O complexo telomérico, responsável pela integridade genômica, é formado pela interação de DNA com proteínas, que são responsáveis pela proteção desses terminais. O complexo RPA de eucariotos compreende um heterotrímero, que cumpre diversas funções vitais na célula, sendo uma peça fundamental na replicação, reparo e recombinação. A ausência de homólogos de proteínas que protegem o telômero em T. cruzi nos fez investigar se o complexo RPA poderia cumprir essa função. Assim, este trabalho teve como objetivo caracterizar a interação TcRPA-1-telômero. Conseguimos verificar a interação in vitro e in vivo da RPA-1 com o telômero em epimastigotas e tripomastigotas. A ausência de homólogos de proteínas que interagem com o overhang telomérico em tripanosomas, a interação específica RPA-1-telômero, e a sua presença nos telômeros da forma de vida não replicativa, bem como as peculiaridades estruturais da TcRPA-1, levam-nos a propor que essa proteína pode estar envolvida com a proteção dos telomérica neste organismo. / The telomeric complex, responsible for genomic integrity and stability , is formed by the interaction of DNA with proteins that are responsible for maintaining and protecting these terminals. The eukaryotic RPA complex comprises a heterotrimer, which fulfills several vital functions in the cell , being a fundamental player in replication , repair and recombination. The absence of homologous proteins that protect telomeres in Trypanosoma cruzi lead us to hypothesize that RPA complex could fulfill this function. Thus, this study aimed to characterize TcRPA-1-telomere interaction. We could verify in vitro and in vivo interaction of RPA - 1 with telomeres in epimastigote and trypomastigote lifeforms. The absence of homologs of proteins that interact with telomeric overhang in trypanosomes, the specific interaction RPA-1- telomere , its presence in non- replicative lifeform as well as structural peculiarities of TcRPA-1, lead us to propose that this protein may be involved in telomere protection in this organism.

Nos indivíduos com obesidade, o excesso de tecido adiposo, exerce um papel fundamental induzindo um estado inflamatório crônico e sistêmico. A obesidade mimetiza processos celulares semelhantes aos do envelhecimento tais como a deterioração de tecidos e órgãos e diminuição na capacidade de reparo dos danos induzidos ao DNA. Nesse contexto, as citocinas pró-inflamatórias induzem atritos ao DNA que impactam, principalmente nas regiões mais susceptíveis dos cromossomos, os telômeros. Os telômeros, presentes nas extremidades dos cromossomos, estão associados a um complexo proteico denominado complexo shelterin. O complexo shelterin é formado por 6 proteínas (TRF1, TRF2, TIN2, POT1, TPP1 e RAP1), que junto com proteínas acessórias como a disquerina (DKC1), participam da regulação do comprimento dos telômeros e protegem os cromossomos dede atividades indesejadas de erosão enzimática, recombinação não-homóloga e fusão das terminações cromossômicas. Nos últimos anos, foram estabelecidas relações positivas entre condições patológicas clinicamente diferentes, como as moduladas por inflamação, e o comprimento dos telômeros. Recentemente, nosso grupo demonstrou telômeros encurtados em indivíduos portadores de obesidade mórbida. Assim o objetivo do presente trabalho foi explorar fatores adicionais associados ao metabolismo telomérico, como a expressão gênica das proteínas do complexo shelterin e citocinas pró-inflamatórias, as quais podem contribuir para o encurtamento acelerado de telômeros. Utilizamos amostras de células mononucleares de sangue periférico (PBMC) de indivíduos adultos saudáveis (n = 27) e indivíduos adultos portadores de obesidade (n = 39). Quantificamos a expressão gênica por transcrição reversa e PCR quantitativa (RT-qPCR) de todos os genes do complexo shelterin, DKC1, IL-1β e TNF-α. Nossos resultados demonstram um perfil de expressão gênica alterado quando comparada a expressão gênica das proteínas analisadas nos dois grupos estudados, controles e portadores de obesidade. Os indivíduos portadores de obesidade mostraram um perfil significativamente elevado dos genes TRF1, POT1, RAP1 e DKC1 (P < 0,05). Não observamos correlação de expressão gênica entre os diferentes genes e o comprimento dos telômeros nos grupos estudados, mas sim com a DKC1 na obesidade. Entretanto, quando analisamos as associações entre os genes de complexo shelterin observamos mudanças significativas nas associações intra-grupo dependentes da condição de obesidade. Nossos resultados salientam a complexa e intrincada rede de fatores associados e desregulados durante o processo fisiopatológico da obesidade. Estudos adicionais serão necessários acrescentando novos fatores para tentar dissecar a regulação coordenada do comprimento dos telômeros na homeostase e no processo patológico da obesidade. / In individuals with obesity, the excess of adipose tissue plays a key role in inducing a chronic and systemic inflammatory state. Like aging, obesity mimics cellular processes such as deterioration of tissues and organs and decreased ability to repair age-dependent DNA damages. In this context, the proinflammatory cytokines induce DNA damage that impact, especially in the most susceptible regions of the chromosomes, the telomeres. The telomeres, present at the ends of the chromosomes, are associated with a protein complex called the shelterin complex. The shelterin complex consists of 6 proteins (TRF1, TRF2, TIN2, POT1, TPP1 and RAP1), which together with accessory proteins such as dyskerin (DKC1), participate in telomere’s length regulation and protect chromosomes from undesired erosion, enzymatic activities, non-homologous recombination and fusion of chromosomal terminations. In recent years, positive relationships have been established between clinically different pathological conditions, such as those modulated by inflammation, and telomeres’ length. Recently, our group demonstrated shortened telomeres in individuals with morbid obesity. Thus, the aim of the present study was to explore additional factors associated with telomeres’ metabolism, such as gene expression of the shelterin complex components and proinflammatory cytokines, which may contribute to the accelerated shortening of the telomeres. We used peripheral blood mononuclear cells (PBMC) samples from healthy adults (n = 27) and adults with obesity (n = 39). We quantified gene expression by reverse transcription and quantitative PCR (RT-qPCR) of all shelterin complex genes, DKC1, IL-1β and TNF-α. Our results demonstrate an altered gene expression profile when compared to the gene expression of the proteins analyzed in the two studied groups, controls and individuals with obesity. Individuals with obesity showed a significantly elevated profile of TRF1, POT1, RAP1 and DKC1 (P < 0.05) genes. We did not observe correlation of gene expression between the different shelterin genes and the length of telomeres in the studied groups, but with DKC1 in obesity. However, when we analyzed the associations between the shelterin complex genes we observed significant changes in the intra-group associations dependent on the obesity condition. Our results highlight the complex and intricate network of associated and deregulated factors during the pathophysiological process of obesity. Further studies are needed together with the inclusion of additional factors to try to dissect the coordinated regulation of telomeres’ length in homeostasis and in the pathological process of obesity.

Leucemia Linfóide Crônica (LLC) é a leucemia mais comum em países ocidentais, tem apresentação clínica e evolução heterogênea. Especula-se que todos os casos sejam precedidos por Linfocitose B Monoclonal (LBM). Não são bem conhecidos os mecanismos moleculares responsáveis por esta evolução. Alterações na expressão de miRNAs e em comprimento telomérico podem contribuir para desencadear esta neoplasia. O objetivo deste estudo foi identificar diferenças em comprimento telomérico e expressão de miRNAs entre pacientes com LLC, portadores de LBM clínica e populacional e controles saudáveis. Estudamos 21 pacientes com LLC, 11 portadores de LBM clínica, 6 de LBM populacional e 10 voluntários saudáveis. Para o controle de comprimento telomérico, utilizamos dados de estudo anterior do nosso serviço com grupo de 261 voluntários saudáveis de 0 a 86 anos. Realizamos separação de células CD19+CD5+ por citometria de fluxo nos grupos de estudo e de linfócitos B no grupo controle. Analisamos expressão dos miRNAs 15a, 16-1, 29b, 34a, 155, 181a e 181b por RT-qPCR e comprimento telomérico por qPCR. O miR- 155 foi o único que demonstrou expressão diferente entre os grupos LLC e LBM, sendo maior nos pacientes com LLC. Este miRNA e o miR-34a têm aumento de expressão nas células com fenótipo anormal, apesar desta diferença não ter tido significância estatística quando considerada a expressão do miR-155 na LBM. Os miRNAs 15a, 16-1, 181a e 181b são hipoexpressos nas células com fenótipo anormal. O miR-29b teve expressão semelhante nos grupos estudados. O comprimento telomérico foi semelhante nos 3 grupos de estudo e menor quando comparados ao grupo controle. O miR-155 tem diferente expressão em LBM e LLC, podendo ser um dos responsáveis por esta evolução. Alterações nos miRNAs 34a, 15a, 16-1, 181a e 181b contribuem para expansão clonal de linfócitos B CD5+. O papel do miR-29b na fisiopatogênese e evolução da LLC ainda não está bem definido. O comprimento telomérico diminuído em LLC e LBM pode fazer parte dos eventos iniciais da fisiopatogênese desta leucemia. / Chronic Lymphocytic Leukemia (CLL) is the most common leukemia on Western countries, it has an heterogeneous clinical presentation and outcome. Monoclonal BCell Lymphocytosis (MBL) may precede all CLL cases. The molecular mechanisms responsible for this evolution are not known. Aberrant miRNA expression and telomere shortening may contribute for the pathophysiology of this disease. The objective of this study was to identify differences on telomere length and miRNA expression between CLL patients, subjects with clinical and population-screening MBL and healthy volunteers. 21 CLL patients, 11 subjects with clinical MBL, 6 with population-screening MBL and 10 healthy volunteers were enrolled on this study. As control for telomere length, we used a group of 261 healthy volunteers aged 0 to 86 years old that had been enrolled on a previous study from our group. After diagnosis confirmation, it has been done a flow citometry CD19+CD5+ cell sorting for the study groups and CD19+ cell sorting for the control group. The expression of the miRNAs 15a, 16-1, 29b, 34a, 155, 181a and 181b was determined by RT-qPCR. The telomere length was determined by qPCR. miR-155 was the only one with different expression between the CLL and MBL groups, presenting higher expression on the CLL group. This miRNA and the miR-34a are overexpressed on the study groups when compared to the control group, although this difference did not reach statistical significance when the miR-155 expression in MBL is considered. miRNAs 15a, 16-1, 181a and 181b are underexpressed on the study groups. The miR-29b was the only one with similar expression on all groups. The telomere length was similar on the 3 study groups and shorter on these groups when compared to normal subjects. The expression of miR-155 is different in CLL and MBL, it may contribute for this evolution. Aberrant expression of miR 34a, 15a, 16-1, 181a and 181b may contribute for the clonal expansion of CD5+ B lymphocytes. The role of miR-29b on the CLL pathogenesis and evolution is still not understood. The reduced telomere length on CLL and MBL may be part of the initial events of this leukemia pathogenesis.

Dans le cancer du sein, la recherche de nouveaux biomarqueurs, permettant de prédire la réponse thérapeutique et de déterminer le pronostic d’une patiente, est importante pour adapter au mieux les traitements mais aussi pour améliorer la compréhension des phénomènes physiopathologiques. Nous nous sommes particulièrement intéressés aux cancers du sein traités par chimiothérapie néoadjuvante. Nous avons étudié, dans des biopsies tumorales réalisées avant traitement et dans des résidus tumoraux, à la fois les paramètres télomériques et la réparation des lésions d’ADN puisque ces 2 mécanismes, lorsqu’ils sont dysfonctionnels, sont à l’origine d’une forte instabilité génomique. Nous avons corrélés ces paramètres à la réponse à la chimiothérapie néoadjuvante et à la survie des patientes. Dans un 1er temps, nous avons montré que des télomères courts, une surexpression de la télomérase (TERT) et une sous-expression d’une protéine impliquée dans différents mécanismes de réparation de l’ADN, ERCC1, sont des marqueurs de mauvais pronostic. La dysfonction simultanée des télomères et des mécanismes de réparation de l’ADN peut contribuer de façon synergique à la progression tumorale et à la résistance thérapeutique. Nous avons ensuite étudié une population de tumeurs du sein triple négatives. Nous avons montré que des télomères courts sont associés aux tumeurs les plus agressives et les plus résistantes. De plus, une résistance thérapeutique est retrouvée associée à une instabilité génomique plus importante. Nous avons enfin analysé les altérations génomiques caractéristiques permettant d’identifier le statut BRCA1-like. Le profil BRCA1-like est corrélé à une résistance thérapeutique et à une forte instabilité génomique.Enfin, nous avons réalisé une étude plus fondamentale visant à identifier les mécanismes à l’origine de la réactivation de la télomérase dans le cancer du sein. Nous avons démontré que la surexpression de TERT, dans le cancer du sein, n’est pas liée à la présence de mutations somatiques activatrices mais plutôt à celle de gain du locus TERT. Ces gains sont associés à une résistance thérapeutique et un risque de rechute plus important. Enfin, la présence de gain de TERT combinée à la surexpression de MYC, permet de définir un sous groupe de très mauvais pronostique et pourrait être utilisé pour évaluer le risque de récidives. Les paramètres télomériques et l’instabilité génomique semblent donc être des biomarqueurs prédictifs de la réponse à la chimiothérapie néoadjuvante dans le cancer du sein triple négatif. Ces paramètres ont également une forte valeur pronostique dans le cancer du sein en général et pourraient être utilisés cliniquement comme biomarqueurs utiles au choix des traitements. / In breast cancer, discovering new biomarkers, that can predict therapeutic response and prognosis, is important to find the best therapeutic options and improve our understanding of physiopathology. Our particular interest is in breast cancer treated by neoadjuvant chemotherapy (NCT). In pre-NCT biopsies and post-NCT tumors, we studied both telomeric parameters and DNA damage repair (DDR), because when these are dysfunctional, both result in high genomic instability. We correlated these parameters to neoadjuvant chemotherapy response and patient outcomes. First, we demonstrated that short telomeres, high telomerase (TERT) expression and low expression of ERCC1 (a protein involved in a number of DNA repair mechanisms) are markers of poor prognosis. Telomere and DDR dysfunction can contribute synergistically to tumor progression and chemoresistance. We then studied a triple negative breast cancer population. We demonstrated that short telomeres were associated with tumor aggressiveness and chemoresistance. Chemoresistance was also associated with high genomic instability. We analyzed genomic alterations specific to BRCA1-like status and demonstrated that BRCA1-like profile correlated with chemoresistance and high genomic instability. Finally, we performed a comprehensive study of telomerase reactivation in breast cancer. We demonstrated that high TERT expression in breast cancer is not associated with somatic enhancer mutations but more probably to TERT locus gains. These gains were correlated to chemoresistance and increased risk of relapse. TERT gain, combined with high MYC expression, was able to isolate a subgroup with a very poor prognosis, and this could be used to evaluate risk of relapse. Telomeric parameters and genomic instability seem to be predictive biomarkers for neoadjuvant chemotherapy response in triple negative breast cancer. These parameters also have strong prognostic value in breast cancer and could be used clinically as biomarkers for tailoring treatment.

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.

Bipolar disorders and recurrent depressions are two common psychiatric disorders with a life time prevalence of approximately 1% and 8%, respectively. Despite treatment these patients suffer from affective symptoms up to 50% of the time, resulting in lower well-being. The average life length is also reduced with 10-15 years, mainly attributable to suicide and cardiovascular disease. Increased stress is one of many factors that have been shown to be linked to an increased risk for developing affective disorders and some comorbid somatic conditions such as metabolic disturbances and cardiovascular disease. An increased stress level is known to cause hyperactivity of the hypothalamic-pituitary-adrenal-axis (HPA-axis) with increased cortisol secretion. Hyperactivity of the HPA-axis (or hypercortisolism) is one of the most replicated neurobiological finding in depression. In other stress related disorders it has however been shown that prolonged stress over long periods of time can lead to a state of low HPA-axis activity, hypocortisolism. Since persons with recurrent affective disorders such as bipolar disorder and recurrent depression are exposed to a high degree of recurrent and chronic stress it could be expected that in addition to hypercortisolism, a state of hypocortisolism could also develop in these disorders, potentially exerting an influence upon the psychological and somatic wellbeing among these patients. The major aim of this thesis was to evaluate whether hypocortisolism is related to relevant psychiatric and somatic phenotypes in recurrent affective disorders. In bipolar disorder, individuals with hypocortisolism exhibited a higher degree of depression and low quality of life compared to patients with normal HPA-axis activity. In recurrent depression, individuals with hypocortisolism exhibited shorter leukocyte telomere length than patients with normal or high HPA-axis activity, which is an indication of an accelerated aging process. In a sample of both bipolar and recurrent depression patients, hypocortisolism was associated with an increased proportion of obesity, dyslipidemia and metabolic syndrome compared with patients with normal or high HPA-axis activity. Patients with recurrent depression showed a higher occurrence of hypocortisolism than the control sample representative of the general population. Patients with bipolar disorder showed a similar occurrence of hypocortisolism as the control sample. Among bipolar disorder patients with a low degree of lifetime with lithium prophylaxis, there was an inverse correlation between age and HPA-axis activity. In contrast, among patients with a higher degree of lifetime with lithium prophylaxis as well as among the controls, there was no correlation between age and HPA-axis activity. Accordingly, hypocortisolism was most common among older patients with a low degree of lifetime with lithium prophylaxis. In conclusion, hypocortisolism in both recurrent depression and bipolar disorder was associated with multiple clinically-relevant phenotypes. Additionally it was shown for bipolar disorder patients that increasing age was a risk factor for hypocortisolism and that prophylactic lithium treatment was a protective factor. It is argued that the protective effect of lithium towards the HPA-axis is attributable to its mood-stabilizing effect, which in turn reduces the chronic stress level. These results provide new insight into the role of hypocortisolism and chronic stress in recurrent affective disorders warranting further studies and hopefully providing clues to improved treatment strategies.

Nos indivíduos com obesidade, o excesso de tecido adiposo, exerce um papel fundamental induzindo um estado inflamatório crônico e sistêmico. A obesidade mimetiza processos celulares semelhantes aos do envelhecimento tais como a deterioração de tecidos e órgãos e diminuição na capacidade de reparo dos danos induzidos ao DNA. Nesse contexto, as citocinas pró-inflamatórias induzem atritos ao DNA que impactam, principalmente nas regiões mais susceptíveis dos cromossomos, os telômeros. Os telômeros, presentes nas extremidades dos cromossomos, estão associados a um complexo proteico denominado complexo shelterin. O complexo shelterin é formado por 6 proteínas (TRF1, TRF2, TIN2, POT1, TPP1 e RAP1), que junto com proteínas acessórias como a disquerina (DKC1), participam da regulação do comprimento dos telômeros e protegem os cromossomos dede atividades indesejadas de erosão enzimática, recombinação não-homóloga e fusão das terminações cromossômicas. Nos últimos anos, foram estabelecidas relações positivas entre condições patológicas clinicamente diferentes, como as moduladas por inflamação, e o comprimento dos telômeros. Recentemente, nosso grupo demonstrou telômeros encurtados em indivíduos portadores de obesidade mórbida. Assim o objetivo do presente trabalho foi explorar fatores adicionais associados ao metabolismo telomérico, como a expressão gênica das proteínas do complexo shelterin e citocinas pró-inflamatórias, as quais podem contribuir para o encurtamento acelerado de telômeros. Utilizamos amostras de células mononucleares de sangue periférico (PBMC) de indivíduos adultos saudáveis (n = 27) e indivíduos adultos portadores de obesidade (n = 39). Quantificamos a expressão gênica por transcrição reversa e PCR quantitativa (RT-qPCR) de todos os genes do complexo shelterin, DKC1, IL-1β e TNF-α. Nossos resultados demonstram um perfil de expressão gênica alterado quando comparada a expressão gênica das proteínas analisadas nos dois grupos estudados, controles e portadores de obesidade. Os indivíduos portadores de obesidade mostraram um perfil significativamente elevado dos genes TRF1, POT1, RAP1 e DKC1 (P < 0,05). Não observamos correlação de expressão gênica entre os diferentes genes e o comprimento dos telômeros nos grupos estudados, mas sim com a DKC1 na obesidade. Entretanto, quando analisamos as associações entre os genes de complexo shelterin observamos mudanças significativas nas associações intra-grupo dependentes da condição de obesidade. Nossos resultados salientam a complexa e intrincada rede de fatores associados e desregulados durante o processo fisiopatológico da obesidade. Estudos adicionais serão necessários acrescentando novos fatores para tentar dissecar a regulação coordenada do comprimento dos telômeros na homeostase e no processo patológico da obesidade. / In individuals with obesity, the excess of adipose tissue plays a key role in inducing a chronic and systemic inflammatory state. Like aging, obesity mimics cellular processes such as deterioration of tissues and organs and decreased ability to repair age-dependent DNA damages. In this context, the proinflammatory cytokines induce DNA damage that impact, especially in the most susceptible regions of the chromosomes, the telomeres. The telomeres, present at the ends of the chromosomes, are associated with a protein complex called the shelterin complex. The shelterin complex consists of 6 proteins (TRF1, TRF2, TIN2, POT1, TPP1 and RAP1), which together with accessory proteins such as dyskerin (DKC1), participate in telomere’s length regulation and protect chromosomes from undesired erosion, enzymatic activities, non-homologous recombination and fusion of chromosomal terminations. In recent years, positive relationships have been established between clinically different pathological conditions, such as those modulated by inflammation, and telomeres’ length. Recently, our group demonstrated shortened telomeres in individuals with morbid obesity. Thus, the aim of the present study was to explore additional factors associated with telomeres’ metabolism, such as gene expression of the shelterin complex components and proinflammatory cytokines, which may contribute to the accelerated shortening of the telomeres. We used peripheral blood mononuclear cells (PBMC) samples from healthy adults (n = 27) and adults with obesity (n = 39). We quantified gene expression by reverse transcription and quantitative PCR (RT-qPCR) of all shelterin complex genes, DKC1, IL-1β and TNF-α. Our results demonstrate an altered gene expression profile when compared to the gene expression of the proteins analyzed in the two studied groups, controls and individuals with obesity. Individuals with obesity showed a significantly elevated profile of TRF1, POT1, RAP1 and DKC1 (P < 0.05) genes. We did not observe correlation of gene expression between the different shelterin genes and the length of telomeres in the studied groups, but with DKC1 in obesity. However, when we analyzed the associations between the shelterin complex genes we observed significant changes in the intra-group associations dependent on the obesity condition. Our results highlight the complex and intricate network of associated and deregulated factors during the pathophysiological process of obesity. Further studies are needed together with the inclusion of additional factors to try to dissect the coordinated regulation of telomeres’ length in homeostasis and in the pathological process of obesity.

O complexo telomérico, responsável pela integridade genômica, é formado pela interação de DNA com proteínas, que são responsáveis pela proteção desses terminais. O complexo RPA de eucariotos compreende um heterotrímero, que cumpre diversas funções vitais na célula, sendo uma peça fundamental na replicação, reparo e recombinação. A ausência de homólogos de proteínas que protegem o telômero em T. cruzi nos fez investigar se o complexo RPA poderia cumprir essa função. Assim, este trabalho teve como objetivo caracterizar a interação TcRPA-1-telômero. Conseguimos verificar a interação in vitro e in vivo da RPA-1 com o telômero em epimastigotas e tripomastigotas. A ausência de homólogos de proteínas que interagem com o overhang telomérico em tripanosomas, a interação específica RPA-1-telômero, e a sua presença nos telômeros da forma de vida não replicativa, bem como as peculiaridades estruturais da TcRPA-1, levam-nos a propor que essa proteína pode estar envolvida com a proteção dos telomérica neste organismo. / The telomeric complex, responsible for genomic integrity and stability , is formed by the interaction of DNA with proteins that are responsible for maintaining and protecting these terminals. The eukaryotic RPA complex comprises a heterotrimer, which fulfills several vital functions in the cell , being a fundamental player in replication , repair and recombination. The absence of homologous proteins that protect telomeres in Trypanosoma cruzi lead us to hypothesize that RPA complex could fulfill this function. Thus, this study aimed to characterize TcRPA-1-telomere interaction. We could verify in vitro and in vivo interaction of RPA - 1 with telomeres in epimastigote and trypomastigote lifeforms. The absence of homologs of proteins that interact with telomeric overhang in trypanosomes, the specific interaction RPA-1- telomere , its presence in non- replicative lifeform as well as structural peculiarities of TcRPA-1, lead us to propose that this protein may be involved in telomere protection in this organism.

Nos indivíduos com obesidade, o excesso de tecido adiposo, exerce um papel fundamental induzindo um estado inflamatório crônico e sistêmico. A obesidade mimetiza processos celulares semelhantes aos do envelhecimento tais como a deterioração de tecidos e órgãos e diminuição na capacidade de reparo dos danos induzidos ao DNA. Nesse contexto, as citocinas pró-inflamatórias induzem atritos ao DNA que impactam, principalmente nas regiões mais susceptíveis dos cromossomos, os telômeros. Os telômeros, presentes nas extremidades dos cromossomos, estão associados a um complexo proteico denominado complexo shelterin. O complexo shelterin é formado por 6 proteínas (TRF1, TRF2, TIN2, POT1, TPP1 e RAP1), que junto com proteínas acessórias como a disquerina (DKC1), participam da regulação do comprimento dos telômeros e protegem os cromossomos dede atividades indesejadas de erosão enzimática, recombinação não-homóloga e fusão das terminações cromossômicas. Nos últimos anos, foram estabelecidas relações positivas entre condições patológicas clinicamente diferentes, como as moduladas por inflamação, e o comprimento dos telômeros. Recentemente, nosso grupo demonstrou telômeros encurtados em indivíduos portadores de obesidade mórbida. Assim o objetivo do presente trabalho foi explorar fatores adicionais associados ao metabolismo telomérico, como a expressão gênica das proteínas do complexo shelterin e citocinas pró-inflamatórias, as quais podem contribuir para o encurtamento acelerado de telômeros. Utilizamos amostras de células mononucleares de sangue periférico (PBMC) de indivíduos adultos saudáveis (n = 27) e indivíduos adultos portadores de obesidade (n = 39). Quantificamos a expressão gênica por transcrição reversa e PCR quantitativa (RT-qPCR) de todos os genes do complexo shelterin, DKC1, IL-1β e TNF-α. Nossos resultados demonstram um perfil de expressão gênica alterado quando comparada a expressão gênica das proteínas analisadas nos dois grupos estudados, controles e portadores de obesidade. Os indivíduos portadores de obesidade mostraram um perfil significativamente elevado dos genes TRF1, POT1, RAP1 e DKC1 (P < 0,05). Não observamos correlação de expressão gênica entre os diferentes genes e o comprimento dos telômeros nos grupos estudados, mas sim com a DKC1 na obesidade. Entretanto, quando analisamos as associações entre os genes de complexo shelterin observamos mudanças significativas nas associações intra-grupo dependentes da condição de obesidade. Nossos resultados salientam a complexa e intrincada rede de fatores associados e desregulados durante o processo fisiopatológico da obesidade. Estudos adicionais serão necessários acrescentando novos fatores para tentar dissecar a regulação coordenada do comprimento dos telômeros na homeostase e no processo patológico da obesidade. / In individuals with obesity, the excess of adipose tissue plays a key role in inducing a chronic and systemic inflammatory state. Like aging, obesity mimics cellular processes such as deterioration of tissues and organs and decreased ability to repair age-dependent DNA damages. In this context, the proinflammatory cytokines induce DNA damage that impact, especially in the most susceptible regions of the chromosomes, the telomeres. The telomeres, present at the ends of the chromosomes, are associated with a protein complex called the shelterin complex. The shelterin complex consists of 6 proteins (TRF1, TRF2, TIN2, POT1, TPP1 and RAP1), which together with accessory proteins such as dyskerin (DKC1), participate in telomere’s length regulation and protect chromosomes from undesired erosion, enzymatic activities, non-homologous recombination and fusion of chromosomal terminations. In recent years, positive relationships have been established between clinically different pathological conditions, such as those modulated by inflammation, and telomeres’ length. Recently, our group demonstrated shortened telomeres in individuals with morbid obesity. Thus, the aim of the present study was to explore additional factors associated with telomeres’ metabolism, such as gene expression of the shelterin complex components and proinflammatory cytokines, which may contribute to the accelerated shortening of the telomeres. We used peripheral blood mononuclear cells (PBMC) samples from healthy adults (n = 27) and adults with obesity (n = 39). We quantified gene expression by reverse transcription and quantitative PCR (RT-qPCR) of all shelterin complex genes, DKC1, IL-1β and TNF-α. Our results demonstrate an altered gene expression profile when compared to the gene expression of the proteins analyzed in the two studied groups, controls and individuals with obesity. Individuals with obesity showed a significantly elevated profile of TRF1, POT1, RAP1 and DKC1 (P < 0.05) genes. We did not observe correlation of gene expression between the different shelterin genes and the length of telomeres in the studied groups, but with DKC1 in obesity. However, when we analyzed the associations between the shelterin complex genes we observed significant changes in the intra-group associations dependent on the obesity condition. Our results highlight the complex and intricate network of associated and deregulated factors during the pathophysiological process of obesity. Further studies are needed together with the inclusion of additional factors to try to dissect the coordinated regulation of telomeres’ length in homeostasis and in the pathological process of obesity.

Leucemia Linfóide Crônica (LLC) é a leucemia mais comum em países ocidentais, tem apresentação clínica e evolução heterogênea. Especula-se que todos os casos sejam precedidos por Linfocitose B Monoclonal (LBM). Não são bem conhecidos os mecanismos moleculares responsáveis por esta evolução. Alterações na expressão de miRNAs e em comprimento telomérico podem contribuir para desencadear esta neoplasia. O objetivo deste estudo foi identificar diferenças em comprimento telomérico e expressão de miRNAs entre pacientes com LLC, portadores de LBM clínica e populacional e controles saudáveis. Estudamos 21 pacientes com LLC, 11 portadores de LBM clínica, 6 de LBM populacional e 10 voluntários saudáveis. Para o controle de comprimento telomérico, utilizamos dados de estudo anterior do nosso serviço com grupo de 261 voluntários saudáveis de 0 a 86 anos. Realizamos separação de células CD19+CD5+ por citometria de fluxo nos grupos de estudo e de linfócitos B no grupo controle. Analisamos expressão dos miRNAs 15a, 16-1, 29b, 34a, 155, 181a e 181b por RT-qPCR e comprimento telomérico por qPCR. O miR- 155 foi o único que demonstrou expressão diferente entre os grupos LLC e LBM, sendo maior nos pacientes com LLC. Este miRNA e o miR-34a têm aumento de expressão nas células com fenótipo anormal, apesar desta diferença não ter tido significância estatística quando considerada a expressão do miR-155 na LBM. Os miRNAs 15a, 16-1, 181a e 181b são hipoexpressos nas células com fenótipo anormal. O miR-29b teve expressão semelhante nos grupos estudados. O comprimento telomérico foi semelhante nos 3 grupos de estudo e menor quando comparados ao grupo controle. O miR-155 tem diferente expressão em LBM e LLC, podendo ser um dos responsáveis por esta evolução. Alterações nos miRNAs 34a, 15a, 16-1, 181a e 181b contribuem para expansão clonal de linfócitos B CD5+. O papel do miR-29b na fisiopatogênese e evolução da LLC ainda não está bem definido. O comprimento telomérico diminuído em LLC e LBM pode fazer parte dos eventos iniciais da fisiopatogênese desta leucemia. / Chronic Lymphocytic Leukemia (CLL) is the most common leukemia on Western countries, it has an heterogeneous clinical presentation and outcome. Monoclonal BCell Lymphocytosis (MBL) may precede all CLL cases. The molecular mechanisms responsible for this evolution are not known. Aberrant miRNA expression and telomere shortening may contribute for the pathophysiology of this disease. The objective of this study was to identify differences on telomere length and miRNA expression between CLL patients, subjects with clinical and population-screening MBL and healthy volunteers. 21 CLL patients, 11 subjects with clinical MBL, 6 with population-screening MBL and 10 healthy volunteers were enrolled on this study. As control for telomere length, we used a group of 261 healthy volunteers aged 0 to 86 years old that had been enrolled on a previous study from our group. After diagnosis confirmation, it has been done a flow citometry CD19+CD5+ cell sorting for the study groups and CD19+ cell sorting for the control group. The expression of the miRNAs 15a, 16-1, 29b, 34a, 155, 181a and 181b was determined by RT-qPCR. The telomere length was determined by qPCR. miR-155 was the only one with different expression between the CLL and MBL groups, presenting higher expression on the CLL group. This miRNA and the miR-34a are overexpressed on the study groups when compared to the control group, although this difference did not reach statistical significance when the miR-155 expression in MBL is considered. miRNAs 15a, 16-1, 181a and 181b are underexpressed on the study groups. The miR-29b was the only one with similar expression on all groups. The telomere length was similar on the 3 study groups and shorter on these groups when compared to normal subjects. The expression of miR-155 is different in CLL and MBL, it may contribute for this evolution. Aberrant expression of miR 34a, 15a, 16-1, 181a and 181b may contribute for the clonal expansion of CD5+ B lymphocytes. The role of miR-29b on the CLL pathogenesis and evolution is still not understood. The reduced telomere length on CLL and MBL may be part of the initial events of this leukemia pathogenesis.