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The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres. / Dissertation

INTRODUÇÃO: O Transtorno Bipolar (TB) é um transtorno psiquiátrico crônico e debilitante e sua etiologia e patologia ainda não são completamente conhecidos, apesar de um componente genético importante ser evidenciado em estudos de família, adoção e gêmeos. Recentemente, o TB tem sido relacionado a um processo de envelhecimento acelerado, com alguns estudos mostrando telômeros encurtados nesta população. O objetivo do presente estudo foi investigar a associação entre o comprimento telomérico, um dos parâmetros do processo de envelhecimento celular, com a ausência ou presença de TB em famílias com muitos membros afetados, além de associar a sintomatologia clínica e outras variáveis a esse parâmetro. Procurou-se também avaliar as influências genéticas e ambientais sobre o comprimento telomérico nessas famílias, estimando-se a herdabilidade desta característica. MÉTODOS: O comprimento telomérico (T) foi mensurado em uma amostra de 143 indivíduos de 22 famílias (60 deles com TB), em relação a um gene de cópia única (S) - beta-globina, através do método de PCR (Polymerase Chain Reaction) em tempo real quantitativo, no qual forneceu uma proporção do número de cópias de T por S (razão T/S). Considerando a estrutura familiar na análise estatística foi ajustado para cada análise o modelo misto poligênico. RESULTADOS: O efeito do TB no comprimento dos telômeros foi pequeno, não tendo sido observada uma associação estatisticamente significante entre TB e comprimento telomérico quando comparado com familiares saudáveis (p > 0,05). No entanto, observou-se associação do comprimento telomérico à covariável ideação suicida (p = 0,02) e à interação entre ideação suicida e curso da doença (p = 0,02). Associação do comprimento telomérico com idade materna e TB também foi observada (p < 0,05). Por fim, estimou-se em 68% a herdabilidade do comprimento telomérico nas 22 famílias do estudo. CONCLUSÕES: A teoria do envelhecimento acelerado em TB, vista pela óptica do comprimento dos telômeros, não pôde ser confirmada no presente estudo, pois não foi encontrada diferença no comprimento telomérico entre indivíduos saudáveis e com TB nas famílias. Por outro lado, covariáveis que indicam gravidade da doença, como a ideação suicida e a interação entre ideação suicida e curso da doença foram associadas ao comprimento telomérico (p < 0,05), ou seja, um encurtamento telomérico foi correlacionado à gravidade clínica do TB. Associação do comprimento telomérico com idade materna e TB (p < 0,05) sugeriu que a idade materna avançada não só pode ser um marcador de longevidade, como também o fenótipo TB pareceu reforçar essa condição. Por fim, a alta herdabilidade estimada do comprimento telomérico (0,68) revelou uma importante variabilidade genética desse fenótipo entre as famílias do estudo. Em súmula, este é o primeiro estudo que relatou uma associação entre ideação suicida, curso da doença, idade materna e comprimento telomérico em famílias com vários membros afetados pelo TB. Outras investigações independentes são necessárias para confirmar esses resultados preliminares / BACKGROUND: Bipolar Disorder (BD) is a debilitating and chronic mental illness. It is etiology and pathology are not completely known yet, despite the evidence of an important genetic component from family, twin and adoption studies. Recently, BD has been related to a process of accelerated aging, with some studies showing shortened leukocyte telomeres in this population. The purpose of the present study was to investigate the association between leucocyte telomere length (LTL) in BD patients compared with healthy relatives of 22 families with several affected members by this illness, besides associating clinical symptomatology and other covariates with this parameter. It was also examined the genetic and environmental influences on telomere length trait in these BD families, using a variance component approach, by estimating the heritability of this trait as well as covariate effects. METHODS: Telomere length (T) was estimated in a sample of 143 individuals, including 60 BD patients from 22 families, which was measured in relation to the single copy gene (S) - beta-globin gene, using a singleplex real time PCR (Polymerase Chain Reaction), providing a ratio of number of copies of T by S (T/S ratio). Taking in consideration the family structure, the statistical analysis was adjusted for the polygenic mixed model. RESULTS: The effect of BD illness in telomere length was small and we found no association between BD group and LTL (p > 0.05). However, LTL was associated with the variable suicidal ideation (p = 0.02) and interaction between suicidal ideation and course of disorder (p = 0.02). Association of LTL and maternal age and BD was also observed (p < 0.05). In addition, an important genetic component for telomere length was also observed (heritability = 0.68) in these families. CONCLUSIONS: The hypothesis of accelerated aging in BD, investigating the telomere length as one of its components, was not confirmed in our study. We found no difference between LTL and BD in our family group. However, using covariates that indicate severity of disease, both suicidal ideation and interaction between suicidal ideation and course of disorder were statistically significant with LTL, showing that shorter LTL was associated with worse clinical course (p < 0.05) and suicidal ideation (p < 0.05) in BD patients. Association of LTL with maternal age and BD (p < 0.05) suggests that advanced maternal age may not only be a marker of longevity, but also the BD phenotype may reinforce this condition. A high heritability for telomere length (0.68) also suggests an important genetic variability of this trait presented among those families. To our knowledge, this is the first study that found association between suicidal ideation, course of disorder, maternal age and LTL in families with several members affected by BD. Further investigations, including replication studies in other BD families, are needed to confirm these new findings

Orientador: Maria Isabel Nogueira Cano / Resumo: Entre as espécies do gênero Leishmania estão os protozoários que causam leishmaniose, uma doença tropical negligenciada endêmica em muitos países, incluindo o Brasil. Métodos de controle e tratamento ainda são ineficientes e a resistência a drogas é um desafio. Por isso, pesquisas para entender melhor a biologia molecular desses parasitos são encorajadas. Uma possível estratégia para isso, é o estudo dos telômeros, estrutura fundamental para a homeostase do genoma. Os telômeros são estruturalmente diferentes do resto do cromossomo, e contam com proteínas específicas que realizam sua manutenção. A Replication Protein A subunit 1 (RPA-1) é uma proteína que interage de DNA de simples fita que tem diversas funções relacionadas com o metabolismo do DNA eucarioto, incluindo os telômeros. A RPA-1 é parte de um complexo heterotrimérico conservado nos eucariotos, incluindo Leishmania spp.. Recentemente nós mostramos por modelagem molecular que a estrutura terciária da LaRPA-1 difere dos seus ortólogos em humanos e leveduras, além de mostrar interações específicas nos telômeros dos parasitos, que na ausência de homólogos canônicos para telomere-end binding protein (TEP) elegem a LaRPA-1 como um potencial candidato para essa função. Neste trabalho, avaliamos a capacidade da LaRPA-1 como uma TEP, cujo papel principal é proteger a extremidade 3' dos telômeros de ataques por exonucleases. Uma busca estrutural por proteínas que compartilham com as TEP domínios de interação proteína-DNA, mos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Among the protozoa parasites of the Leishmania genus are the causative agents of leishmaniasis, a neglected tropical disease endemic in many countries, including Brazil. Disease control and treatment are still inefficient and parasite drug resistance is a challenge. Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged. One possible strategy is to study parasite telomeres, a vital chromosome structure important to maintain genome homeostasis. Telomeres are significantly different from the rest of the chromosome and are associated with proteins involved in their maintenance. Replication Protein A subunit 1 (RPA1), a single-stranded DNA-binding protein that plays multiple roles in eukaryotic DNA metabolism, including telomeres, is part of a conserved heterotrimeric complex which is present in most eukaryotes including Leishmania spp. Recently, using molecular dynamics simulations we have shown that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1 and that it also shows parasitespecific interactions with telomeric DNA. In the absence of real homologues to telomere-end binding proteins, LaRPA-1 could be considered a potential candidate. If LaRPA-1 is a telomere-end binding protein, one of its main role would be to protect the telomeric 3`-end termini from nuclease attack. A structural search for proteins that share with the TEP domains of protein-DNA interaction, showed that ... (Complete abstract click electronic access below) / Mestre

Submitted by Gabriel Arantes Galvão Dias dos Santos (arantes_gabriel@hotmail.com) on 2018-05-14T15:21:05Z No. of bitstreams: 1 Dissertação pós defesa.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-14T17:46:54Z (GMT) No. of bitstreams: 1 santos_gagd_me_bot.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) / Made available in DSpace on 2018-05-14T17:46:54Z (GMT). No. of bitstreams: 1 santos_gagd_me_bot.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Entre as espécies do gênero Leishmania estão os protozoários que causam leishmaniose, uma doença tropical negligenciada endêmica em muitos países, incluindo o Brasil. Métodos de controle e tratamento ainda são ineficientes e a resistência a drogas é um desafio. Por isso, pesquisas para entender melhor a biologia molecular desses parasitos são encorajadas. Uma possível estratégia para isso, é o estudo dos telômeros, estrutura fundamental para a homeostase do genoma. Os telômeros são estruturalmente diferentes do resto do cromossomo, e contam com proteínas específicas que realizam sua manutenção. A Replication Protein A subunit 1 (RPA-1) é uma proteína que interage de DNA de simples fita que tem diversas funções relacionadas com o metabolismo do DNA eucarioto, incluindo os telômeros. A RPA-1 é parte de um complexo heterotrimérico conservado nos eucariotos, incluindo Leishmania spp.. Recentemente nós mostramos por modelagem molecular que a estrutura terciária da LaRPA-1 difere dos seus ortólogos em humanos e leveduras, além de mostrar interações específicas nos telômeros dos parasitos, que na ausência de homólogos canônicos para telomere-end binding protein (TEP) elegem a LaRPA-1 como um potencial candidato para essa função. Neste trabalho, avaliamos a capacidade da LaRPA-1 como uma TEP, cujo papel principal é proteger a extremidade 3' dos telômeros de ataques por exonucleases. Uma busca estrutural por proteínas que compartilham com as TEP domínios de interação proteína-DNA, mostrou que no genoma de Leishmania spp. não existem homólogos estruturais para as mesmas. Aqui mostramos por diferentes abordagens que a LaRPA-1 tem a capacidade de interagir com no mínimo uma repetição telomérica e também é capaz de proteger in vitro a simples fita telomérica rica em G (5’ TTAGGG 3’) da digestão por Exonuclease I bacteriana cuja atividade é no sentido 3’-5’. Somando esses dados, com dados anteriores que mostram que a LaRPA-1 tem preferência pela fita telomérica rica em G e o fato dela ter sido co-purificada com a atividade de telomerase sugerem fortemente que ela está diretamente relacionada com a manutenção da maquinaria telomérica, podendo inclusive ser considerada a principal ligante de simples fita telomérica rica em G (3’ G-overhang) em Leishmania spp. / Among the protozoa parasites of the Leishmania genus are the causative agents of leishmaniasis, a neglected tropical disease endemic in many countries, including Brazil. Disease control and treatment are still inefficient and parasite drug resistance is a challenge. Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged. One possible strategy is to study parasite telomeres, a vital chromosome structure important to maintain genome homeostasis. Telomeres are significantly different from the rest of the chromosome and are associated with proteins involved in their maintenance. Replication Protein A subunit 1 (RPA1), a single-stranded DNA-binding protein that plays multiple roles in eukaryotic DNA metabolism, including telomeres, is part of a conserved heterotrimeric complex which is present in most eukaryotes including Leishmania spp. Recently, using molecular dynamics simulations we have shown that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1 and that it also shows parasitespecific interactions with telomeric DNA. In the absence of real homologues to telomere-end binding proteins, LaRPA-1 could be considered a potential candidate. If LaRPA-1 is a telomere-end binding protein, one of its main role would be to protect the telomeric 3`-end termini from nuclease attack. A structural search for proteins that share with the TEP domains of protein-DNA interaction, showed that in the genome of Leishmania spp. there are no structural homologues for them. In this work, we show by different methods, that in vitro LaRPA-1 can bind at least one telomeric repeat and it can also protect the telomeric G-rich sequence (5’ TTAGGG 3’) from the bacterial 3’-5’Exonuclease I digestion. These data compiled to previous data showing that LaRPA-1 preferentially binds the G-rich telomeric DNA and that it co-purifies with telomerase activity strongly suggest that LaRPA-1 is directly involved with parasite telomere maintenance and, possibly, is the main G-rich single-stranded (3’ G-overhang) telomere-binding protein in Leishmania spp.

INTRODUÇÃO: O Transtorno Bipolar (TB) é um transtorno psiquiátrico crônico e debilitante e sua etiologia e patologia ainda não são completamente conhecidos, apesar de um componente genético importante ser evidenciado em estudos de família, adoção e gêmeos. Recentemente, o TB tem sido relacionado a um processo de envelhecimento acelerado, com alguns estudos mostrando telômeros encurtados nesta população. O objetivo do presente estudo foi investigar a associação entre o comprimento telomérico, um dos parâmetros do processo de envelhecimento celular, com a ausência ou presença de TB em famílias com muitos membros afetados, além de associar a sintomatologia clínica e outras variáveis a esse parâmetro. Procurou-se também avaliar as influências genéticas e ambientais sobre o comprimento telomérico nessas famílias, estimando-se a herdabilidade desta característica. MÉTODOS: O comprimento telomérico (T) foi mensurado em uma amostra de 143 indivíduos de 22 famílias (60 deles com TB), em relação a um gene de cópia única (S) - beta-globina, através do método de PCR (Polymerase Chain Reaction) em tempo real quantitativo, no qual forneceu uma proporção do número de cópias de T por S (razão T/S). Considerando a estrutura familiar na análise estatística foi ajustado para cada análise o modelo misto poligênico. RESULTADOS: O efeito do TB no comprimento dos telômeros foi pequeno, não tendo sido observada uma associação estatisticamente significante entre TB e comprimento telomérico quando comparado com familiares saudáveis (p > 0,05). No entanto, observou-se associação do comprimento telomérico à covariável ideação suicida (p = 0,02) e à interação entre ideação suicida e curso da doença (p = 0,02). Associação do comprimento telomérico com idade materna e TB também foi observada (p < 0,05). Por fim, estimou-se em 68% a herdabilidade do comprimento telomérico nas 22 famílias do estudo. CONCLUSÕES: A teoria do envelhecimento acelerado em TB, vista pela óptica do comprimento dos telômeros, não pôde ser confirmada no presente estudo, pois não foi encontrada diferença no comprimento telomérico entre indivíduos saudáveis e com TB nas famílias. Por outro lado, covariáveis que indicam gravidade da doença, como a ideação suicida e a interação entre ideação suicida e curso da doença foram associadas ao comprimento telomérico (p < 0,05), ou seja, um encurtamento telomérico foi correlacionado à gravidade clínica do TB. Associação do comprimento telomérico com idade materna e TB (p < 0,05) sugeriu que a idade materna avançada não só pode ser um marcador de longevidade, como também o fenótipo TB pareceu reforçar essa condição. Por fim, a alta herdabilidade estimada do comprimento telomérico (0,68) revelou uma importante variabilidade genética desse fenótipo entre as famílias do estudo. Em súmula, este é o primeiro estudo que relatou uma associação entre ideação suicida, curso da doença, idade materna e comprimento telomérico em famílias com vários membros afetados pelo TB. Outras investigações independentes são necessárias para confirmar esses resultados preliminares / BACKGROUND: Bipolar Disorder (BD) is a debilitating and chronic mental illness. It is etiology and pathology are not completely known yet, despite the evidence of an important genetic component from family, twin and adoption studies. Recently, BD has been related to a process of accelerated aging, with some studies showing shortened leukocyte telomeres in this population. The purpose of the present study was to investigate the association between leucocyte telomere length (LTL) in BD patients compared with healthy relatives of 22 families with several affected members by this illness, besides associating clinical symptomatology and other covariates with this parameter. It was also examined the genetic and environmental influences on telomere length trait in these BD families, using a variance component approach, by estimating the heritability of this trait as well as covariate effects. METHODS: Telomere length (T) was estimated in a sample of 143 individuals, including 60 BD patients from 22 families, which was measured in relation to the single copy gene (S) - beta-globin gene, using a singleplex real time PCR (Polymerase Chain Reaction), providing a ratio of number of copies of T by S (T/S ratio). Taking in consideration the family structure, the statistical analysis was adjusted for the polygenic mixed model. RESULTS: The effect of BD illness in telomere length was small and we found no association between BD group and LTL (p > 0.05). However, LTL was associated with the variable suicidal ideation (p = 0.02) and interaction between suicidal ideation and course of disorder (p = 0.02). Association of LTL and maternal age and BD was also observed (p < 0.05). In addition, an important genetic component for telomere length was also observed (heritability = 0.68) in these families. CONCLUSIONS: The hypothesis of accelerated aging in BD, investigating the telomere length as one of its components, was not confirmed in our study. We found no difference between LTL and BD in our family group. However, using covariates that indicate severity of disease, both suicidal ideation and interaction between suicidal ideation and course of disorder were statistically significant with LTL, showing that shorter LTL was associated with worse clinical course (p < 0.05) and suicidal ideation (p < 0.05) in BD patients. Association of LTL with maternal age and BD (p < 0.05) suggests that advanced maternal age may not only be a marker of longevity, but also the BD phenotype may reinforce this condition. A high heritability for telomere length (0.68) also suggests an important genetic variability of this trait presented among those families. To our knowledge, this is the first study that found association between suicidal ideation, course of disorder, maternal age and LTL in families with several members affected by BD. Further investigations, including replication studies in other BD families, are needed to confirm these new findings

Orientador: Maria Isabel Nogueira Cano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T06:26:57Z (GMT). No. of bitstreams: 1 DaSilva_MarceloSantos_D.pdf: 9189361 bytes, checksum: 79ecad917f6be7c8b200b549dafc493e (MD5) Previous issue date: 2014 / Resumo: A leishmaniose é um espectro de doenças causadas por parasitos do gênero Leishmania, que afeta milhões de pessoas em todo o mundo. Durante a infecção, os parasitos usam diferentes estratégias para sobreviver as defesas do hospedeiro, incluindo superar a exposição intensa a espécies reativas de oxigênio (ROS), principais responsáveis por causar danos no DNA, sobretudo nos telômeros, induzindo instabilidade genômica, senescência e morte celular. Telômeros são estruturas nos terminais dos cromossomos compostos por sequências de DNA repetitivas e proteínas, cuja função é proteger as extremidades dos cromossomos, evitando fusões terminais e degradação nucleolítica. Neste trabalho nós induzimos estresse oxidativo agudo em formas promastigotas de L. amazonensis através do tratamento com 2 mM de peróxido de hidrogênio (H2O2) por 1h, o qual foi capaz de aumentar os níveis de ROS intracelular, como demonstrado pela reação utilizando CM-H2DCFDA. Além disso, o estresse oxidativo induziu danos no DNA, como mostrado por análise quantitativa de 8-oxodG e núcleos positivos para o ensaio TUNEL. Observamos também, através de parâmetros qualitativos e quantitativos (Southern blot, telomere-PCR e flow-FISH), que o estresse oxidativo, assim como em mamíferos, induziu encurtamento dos telômeros. Analisando a co-localização e interação proteína:DNA por FISH-IIF e ensaios ChIP, foi possível demostrar que o estresse oxidativo causou erosão da extremidade 3¿G overhang, fazendo com que a proteína LaRPA-1 perdesse seu sítio de interação nos telômeros. Além disso, pudemos observar uma maior afinidade de LaRPA-1 para com a fita telomérica rica em C, nesse caso uma região de simples-fita gerada dentro da dupla fita telomérica, provavelmente como consequência do reparo de DNA, sugerindo a participação de LaRPA-1 na resposta a danos oxidativos. Por análise de curvas de crescimento e incorporação de EdU, foi possível observar que o estresse oxidativo induziu diminuição acentuada no número de parasitos em cultura, enquanto os sobreviventes continuaram proliferando e replicando DNA. Observamos também que o estresse oxidativo agudo provocou arrest de ciclo celular na fase G2/M em parte da população em crescimento exponencial. Em conjunto, esses resultados sugerem a presença de um sistema muito eficiente de resposta a danos oxidativos no DNA telomérico, que permite que os parasitos sobrevivam e repliquem DNA mesmo após um estresse agudo / Abstract: Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects million people around the world. During infection, parasites use different strategies to survive host defenses including overcoming exposure to Reactive Oxygen Species (ROS), mainly responsible for causing DNA damage, especially at telomeres which frequently results in genome instability, senescence and cell death. Telomeres are chromosomes end termini structures composed by repetitive DNA coupled with proteins whose function is to protect chromosome ends and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H2O2) for 1 hour, which was able to increase intracellular ROS levels, as demonstrated by CM-H2DCFDA reaction. In addition, oxidative stress induced DNA damage, as confirmed by quantitative analysis of 8-oxodG and TUNEL-positive nuclei. We have also observed using qualitative and quantitative parameters (Southern blot, telomere-PCR and flow-FISH) that oxidative stress, as in mammals, induced telomere shortening. Analysing the protein:DNA co-localization and interaction by FISH-IIF and ChIP assays, it was possible to show that oxidative stress is able to induce erosion of the 3¿G overhang, inducing a displacement of LaRPA-1 from its telomeric interaction site. In addition, we observed an increase in the affinity between LaRPA-1 and the telomeric C-rich strand, in this case, a single-strand region inside the double-strand telomeric DNA generated probably as a consequence of DNA repair, suggesting the participation of LaRPA-1 in oxidative DNA damage response. Analysis of growth curves and EdU incorporation showed that oxidative stress induced a decrease in the number of parasites in culture, while the survivors continued proliferating and replicating DNA. Moreover, as result of acute oxidative stress, part of the parasites in exponential growth shows a G2/M cell cycle arrest. Taken together, these results suggest the presence of a very efficient oxidative damage response in the telomeres that allows parasites to survive and to replicate DNA even after acute stress / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.

Overwhelming evidence for senescence, the within-individual decline in performance at advanced age, has now been documented in the natural populations of many taxa. As such, the focus of senescence research is shifting from simply documenting its existence, towards understanding the fundamental mechanisms underpinning it and determining which environmental factors give rise to the considerable variation in senescence rates observed in nature. In this thesis I use a wild population of European badgers (Meles meles) to investigate three important traits implicated in, or arising as a direct product of, senescence; immune cell telomere length, pro-inflammatory cytokine response and body mass declines in late life. My work reveals rare longitudinal evidence for the existence of senescence in immune traits in a wild mammal. First, I show that within-individual declines in immune cell telomere length occur with increasing age (Chapter 2). Second, after demonstrating that immune cell telomere length displays repeatable between-individual differences in adulthood, I show that the environmental conditions experienced in early-life contribute to such between-individual variation. Individuals that experienced harsh early-life environmental conditions had shorter immune cell telomere lengths than those that experienced favourable conditions (Chapter 3). Third, I show that within-individual declines in a second immune trait, pro-inflammatory cytokine response, also occur with age (Chapter 4). However, the declines in immune cell telomere length and pro-inflammatory cytokine response occur independently of one another (Chapter 4). Finally I take advantage of a 37 year longitudinal dataset to reveal that sex differences in body mass senescence arise as a consequence of the scale of intra-sexual competition experienced in early adulthood (Chapter 5). Taken together this work provides novel evidence suggesting that age-related declines in immunocompetence can contribute to whole organism senescence in the wild. Furthermore, evidence that early life environmental and social conditions can markedly influence senescence rates has important implications for our understanding of the drivers of variation in senescence rates observed within natural populations.