreased in Day-14 cells (expanded and differentiated 14 days) relative to post-sep-2 (from 111.8 ± 19.6% to 54 ± 21.2%, p = 0.001, n = 3 for the TRAP, and from 14 kb to 9 kb, p = 0.0001, n = 2 for the TLA). Those two parameters also decreased in pre-sep-3 cells (terminally differentiated by treatment with All Trans Retinoic Acid for 14 days) relative to post-sep-2 (from 111.3 ± 4.9% to 14.8 ± 1.7%, p = 0.0001, n = 6 for the TRAP, and from 14 kb to 7.5 kb, p = 0.001 for the TLA), supporting our second hypothesis. Telomerase activity was found to not directly correlate with CD34 + CD38 - content, supporting recent observations that a significant portion of HSCs reside outside this population."

Telomeres are the physical ends of linear chromosomes in eukaryotes. Telomeres not only protect chromosome ends from being recognized as double-strand breaks but also maintain the chromosome terminal sequences. These processes involve a number of telomere-related proteins. A major challenge in the field is to elucidate the full constitution of telomere-associated proteins and to understand how different protein complexes are regulated at chromosome termini. Here, I report the identification and characterization of STN1 (Suppressor of cdc thirteen, 1), CTC1 (Conserved Telomere maintenance Component 1) and TEN1 (Telomeric pathways in association with Stn1, 1) in Arabidopsis. CTC1/STN1/TEN1 (CST) forms a trimeric complex that specifically associates with telomeres. Loss of any component of the CST induces catastrophic telomere loss, disrupted telomere end architecture, and massive chromosome end-to-end fusions. Thus, CST plays an essential role in chromosome end protection. I also show that CST function at telomeres is independent of a previously characterized capping complex KU70/KU80, and that ATR is responsible for a checkpoint response in plants lacking CTC1/STN1. Additionally, I present data showing that Arabidopsis POT1a (Protection Of Telomere 1, a) has evolved as a telomerase recruitment factor. Unlike POT1 in other eukaryotes which binds and protects ss telomeric DNA, AtPOT1a interacts with telomerase RNA (TER). Based on an evolutionary analysis, we found that the POT1a lineage is under positive selection in the Brassicaceae family in which Arabidopsis belongs. Mutations of two positive selection sites significantly reduce POT1a?s activity in vivo. These data suggest POT1a is under pressure to evolve from a telomeric DNA binding protein to a TER binding protein. I also discovered that POT1a interacts with the novel telomere capping protein CTC1 in vitro and in vivo. Thus, I hypothesize that POT1a acts as a telomerase recruitment factor linking this enzyme to the chromosome termini via interacting with TER and CTC1. Finally, I dissected the functional domains of POT1a and demonstrated that both the N-terminus and the C-terminus of POT1a are required for its function in vivo. In summary, my work has uncovered several new and essential telomereassociated proteins that provide new insight into mechanisms of chromosome end protection and maintenance.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statment inserted. Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 127-154.