Introdução: As síndromes de Li-Fraumeni (LFS) e Li-Fraumeni Like (LFL) são síndromes hereditárias de predisposição a câncer frequentemente associadas à mutações germinativas no gene TP53. Devido à importância de p53 e diversidade de processos celulares que ela regula, várias vias de sinalização podem ser afetadas pela doença. Nesse estudo discutimos o impacto da mutação p.R337H na proliferação, senescência, autofagia, população e funcionalidade mitocondrial. Métodos: As taxas de proliferação foram avaliadas pelo ensaio de Population Doubling. Os experimentos de senescência, autofagia, massa total e funcionalidade mitocondrial foram realizados por citometria de fluxo. Resultados: As células contendo a mutação proliferaram mais do que as células controle. Além disso, as células mutadas não ativaram autofagia sob tratamento de Rapamicina nem senescência sob tratamento de Doxorubicina ou Cisplatina e exibiram maior população mitocondrial, mas com funcionalidade inalterada após os tratamentos. Conclusão: os dados sugerem que a mutação p.R337H em TP53 afeta a indução de senescência realizada por p53 e suas funções pró-autofágicas, bem como seu controle. As células mutadas proliferam mais do que células sem mutação em TP53 e exibiram maior massa mitocondrial sem perda de funcionalidade após o tratamento com Doxorubicina. / Background: Li-Fraumeni (LFS) and Li-Fraumeni Like (LFL) syndromes are hereditary cancer predisposition syndromes frequently associated with germline mutation in TP53. Due to the importance of the protein p53 and its regulation of several important cellular processes, impairment in some pathways can be implicated. Here we discuss the impact of p.R337H TP53 mutation on proliferation, senescence, autophagy, mitochondrial population and functionality. Methods: Growth rates were assayed with Population Doubling assay. Senescence and autophagy were assessed through flow cytometry and functionality and total population of mitochondria were also analyzed through flow cytometry. Results: mutated cells proliferated more than control cells. TP53 mutated cells didn’t build up autophagy under Rapamycin treatmend nor senescence under Doxorubicin or Cisplatin treatments and showed more mitochondrial mass, but no alterations in mitochondrial functionality after Doxorubicin treatment. Conclusion:data suggests that p.R337H TP53 mutation affect senescence induction by p53 and pro-autophagic actions of p53. Mutated cells proliferate more than control cells and exhibited larger mitochondrial mass without effects in their functionality in response to Doxorubicin treatment.

A descondensação da cromatina exerce um papel central nas diversas etapas do processo de carcinogênese abrindo o genoma para a ação de fatores de transcrição, exercendo papel na progressão e resistência tumoral. Bromodomínios e proteínas com terminal extra, como o BRD4, são leitores epigenéticos que regulam a expressão gênica e, portanto, também estão envolvidos na patogênese do câncer. O objetivo do presente estudo foi estudar o efeito da inibição do BRD4 no carcinoma espinocelular de cabeça e pescoço (CECP). Para esse propósito, foi utilizado JQ1, inibidor de BRD4, em concentração de 1uM, nas linhagens de carcinoma de cabeça e pescoço HN6, HN12 e HN13. Foi analisado os níveis de BRD4, H4 acetilada e SIRT1 fosforilado através de reações de imunofluorecência e p16ink4 por imunohistoquímica. Foi realizado western blot para checar os níveis de p53 e p53 acetilado. Ensaio de formação de colônias e câmera de invasão foram realizados para testar o efeito do inibidor na proliferação e invasão celular. Através da citometria de fluxo foi analisado o efeito da apoptose com a marcação de caspase-3 clivada, do ciclo celular através da reação por iodeto de propídio e ainda da população de células tronco tumorais pela análise de ALDH e CD44. Por fim, foi realizado modelo xenográfico subcutâneo para analisar o efeito do JQ1. Os resultados mostraram diminuição significativa da expressão de BRD4 e H4ac após tratamento com JQ1. As linhagens celulares mostraram redução na capacidade de invasão e de formação de colônias quando submetidas ao JQ1. Não foram encontradas diferenças em relação ao número de células caspase-3 clivada positivas. Por outro lado, foi encontrado um maior número de células na fase G1 do ciclo celular após o uso do inibidor estudado. As células tratadas com JQ1 mostraram menor expressão de p-SIRT1 o que levou a uma diminuição da acetilação do p53 e um aumento na expressão de p16ink4. Paralelamente, foi encontrado uma diminuição na população de células positivas para ALDH e CD44. Houve diminuição do crescimento do tumor no modelo xenográfico tratado com JQ1 quando comparado ao veículo. Nos tecidos derivados do ensaio in vivo, houve uma diminuição nos marcadores p16ink4, pSIRT1 além de acúmulo de H2AX. Conclui-se que o uso de JQ1 resulta na disrupção do crescimento do CECP associado a ativação de senescência, indução de dano de DNA além de reduzir a população de células tronco tumorais. Esses novos achados indicam que o BRD4 é um importante modificador epigenético nos CECP sendo um viável alvo terapêutico. / Chromatin descondensation plays a central step in the various stages of the carcinogenesis process opening the genome for transcription factors playing a role in tumor progress and resistance. Bromodomains and extra terminal family, as BRD4, are epigenetics readers that regulate gene expression thus they are also involved in cancer pathogenesis. The objective of this project was studied the effect of BRD4 in head and neck squamous cell carcinoma (HNSCC). For this purpose, JQ1, a BRD4 inhibitor, was used in 1uM concentration, in HN6, HN12 and HN13 head and neck carcinoma cell lines. The levels of BRD4, acetylates h4 and phosphorylated SIRT1 were analyzed by immunofluorescence and p16ink4 labeling by immunohistochemistry. Western blot was performed to check the levels of p53 and acetylated p53. Colony assay and invasion chamber were performed to test the inhibitory effect on cell proliferation and invasion. The effect of apoptosis with the cleaved caspase-3 labeling, the cell cycle by propidium iodide and of the population of tumor stem cells by the analysis of ALDH and CD44 was analyzed through flow cytometry. Finally, a subcutaneous xerographic model was performed to analyze the effect of JQ1. A significant decrease in the expression of BRD4 and H4ac was found after application of JQ1. The cell lines results showed a reduction in the capacity of invasion and also formation of colonies when submitted to JQ1. No differences were found in relation to the number of cells caspase-3 cleaved positives. On the other hand, a large number of cells were found in G1 arrest of cell cycle after use of the BRD4 inhibitor studied. Cells treated with JQ1 showed lower expression of p-SIRT1 which led to a decrease in p53 acetylation and an increase in p16ink4 expression. In parallel, a decrease of ALDH and CD44 positive cells population was found. A decrease in tumor growth was discovered when treated by JQ1 if compared to the vehicle. In tissues samples derived from the in vivo assay, there was a decrease in p16ink4, pSIRT1 markers in addition to -H2Ax accumulation. In conclusion JQ1 causes HNSSC tumor growth disruption associated a senescence activation, DNA damage and a reduce number of cancer stem cells. These new findings indicate that BRD4 is an important genetic modifier in HNSSC and is a viable therapeutic target.

O interesse sobre o papel dos elementos traço na gênese de doenças, na mortalidade e na manutenção da saúde vem sendo cada vez maior. Este trabalho tem como objetivo determinar as concentrações séricas dos elementos: Br, Ca, Cl, Fe, Na, Rb, Se e Zn e correlacioná-las com os aspectos clínicos de uma população de idosos saudáveis. Foram considerados saudáveis os idosos sem senilidade sistêmica e sintomática, sendo selecionamos 101 idosos saudáveis, 33 (32,67%) homens e 68 (67,33%) mulheres com idade média de 71,7 ± 7,1 (60-98). O nível sérico de Se foi significativamente menor em pacientes mais idosos (p < 0.001). Em comparação com os valores de referência, o Br, Cl e Na apresentaram médias menores. Em relação ao sexo, os homens tiveram menores níveis de Br (p < 0,001) e de Se (p = 0,005) com maiores níveis de Fe (p < 0,001). Menores níveis de Se foram relacionados com menor escolaridade (p= 0.013). Os valores de Br estiveram muito abaixo dos valores de referência e a prática da atividade física correlacionou-se com menores níveis de Br (p= 0,008). Poderíamos questionar se os altos níveis de Br seriam prejudiciais ao envelhecimento saudável, se níveis baixos de bromo seriam marcadores de saúde em idosos ou até se existe relação entre hábitos de vida saudáveis com os níveis de Br. A partir dos 71,2 anos, os níveis séricos de selênio começam a cair mesmo em indivíduos saudáveis. Estudos adicionais deverão determinar se o monitoramento dos níveis séricos de selênio pode ser utilizado como marcador precoce do desenvolvimento de doenças e mortalidade / Concern about the role of trace elements in the genesis of disease, mortality and health maintenance has been increasing. This study aims to determine serum concentrations of the elements: Br, Ca, Cl, Fe, Na, Rb, Se and Zn and correlate them with clinical features of a population of healthy elderly. Were considered healthy elderly people without senile systemic symptoms. We selected 101 healthy elderly, 33 (32.67%) men and 68 (67.33%) women with a mean age of 71.7 ± 7.1 (60-98). The serum level of Se was significantly lower in older patients (p <0.001). Compared with the reference values Br, Cl and Na diminished. Longer averages about sex men had lower levels of Br (p <0.001) and Se (p = 0.005) with higher levels of Fe (p <0.001). If lower levels were associated with less education (p = 0.013). Br values were well below the reference values and physical activity correlated with lower levels of Br (p = 0.008). One might question whether high levels of Br would be detrimental for healthy aging, if low levels of bromine were markers of health in the elderly or even if there is a relationship between healthy lifestyle habits with the levels of Br. From 71.2 years, serum levels of selenium begin to fall even in healthy individuals. Additional studies will determine if monitoring of serum levels of selenium can be used as a marker of early development of disease and mortality

Orientadores: Inacia Maria Dal Fabbro, Roberto Alves Braga Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agricola / Made available in DSpace on 2018-08-09T03:56:10Z (GMT). No. of bitstreams: 1 Rodrigues_Silvestre_D.pdf: 5874424 bytes, checksum: f82d98473ad716d4bbdd08a46b90d886 (MD5) Previous issue date: 2007 / Resumo: Este trabalho tem por objetivo contribuir para o desenvolvimento de uma metodologia de análise de vitalidade de tecidos vivos, por meio de um modelo estocástico destinado a interpretar e quantificar Padrão associadas ao fenômeno conhecido como biospeckle ou speckle dinâmico, e geradas a partir da interação de luz coerente com materiais biológicos ou com sistemas particulados passíveis de fenômenos dinâmicos, à semelhança do movimento browniano. O biospeckle é formado pela mudança do padrão de interferência quando a luz coerente incide no tecido biológico ou no sistema particulado, gerando Padrão que podem ser observadas e apropriadamente capturadas. O material biológico tanto quanto o sistema particulado exibem transformações superficiais e internas ao longo do tempo, apresentando-se à luz coerente como uma rede de difração dinâmica. Através de tratamento digital das Padrão coletadas para retratação do fenômeno, é possível diferenciar níveis de atividades biológicas nos tecido em estudo. A análise de Padrão associadas ao biospeckle apresenta um comportamento típico estocástico, sugerindo um estudo estatístico probabilístico do comportamento da padrão, seja por movimento browniano, entropia ou outro modelo aplicável a fenômenos aleatórios. Para o estudo desses fenômenos, este trabalho abordou a viabilidade de sementes, senescência de tecidos biológicos, além de uma simulação de movimento browniano com sistemas particulados. Os resultados mostraram que as Padrão geradas pelo método STS (Spatial Temporal Speckle) se comportam de maneira totalmente aleatória, sendo difícil eleger um modelo que possa quantificar essas Padrão. Ao contrário, nas Padrão geradas pelo método MOC (Matriz de Ocorrência), o efeito ¿caos¿ observado nas Padrão anteriores é minimizado, tornando-se então passíveis de serem quantificadas pela entropia, que gerou um padrão semelhante ao apresentado pelo método denominado ¿Momento de Inércia¿. Palavras Chaves: Imagens, sementes, senescência / Abstract: This research work had the objective of contributing to the development of a methodology applicable to biological tissues vitality analysis by means of a stochastic model. The conceived and tested model is able to interpret as well as to quantify the biospeckle images generated on living tissues. The biospeckle or dynamic speckle phenomenon is generated from the interaction of a coherent light with living tissues or with body surface exhibiting certain kinds of activities. In other words, the biospeckle phenomenon is observed when interfering patterns generated by the incidence of coherent light on a surface exhibiting some kind of dynamic or biological activities change at certain rate. Biological tissues, as well as particles in suspension exhibit dynamic activities, similar to brownian motion, acting as a dynamic diffraction grid to the coherent light. By capturing and processing biospeckle images it is possible to differentiate levels of biological or dynamic activities in the body under study. Dynamic speckle image analysis presents a typical stochastic behavior, suggesting a probabilistic statistical study of image behavior, as brownian motion, entropy or other kind of model associated to random phenomenon. Toward that sense, seed viability analysis, vegetative tissue senescence, as well as brownian motion simulation tests had been carried out. Results indicate that STS (Spatial Temporal Speckle) images show random behavior, impeding quantitative analysis. In opposition, MOC (Matrix Occurrence) images or occurrence matrix, where the chaos effect is minimized, are susceptible to quantifying analysis, similarly to the ¿moment of inertia¿ method. Key words: Image, seed, senescence / Doutorado / Maquinas Agricolas / Doutor em Engenharia Agrícola

Neste trabalho analisamos a influência da variabilidade climática sobre um ecossistema representado principalmente por cerrado sensu stricto, na Gleba Pé de Gigante, em Santa Rita do Passa Quatro, SP, durante o período de 2001 a 2007. Os dados coletados para esta análise são provenientes da torre micrometeorológica localizada no Parque Estadual de Vassunuga, cuja instalação está associada ao desenvolvimento do projeto temático Interação Biosfera- Atmosfera Fase 2: Cerrados e Mudanças de Uso da Terra. As propriedades físicas do clima utilizadas para análise foram a precipitação, a temperatura do ar e a radiação solar. Um levantamento teórico da biomassa acima e abaixo do solo foi realizado para caracterizar a vegetação quanto ao potencial de estoque de carbono existente. A biomassa da área coberta por campo cerrado foi de 67,1 Mg.ha-1, da área coberta por cerrado sensu stricto, 185,6 Mg.ha-1 e da área coberta por cerrado denso, 242,7 Mg.ha-1. Uma relação entre estoques de carbono e fluxos de CO2 foi estabelecida, onde uma tonelada de carbono em cerrado sensu stricto é capaz de assimilar, em média, 0,27 KgC.ha-1.dia-1 da atmosfera. A combinação de diferentes intensidades das propriedades climáticas formam condições ambientais variadas que contribuem para o estado da vegetação e sua produtividade. O principal parâmetro usado para avaliar o estado da vegetação foi o albedo, tanto para a faixa espectral da radiação visível (albedo solar) como para a faixa da radiação fotossintéticamente ativa (albedo RFA). O comportamento sazonal do albedo permitiu verificar que a vegetação apresentou-se fortemente condicionada pela variabilidade climática, que ditou o ritmo da funcionalidade ecossistêmica. De maneira geral, a precipitação, a temperatura do ar e a oferta de energia solar oscilam de forma proporcional ao longo das estações, caracterizando dois períodos distintos: um período com condições favoráveis ao desenvolvimento vegetal, de outubro a março, e um período de estresse, de abril a setembro. Os valores mínimos e máximos de albedo solar sobre o cerrado sensu stricto, durante o período analisado, oscilou entre 15% (novembro/dezembro) e 9% (setembro/outubro) e, o albedo RFA oscilou entre 2% (fevereiro/março) e 6% (setembro/outubro). Na escala interanual, observou-se o aumento do albedo RFA em 2006 após um período de três anos de queda contínua da precipitação, entre 2003 e 2006, sendo 2006 o ano menos chuvoso de toda a série considerada. Em 2007, os valores de albedo RFA foram bem mais baixos do que os calculados para os demais anos, respondendo rapidamente ao alto índice de precipitação ocorrido na estação chuvosa entre 2006 e 2007. Embora tenha sido observado uma resposta relativamente rápida do albedo RFA à recuperação do estresse hídrico na escala sazonal, o padrão do albedo na escala interanual é distinto: entre 2003 e 2006, período em que se observou taxas negativas de precipitação consecutivas, o albedo RFA diminuiu ou ficou com valores aparentemente constantes, apresentando valores mais altos somente em 2006. Desta forma, conclui-se que o estado da vegetação é condicionado principalmente pelo índice de precipitação, uma vez que a temperatura do ar e a quantidade de radiação solar não apresentam variações bruscas na região considerada. Considerando-se a importância da estimativa de albedo RFA como um parâmetro para estimar a variação sazonal do estado da vegetação, sugeriu-se um ajuste linear simples para a estimativa de albedo RFA em cerrado sensu stricto com base nos valores de IVDN, cuja variância explicada foi igual a 0,68. / In this work we analyze the climatic variability influence over a woodland savannah ecosystem at Gleba Pe de Gigante, Santa Rita do Passa Quatro, SP, during the 2001-2007 period. The data collected for this analysis are from a micrometeorological tower located at Vassununga State Park, which was installed under the thematic project called Biosphere- Atmosphere Interaction Phase 2: Savannah and Land Use Change. The physical climate properties used for this analysis were precipitation, air temperature and solar radiation. A theoretical survey for above and below ground biomass was made to characterize the existing carbon stock potential related to the vegetation. The total biomass estimated at grassland savannah was 67.1 Mg.ha-1, at woodland savannah was 185.6 Mg.ha-1 and at dense savannah was 242.7 Mg.ha-1. A relationship between carbon stocks and CO2 fluxes was established where one tone of carbon in woodland savannah absorbs an average of 0.27 KgC.ha-1.day-1 from the atmosphere. The combination of different climate properties and intensities generates different environmental conditions that lead to the vegetation state and its productivity. The main physical parameter considered to evaluate vegetation state was the albedo, which was shared in two spectral bands: visible spectrum (solar albedo) and photosynthetic active radiation (PAR albedo). The seasonal pattern of albedo allows checking that vegetation was strongly conditioned by climatic variability, which dictates the ecosystem functionality rhythm. Generally, precipitation, air temperature and solar radiation vary in a proportional way along the year, providing two different periods related to vegetation status: one period characterized by favorable conditions to vegetal development (October-March) and another by stressing conditions (April-September). Maximum and minimum values for solar albedo at woodland savannah varied, respectively, between 15% (November/December) and 9% (September/October); for PAR albedo, maximum and minimum values varied between 6% (September/October) and 2% (February/March). At annual scale, PAR albedo rose in 2006, after a four years period of falling precipitation rate, between 2003 and 2006. 2006 was the drier year among the others. In 2007, the PAR albedo values were much lower than those calculated for the remaining years, promptly responding to the high precipitation rate observed in the previous rainy season, 2006-2007. Even though a quick response in PAR albedo was noticed due to the recovered water stress in seasonal scale, the albedo pattern in annual scale held a different way: between 2003 and 2006, period characterized by consecutive and negative precipitation rates, vegetation was apparently associated to stable values of PAR albedo, presenting higher values only in 2006. Considering these results, we conclude that the vegetation state is mainly conditioned by precipitation rate, once the air temperature and solar radiation had not presented high variation in the study region. Based on the importance of PAR albedo as a parameter to estimate seasonal vegetation status, a simple linear adjustment according for woodland savannah PAR albedo based on NDVI values was suggested, which explained variance by NDVI was equal to 0.68.

O objetivo desta pesquisa foi avaliar a influência do envelhecimento e da perda dos elementos dentais nos níveis totais de imunoglobulina secretória do tipo A (SIgA) na saliva. Foram selecionados 76 pacientes (entre 20 e 87 anos), os quais foram divididos em três grupos de acordo com sua faixa etária e condição bucal: adultos jovens com idades de 20 a 40 anos (Grupo I ou Grupo controle); idosos com idade entre 65 e 78 anos, desdentados parciais, portadores de prótese total unimaxilar (Grupo II) e idosos com idade entre 65 e 87 anos, desdentados totais, portadores de prótese total bimaxilar (Grupo III). Os níveis totais de imunoglobulina secretória do tipo A na saliva foram determinados por meio da técnica de ensaio imunoenzimático em fase sólida ( ELISA – Enzyme-linked Imunosorbent Assay). Após obtenção dos dados experimentais foi empregada a análise de variância de ANOVA com dois fatores (sexo e grupo) para verificar o efeito significante da interação destes fatores. Os níveis totais de imunoglobulina do tipo A secretória na saliva não apresentaram, em média, diferenças significantes entre os três grupos. Em relação ao fator gênero, ou sexo, em média, homens e mulheres apresentaram comportamentos de SIgA diferentes nos grupos. Para o grupo controle o nível total de SIgA dos homens foi maior que o das mulheres enquanto que para o grupo III o nível total de SIgA das mulheres foi maior que dos homens e para o grupo II não foi observada diferença significante dos níveis de SIgA entre homens e mulheres. Pela análise comparativa dos grupos I e III foi observada diferença significante no sexo feminino, o que não foi observado quando comparados os dois grupos experimentais (Grupos II e III). Os resultados desta pesquisa sugerem que não há influência direta dos fatores envelhecimento e perda dental sobre os níveis totais de imunoglobulina secretória do tipo A na saliva. Estes resultados mostraram a influência do gênero sobre os níveis de imunoglobulina secretória do tipo A. Entretanto, a influência do gênero não é bem conhecida e merece mais estudos. / The aim of this study was to evaluate the influence of senescence and teeth loss on secretory immunoglobulin A (SIgA) levels in saliva. Seventy-six patients (20 to 87 years old) were selected and classified in three groups according to their age and oral dental state: young adults were aged 20-40 years (Group I or Control group); elderly subjects were aged 65-78 years and wore maxillary or mandibular denture (Group II); and edentulous old subjects were aged 65-87 years and wore maxillary and mandibular denture (Group III). The secretory immunoglobulin A levels were determined by the Enzyme-linked imunosorbent assay (ELISA method). All results were correlated using ANOVA statistical analysis with two factors (sex and group) to verify the significant effect of these factors. The secretory immunoglobulin A levels were not significant differences among the average values of the three groups. In gender relation , men and women showed the mean rate of SIgA levels different in the groups. The men SIgA levels of control group showed greater when compared to women levels. In Group III the women levels were greater when compared to men levels. And to Group II statistical analysis demonstrated no significant difference between the SIgA levels of men and women. The analysis showed significant differences in the women levels when compared to Groups I and III. No differences of levels were demonstrated when compared to Groups II and III. These results suggests that the senescence and teeth loss do not have a direct relationship to the secretory immunoglobulin A levels in whole saliva. The se results showed that there is influence of gender in the secretory immunoglobulin levels. However, the influence of gender is not well known and further studies are still necessary.

Ce travail est consacré au rôle pléotropique des microparticules endothéliales dans la réponse et l'homéostasie vasculaire. Un modèle de sénescence réplicative a été caractérisé en utilisant des cellules endothéliales de coronaires de porc en culture primaire. Ce modèle a mis en évidence des changements drastiques du phénotype endothélial avec la production de ROS, la dépolarisation de la membrane mitochondriale et la surexpression de régulateurs clés du cycle cellulaire incluant p53, p21 et p16. La sénescence a transformé le phénotype endothélial vers un statut procoagulant indiqué par la libération de microparticules (MP). L'induction d'activité Facteur Tissulaire (FT) et une réduction drastique de l'inhibition de l'agrégation plaquettaire due à la sécrétion réduite de NO endothélial. Simultanément, une augmentation importante des protéines du système de l'angiotensine à la surface des cellules sénescentes et des MPs qu'elles émettent a été mesurée. D'autres résultats obtenus avec les MPs circulantes de patients transplantés ou atteints de syndrome coronarien suggèrent l'existence dune boucle d'amplification des effets délétères des MPs au travers de la signalisation Redox et l'altération des fonctions vasculaires résultant d'une sénescence exacerbée. En plus de ses qualités reconnues d'immunosuppresseur, la cyclosporine A (CsA) est un inhibiteur puissant de l'ouverture des pores mitochondriaux (mPTP). Certaines études ont présenté le traitement bref et contrôlé par CsA comme un moyen de limiter les dommages vasculaires de l'ischémie reperfusion.Nos données suggèrent une possible modulation de la sénescence endothéliale induite par les MPs grâce au préconditionnement avec des concentrations faibles de CsA. nos résultats suggèrent aussi que de faibles doses de Cs A peuvent avoir un effet bénéfique dans les pathologies cardiovasculaires lorsque la sénescence est exacerbée et contribue les fonctions vasculaires de l’endothélium. / This scientific work has tackled the issue of the pleitropic role mediated by endothelial microparticles function and homeostasis. A replicative model of senescence using coronary endothelial cells was set showing drastic phenotype changes characterized by ROS production, mitochondrial membrane depolarization and the up-regulation of key regulators of cell cycle arrest including p53, p21 and p16.Replicative senescence shifted the coronary endothelial phenotype toward a procoagulant status as evidenced by (i) procoagulant MP shedding (ii) enhanced tissue factor (TF) expression and (iii) a marked decrease in the endothelial NO-mediated inhibition of platelet aggregation. In parallel, a drastic up regulation of the angiotensin system could be evidenced at the surface of senescent cells or derived MP. Results obtained with MPs from patients with acute coronary artery syndrome and from grafted patients,suggested a feedback loop disseminating the deleterious effect of circulating MPs redox signaling and alteration of vascular function owing to exaggerated senescence. In addition to its well-known immunosuppressive properties, cyclosporine A (Cs A) is a potent inhibitor of the opening of the mitochondrial permeability transition pore (mPTP), and several reports have indicated that a brief and timely administration of Cs A can limit ischemia-reperfusion injuries. Our data evidenced the possible pharmacological modulation of endothelial MP-mediated senescence by cell preconditioning with low concentrations of Cs A. our data are thus suggestive of a beneficial effect of CsA in cardiovascular disorders where senescence is altering the endothelial vascular functions.

La leucémie myéloïde chronique (LMC) est un syndrome myéloprolifératif lié à l’acquisition d’une anomalie chromosomique t(9;22) conduisant à l’expression d’une protéine de fusion p210 Bcr-Abl dont l’activité tyrosine kinase dérégulée est nécessaire et suffisante pour engendrer la maladie.Cette pathologie bénéficie depuis 2002 d’une avancée thérapeutique : les inhibiteurs de tyrosine kinase (ITK). Cette thérapeutique dite ciblée, dont le chef de file est l’imatinib, est très efficace puisque 80% des patients entre en rémission. Malheureusement, 20% des patients traités développent des résistances primaires ou secondaires, dépendantes ou non de l’oncogène Bcr-Abl dont certaines ont été caractérisées. A ce titre, la LMC est devenue un modèle d’étude à la fois des mécanismes oncogéniques mais aussi des résistances.La résistance aux ITK dans la LMC peut être considérée sur deux plans. D’une part la résistance qui permet à la cellule leucémique d’échapper à la pression thérapeutique des ITK et d’autre part la résistance « intrinsèque » de la cellule souche leucémique par des mécanismes certainement multiples. Ce second niveau de résistance est à l’origine de la récurrence de la LMC lors de l’arrêt du traitement.Cette thèse a consisté à déterminer comment pouvait mourir les cellules de LMC en réponse aux ITK pour mettre en évidence les morts induites et les régulations qui existent entre-elles. De plus, cela a permis d’utiliser les morts non-apoptotiques pour contourner les mécanismes de résistance aux ITK.Nous avons montré pour la première fois en utilisant différents modèles cellulaires de LMC (cellules K562, Lama-84 et AR-230), que l’imatinib (ainsi que les autres ITK nilotinib et dasatinib) induit de la sénescence en plus d’une réponse apoptotique. En absence d’apoptose, par inhibition de cette dernière, la réponse sénescente devient une réponse majeure des cellules de LMC suggérant que l’apoptose a un rôle de « frein » sur la sénescence. L’autophagie activée par les ITK régule négativement la réponse apoptotique alors qu’elle est nécessaire pour une réponse sénescente majeure. Nous avons pu mettre en évidence deux types de sénescences induites par l’imatinib : une sénescence dépendante et une indépendante de l’autophagie. L’autophagie semble donc au cœur de la régulation des morts cellulaires. Puisque les cellules de LMC peuvent mourir par des morts non-apoptotiques, nous avons cherché à éliminer les cellules résistantes par des morts non-apoptotiques. Pour cela différentes molécules ont été utilisées telles que l’acide mycophénolique (MPA), un immunosuppresseur déjà utilisé en clinique. Le MPA en inhibant la synthèse de GTP permet d’induire des dommages à l’ADN et une réponse apoptotique et/ou sénescente. Dans ce contexte, l’autophagie protège la cellule de la réponse apoptotique mais ne protège pas la cellule de la sénescence. Le MPA est au contraire un puissant inducteur d’apoptose sur les cellules primaires. En effet, il induit une apoptose massive des cellules primaires résistantes aux ITK quelque soit le mécanisme impliqué (surexpression de tyrosine kinase, mutation de Bcr-Abl). Le MPA est l’exemple parfait des molécules qu’il nous faut rechercher pour éliminer les cellules résistantes de LMC notamment dans le cas où les patients sont en crise blastique et donc résistants aux thérapeutiques.Ces résultats suggèrent que la sénescence est une des morts qui peut être induite pour dépasser la résistance des cellules cancéreuses. / Chronic Myeloid Leukemia is a myeloproliferative syndrome connected to the acquisition of a chromosomal abnormality t(9;22) leading to the expression of a fusion protein p210 Bcr-Abl of whom the tyrosine kinase activity deregulated is necessary and sufficient to engender the disease.This pathology benefits since 2002 of a therapeutic advance: the tyrosine kinase inhibitors (TKI). This targeted therapeutics, from which imatinib is the front-line, is very effective because 80 % of patients enters in remission. However, 20 % of the treated patients develop primary or secondary resistances which can be dependent or not to the Bcr-Abl oncogene among which some have been characterized. Indeed, CML is now a model to study both oncogenic and resistances mechanisms.Resistance to TKI in CML can be considered on two sides. On one hand the resistance allowing the leukemic cell to escape the therapeutic pressure of TKI and on a second hand the “intrinsic” resistance of Leukemic stem cells by multiple mechanisms. This second level of resistance is at the origin of the CML recurrence.This thesis consisted in determining how could die the CML cells in response to TKI to bring to light cell deaths induced and the regulations existing between them. Furthermore, it allowed exploring the use of non-apoptotic cell deaths to overcome resistance to TKI.We showed for the first time by using CML cell lines (K562, Lama-84 and AR-230), that imatinib (as well as nilotinib and dasatinib) induced senescence besides an apoptotic response. In absence of apoptosis, by its inhibition, senescence becomes a major response of CML cells suggesting that apoptosis is limiting senescence. Autophagy activated by TKI negatively regulates apoptosis while it is necessary for a major senescent response. We were able to bring to light two types of senescence in response to TKI : a senescence dependent and a senescence independent of autophagy suggesting it plays a critical role in cell death regulation.Because CML cells can die by non-apoptotic cell deaths, we used them to eliminate TKI resistant cells. Mycophenolic acid (MPA), an immonusuppressor already used in therapeutic as an immunosuppressive agent has been extensively used. MPA by inhibiting the synthesis of GTP induces DNA damage and apoptotic and\or senescent response. In this context, autophagy protects the cells from apoptotic response but do not from senescence. Conversely, MPA is a powerful inductor of apoptosis on hematopoietic primary cells. Indeed, it induces apoptosis of TKI resistant primary cells whatever the mechanism involved (overexpression of tyrosine kinases or mutation of Bcr-Abl). MPA illustrates the need to look for new molecules to eliminate TKI resistant CML cells, particularly when patients are in the evolved blastic phase of the disease.These results suggest that senescence is one of the deaths which can be used to overcome resistance of cancer cells.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Phloem-feeding insects like aphids feed on a variety of crop plants and limit plant productivity. In addition they are vectors for important plant viruses. Efforts to enhance plant resistance to aphids have been hampered by lack of sufficient understanding of mechanisms of plant defense against aphids. I have utilized a plant-aphid system consisting of the model plant Arabidopsis thaliana and the generalist aphid, Myzus persicae Sulzer (green peach aphid [GPA]), to study plant response to aphids. These studies have demonstrated an important role of premature leaf senescence in controlling aphid growth in Arabidopsis. Molecular and physiological studies suggest that the Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT 4) gene modulates the GPA feeding-induced senescence process. Furthermore, in comparison to the wild type plants, GPA growth was higher on pad4 mutant plants, suggesting an important role for PAD4 in plant defense against GPA. In contrast, constitutive expression of PAD4 in transgenic Arabidopsis enhanced basal resistance against GPA. Unlike its involvement in plant defense against pathogens, the role of PAD4 in Arabidopsis resistance to GPA is independent of its involvement in phytoalexin biosynthesis and of its interaction with EDS1, a PAD4-interacting protein. Instead, the heightened resistance to GPA in these PAD4 constitutively expressing plants was associated with the rapid activation of leaf senescence. The association of premature leaf senescence in basal defense against GPA is supported by our observation that in comparison to the wild type plant, GPA growth was restricted on the Arabidopsis hypersenescence mutants, ssi2 and cpr5. Gene expression studies suggested some overlap between plant responses to pathogens and aphids, for example, activation of genes associated with the salicylic acid (SA) signaling pathway. However, the characterization of aphid performance on Arabidopsis SA biosynthesis and signaling mutants have ruled out the involvement of SA signaling in controlling aphid growth.

Abstract: By differentiating chromosomal ends from internal breaks, telomeres prevent DNA damage checkpoint activation and provide protection from inappropriate DNA repair activity that could create genomic instability. In Saccharomyces cerevisiae, a large number of genes have been identified that are implicated in telomerase and telomere structure and/or function. However, a comprehension of the mechanism of action of these genes and how they relate to other genes is lacking. The function of end protection is based on the telomeric repeats and associated proteins, but evidence is accumulating that the subtelomeric region also plays a role. This region contains binding sites for various proteins, notably Tbf1. TBF1 is an essential gene and the protein has been implicated in telomere homeostasis, chromatin remodelling, and the DNA damage response. My master’s project is based on the observation that cells harbouring a thermosensitive (tbf1-ts) allele have abnormally short telomeres. However, all four known mutant tbf1 alleles have multiple point mutations, which renders their analyses difficult. In order to be able to more precisely determine the origin of the phenotypic variations, we used site-directed mutagenesis to create single point mutation tbf1 alleles. These experiments yielded two particular mutations, tbf1-82 and tbf1-453, which were found to have growth defects at various temperatures as well as increased sensitivity to DNA damaging drugs. Although the alleles had only minor telomere length phenotypes, it was discovered that Tbf1 could have a direct role in telomere stability in special situations. For example, in the absence of telomerase, which normally maintains telomeres, cells enter replicative senescence after about 60 population doublings and stop dividing. A small subset of the cellular population is able to evade this growth arrest by maintaining telomeres via a recombination-dependent process. An introduction of the tbf1-82 or tbf1-453 mutation into strains that also lacked telomerase caused a dramatic advance in time of onset of senescence. Thus this work uncovered that Tbf1 is a previously unknown regulator of senescence. Various genetic assays with homologous recombination genes and chromatin regulators were performed to help further characterize TBF1 and its interactors. Characterization of these novel tbf1 alleles has given new insights into the multiple roles of Tbf1. / En différenciant les extrémités chromosomiques des cassures d’ADN internes, les télomères empêchent l'activation de la signalisation d’un dommage à l'ADN et fournissent une protection contre des activités inappropriées qui sont associées à une réparation de l'ADN. Une telle réparation pourrait en fait créer une instabilité génomique. Chez Saccharomyces cerevisiae, un nombre de protéines sont impliquées dans la structure du télomère et / ou la fonction de la élomérase. On pense que la protection des télomères est gérée par les répétitions télomériques et les protéines associées, mais il y a de plus en plus d’indices que la région sous-télomérique joue également un rôle. Cette région contient des sites de liaison pour plusieures protéines, notamment pour Tbf1. TBF1 est un gène essentiel et la protéine est impliquée dans l'homéostasie des télomères et dans la réponse aux dommages de l’ADN. Toutefois, les mécanismes moléculaires restent à être précisés. Mon projet de Maîtrise est basé sur l’observation que dans les cellules qui ont un allèle thermosensible (tbf1-ts), les télomères sont anormalement courts. Malheureusement, les 4 allèles mutants de tbf1 connus présentent tous des mutations ponctuelles multiples ce qui rend leur analyse difficile. Pour clarifier l'origine des variations phénotypiques de ces mutations, la mutagenèse dirigée a été utilisée pour créer des allèles tbf1 avec une seule mutation. Mes résultats montrent que deux mutations spécifiques, tbf1-82 et tbf1-453, causent des défauts de croissance cellulaires, ainsi qu'une sensibilité aux drogues qui endommageant l'ADN. Une analyse détaillée de ces nouveaux allèles de tbf1 a montré que la protéine pourrait avoir un rôle direct dans le maintien de la stabilité des télomères. Par exemple, en absence de la télomérase qui est responsable du maintien des télomères, les cellules entrent en sénescence réplicative après environ 60 générations et arrêtent de se diviser. Par contre, une petite fraction de la population est capable de contourner cet arrêt de croissance car ces cellules maintiennent les télomères par un processus dépendant de la recombinaison homologue. L'introduction de mutations tbf1 dans des souches sans télomérase provoque une accélération d’entrée en sénescence; donc Tbf1 est un régulateur précédemment inconnu de la sénescence. Divers tests génétiques avec des gènes de recombinaison homologue et des régulateurs de chromatine ont été effectués pour aider à caractériser TBF1 et ses interactions. La caractérisation de ces nouveaux allèles a permis de mieux comprendre les multiples rôles de Tbf1.