No description available.

No description available.

Thesis (Ph. D. in Biological Sciences)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Thesis (Ph. D.)--University of Hong Kong, 1997. / Includes bibliographical references.

Thesis (M.Med.Sc.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 65-85). Also available in print.

Telomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.

There remains a pressing need for the development of effective drugs that meet the clinical needs for cancer treatment, and inhibition of telomere length maintenance by disrupting human telomerase is a proven and tractable target for suppression of cancer cell growth. In response to the lack of currently available small molecules with efficacy against human telomerase, we developed a genetically and chemically tractable cell-based system in which S. cerevisiae is used to streamline the search for novel human telomerase inhibitors. Our results confirmed that yeast cell growth was rapidly inhibited upon induction of functional human telomerase at the telomere. This inducible growth arrest was used as a read-out for a high-throughput chemical screen for human telomerase inhibitors based on their ability to restore growth in the yeast system. From a library consisting of small, bioactive and cell-permeable compounds of diverse structure, we identified three novel “drug-like” compounds that inhibited the activity of native and recombinant telomerase complexes in vitro. “Validation assays” also confirmed the novel inhibitors were free of uncharacterized adverse effects against yeast and human cell models, thus confirming the specificity of these novel inhibitors against human telomerase target. This surrogate yeast model has therefore proven to be a cost-effective alternative to accelerate the search for human telomerase inhibitors, which we hope will serve to streamline the identification of further lead compounds effective against human cancer.

Telomerase is an enzyme that helps maintain the telomeric ends of chromosomes during DNA replication. Telomere lengths represent a balance between telomerase activity attempting to elongate their ends, and cell division that causes telomere shortening. As cells age, diminished telomerase activity allows a shortening of telomere lengths until they reach a target length that stimlulates apoptosis. Identifying a drug capable of upregulating telomerase activity may help increase cell (and even organismal) lifespan. The purpose of this thesis was to determine whether treatment of human primary foreskin fibroblast cultures with a 14 amino acid (aa) ependymin peptide mimetic upregulates (or at least maintains) telomerase activity and telomere lengths during cellular ageing. The 14aa peptide was previously shown to significantly increase the murine lifespan by 25%, so its activity was a logical candidate to test in this thesis. In a preliminary set of experiments, the human primary fibroblast cells were shown to respond to the 14aa drug by upregulating the antioxidative enzyme superoxide dismutase (SOD), thus human fibroblast cells likely contain the appropriate receptor for binding this drug. This same dose proved optimal for upregulating telomerase activity in the fibroblast cells an average of 57% relative to untreated cells (p value = 0.003). The upregulation appears to be specific for the sequence of aa in the 14aa drug since a“scrambled" peptide containing the same aa but in a different order showed no upregulation, even at doses 10-fold higher. Treatment of mice once per day or twice per day with the 14aa peptide was also found to upregulate telomerase activity in vivo in brain and heart. The activity was optimal at a 3.3 mg/kg dose for each aged organ, and was generally high in young organs. The activity observed in heart was a total surprise since heart cells are generally thought to be quiescent, and telomerase is usually associated with cell division, so perhaps telomerase has a function other than in cell division. The second part of the hypothesis tested whether treatment of fibroblast cells with the 14aa drug elongated (or prevented from shortening) telomere lengths in aged cells. A telomere length assay (TLA) based on a Southern hybridization approach using a telomere probe appeared to work well, since marker DNAs showed appropriate differences in their“telomere smears", and aged fibroblast cells showed shorter smears than young cells. However, no difference was observed between drug-treated versus vehicle-treated cells, even at the 10 ng/ml dose previously shown to strongly upregulate telomerase activity. So perhaps the upregulation of telomerase activity was not sufficient to provide a measurable increase in telomere lengths. Telomerase has been shown to extend the lifespan of virus-transformed human cells without showing any visible telomere lengthening (Blackburn et al, 1999), so perhaps telomerase can increase cell lifespan without increasing telomere lengths. To our knowledge, this is the only drug demonstrated to upregulate telomerase activity. Transforming cells with the viral T-antigen can upregulate telomerase, but T-antigen is not a therapeutic drug since it also causes cancer. Telomerase upregulation is known to occur during oncogenesis, but telomerase itself is not an oncogene since oncogenesis also requires the upregulation of oncogenes. Our lab previously showed this peptide does not upregulate the potent oncogene myc. If this proves to be the case for other oncogenes, using this 14aa drug to upregulate telomerase activity without activating oncogenes could prove extremely useful for helping prove telomerase is not an oncogene, and for extending cell lifespan.