Les télomères sont les extrémités des chromosomes et sont protégés par le complexe shelterin pour ne pas être reconnus comme des cassures accidentelles d'ADN double brin. Un enjeu majeur dans la recherche sur les télomères est de comprendre comment l'intégrité des extrémités des chromosomes au cours de l'oncogenèse est modifiée. La perte de répétitions télomériques à chaque division cellulaire peut être compensée par l’activité de l’enzyme télomérase qui ajoute "de novo" des motifs télomériques. Bien que la télomérase soit surexprimée dans la majorité des cellules cancéreuses, l’impact de modifications des protéines shelterin qui coiffent les télomères dans le processus oncogénique n'est pas clair. Dans la première étude, nous avons analysé l'état des télomères de patients atteints de la Leucémie Lymphoïde Chronique (LLC) uniquement au stade précoce de la LLC, stade A de Binet. La LLC est une leucémie commune aux pays occidentaux et se développe chez les personnes âgées. Nous avons montré que la majorité des patients atteints de la maladie présente des télomères dysfonctionnels. Les dommages télomériques ne corrèlent pas avec la longueur des télomères ou le statut mutationnel des patients, mais corrèlent avec la faible expression des protéines télomériques TIN2 et TPP1. Dans la seconde étude, nous avons montré que l'expression de l'oncogène H-RasV12 confère une résistance accrue vis-à-vis des télomères dysfonctionnels et aucun défaut de croissance n’est observable lorsque TRF2, une protéine télomérique est inhibée. Cet effet dépend de l'expression de l'interleukine 6 (IL-6), révélant un rôle inattendu de cette cytokine dans la fonction des télomères. De plus, la co-inhibition de TRF2 et IL-6 dans les cellules RasV12 conduit à blocage G2 / M associé à une séparation des chromatides sœurs. Ces résultats suggèrent que l'induction de la tumorigénicité par l’oncogène Ras protège les télomères contre les dommages et que la ségrégation des chromosomes est dépendante de TRF2 et IL-6. / Telomeres protect the chromosome extremities from being repaired and recognized as accidental DNA breaks double-strand. A major issue in telomere research is to understand how systems monitoring the integrity of chromosomes ends change during oncogenesis. Although telomerase overexpression occurs in the majority of cancer cells, whether other types of telomere changes play roles in the oncogenic process is unclear. In the first study, we analyzed the status of telomeres in patients with early stage Chronic Lymphocytic Leukemia (B-CLL), which is a common leukemia in Western countries that develops in the elderly. We showed that the majority of CLL patients exhibit telomeric dysfunction. It does not correlate with telomere length or mutation status of patients, but correlates with low expression of telomeric proteins TIN2 and TPP1. In the second study, we showed that expression of the H-RasV12 oncogene confers increased resistance to telomere uncapping and protects against to growth defects when TRF2, a telomeric protein, is depleted. This effect depends upon the expression of interleukin 6 (IL-6), revealing an unexpected role for this cytokine in telomere function. Notably, the co-inhibition of TRF2 and IL-6 in RasV12 cells led to G2/M block associated with an increased incidence of premature sister chromatid separation. These findings suggest that the induction of a tumorigenic state by oncogenic Ras protects against telomere damage and chromosome segregation defects in TRF2-compromised cells through an increase of IL-6 expression

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

La protéine p53 est surtout étudiée pour sa capacité à empêcher la prolifération de cellules dont le génome est endommagé. Toutefois, en analysant les souris p53Δ31/Δ31 qui expriment une protéine p53 hyperactive, notre laboratoire a découvert un rôle inattendu de p53 dans la régulation du métabolisme des télomères. Ces souris modélisent la dyskératose congénitale (DC), un syndrome d’insuffisance médullaire héréditaire (SIMH) causé par un dysfonctionnement télomérique. Mon projet de thèse a combiné les analyses de modèles murins et de lignées cellulaires humaines afin d’approfondir l'étude de cette nouvelle fonction de p53 et de mieux comprendre le rôle de la voie p53 dans le développement des SIMH.En poursuivant notre analyse du modèle murin p53Δ31, nous avons montré que les cellules p53Δ31/Δ31 présentent une diminution d’expression de 12 gènes mutés dans l’anémie de Fanconi (AF), un autre SIMH étroitement lié à la DC. De plus, ces cellules montrent une capacité réduite à réparer les ponts inter-brins de l’ADN, une caractéristique typique de l’AF. Notre étude suggère que l’activation soutenue de p53 pourrait ainsi contribuer aux similitudes cliniques entre ces deux syndromes. Par ailleurs, nous avons identifié par quel mécanisme p53 régule négativement le métabolisme des télomères et la voie Fanconi de réparation de l’ADN. Ce mécanisme de régulation indirect, via p21 et E2F4, est très conservé entre la souris et l’Homme. De fait, les cellules p53Δ31/Δ31 constituent un outil puissant pour identifier de nouvelles cibles de p53 réprimées par ce mécanisme. Ainsi, elles nous ont permis de montrer que deux gènes essentiels pour la structure des centromères sont réprimés par p53.L’activation de p53 est clairement impliquée dans les étapes ultimes d’une insuffisance médullaire, mais le rôle d’une suractivation de p53 dans l’initiation d’un SIMH chez l'Homme reste, à ce jour, controversé. En effet, une mutation inactivatrice de la ribonucléase PARN a été trouvée chez des patients atteints de DC. Il a été proposé que cette protéine déstabilise l’ARNm de p53 tout en favorisant la maturation de l’ARN de la télomérase, si bien que les conséquences d’une inactivation de PARN restent mal comprises. Au cours de ma thèse, j'ai également étudié de nouveaux modèles murins et établi des modèles cellulaires humains pour déterminer l’implication d’une activation de p53 dans l’initiation d’un SIMH chez l’homme. Les résultats en cours, particulièrement prometteurs, devraient notamment déterminer l’impact d’une déficience en Parn sur la voie de régulation de p53.En conclusion, nous avons montré qu’une activation soutenue de p53 conduit à des phénotypes principalement associés aux SIMH. Par ailleurs, nous avons montré que p53 réprime de nombreux gènes impliqués dans la maintenance du génome, ce qui peut sembler surprenant au regard du concept de « gardien du génome » communément attribué à p53, et nous a amené à rediscuter ce concept. Ainsi, les résultats obtenus pendant ma thèse devraient permettre de mieux comprendre les mécanismes impliqués dans l’insuffisance médullaire, la suppression tumorale et le vieillissement. / The p53 protein is mostly studied for its capacity to prevent the proliferation of cells with damaged genome. However, while studying p53Δ31/Δ31 mice that express a hyperactive p53 protein, our laboratory uncovered an unexpected role of p53 in the regulation of telomere metabolism. These mice model dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) caused by defects in telomere maintenance. My PhD project combined analyses of mouse models and human cell lines to extend the study of this new function for p53 and to understand the role of the p53 pathway in the development of IBMFS.By extending our analysis of the p53Δ31 mouse model, we revealed that the p53Δ31/Δ31 cells exhibit decreased expression levels for 12 genes mutated in Fanconi anemia (FA), another IBMFS closely related to DC. Furthermore, these cells display a reduced capacity to repair DNA inter-strand crosslinks, a typical feature of FA cells. Our study suggests that a sustained activation of p53 might actually contribute to the clinical overlap between both syndromes. Importantly, we identified the mechanism used by p53 to downregulate telomere metabolism and the FA DNA repair pathway. This regulatory mechanism is indirect, via p21 and E2F4, and largely conserved between mice and humans. In fact, the p53Δ31/Δ31 cells constitute a powerful tool to find p53 target genes downregulated through this regulatory pathway. Accordingly, they were useful to show that two genes essential for centromere structure are also downregulated by p53.p53 activation is clearly involved in the final stages of bone marrow failure, but to-date, the role of p53 hyperactivation in the initiation of IBMFS remains controversial. Indeed, an inactivating mutation of the PARN gene, encoding a ribonuclease, was found in patients with DC. This protein was proposed to destabilize the p53 mRNA while promoting the maturation of the telomerase RNA, so that the consequences of PARN inactivation remain poorly understood. During my thesis, I also studied new mouse models and established human cellular models to determine the contribution of p53 activation in initiating IBMFS in humans. The current results, which are particularly promising, should notably determine the impact of Parn deficiency on the p53 regulatory pathway.In conclusion, we demonstrated that a sustained activation of p53 leads to phenotypes mainly associated with IBMFS. In addition, we showed that p53 suppresses many genes involved in genome maintenance, which may seem surprising in view of the concept of "guardian of the genome" commonly attributed to p53, leading us to revisit this concept. Hence, the results obtained during my thesis should help to better understand the mechanisms involved in bone marrow failure, tumor suppression and aging.

<p>B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. </p><p>Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.</p><p>In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases. </p>

B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (VH) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by VH3-21/Vλ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the VH3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin. In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases.

Orientador: Maria Isabel Nogueira Cano / Resumo: O câncer de pulmão é o câncer que apresenta maior mortalidade no mundo: >1,6 milhões de óbitos por ano segundo a Organização Mundial de Saúde (OMS), sendo o câncer de pulmão de células não pequenas (NSCLC) o tipo mais frequente (~ 85%) de carcinoma pulmonar. O NSCLC apresenta dois subtipos histológicos principais: adenocarcinoma (LUAD) e carcinoma de células escamosas (LUSC). Pesquisas têm sido realizadas objetivando identificar biomarcadores moleculares úteis como alvo terapêuticos em NSCLC e os telômeros têm sido considerado alvos promissores para terapias antitumoral, devido ao seu papel crucial na integridade, estabilidade e manutenção genômica. Telômeros humanos consistem em DNA repetitivo rico em G associado a proteínas do complexo shelterin e mantido pela ação da telomerase. Estudos recentes mostraram que a expressão de TERT (subunidade catalítica da telomerase) e de alguns componentes do complexo shelterin estão alterados em câncer de pulmão. No entanto, a correlação entre alterações na função telomérica e o desenvolvimento e progressão de NSCLC não foi elucidada. Portanto, o objetivo do presente estudo foi verificar alterações na expressão de genes associados aos telômeros, incluindo os ncRNAs associados à regulação e manutenção dos telômeros em NSCLC, no intuito de identificar novos biomarcadores associados ao desenvolvimento e progressão do NSCLC. Para tanto, foram realizadas análises de expressão de 50 genes associados aos telômeros em amostras de tecido tumoral e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: According to the World Health Organization, lung cancer has a high mortality rate associated with >1.6 million deaths per year, being Non-small cell lung cancer (NSCLC) the most frequent type (~ 85%) of lung carcinoma. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the two major histological subtypes of NSCLC. Research efforts have been made to identify molecular biomarkers useful as therapeutic targets in NSCLC and telomere have been considered very promising targets for anticancer therapies due to their crucial role in genome integrity maintenance and stability. Human telomeres consist of repetitive G-rich DNA associated with proteins of the shelterin complex, maintained by the action of telomerase. Recent studies showed that expression of TERT (telomerase reverse transcriptase component) and of some of the shelterin components are altered in lung cancer. However, the mechanisms of telomere deregulation associated with NSCLC development and progression have not been elucidated. Therefore, our aim was to study expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. Such alterations may represent novel biomarkers associated with NSCLC development and progression. For this purpose, expression analyzes of 50 telomere-associated genes were performed in samples of tumor tissue and normal lung tissue, adjacent to the tumor, from patients with NSCLC. The following techniques were used: 1... (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.

<p>Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance <i>via</i> inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates <i>in vitro</i>. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.</p>

Squamate reptiles in general possess an unusual variability in karyotype and sex determining mechanisms. However, in these two aspects, iguanas (Pleurodonts) are considered as a relatively conservative group of lizards. So far only genotypic sex determination with male heterogamety has been detected in this lineage. However, the sex chromosomes have not been revealed in many species by classical cytogenetics, probably due to their homomorphy. Significant variability in karyotype was observed only in the species-rich genera Anolis, Sceloporus and Liolaemus. The aim of this thesis was to investigate the genome of available species from the main iguanid lineages using classical and molecular cytogenetic methods. As well as finding the karyotype characteristics, which may represent synapomorphies of main iguanid lineages, the other aim was to try to identify sex chromosomes. This study confirmed previously published karyotypes of 13 species and established new descriptions of karyotype for eight species. The chromosomes of all studied specimens were examined by methods of classical and molecular cytogenetics, 21 species covering eight iguanid families were analysed in this thesis. The majority of studied species shared the apparently ancestral karyotype of the group, with diploid chromosome number...

La mastocytose se définit par une accumulation excessive de mastocytes dans un ou plusieurs organes ou tissus. Si dans la plupart des cas la maladie ne diminue pas l’espérance de vie, elle reste néanmoins associée à un handicap souvent mal connu et mal compris. Malgré l’importance des manifestations neuropsychiatriques accompagnant la mastocytose, les efforts concentrés sur leur compréhenson et celle de leurs liens avec la maladie ont été longtemps mis au second plan. L’objectif de cette thèse était de mieux connaître ces troubles neuropsychologiques, notamment la dépression et les troubles de l’attention/ mémoire, d’investiguer les liens entre ces troubles et des aspects biologiques et émotionnels comme l’effet d’un inhibiteur de tyrosine kinase bloquant les mastocytes sur la dépression ; les liens entre le stress perçu, la dépression et la longueur des télomères ; et les liens entre dépression et compétences de régulation émotionnelle. Nos résultats montrent que la dépression est un symptôme fréquent dans la mastocytose, ainsi que les troubles de la mémoire. Ces derniers ne semblent pas liés à la dépression. Par ailleurs, la dépression est améliorée par un traitement visant les mastocytes ; le stress perçu chez ces patients est lié à la longueur des télomères et le métaolisme du tryptophane, en particulier l’activité de l’IDO est associée aux troubles neuropsychologiques chez ces patiens. Ces résultats ouvrent des nouvelles perspectives dans la compréhension des symptômes psychologiques associés à la mastocytose. / Mastocytosis is defined as an excessive accumulation of mast cells in one or more organs or tissues. While in most cases the disease does not reduce life expectancy, it is associated with a disability often poorly known and poorly understood relate to the symptoms due to the release of mast cell mediators. Despite the importance of these neuropsychiatric manifestations, efforts focused on their understanding and their relationship to the disease has been put into the background. The aim of this thesis was to better know these neuropsychological disorders present in mastocytosis, including depression and attention deficit disorder and to investigate the relationship between these disorders and biological and emotional aspects as effect of an inhibitor of tyrosine kinase locking mast cells, the relationship between perceived stress, depression, and telomere length, and the links between depression and emotional regulation skills. Our results show that depression and memory disorders are common symptoms in mastocytosis. Attention impairment do not seem related to depression. Furthermore, depression is improved by masitinib treatment. Perceived stress in these patients is related to telomere length and tryptophan metabolism; in particular the activity of IDO is associated with neuropsychological disorders. These results open new perspectives in understanding the psychological symptoms associated with mastocytosis.