As sirtuínas 1-7 (SIRT) constituem uma família altamente conservada de desacetilases de histonas que, de modo geral, participam da regulação da longevidade em diversos organismos, modulando a resposta celular frente ao stress oxidativo e promovendo mecanismo de reparo de DNA, parada do ciclo celular, estabilidade telomérica, senescência e apoptose celulares. O envolvimento das SIRTs no processo tumorigênico tem sido bastante investigado, contudo ainda não existe descrição do estudo desses genes nos adenomas hipofisários. O objetivo desse estudo foi avaliar a expressão gênica das SIRT1-7 nos somatotropinomas e adenomas hipofisários clinicamente não funcionantes (ACNF) e sua relação com o tamanho e a invasividade do tumor. A expressão das sirtuínas foi ainda correlacionada à expressão dos marcadores de senescência CDKN1A (p21) e CDKN2A (p16) e do proto-oncogene PTTG (pituitary tumor transforming gene). Foram selecionados 68 pacientes, 37 somatotropinomas e 31 portadores de ACNF. Desses casos, 33 apresentavam tumores invasivos e 35 eram não invasivos. A quantificação do RNAm das SIRT1-7, CDKN1A, CDKN2A e PTTG foi realizada nas amostras tumorais pela técnica de PCR em tempo real utilizando o método de quantificação relativa 2-??Ct. A hiperexpressão da SIRT1 foi observada em 86,5% dos somatotropinomas versus 41,9% dos ACNF (P < 0.01), não sendo observada perda de expressão desse gene. A SIRT3 foi mais hipoexpressa nos ACNF em relação aos somatotropinomas (77,4% e 40,5%, respectivamente; P < 0.01). A SIRT4 foi hipo e hiperexpressa, respectivamente, em 45,2% e 12,9% dos ACNF e 16,2% e 24,3% dos somatotropinomas (P=0.03). A hipoexpressão da SIRT7 também foi maior nos ACNF (67,7%) versus somatotropinomas (32,4%; P=0.01) e, para ambos os subtipos, o percentual de casos apresentando hiperexpressão desse RNAm foi baixo. O padrão de expressão das SIRT2 e 5 não diferiu entre os subtipos tumorais e não se mostrou alterado em relação ao pool de hipófises normais. Não foi observada diferença estatisticamente significante na expressão dos genes das sirtuínas entre os grupos de tumores invasivos e não invasivos. Contudo, a expressão das SIRT1 e 3 foi relacionada ao tamanho tumoral; nos casos com hiperexpressão da SIRT1 a média do maior diâmetro tumoral foi 2.4 ± 1.1 enquanto nos pacientes com expressão normal foi de 3.3 ± 1.3 (P < 0.01). Já os casos com perda de expressão da SIRT3 apresentaram tumores maiores (3.1 ± 1.2) em relação aos casos com expressão normal (2.2 ± 1.1; P < 0.01). A expressão de todas as SIRTs apresentou correlação positiva moderada (SIRT1-5,7) ou forte (SIRT6) com a expressão do CDKN1A. Uma correlação positiva foi observada também em relação a expressão do CDKN2A. Contudo, essa foi fraca e presente apenas para as SIRTs 3-5. Em relação ao PTTG, foi observado apenas uma fraca correlação com a expressão da SIRT1 e SIRT3. Em conclusão, esses resultados sugerem que a hiperexpressão de SIRT1 e a hipoexpressão das SIRTs 3, 4 e 7 podem estar relacionadas ao processo tumorigênico nos somatotropinomas e ACNFs, respectivamente e, em especial as SIRT1 e 3, ao controle da proliferação celular nesses adenomas / Sirtuins 1-7 (SIRT) are a highly conserved family of histone deacetylases. In general, these proteins are involved in the regulation of longevity in several organisms, modulating the cellular response to oxidative stress. SIRTs can also regulate DNA repair, telomeric stability, cell senescence and apoptosis. Due to their functions, there is a growing interest in the role of sirtuins in tumorigenesis. However, these genes were not investigated in pituitary tumors so far. In this study, SIRT1-7 gene expression was evaluated in somatotropinomas and nonfunctioning pituitary adenomas (NFPA) and related to tumor size and invasiveness. SIRT1-7 expression was also correlated with cellular senescence markers CDKN1A (p21) e CDKN2A (p16) and with the proto-oncogene PTTG (pituitary tumor transforming gene). Sixty-eight patients were selected, 37 with somatotropinomas and 31 with NFPA. Tumor invasion was observed in 33 patients. SIRT1-7, CDKN1A, CDKN2A and PTTG mRNA levels was evaluated from pituitary tumor samples by the real-time PCR using 2-??Ct relative quantification. Pronounced differences in SIRT1, 3, 4 and 7 expressions were identified between somatotropinomas and NFPA. Overexpression of SIRT1 was observed in 86.5% of somatotropinomas versus 41.9% of NFPA (P < 0.01) whereas underexpression was not detected. SIRT3 was more underexpressed in NFPA than somatotropinomas (77.4% and 40.5%, respectively, P < 0.01). SIRT4 was under and overexpressed, respectively, in 45.2% and 12.9% of NFPA and 16.2% and 24.3% of somatotropinomas (P=0.03). SIRT7 underexpression was also higher in NFPAs (67.7%) versus somatotropinomas (32.4%; P=0.01) with few cases showing overexpression. SIRT2 and 5 expressions did not differ between tumors subtypes and was not altered in relation to the normal pituitary gland pool. No statistically significant difference was observed in the expression of these genes between invasive and non-invasive tumor groups. However, SIRT1 and 3 expressions were related to tumor size. Mean of the largest tumor diameter was 2.4 ± 1.1 and 3.3 ± 1.3 (P < 0.01) in adenomas with SIRT1 over- and normal expression, respectively. On the other hand, cases with SIRT3 underepression exhibited larger tumors (3.1 ± 1.2) compared to cases with SIRT3 normal expression (2.2 ± 1.1, P < 0.01). Moderated (SIRT1-5.7) or strong (SIRT6) positive correlation was observed between sirtuins and CDKN1A expression. A weak correlation was observed with respect to CDKN2A expression and SIRTs 3-5. Regarding PTTG mRNA, only a weak correlation with SIRT1 and SIRT3 expression was observed. In conclusion, these results suggest that overexpression of SIRT1 and underexpression of SIRTs 3, 4 and 7 could be related to the tumorigenic process in somatotropinomas and NFPAs, respectively. SIRT1 and 3 could also play a role in control of pituitary adenomas cell proliferation

Les G-quadruplexes (G4) sont des structures non canoniques des acides nucléiques qui peuvent être formés dans des régions d’ADN ou d’ARN riches en guanines. Les ligands G4 (LG4), sont des molécules capables d’interagir et de stabiliser les structures G4, qui présentent de nombreuses propriétés anti-cancéreuses. Nous avons travaillé avec le LG4 20A, appartenant à la famille des triarylpyridines, qui stabilise efficacement les structures G4 in vitro. Les objectifs de ce travail ont été de déterminer les mécanismes moléculaires et cellulaires responsables des effets anti-prolifératifs du 20A dans des cellules cancéreuses. Dans cette étude, nous avons montré que le 20A induit un arrêt de la croissance cellulaire de cellules en culture et dans un modèle de xénogreffe tumorale, grâce à l’induction de la sénescence et de la mort cellulaire par apoptose. Ces réponses sont associées à l’activation de la voie des réponses aux dommages à l’ADN (DDR) via la kinase ATM, qui favorise l’autophagie (un processus catabolique) et la sénescence, tout en protégeant les cellules de l’apoptose. De plus, nous avons observé que le 20A induit un échec de la cytokinèse, conduisant à l’accumulation de cellules binucléées qui présentent une résistance à la mort cellulaire. De façon inattendue, nous avons trouvé que le 20A s’accumule dans les lysosomes, induisant une augmentation de la taille de ces derniers. La combinaison du 20A et de l’agent lysomotropique chloroquine, potentialise de façon importante la perméabilisation de la membrane lysosomale (LMP) et la mort cellulaire. En particulier, cette combinaison sensibilise de façon notable ces cellules binucléées à la mort cellulaire. L’ensemble de ces résultats révèle une relation entre les processus de mort cellulaire et de sénescence induits par le LG4 20A, et les voies de DDR et lysosomales. Ces régulations devraient être prises en considération lors de l’utilisation d’agents antiprolifératifs susceptibles d’interférer avec les fonctions lysosomales. / G-quadruplexes (G4) are unusual nucleic acid structures that can be formed by guanine-rich DNA and RNA. Through their ability to stabilize G4 structures, G4 ligands (G4L) have been described to display potent anticancer properties. Here, we studied the G4L 20A belonging to the triarylpyridine family of compounds that have the ability to efficiently bind to and stabilize G4 structures in vitro. The objectives of this work were to determine the molecular and cellular mechanisms responsible for the anti-proliferative effects of 20A in cancer cells. In this study, we showed that 20A causes cancer cell growth arrest in cell culture and a mice tumour xenograft model, through induction of senescence and apoptotic cell death. These cellular responses are associated with the induction of the DNA damage response pathway (DDR), in particular ATM activation, which promotes the induction of both autophagy (a lysosomal catabolic pathway) and senescence, while protecting cells against apoptosis. Furthermore, we found that 20A induces failure of cytokinesis which results in the accumulation of binucleated cells that display marked resistance to 20A-induced cell death. Unexpectedly, we found that 20A accumulates in the lysosomal compartment and causes lysosome enlargement. The combination of a lysosomotropic agent, chloroquine, and 20A promotes a significant induction of lysosomal membrane permeabilization (LMP) and a robust cell death. In particular, this combination significantly sensitizes binucleated cells to cell death. Altogether, our results uncover the relationship of the DDR and lysosomal pathways to cell death and senescence induced by the G4L 20A. Such regulation should also be taken into account when using antiproliferative drugs susceptible to interfere with the lysosomal functions.

Made available in DSpace on 2016-08-19T17:47:11Z (GMT). No. of bitstreams: 1 ANILDES IRAN PEREIRA SOUSA.pdf: 116617 bytes, checksum: 14ba38e42bfb1dbd3eba6477cff88f65 (MD5) Previous issue date: 2008-04-16 / The babassu mesocarp (Orbignya phalerata, Arecaceae) is popularly used in Maranhão, northeast of Brazil, as food and as medicine. It was the aim of this work to evaluate the effect of the babassu mesocarp extract (BME) on the biochemical parameters in mice bearing Ehrlich Ascitic tumor. C3H/HePas (N = l0 for group) with age of 120 and 240 days, were treated orally with BME (2mg/mL) during 15 or 30 days. At the end of this period the animals received, by intraperitoneal route, l06 cells of Ehrlich tumor. The animals were sacrificed ten days after the tumor implantation, when it was obtained the serum and the tumoral cells. The total number of tumoral cells was quantified in Neubauer chamber with aid of an optic microscope. The concentration of cholesterol, LDL, HDL, VLDL, triglicérides, total proteins and albumin was determined in serum, by colorimetric assay. The oral treatment with BME significantly increases on the number of tumoral cells. BME also affect the lipidic profile due to a strong reduction on the concentration of total cholesterol and HDL, LDL fractions. It was also observed a significant increase on the triglycerides concentration. Besides, the values of VLDL and total proteins were larger than the control in animals treated with EAB. Based on this, it is reasonable to propose that the reduction of cholesterol seems to be increased the tumor virulence. Additionally it was observed that BME induced a variable effect on blood biochemistry. Those results altogether makes BME unfeasible for using in the treatment of tumors with the same characteristics of the Ehrlich ascitic tumor. / O mesocarpo de babaçu (Orbignya phalerata, Arecaceae) é popularmente usado no Maranhão, nordeste do Brasil, como alimento e medicamento. Esse trabalho avaliou o efeito do tratamento com extrato bruto aquoso do mesocarpo de babaçu (EAB) na bioquímica sanguinea de camundongos C3H/HePas que desenvolveram o tumor de Ehrlich. Camundongos C3H/HePas (n= l0 por grupo), com idade de 120 e 240 dias, receberam, via oral, ad libitum, EAB (2mg/mL) durante 15 ou 30 dias. Em seguida ao último dia do tratamento os animais receberam, via intraperitoneal, l06 células de tumor de Ehrlich. Os animais foram sacrificados dez dias após a implantação do tumor, quando foram obtidos o soro e as células tumorais. As células foram quantificadas em câmara de Neubauer com auxílio de microscópio ótico de luz comum e o soro foi utilizado para determinar, por ensaios colorimétricos, a concentração de colesterol, triglicérides, proteínas totais e albumina. O tratamento com EAB induziu aumento significativo no número de células tumorais em camundongos C3H/HePas. A determinação do perfil lipídico mostrou que o tratamento com EAB resultou na redução da concentração de colesterol total e das frações HDL, LDL e no aumento da concentração de triglicérides . Além disso, os valores de VLDL e de proteínas totais foram maiores do que o controle em animais tratados previamente com EAB. Assim, é possível concluir que o tratamento oral com EAB aumenta a invasividade do tumor, possivelmente por reduzir a concentração de colesterol. Adicionalmente, o consumo de EAB tem efeitos variáveis sobre alguns parâmetros bioquímicos do sangue, dependendo da idade e do tempo de tratamento, o que inviabiliza o seu uso no tratamento de tumores com as mesmas características do tumor ascítico de Ehrlich.

La sénescence cellulaire est une réponse à un stress des cellules de mammifère caractérisée par un arrêt durable du cycle cellulaire. Celle-ci peut être déclenchée par un dysfonctionnement des télomères, des stress génotoxiques et l’activation d’oncogènes. La sénescence constitue une puissante ligne de défense contre le développement de cancers et intervient aussi dans le vieillissement. Les cellules en sénescence réorganisent leur génome par l’assemblage en hétérochromatine sous forme de SAHFs (senescence-associated heterochromatin foci). Nous avons mis en évidence que la désacétylation globale de H4-K16Ac par la désacétylase SIRT2 est impliquée dans l’assemblage de l’hétérochromatine en sénescence. De plus, nous avons identifié une accumulation de variants d’histones H2A et H2B spécifiquement dans des cellules en sénescence présentant des dommages persistants à l’ADN. Ces variants d’histone pourraient avoir des fonctions spécifiques dans ces cellules et pourraient représenter un biomarqueur du vieillissement in vivo.Mes travaux apportent des éléments pour la compréhension des rôles de l’information épigénétique dans la sénescence cellulaire. / Cellular senescence is a stress response of mammalian cells characterized by a stable cell proliferation arrest. It can be triggered by telomere dysfunction, genotoxic stress and oncogene activation. Cellular senescence acts as a natural barrier against cancer development and is involved in ageing. Senescent cells reorganize their genome by the assembly of chromatin into senescence-associated heterochromatin foci (SAHF). We showed that SIRT2-mediated global deacetylation of H4-K16Ac is involved in heterochromatin assembly in senescence. Moreover, we identified the accumulation with time of specific H2A and H2B variants in senescence triggered by persistent DNA damage signaling. These histone variants could have specific functions in senescent cells and could be a useful ageing biomarker in vivo.This work provides novel insights into chromatin modification and epigenetic regulation in cellular senescence.

O acúmulo de forragem é um processo dinâmico, mediado por alterações em respostas morfogênicas e demografia de perfilhos, que envolve o balanço entre crescimento e senescência. O manejo do pastejo pode alterar esse balanço, afetando a produção e a eficiência de utilização da forragem produzida. Objetivo deste trabalho foi estudar os padrões de respostas morfogênicas e a dinâmica do acúmulo de forragem, caracterizados pelo crescimento e senescência de tecidos, em pastos de capim-marandu submetidos a estratégias de manejo do pastejo sob lotação rotacionada como forma de compreender e permitir o planejamento e manipulação do processo de desfolhação de forma eficiente, assegurando o uso adequado dessa planta forrageira. O experimento foi implantado e conduzido no Departamento de Zootecnia da USP/ESALQ, entre dezembro de 2004 e dezembro de 2005. Os tratamentos experimentais compreenderam a combinação entre duas intensidades (altura de resíduo de 10 e 15 cm) e dois intervalos entre pastejos (período de tempo necessário para atingir 95 e 100% de interceptação luminosa pelo dossel durante a rebrotação - IL), e foram alocados às unidades experimentais (piquetes de 1.200 m2) segundo um delineamento de blocos completos casualizados e arranjo fatorial 2 x 2, com 4 repetições. Foram avaliadas as seguintes variáveis-resposta: número de folhas vivas (NFV), em senescência (NFS) e em expansão (NFE) por perfilho; taxa de aparecimento (TApF), filocrono e duração de vida das folhas (DVF); taxa de alongamento de folhas (TAlF) e de colmos (TAlC), além das taxas de crescimento e senescência dos pastos, tanto para perfilhos basais quanto para perfilhos aéreos. De uma maneira geral, para a média do dossel, os tratamentos de 95% de IL resultaram em valores maiores para o NFV, assim como para as variáveis NFS, NFE e DVF, em relação aos tratamentos de 100% de IL. Por outro lado, os tratamentos de 100% de IL resultaram em um maior alongamento de colmo quando comparados aos de 95% de IL. Para as demais variáveis, o tratamento 95/15 apresentou um padrão de resposta diferenciado dos demais tratamentos, caracterizado pelos maiores valores de TApF e de TAlF e a menor proporção de perfilhos aéreos. Como média do período experimental, as taxas de crescimento e de acúmulo líquido total não foram afetadas nem pelo resíduo pós-pastejo e nem pela interceptação luminosa pré-pastejo, mas os tratamentos de 95% de IL apresentaram as maiores taxas de senescência. Houve, no entanto, efeito das interações IL x época do ano, resíduo x época do ano e IL x resíduo x época do ano, que conferiram um padrão alternado de respostas ao longo do período experimental para o crescimento, senescência e acúmulo líquido de forragem, com os tratamentos de 95% de IL desempenhando melhor durante as épocas de verão/início do outono e final de primavera. A época do ano afetou significativamente a dinâmica do acúmulo de forragem, sendo que a época de final do inverno/início de primavera correspondeu a um período crítico para o restabelecimento da produção dos pastos na nova estação de crescimento. Pastos manejados a 95% de IL apresentaram menor massa de forragem, com menores quantidades de material morto e de colmos, o que favoreceu seu retorno mais rápida e precocemente à produção. Pastos de capim-marandu submetidos a pastejo rotacionado devem ser pastejados quando o dossel intercepta 95% da luz incidente durante a rebrotação, ou seja, cerca de 25 cm de altura pré-pastejo, e os animais removidos quando com um resíduo de 15 cm. / Herbage accumulation is a dynamic process mediated by modifications in morphogenetic responses and tiller demography that involves the balance between growth and senescence. Grazing management can alter this balance, affecting the production and the efficiency of utilisation of the produced herbage. The objective of this experiment was to evaluate the patterns of morphogenetic responses and the dynamics of herbage accumulation, characterised by growth and senescence, in marandu grass swards subjected to strategies of rotational grazing management in order to understand and enable the efficient planning and manipulation of the defoliation process, ensuring the adequate use of the this forage plant. The experiment was carried out at Departamento de Zootecnia, USP/ESALQ, from December 2004 to December 2005. Treatments corresponded to combinations between two grazing intensities (post-grazing residues of 10 and 15 cm) and two grazing frequencies (equivalent to the period of time necessary for swards to reach 95 and 100% interception of the incident light during regrowth ? LI), and were allocated to experimental units (1200 m2 paddocks) according to a complete randomised block design and a 2 x 2 factorial arrangement, with 4 replications. The following response variables were analysed: number of live (NLL), senescing (NSL) and expanding (NEL) per tillers; leaf appearance rate (LAR), phyllochron and leaf lifespan (LLS); rates of leaf (LER) and stem (SER) elongation, and rates of sward growth and senescence considering both basal and aerial tillers. In general, considering the entire sward tiller population (basal and aerial), the 95% LI treatments resulted in higher values of NLF as well as NSL, NEL and LLS than the 100% LI treatments. On the other hand, the 100% LI treatments resulted in higher rates of stem elongation than the 95% LI treatments. As for the remaining variables, treatment 95/15 showed a particular pattern of response in relation to the other treatments characterised by higher values of LAR and LER and lower proportion of aerial tillers in sward tiller population. Considering the entire experimental period, both post-grazing residue and sward light interception pre-grazing did not affect the rates of growth and senescence, but the 95% LI treatments showed the highest senescence rates. There was, however, an effect of the LI x season of the year, residue x season of the year and LI x residue x season of the year interactions, which determined an alternate pattern of responses throughout the year for growth, senescence and net herbage accumulation of the sward, with the 95% LI treatments performing better than other treatments during the summer/early autumn and late spring periods. Season of the year had a strong effect on the dynamics of herbage accumulation, with the late winter/early spring corresponding to a critical period for reestablishing conditions for high herbage production during the new pasture growth season. Swards grazed at 95% LI showed lower herbage mass, with low amounts of accumulated dead material and stem, favouring a fast and early return to production early during the new growth season. Marandu grass subjected to rotational grazing management should be grazed when swards reach 95% interception of the incident light during regrowth, around 25 cm pre-grazing height, and animals removed with a post-grazing residue of 15 cm.

Cellular senescence a specialized form of growth arrest that contributes to the pathogenesis of several aging-related disorders including cancer. While by definition tumor cells are considered immortalized, they can undergo senescence when exposed to conventional and targeted cancer therapy. Therapy-Induced Senescence (TIS) represents a fundamental response to therapy and impacts its outcomes. However, TIS has been considered a positive therapeutic goal since senescent tumor cells are expected to enter a state of permanent growth abrogation. In this work we examined the hypothesis that a subpopulation of senescent cells can re-acquire proliferative potential after a state of senescent dormancy, indicating that senescence is not obligatorily an irreversible process. Our observations indicate that H460 non-small cell lung cancer cells induced into senescence by exposure to etoposide, and enriched based on β-galactosidase staining and size, were shown to recover reproductive capacity, which was accompanied by resolution of the DNA-damage-response (downregulation of p53 and p21Cip1 induction), attenuation of the Senescence-associated Secretory Phenotype (SASP). To overcome the reservation that the newly dividing cells may not have been derived from the senescent population and in an effort to establish that escape from TIS is feasible, tumor cells induced into senescence by chemotherapy were enriched for senescence by flow cytometry; the subsequent division of senescent cells was demonstrable utilizing both real-time, live microscopy and High Speed Live Cell Interferometry (HSLCI). Furthermore, sorted senescent cells were observed to form tumors when implanted in immune deficient mice and with a significant delay in immunecompetent mice. As chemotherapy induced senescent cells have been identified in patient tumors, it is reasonable to propose that tumor cells that escape from senescence could contribute to disease recurrence. In addition, therapy-induced senescence could prove to reflect one form of tumor dormancy. Recently, ABT263 has been used as a senolytic drug, effectively eliminating senescent cells from aging-related animal models. Here, we utilize ABT263 in a two-hit approach to eliminate senescent tumor cells that persistent after exposure to chemotherapy. ABT263 results in the killing of senescent tumor cells in a concentration-dependent manner and shifts the response towards apoptotic cell death. Furthermore, sequential administration of ABT263 interferes with the ability of senescent tumor cells to recover growth potential. These results indicate that senescent tumor cells can contribute to cancer relapse by acquiring proliferative properties and that senolytic therapy allows for the clearance of dormant senescent tumor cells and will potentially decrease cancer recurrence rates.

The preferred method of communication for most prelingual/culturally Deaf individuals is American Sign Language (ASL), and members of this linguistic/cultural minority community are often not recognized as being bilingual. Many prelingually/culturally Deaf individuals have limitations and deficits in English proficiency; which can lead to deficits in general knowledge of health-related terminology. Current projections are that older adults are expected to live longer, and will also experience the development of, increases in and more extended periods of living with senescence/age-related health disorders, also includes prelingual/culturally Deaf individuals. This quantitative research project, utilizing the theoretical framework of health literacy and a modified version of the REALM (Rapid Estimate of Health Literacy in Medicine), utilizing American Sign Language (ASL) graphics; analyzed the convergence of prelingual/cultural Deafness and health literacy related to senescence/age-related disorders. An evaluation of a sample population of 27 Deaf participants, on health-related items of medical words, medical conditions medical procedures, and medical/numeracy instructions revealed significant deficits in all areas of health literacy. These deficits are critical and impact one's ability to manage effectively, age-related disorders. The results of this study will inform the health care community of the unrecognized magnitude, implication, and the need for positive social change in health care policies and procedures related to the appropriate provision of medical, health care, and health-related information for prelingual/culturally Deaf individuals.

Le mélanome est le cancer cutané le plus meurtrier. Il est issu de la transformation maligne des mélanocytes et se dissémine rapidement dans l'organisme sous forme de métastases. A ce stade, ce cancer est réfractaire à pratiquement toutes les thérapies. Ainsi, l'identification de nouvelles cibles thérapeutiques est donc incontournable pour la mise en place de biothérapies spécifiques dans le mélanome. Dans ce contexte, nous nous sommes intéressés au facteur de transcription E2F1 qui joue un rôle prépondérant dans le cycle cellulaire. Plus récemment, il lui a été identifié divers rôles dans les fonctions cellulaires. Ainsi, nous avons cherché à caractériser son implication dans le mélanome. Nous avons observé que E2F1 est faiblement exprimé dans les cellules saines de la peau. En revanche, elle est fortement exprimée dans le mélanome, et est corrélée à un mauvais pronostic clinique. Ainsi, nous avons montré que son inhibition réduisait la viabilité de cellules de mélanomes in vitro et in vivo dû à un arrêt du cycle cellulaire de la sénescence et d'une apoptose. Ces processus semblent être dépendants de la voie p53. Ce travail a permis de caractériser E2F1 comme une potentielle cible thérapeutique dans le mélanome non muté p53. En parallèle, nous avons initié une collaboration avec le Dr Slama-Schwok dont l'étude portait sur un composé appelé NS1, un inhibiteur de la NO-Synthase. Ce composé présente une activité anti-mélanome in vitro. En effet, il induit un stress du réticulum endoplasmique lui même conduisant à une autophagie partielle et une mort des cellules par apoptose. Ce projet ouvre de nouvelles perspectives pour le traitement du mélanome métastatique. / Melanoma is the most deadly form of skin cancer. It originates from malignant transformation of melanocytes and quickly disseminates as metastasis through the body. At this stage, this cancer is refractory to almost all therapies. Thus, new therapeutic target identification is needed for setting up specific biotherapies against melanoma. In this context, we focused on E2F1 transcription factor which plays a critical role in cell cycle. Recently, it was also implicated in several cell functions. So we aimed at characterizing its implication in melanoma. We observed that E2F1 is weakly expressed in normal skin cells. On the contrary, it is strongly expressed in melanoma and its expression correlates with a bad clinical prognosis. We also showed that E2F1 inhibition decreased melanoma cell viability in vitro and in vivo, as a result of cell cycle arrest, senescence and apoptosis. These processes seem to depend on p53 pathway. With this work we characterized E2F1 as a potential therapeutic target in non-mutated p53 melanoma. In parallel, we initiated a collaboration with Dr Slama-Schwok for studying NS1 compound, a NO-synthase inhibitor. This compound presents an in vitro anti-melanoma activity. Indeed, it induces endoplasmic reticulum stress, which leads to partial autophagy and cell death by apoptosis. This work opens new perspectives for metastatic melanoma treatment.

El síndrome de Down constituye la cromosomopatía más frecuente que ocurre en uno de cada 700 a 1000 nacimientos y está causado por la trisomía completa o por una parte del cromosoma 21 humano (HSA21). Aún se desconoce cómo la presencia del cromosoma 21 extra da lugar al fenotipo característico de este síndrome. En este sentido la participación de las especies reactivas de oxígeno (ROS) ha sido propuesta como uno de los mecanismos que intervienen en la patogénesis del mismo. Dicho mecanismo se fundamenta en la sobreexpresión de al menos 16 genes del HSA21 relacionados con el metabolismo de las especies reactivas de oxígeno (ROS) y con la generación de energía mitocondrial. Uno de estos genes es el que codifica una importante enzima del sistema antioxidante celular, el gen SOD1, propuesto como potencial culpable del estrés oxidativo inusual en los individuos con SD. En condiciones normales, los ROS, producidos in vivo principalmente por la respiración aeróbica, se eliminan de la célula por la acción de las enzimas antioxidantes, superóxido dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx). La Cu/Zn superóxido dismutasa (SOD1) convierte el radical superóxido a peróxido de hidrógeno, el cual es eliminado por la glutatión peroxidasa y/o catalasa a agua y oxígeno. La sobreexpresión de la SOD1 puede producir un desequilibrio en la relación de las enzimas antioxidantes (SOD1, GPx y CAT) generando estrés oxidativo y podría resultar en el daño oxidativo a biomoléculas tales como ácidos grasos poliinsaturados en los lípidos de las membranas, proteínas esenciales y el DNA, ya que existe una variabilidad en los niveles de enzimas antioxidantes dentro de la población con SD, lo que puede estar relacionado con una desregulación compleja que afecte no sólo a los genes del HSA21 sino también en otros cromosomas. Así, el daño celular puede ser inducido por los ROS y asociarse a algunas de las alteraciones celulares en el SD, causando diversas patologías y conducir a un envejecimiento prematuro. Se obtuvieron 18 muestras de fibroblastos primarios fetales humanos, 9 de ellos con síndrome de Down (FT21) y 9 controles (FC), en los cuales se evaluó la disminución de la capacidad endógena antioxidante debido a la sobreexpresión de la SOD1, causando un exceso en la producción intracelular de ROS y el origen prematuro de estrés oxidativo asociado al daño oxidativo a lípidos y proteínas, así como a una disfunción mitocondrial. Se analizaron varios marcadores de senescencia celular con la finalidad de contribuir al conocimiento de un nuevo aspecto de la patología de este síndrome, el envejecimiento prematuro. Estos mecanismos fisiopatológicos podrían estar relacionados con la aparición y establecimiento de la senescencia celular prematura en los fibroblastos con trisomía del cromosoma 21 (FT21). / Down syndrome is the most common chromosomal disorder that occurs in 1 in 700 to 1,000 births and is caused by trisomy full or part of human chromosome 21 (HSA21). It is still unknown how the presence of the extra chromosome 21 results in the phenotype of this syndrome. In this sense the involvement of reactive oxygen species (ROS) has been proposed as one of the mechanisms involved in the pathogenesis of same. This mechanism is based on the overexpression of at least 16 genes related HSA21 metabolism of reactive oxygen species (ROS) and mitochondrial energy generation. One of these genes encoding is an important cellular antioxidant enzyme system, the SOD1 gene, proposed as a potential culprit unusual oxidative stress in individuals with DS. Under normal conditions, the ROS produced in vivo mainly by aerobic respiration of the cells are removed by the action of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). The Cu/Zn superoxide dismutase (SOD1) converts the superoxide radical to hydrogen peroxide, which is eliminated by glutathione peroxidase and/or catalase into water and oxygen. SOD1 overexpression can produce an imbalance in the ratio of antioxidant enzymes (SOD1, GPx and CAT) generating oxidative stress and may result in oxidative damage to biomolecules such as polyunsaturated fatty acids in the membrane lipids, proteins and essential DNA, since there is a variability in the levels of antioxidant enzymes in the DS population, which can be related to a complex deregulation affects not only Hsa21 genes but also on other chromosomes. Thus, cell damage can be induced by ROS and associated with some of the cellular changes in the DS, causing various diseases and lead to premature aging. Eighteen samples were obtained from primary human fetal fibroblasts, 9 with Down syndrome (TF21) and 9 normal (NF), which was evaluated in decreasing endogenous antioxidant capacity due to overexpression of the SOD1, causing excess in intracellular production of ROS and oxidative stress origin associated premature oxidative damage to lipids and proteins, as well as mitochondrial dysfunction. We analyzed several markers of cellular senescence in order to contribute to the knowledge of a new aspect of the pathology of this syndrome, premature aging. These pathophysiological mechanisms may be related to the emergence and development of premature cellular senescence in fibroblasts with trisomy 21 (FT21).

Μηχανισμοί οι οποίοι εξασφαλίζουν τη σωστή διαδοχή των φάσεων του κυτταρικού κύκλου συμβάλλουν στη διασφάλιση της γονιδιωματικής σταθερότητας των κυττάρων. Η αδειοδότηση της αντιγραφής του DNA, η συγκρότηση επί των αφετηριών της αντιγραφής του DNA του προ-ανιγραφικού συμπλόκου, καθορίζει τη σωστή χρονικά και τοπικά έναρξη της αντιγραφής. Βασικό συστατικό αυτού του συμπλόκου είναι ο παράγοντας Cdt1. Η Geminin προσδένεται στο Cdt1, αναστέλοντας τη δράση του από την S μέχρι και την Μ φάση, παρεμποδίζοντας, έτσι, την αδειοδότηση της αντιγραφής. Παρά το οτι φυσική αλληλεπίδραση των δύο πρωτεϊνών έχει δειχθεί τόσο in vitro όσο και in vivo, προηγούμενες μελέτες δείχνουν οτι έκφραση των Cdt1 και Geminin εντοπίζεται σε διαφορετικές φάσεις του κυτταρικού κύκλου. Τα φυσιολογικά κύτταρα, ανάλογα με τα μηνύματα που δέχονται, είτε παραμένουν σε μιτωτικό κύκλο, είτε εξέρχονται από αυτόν προς φάση ηρεμίας (ή G0), διαφοροποίηση ή γήρανση. Αυστηρός συντονισμός των παραπάνω διαδικασιών είναι απαραίτητος προκειμένου να διασφαλιστεί η ομοιόσταση των πολύπλοκων δομών των ιστών των μεταζώων. Προηγούμενες μελέτες προτείνουν το σύστημα της αδειοδότησης της αντιγραφής του DNA ως έναν βασικό ρυθμιστή της εξόδου από τον κυτταρικό κύκλο και της επανεισόδου στη G1. Οι παράγοντες Cdt1 και Geminin ρυθμίζονται αρνητικά κατά την έξοδο των κυττάρων σε G0, ενώ έκφρασή τους χαρακτηρίζει διαιρούμενα κύτταρα. Σε αντίθεση με τις άλλες καταστάσεις εκτός κυτταρικού κύκλου, λίγα είναι γνωστά αναφορικά με τη ρύθμιση των Cdt1 και Geminin κατά την κυτταρική γήρανση. Στο πρώτο μέρος της διατριβής εστιαστήκαμε στη μελέτη του προτύπου έκφρασης των Cdt1 και Geminin κατά τη διάρκεια του κυτταρικού κύκλου πρωτογενών ανθρώπινων ινοβλαστών, και στη σύγκρισή του με εκείνο των καρκινικών κυττάρων. Διαπιστώσαμε οτι τόσο η ενδοκυτταρική εντόπιση όσο και η ικανότητα των Cdt1 και Geminin να εκφράζονται σε συγκεκριμένες φάσεις του κυτταρικού κύκλου, δεν διαφοροποιούνται στους πρωτογενείς φυσιολογικούς ινοβλάστες σε σχέση με κύτταρα που προέρχονται από καρκινικό ιστό. Επιπλέον, δείξαμε οτι ο παράγοντας Cdt1 εκφράζεται αποκλειστικά σε BrdU-αρνητικά κύτταρα, σε αντίθεση με την Geminin, η οποία δείχνει να συσσωρεύεται σταδιακά μετά την έναρξη της S φάσης, ενώ δεν εντοπίστηκε συνέκφραση των δύο πρωτεϊνών στο χρονικό παράθυρο της G1/S μετάβασης. Στο δεύτερο μέρος της εργασίας εστιαστήκαμε στη μελέτη της έκφρασης του παράγοντα αδειοδότησης Cdt1 και του αρνητικού ρυθμιστή αυτού, Geminin, κατά την είσοδο των κυττάρων σε κυτταρική γήρανση και εξετάσαμε την πιθανή λειτουργική εμπλοκή τους στην εξέλιξη του φαινομένου. Δείξαμε οτι, ενώ οι παράγοντες Cdt1 και Geminin διατηρούν τη σωστή ενδοκυτταρική εντόπιση και το σωστό πρότυπο έκφρασης κατά τη διάρκεια του κυτταρικού κύκλου, υφίστανται αρνητική ρύθμιση σε κύτταρα που εισέρχονται σε κυτταρική γήρανση, τόσο αναπαραγωγική όσο και πρόωρη, επαγόμενη από οξειδωτικό στρες. Το γεγονός οτι η μείωση της έκφρασης της Geminin προηγήθηκε της εμφάνισης του γηρασμένου φαινοτύπου, μας ώθησε στην περαιτέρω διερεύνιση του λειτουργικού ρόλου της Geminin στην επαγωγή της κυτταρικής γήρανσης. Για το σκοπό αυτό, απορρυθμίσαμε τα επίπεδα έκφρασης της Geminin σε πρωτογενή φυσιολογικά κύτταρα ανθρώπου και ποντικού, αξιοποιώντας την τεχνολογία του RNAi και ρετροϊικά συστήματα υπερέκφρασης γονιδίων αντίστοιχα. Δείξαμε οτι η μείωση της έκφρασης της Geminin σε ανθρώπινους ινοβλάστες (χρησιμοποιώντας siRNAs αλλά και pSUPER πλασμιδιακούς φορείς αποσιώπησης γονιδίων που κατασκευάστηκαν ειδικά για την Geminin) επάγει αύξηση της κυτταρικής γήρανσης της καλλιέργειας. Επιπλέον, κύτταρα που στερούνταν της έκφρασης της Geminin ήταν πιο επιρρεπή σε γήρανση επαγόμενη από οξειδωτικό στρες, σε σχέση με τα κύτταρα-μάρτυρες. Ετεροζυγώτες για το γονίδιο της Geminin εμβρυικοί ινοβλάστες ποντικού εμφάνιζαν μεγαλύτερα ποσοστά κυτταρικής γήρανσης σε σχέση με τους αντίστοιχους ινοβλάστες αγρίου τύπου. Αντίθετα, αύξηση των επιπέδων της Geminin σε αγρίου τύπου εμβρυικούς ινοβλάστες ποντικού προκάλεσε μείωση της εμφανιζόμενης γήρανσης. Τέλος, η μείωση των επιπέδων έκφρασης του παράγοντα αδειοδότησης Cdt1 σε ανθρώπινα κύτταρα ήταν, επίσης, σε θέση να επάγει ισχυρό φαινότυπο κυτταρικής γήρανσης. Συνοψίζοντας, τα αποτελέσματα μας αναδεικνύουν την κρισιμότητα του ισοζυγίου Cdt1:Geminin στα κύτταρα, και προτείνουμε οτι η διατάραξη της ισορροπίας αυτής είναι ικανή να επάγει κυτταρική γήρανση, μέσω διαδικασιών όπως η υπεραδειοδότηση ή η υποαδειοδότηση της αντιγραφής του DNA. / Genome integrity relies on the strict alternation of S and M phases of the cell cycle, so that one and only round of DNA replication takes place per cell cycle. This is achieved through replication licensing, which involves the formation of a multi-protein complex, the pre-replicative complex, onto origins of replication. Cdt1 is a crucial component of this complex and Geminin, a small protein shown to tightly bind Cdt1, inhibits its licensing function from S to M phase, when licensing is illegitimate. Although previous experimental evidence shows that Cdt1 and Geminin are expressed in different phases of the cell cycle, physical interaction between these two proteins has been demonstrated in vitro as well as in vivo. The fate of a normal cell is not perpetual division. Cells may exit the mitotic cell cycle to enter quiescence, to terminally differentiate or to senesce. These “out-of-cycle-states” must be strictly regulated in order to establish and maintain the hierarchical organization of complex tissues in metazoa. Replication licensing has been proposed to coordinate cell-cycle exit and re-entry in vitro and in metazoan tissues. Cdt1 and Geminin down-regulation during exit to quiescence supports the idea that their expression correlates with cell proliferation. In contrast to other out-of-cycle states, little is known about the regulation of Cdt1 and Geminin expression during cellular senescence. Senescence refers to the irreversible resting state of cells grown for succeeding passages in culture, as a response to DNA damage caused by telomeres erosion. Other stimuli, such as oxidative or oncogenic stress, may force mitotically competent cells to respond similarly, a phenomenon termed as Stress Induced Premature Senescence (SIPS). The first part of this work focused on the study of the expression patterns of Cdt1 and Geminin during the unperturbed cell cycle of primary human fibroblasts and compared to that of tumor-derived cell lines. The cell cycle specific expression and the intracellular localization of both proteins, as assessed at a single-cell level using indirect immunofluorescence and a new monoclonal antibody against Geminin, appear similar in primary fibroblasts compared to the cancer cells examined. Cdt1 is strictly expressed in BrdU-negative cells, whereas Geminin starts accumulating after S phase onset. The two proteins are, therefore, not co-expressed at the ”time-window” of G1/S transition of the cell cycle. We showed that Cdt1 levels, but not those of Geminin, are mainly regulated in a proteasome-dependent way during normal cell cycle of human primary and cancer cells. The second part of this work focused on the investigation of Cdt1 and Geminin during cellular senescence and their possible role in the establishment of the senescence phenotype. To this end, primary human fibroblasts were maintained in culture for succeeding passages in order to induce them to undergo replicative senescence. Alternatively, an H202-induced senescence protocol was applied to force cells to undergo premature senescence (Stress-Induced Premature Senescence/SIPS). We show that, although Cdt1 and Geminin retain their nuclear localization and are correctly expressed during specific phases of the cell cycle during both replicative and Η202-induced premature senescence, their expression levels are down-regulated. In SIPS-experiments, Geminin down-regulation is an early event during the establishment of the senescent-phenotype, as assessed by senescence-associated β-Galactosidase and BrdU incorporation assays. This prompted us to further examine Geminin’s functional significance in the establishment of cellular senescence. To achieve this, we interfered with Geminin expression levels in human and mouse cells. Using RNA interference techniques, we were able to show that Geminin depletion from human cells is able to induce a senescent-phenotype in a fraction of the treated culture. Similarly, Geminin-depleted human cells were more susceptible to Η202-induced premature senescence, compared to control cells. Heterozygotes for Geminin mouse embryonic fibroblasts were more prone to senescence compared to their control counterparts. In contrast, when Geminin was over-expressed in control mouse embryonic fibroblasts cultures, senescent phenotype was reduced. Finally, a strong senescent-phenotype was induced when the licensing regulator, Cdt1, was silenced within human cells. Taken together, we conclude that Cdt1:Geminin balance within cells is crucial, and when disturbed, is able to promote a senescent phenotype, possibly through a mechanism that involves over- or under-licensing of DNA replication.