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.

No description available.

La sénescence réplicative est le phénomène associé à un arrêt de croissance permanent causé par le raccourcissement progressif des télomères à chaque division. Lorsqu’ils atteignent une longueur critique, les télomères perdent leur structure terminale protectrice en t-loop, ce qui révèle l’extrémité du chromosome et déclenche une Réponse aux dommages à l’ADN (RDA) p53-dépendante. Le nombre de télomères ouverts nécessaire à la mise en place de la sénescence n’est pas connu, mais plusieurs évidences suggèrent que la cellule pourrait en tolérer un certain nombre avant de s’arrêter définitivement. Dans ce projet, nous utilisons un dominant négatif de Tin2 (Tin2DN), un membre du complexe nucléo-protéique nommé le télosome qui stabilise la t-loop, pour démontrer que la dysfonction chromatinienne télomérique seule ne suffit pas à déclencher un arrêt de croissance permanent. Lorsqu’il est exprimé, Tin2DN induit la formation de foyers de dommages de 53BP1, la RDA ainsi qu’un arrêt de croissance transitoire. De façon surprenante, nous observons que les cellules qui ont subi ce premier arrêt de croissance ré-entrent dans le cycle cellulaire et se divisent, et ce malgré la présence de foci télomériques. Cette réentrée cause l’apparition de cassures secondaires ainsi qu’une accumulation d’instabilités génomiques, telles que des ponts chromosomiques ou des micro-noyaux. Cet échappement des points de blocages du cycle cellulaire pourrait être expliqué par notre observation que la dysfonction télomérique induite par Tin2DN n’active que très faiblement p53 et p21, et pratiquement pas la kinase chkChk2. Néanmoins, en inhibant directement l’activité de p53, nous n’observons plus aucun arrêt de croissance mais une accumulation de foci et d’instabilités génomiques, avec une forte occurrence de catastrophes mitotiques. L’ensemble de ces résultats propose un nouveau modèle d’entrée en sénescence réplicative : l’ouverture des télomères induits une faible RDA menant à un premier arrêt de prolifération transitoire p53-dépendant. Les cellules échappent à cet arrêt et se divisent, mais l’ouverture des télomères ayant causé des fusions chromosomiques, la division crée alors de nouvelles cassures doubles brins dans le génome qui déclencheront une forte RDA et un nouvel arrêt de croissance permanent, la sénescence réplicative. / Replicative senescence is the physiological permanent growth arrest caused by telomeres shortening, at each round of replication. Once they have reach a critical length, the telomeres lose their t-loop structure, revealing the chromosome extremity that triggers a p53-dependant DNA damage response (DDR) and leads to proliferation arrest. The number of shortened telomeres that are necessary to onset senescence is not known, but accumulating evidences suggest that the cell is able to tolerate a certain level of telomere uncapping before stopping its divisions. Here, we used an inducible dominant negative form of Tin2 (Tin2DN), a member of the shelterin complex that stabilizes the t-loop, to show that telomeres uncapping alone is not sufficient to induce a stable growth arrest. When expressed, Tin2DN leads to the openingverture of the t-loop, creating a DDR with the formation of 53BP1 DNA damage foci (DDF) and a transient growth arrest. Indeed, we observed that the cells were re-entering the cell cycle and dividing, despite their uncapped DDF harbouring telomeres. As telomere uncapping creates chromosome fusions, such division leads to the apparition of secondary DNA breaks, with an accumulation of genomic instabilities, such as chromosomes bridges or micronuclei. We observed that Tin2 DN-induced telomere uncapping leads to a very weak activation of p53 and p21, with almost no phosphorylation of chkChk2. Nevertheless, when we infected our cells with a shp53, the primary growth arrest did not occur, leading to an amplification of the damages, with strong signs of instability and mitotic catastrophe. Altogether, these results propose a new model for replicative senescence: telomere uncapping induces a weak DDR that leads to a transitory growth arrest. The cells divide with fused chromosomes, creating new randomly distributed double strand breaks that trigger a stronger DDR and a permanent growth arrest. In that model, replicative senescence is not directly induced by telomere uncapping, but by an amplification of DNA damages through mitotic catastrophe.