Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis. / Alveoläre Echinokokkose wird durch das Metazestodenstadium des kleinen Fuchsbandwurms Echinococcus multilocularis verursacht und medizinisch als eine schwere Zoonose mit begrenzten Behandlungsmöglichkeiten betrachtet. Um ein besseres Verständnis für die Biologie der Zestoden zu erlangen, wurde das Genom von E. multilocularis, zusammen mit denen anderer Zestoden, bereits sequenziert. Bisher wurden nur wenige Studien zum Transkriptom von E. multilocularis durchgeführt und eine umfassende Analyse der Transkriptionsprofile über verschiedene Stadien des Lebenszyklus hinweg fehlt bislang. Diese Arbeit stellt die bisher umfassendste Untersuchung des Transkriptoms von E. multilocularis dar. Unterschiede in der Genexpression in verschiedenen Stadien des Lebenszyklus und unter experimentellen Bedingungen wurden qualitativ und quantitativ untersucht. Dazu wurden Daten aus RNA-Sequenzierungen in drei biologischen Replikaten verwendet. Die untersuchten Datensätze beruhen auf Proben von Metazestoden, die unter aeroben und anaeroben Bedingungen kultiviert, sowie von Metazestoden, die direkt aus Gerbilen isoliert wurden. Weitere Proben umfassen Stammzellkulturen zu drei verschiedenen Entwicklungszeitpunkten sowie nicht-aktivierte und aktivierte Protoskolizes, das Larvenstadium das sich zu Adulten entwickeln kann. Zusätzlich wurden zwei Datensätze von Metazestoden unter experimentellen Bedingungen, die zur Identifizierung stammzellspezifischer (keimzellspezifischer) Gene geeignet sind, sowie ein Datensatz von einem siRNA-Experiment untersucht. Die Analyse dieser Datensätze führte zu Genexpressionsprofilen für alle annotierten Gene, unter anderem für Gene, die im Tegument des Metazestoden exprimiert werden und eine Rolle spielen bei Wirt-Parasit-Interaktionen und der Modulierung der Immunantwort des Wirts. Genexpressionsprofile liefern zudem Informationen über Gene, die für das infiltrative Wachstum des Parasiten in der Leber verantwortlich sein könnten. Des Weiteren wurden keimzellspezifische Gene identifiziert. Keimzellen sind die einzigen proliferierenden Zellen in E. multilocularis und daher von essentieller Bedeutung für die Entwicklung und das Wachstum des Parasiten. Durch eine Kombination von Keimzelldepletierungs- und Keimzellanreicherungsverfahren wurden Gene mit keimzellspezifischer Expression identifiziert. Wie erwartet, sind viele dieser Gene in der Translation, der Zellzyklusregulation oder DNA-Replikation und –Reparatur involviert. Darüber hinaus wurden keimzellspezifisch exprimierte Transkriptionsfaktoren detektiert, von denen viele in der Festlegung des Zellschicksals eine Rolle spielen. Als Beispiel eines keimzellspezifischen Genes wurde das Gen, das für die reverse Transkriptase (TERT) kodiert, genauer untersucht. Die Expression von E. multilocularis tert in Keimzellen wurde experimentell bestätigt. Zellkulturexperimente weisen darauf hin, dass TERT für die Proliferation und die Entwicklung essentiell ist. TERT ist daher ein potentiell interessantes Wirkstofftarget für die chemotherapeutische Behandlung der alveolären Echinokokkose. Zu den keimzellspezifischen Genen in E. multilocularis gehören auch Gene densoviralen Ursprungs. Es wurden mehr als 20 Densovirusloci mit Informationen für nicht-strukturelle und strukturelle Densovirusproteine im E. multilocularis-Genom identifiziert. Densovirale Elemente wurden auch in vielen anderen Zestodengenomen detektiert. Die genomische Integration dieser Elemente deutet darauf hin, dass densovirus-basierte Vektoren zur genetischen Manipulation von Zestoden geeignet sein könnten. Interessanterweise sind nur drei von mehr als 20 Densovirusloci im E. multilocularis-Genom exprimiert. Da es in Zestoden keinen kanonischen piRNA-Signalweg gibt, stellt sich die Frage nach möglichen Genabschaltungsmechanismen. Die Analyse der RNA-Sequenzierdaten ergab Hinweise auf natürliche Antisense-Transkripte als einen möglichen Genregulationsmechanismus in E. multilocularis. Vorläufige Experimente und bisherige Studien deuten weiterhin darauf hin, dass DNA-Methylierung ein Mechanismus der Genregulation und -abschaltung in Zestoden sein könnte. Die Transkriptionsdaten enthalten auch Informationen zu Genen, die in differenzierten Zellen exprimiert werden, wie zum Beispiel das Serotonintransportergen, das in Nervenzellen exprimiert wird. Zellkulturversuche weisen darauf hin, dass Serotonin und Serotonintransport eine wichtige Rolle bei der Proliferation, der Entwicklung und dem überleben von E. multilocularis spielen. Insgesamt bietet diese Arbeit einen umfassenden Transkriptionsdatenatlas über die Stadien des Lebenszyklus von E. multilocularis. Die Identifizierung von keimzellspezifischen Genen und Genen, die für die Interaktion zwischen Wirt und Parasit wichtig sind, wird die zukünftige Forschung erheblich erleichtern. Ein globaler Überblick über die Genexpressionsprofile wird zudem hilfreich sein bei der Entdeckung geeigneter Wirkstofftargets und bei der Entwicklung neuer Chemotherapeutika gegen die alveoläre Echinokokkose.

Telomeres are the ends of linear chromosomes which are protected by a multi-protein complex called shelterin. The proper maintenance of telomeres involves strict control over the length of the TTAGGG telomeric repeat sequences. In part, this is achieved through the action of the shelterin complex component TRF1. TRF1 binds to duplex telomere DNA and acts as a suppressor of telomerase-dependent telomere elongation, however the exact mechanism by which it achieves this is currently unknown. Recent observations with a phospho-specific TRF1 antibody indicate that phosphorylated (pT371)TRF1 localizes at Cajal bodies. Cajal bodies are subnuclear organelles with myriad functions, one of which is to recruit the subunits of the telomerase holoenzyme for assembly and the subsequent targeting of the enzyme to telomeres for elongation. The results presented here demonstrate that this association of phosphorylated (pT371)TRF1 to Cajal bodies is highly specific, requiring its DNA binding capability, and occurring only in Cajal bodies which are not actively involved with telomere extension. While the function of this association has not been elucidated, the data are suggestive of a telomerase-related role being played by phosphorylated (pT371)TRF1 at Cajal bodies, possibly related to its function in suppressing telomere elongation. CSB is a multifunction protein which is implicated in transcription-coupled repair (TCR), base excision repair (BER), and control of transcription. Certain mutations and truncations of CSB are known to cause Cockayne syndrome (CS) in humans, an autosomal recessive progeria with devastating consequences. Unlike other progeria, CS patients do not display increased cancer incidence. Despite this fact, CSB is upregulated and in many cancers. In these cells, removal of CSB leads to apoptosis and increased sensitivity to chemotherapeutic drugs which suggests a dependency on CSB. The CSB of some non-CS cancer patients has also been found to be mutated at several recurring SNPs through the CSB gene. Preliminary examination of some of these SNPs suggests that they may invoke a change in the efficiency of TCR repair of UV-induced DNA damage. The results presented here demonstrate that, for the SNPs examined, there is no significant change in the repair of UV damage as assessed by colony survival assays post UV-treatment. While this may rule out an effect on TCR by these cancer-associated SNPs, it is possible that they may have an effect on CSB’s involvement in other vital cellular processes. / Thesis / Master of Science (MSc)

Yes / To develop potential next-generation metal anticancer agents, we designed and synthesised five Cu(II) 2-pyridine-thiosemicarbazone complexes by modifying the hydrogen atom at the N-4 position of ligands, and then investigated their structure-activity relationships and anticancer mechanisms. Modification of the N-4 position with different groups caused significant differences in cellular uptake and produced superior antitumor activity. Cu complexes arrested the cell cycle at S phase, leading to down-regulation of levels of cyclin and cyclin-dependent kinases and up-regulation of expression of cyclin-dependent kinase inhibitors. Cu complexes exerted chemotherapeutic effects via activating p53 and inducing production of reactive oxygen species to regulate expression of the B-cell lymphoma-2 family of proteins, causing a change in the mitochondrial membrane potential and release of cytochrome c to form a dimer with apoptosis protease activating factor-1, resulting in activation of caspase-9/3 to induce apoptosis. In addition, Cu complexes inhibited telomerase by down-regulating the c-myc regulator gene and expression of the human telomerase reverse transcriptase. / Natural ScienceFoundation of China (31460232, 21431001, 21561017, 21462004),the Natural Science Foundation of Guangxi (2017GXNSFEA198002,AD17129007), IRT_16R15, Guangxi“Bagui”scholar program to HBSun, and High-Level Innovation Team and Distinguished Scholarprogram of Guangxi universities to F Yang.

Telomerase, discovered in 1985, is considered a near-universal marker of malignancy and therefore has a potential use in cancer therapeutics and diagnostics. In this study, I used several approaches to gain a better understanding of telomerase and its potential applications in the canine context, for both cancer therapeutics and diagnosis. Having already developed an effective siRNA viral vector in vitro, the challenge still remained to deliver it efficiently in vivo. Thus, I initially investigated two possible approaches for in vivo delivery. First, I investigated a cell-based system for direct delivery to the tumours. Specifically I optimised a system for efficient gene-transfer to endothelial cells using a green fluorescent protein plasmid vector, and monitored systemic delivery by ex vivo imaging of dye-labelled cells in a canine xenograft tumour mouse model. In parallel, in vitro I investigated the gene transfer mediated by a novel dendrimer vector that can form nanoparticles with DNA and accumulate in tumour sites in vivo after i.v. administration. In order to utilize these delivery systems, I developed a DNA plasmid-based siRNA vector and tested its efficacy on canine tumour cells. To investigate telomerase as a cancer biomarker, I conducted a study that aimed to detect circulating telomerase reverse transcriptase (TERT) mRNA in serum taken from canine cancer patients. For this I developed several systems for effective RNA isolation from serum and used both conventional and quantitative PCR assays to detect TERT expression. Although for the first time I can confirm the existence of mRNA in serum of canine cancer patients, in this clinical study, I could only detect telomerase transcripts in a very small proportion of canine cancer patients. In a final pilot study to investigate anti-ageing technologies, I looked at the potential for drug-dependant telomerase induction rather than inhibition. For this I investigated the ability of three candidate drugs to induce TERT mRNA activation in canine embryonic fibroblasts. In this study, telomerase induction was measured using the quantitative PCR method that I had developed for serum detection. In summary, I have demonstrated that a cell-based delivery vehicle has a potential application in cancer therapy, but that more development is required before it can be applied clinically. I have also reported here that PPIG3 dendrimer-based gene transfer in vitro is low in canine cancer cells and thus require more optimisation and development before it can be utilised as an efficient systemic delivery vehicle. For the siRNA experiment, unfortunately, I did not observe any telomerase genesilencing in canine cancer cells using the plasmid-based siRNA expression vector, and therefore the gene sequence of cTR that we were targeting as well as the siRNA plasmid-vector that we used needs further validation in canine cells. I also suggest that TERT mRNA may not be a good serum biomarker for canine cancer diagnostics as I did not find TERT transcript in most of our serum samples from canine cancer patients, although circulating mRNA of a housekeeping gene was detected. Finally, in a pilot study, I have demonstrated that telomerase can be induced in normal canine somatic cells using small molecules. However, the long-term effects of telomerase induction on ageing must be determined in future studies.

En hématologie, du fait de leur évolution favorable, les tumeurs spontanément regressives comme lesmodèles myéloïde de la mastocytose, et lymphoïde de la papulose lymphomatoïde sont peu étudiés. Lamastocytose est une hémopathie myéloproliférative clonale dont les lésions cutanées peuvent régresserspontanément chez l’enfant alors que la maladie est chronique chez l’adulte. Les mutations chezl’enfant sont en partie différentes de celles retrouvées chez l’adulte. Nous avons montré in vivo dansles mastocytoses pédiatriques une expression diminuée de la télomérase, associée à des télomèrescourts. In vitro, grâce à deux modèles cellulaires comportant différents mutants de KIT de « typepédiatriques » ou de « type adulte », nous avons montré une augmentation de la longueur destélomères dans le mutant adulte, associée à une moindre sénescence comparées aux mutantspédiatriques sans pour autant mettre en évidence de différence dans l’expression et l’activité de latélomérase. Ces observations permettent en partie d’expliquer la régression des formes pédiatriques.La papulose lymphomatoïde est une lymphoprolifération cutanée T CD30+ dont les lésions régressentspontanément sans traitement. Cependant 10 à 20% des cas sont associés à un lymphome. Nous avonsétudié la physiopathologie d’expression du PDGFRβ dans les cellules tumorales via l’activation deNotch1. L’étude des télomères et de la télomérase in vivo et in vitro est préliminaire, et montrenotamment des télomères courts dans les cellules tumorales. En conclusion, nous montrons d’une partque la longueur des télomères dans les mastocytoses et la papulose lymphomatoïde est corrélée àl’évolution de la maladie, d’autre part, nous identifions un type de mutation potentiellement agressivedans les mastocytoses. Nous recommandons le génotypage systématique de cette pathologie dans lebut d’un suivi clinique attentif lorsque les lésions sont persistantes ou évolutives. / Childhood mastocytosis and lymphomatoid papulosis are mostly spontaneously regressive diseases.Mastocytosis is a clonal myeloproliferative disease; whereas cutaneous forms may regressspontaneously especially in childhood, the disease is chronic among adults. KIT mutations aredifferent between children and adults. We showed in vivo that children with mastocytosis displaydecreased telomerase expression with shorter telomere length. In vitro, using infected cells withdifferent KIT mutants, "paediatric" or "adult " one, we found longer telomere among adult mutant withdecreased senescence compared to paediatric mutant, without significant differences of telomeraseexpression and activity. These observations could explain the regression in paediatric mastocytosis.Lymphomatoid papulosis is a primary cutaneous CD30+ T-cell lymphoproliferative disorder.Cutaneous lesions are spontaneously regressive but association with a T-cell lymphoma is observed in10 to 20% of cases. We studied PDGFRβ expression to explain proliferative phase and telomerebiology to understand the regressive phase of the disease. Our result showed that PDGFRβ expressionin CD30+ cells was associated to short telomeres. In conclusion, we showed for the first time thattelomere length in mastocytosis and lymphomatoid papulosis seemed to be correlated with diseaseoutcome; on the other hand, we identify a mutation potentially aggressive. Taken together, werecommend to systematically look for KIT mutation among each patient with mastocytosis in order tocarefully monitor them when the cutaneous lesions are persistent and/or progressive.

Orientador: Maria Isabel Nogueira Cano / Resumo: A leishmaniose é uma doença crônica, causada por parasitos flagelados do gênero Leishmania, podendo se apresentar nas formas clínicas, tegumentar (cutânea), mucocutânea e visceral (calazar). A doença é considerada negligenciada pela OMS, pois não existem até o momento métodos eficientes de tratamento e controle para a mesma. Os telômeros desse parasito são um dos potenciais alvos no desenvolvimento de novos fármacos para o combate dessa doença e, para tanto, é necessário o entendimento da biologia desta estrutura. Uma enzima de grande interesse para o estudo dos telômeros é a telomerase que é a responsável pela manutenção e elongação dessas estruturas nos terminais dos cromossomos. A manutenção dos telômeros não é unicamente regulada pelo complexo ribonucleoproteico (RNP) da telomerase, mas também por proteínas que se associam ao complexo e ao DNA telomérico, tornando a ação do complexo mais efetiva e estável. Até o momento, o complexo telomérico de Leishmania amazonensis é o melhor caracterizado dentre os tripanosomatídeos, porém pouco se sabe sobre a biogênese e a composição do complexo RNP telomerase deste parasito. HSP83, ortólogo da HSP90 humana em Leishmania é uma chaperona altamente conservada, dependente de ATP e expressa quando as células são submetidas a diferentes tipos de estresse estando envolvida em transdução de sinal, crescimento, diferenciação celular e sobrevivência. Também é de grande importância para patógenos humanos, em particular aqueles cujo ciclo de v... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leishmaniasis is a chronic disease, caused by flagellated parasites of the genus Leishmania, which could be present in different clinical forms such as, tegumentar (cutaneous), mucocutaneous and visceral (kalazar). WHO classifies leishmaniasis as a neglected disease since there are no efficient methods for disease treatment and control. Parasites telomeres are one of the potential targets for the development of new anti-parasitic drugs to combat this disease and, thus, it is necessary to understand the biology of this structure. Telomerase is the enzyme responsible for maintaining and replicating these structures at the chromosomes termini. However, telomeres maintenance is not only regulated by the telomerase ribonucleoprotein complex (RNP), but also by proteins that associate with the complex and with telomeric DNA, making the action of the complex more effective and stable. To date, the telomeric complex of Leishmania amazonensis is the best characterized among trypanosomatids, although little is known about the biogenesis and composition of the RNP telomerase complex of this parasite. HSP90 is a highly conserved, ATP dependent chaperone and expressed when cells are subjected to different types of stress. It is also involved in signal transduction, growth, cell differentiation and survival of the chaperonin is of great importance for human pathogens, particularly those transmitted by insects to a mammalian host, and which suffer from environmental changes such as temperatu... (Complete abstract click electronic access below) / Mestre

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Vithal K. Ghanta, J. Michael Ruppert, Theresa V. Strong, R. Douglas Watson. Description based on contents viewed Oct. 3, 2008; title from PDF t.p. Includes bibliographical references.

Orientador: Maria Isabel Nogueira Cano / Resumo: Parasitos do gênero Leishmania pertencem à família Trypanosomatidae, os quais causam a leishmaniose, doença tropical negligenciada, que pode se apresentar em três formas clínicas: cutânea, mucocutânea e visceral. O Brasil é um dos países mais afetados pela doença, devido principalmente às condições socioeconômicas, às mudanças climáticas e ambientais. Pesquisas relacionadas à biologia da Leishmania contribuem para o entendimento dos mecanismos fisiológicos do parasito, e assim fornecem a possibilidade de encontrar novos alvos terapêuticos. O estudo dos telômeros de Leishmania se mostram promissores, já que estão relacionados com a estabilidade do genoma. Os telômeros estão localizados nas extremidades dos cromossomos e são responsáveis por proteger os cromossomos assegurando que a informação genética seja corretamente copiada durante a duplicação celular. Os telômeros são elongados por uma transcritase reversa especializada denominada telomerase. A telomerase é uma ribonucleoproteína, constituída por duas subunidades, uma proteína com função de transcriptase reversa denominada TERT, e um componente RNA (TER) que contém a sequência do molde da repetição telomérica copiado pela TERT. Estudos recentes mostram que o TER possui outras funções além de conter apenas um molde para elongamento dos telômeros. Sua estrutura secundária possui domínios com funções de controle da inserção de nucleotídeos pelo TERT, reconhecimento da sequência e ligação de proteínas acessórias. Recentemente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Parasites of the Leishmania genus belong to the Trypanosomatide family, which present peculiar and particular characteristics. Among them are the species that cause leishmaniasis, a neglected tropical disease that can be expressed in three different clinical forms: cutaneous, mucocutaneous and visceral. Brazil is one of the most affected countries, due mainly to socioeconomic conditions, climate change and environmental alterations. Research related to the biology of Leishmania contributes to the understanding of the important physiological mechanisms of the parasite, and thus provide new therapeutic targets against the disease. The study of Leishmania telomeres appears promising since they related are to the genome stability. Telomeres are nucleoprotein structures located at the ends of the chromosomes and are responsible for protecting the chromosomes ensuring that the genetic information copied is correctly during cell duplication. DNA polymerase does not elongate telomeres as the rest of the genetic material, and thus maintained are by the action of a specialized reverse transcriptase named telomerase. Telomerase is a ribonucleoprotein minimally composed by two subunits, a protein with reverse transcriptase function TERT, and an RNA component (TER) that contains the telomeric repeat template sequence copied by TERT. Recent studies shown that TER has other functions besides being just a template for telomeres elongation. Its secondary structure has domains with control fun... (Complete abstract click electronic access below) / Mestre

abstract: Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer is the second most common cancer among women in the US after skin cancer. While early detection and improved therapy has led to an overall decline in breast cancer mortality, metastatic disease remains largely incurable, indicating a need for improved therapeutic options for patients. Telomeres are repetitive (TTAGGG)n DNA sequences found at the end of chromosomes that protect the ends from recombination, end to end fusions, and recognition as damaged DNA. The enzyme telomerase acts to stabilize short telomeres, preventing apoptosis or senescence due to genomic instability. Telomerase is active in 85-90% of cancers, and inactive in most normal cells, making telomerase an attractive target for cancer therapy. Use of the telomerase-specific, lipidated oligonucleotide GRN163L can antagonize telomerase activity and telomere maintenance in cancer cells by preventing telomerase from binding to telomeres. GRN163L has been shown by our laboratory to inhibit breast cancer cell growth and metastasis in animal models. However, the mechanisms of cancer cell growth and metastatic inhibition via GRN163L are not completely understood. The overall goal of this research project was to further elucidate the role of telomerase in breast cancer cell survival by: 1) determining the effects of combining telomere dysfunction induced by GRN163L with a DNA damage inducer (irradiation); 2) elucidating the mechanisms underlying the cellular response to GRN163L and the effect of combination therapy with the mitotic inhibitor paclitaxel; and 3) testing the hypothesis that a telomerase inhibitor can augment the effects of trastuzumab in breast cancer cells with HER2 amplification. Results support the central hypothesis that the telomere dysfunction, structural and proliferative changes in breast cancer cells induced by GRN163L can synergize with irradiation, paclitaxel, and trastuzumab to inhibit the tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, GRN163L can restore sensitivity of therapeutically resistant breast cancer cells to trastuzumab. These results provide insight into the role of telomerase in cancer cell growth. Additionally, implications of this research support GRN163L as an important part of therapeutic regimens for primary tumors, recurrence, and metastatic disease.