La sénescence est un mécanisme de défense antiprolifératif dont la cellule est munie afin de prévenir l’accumulation de mutations pouvant mener à sa transformation et l’éventuel développement d’une tumeur. Ce programme consiste en un arrêt permanent du cycle cellulaire. Il peut être activé par de nombreux stimuli tels que le raccourcissement des télomères, le stress oxydatif, ou l’expression d’un oncogène constitutivement actif. Sa régulation requiert l’activation de protéines appelées des suppresseurs de tumeur dont les plus importants sont p53 et RB. De manière plus spécifique, les sénescences induites par l’expression des oncogène RASV12 ou STAT5A(1*6) sont respectivement caractérisées par l’augmentation de l’expression des protéines PML et CHES1/FOXN3. Le but de cette thèse est, dans un premier temps, de mettre en évidence le mécanisme de régulation de la sénescence par PML. PML est un suppresseur de tumeur dont l’expression dans des cellules diploïdes normales est suffisante pour induire la sénescence. Cette protéine forme des corps nucléaires sphériques au sein desquels est recruté, parmi d’autres molécules, la protéine du rétinoblastome RB. RB est un régulateur négatif du cycle cellulaire capable de lier et inhiber les facteurs de transcription E2F dont les gènes cibles sont nécessaires à la prolifération. Nos travaux démontrent que le mécanisme d’induction de la sénescence par PML implique le recrutement du complexe RB/E2F aux corps de PML afin de renforcer l’inhibition de l’activité des E2F par RB. Également, les E2F sont recrutés aux corps de PML en compagnie de leurs promoteurs ce qui favorise la formation d’hétérochromatine au niveau de ces gènes, aidant à leur répression et donc à l’établissement de la sénescence. D’autre part, cette thèse a pour but de caractériser le rôle de CHES1/FOXN3 dans la régulation du cycle cellulaire. CHES1 est un facteur de transcription de la famille des Forkheads. Son expression dans des cellules cancéreuses provoque un ralentissement de leur prolifération. Afin de comprendre son mécanisme de fonctionnement, une analyse sur micropuce d’ADN de l’expression des gènes de cellules cancéreuses exprimant CHES1 a été réalisée. Cette analyse a montré que, dans la cellule, CHES1 joue un rôle de répresseur transcriptionnel. Plus précisément, CHES1 réprime, entre autres, l’expression de gènes nécessaires à la synthèse des protéines tels que PIM2 et DYRK3. De manière intéressante, la réexpression de PIM2 dans des cellules cancéreuses exprimant CHES1 permet de rétablir partiellement la prolifération cellulaire. Également, l’analyse sur micropuce a révélé que CHES1 régule l’expression de nombreux gènes impliqués dans la formation des cilia dont l’une des fonctions semble être de moduler la synthèse protéique. Pris ensemble, ces résultats suggèrent que le mécanisme antitumoral de CHES1 consiste en une inhibition de la synthèse de protéines. / Senescence is an antiproliferative defense mechanism that protects the cell against the accumulation of mutations and, eventually, her transformation and the development of a tumor. This program consists of a permanent cell cycle arrest. It can be activated by many stimuli, such as telomere shortening, oxidative stress, or the expression of a constitutively active oncogene. Senescence regulation requires activation of proteins called tumor suppressors which the most important are p53 and RB. More specifically, senescences induced by the expression of oncogenic RASV12 or STAT5A (1 * 6) are respectively characterized by increased expression of the proteins PML and CHES1/FOXN3. The purpose of this thesis is, firstly, to identify the mechanism of regulation of senescence by PML. PML is a tumor suppressor whose expression in normal diploid cells is sufficient to induce senescence. This protein forms spherical nuclear bodies in which is recruited, among other molecules, the retinoblastoma protein RB. RB is a negative regulator of the cell cycle due to its capacity to bind and inhibit the E2F transcription factors whose target genes are necessary for proliferation. Our work demonstrates that the mechanism of induction of senescence by PML involves the recruitment of the complex RB/E2F to the PML body to enhance the inhibition of E2F activity by RB. Also, the E2Fs are recruited to PML bodies together with their promoters which promotes the formation of heterochromatin in these genes, helping their repression and thus the establishment of senescence. On the other hand, this thesis aims to characterize the role of CHES1/FOXN3 in regulating the cell cycle. CHES1 is a transcription factor of the Forkheads family which expression in cancer cells causes a growth reduction. To understand the antitumoral mechanism of CHES1, a microarray analysis of cancer cells expressing CHES1 was performed. This analysis shows that CHES1 is a transcriptional repressor. Specifically, CHES1 represses, among others, the expression of genes required for the synthesis of proteins such as PIM 2 and DYRK3. Interestingly, re-expression of PIM 2 in cancer cells expressing CHES1 partially restores cell proliferation. Also, microarray analysis revealed that CHES1 regulates the expression of many genes involved in the formation of cilia which one of the functions seems to be to modulate protein synthesis. Taken together, these results suggest that the antitumor mechanism of CHES1 involves inhibition of protein synthesis.

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer. / An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between its α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9, ortholog protein in Caenorhabditis elegans. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cancer cells. When these residues are mutated and proteins expressed in cancer cells, cells harbor mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation, events associated with chromosome instability and aneuploidy. To further analyze the effects of Bcl-xL Ser49 and Ser62 in normal cells, the present studies were performed in normal human diploid cells, and in vivo in Caenorhabditis elegans. First, we studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL(wild type), (S49A), (S49D), (S62A), (S62D) and the dual (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL Ser49 and Ser62 phosphorylation/ dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. Second, we undertook experiments in Caenorhabditis elegans to understand the importance of Bcl-xL Ser49 and Ser62 in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual-site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching, variations in the length of their mitotic regions and transition zones, chromosomal abnormalities at their diplotene stages, and increased germline apoptosis. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. The in vivo observations confirmed the importance of Ser49 and Ser62 within the loop domain of Bcl-xL in maintaining chromosome stability. These studies could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy.

4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....

World population is ageing and in Europe the situation is especially alarming. In the next two decades the ratio of people over 65 years of age is expected to double. This threatens long-term sustainability of welfare systems. The debate about future developments has been going on since the turn of the century. Based on this debate the European Union defined improving the inclusion of people over 50 as the main solution to this problem. The means to achieving this goal have been identified at the level of the labour market, disease prevention and lifelong learning. The specific instruments are: 1) increasing the participation of older workers in the labour market by increasing retirement age, introducing penalties for early retirement, promoting retraining of older and increasing retraining efficiency, 2) restructuring of pension systems and provision of social services and 3) strengthening participation of older people in society, applying the principles of active aging at all levels of social life and increasing the quality and variety of lifelong learning opportunities. This paper analyses the situation of persons over 50 years of age in the Czech Republic in terms of their position in the labour market, participation in social life and quality of life in the context of the strategic concept of...

Diversas pesquisas têm reportado que o aumento da produtividade da cultura do milho se deve muito mais à tolerância a estresses abióticos do que a aumentos per se. O déficit hídrico é um dos mais importantes fatores envolvidos na redução de produtividade nessa cultura, mas devido à dificuldade de seleção direta para tolerância à seca, são empregados caracteres secundários como o caráter stay green ou senescência retardada de folhas e colmos. Todavia, estudos sobre a herança desse caráter são escassos na literatura. Em vista disso, este trabalho teve como objetivo estudar a herança deste caráter em milho tropical, utilizando o delineamento III (Comstock e Robinson, 1952). A partir do cruzamento entre as linhagens endogâmicas L08-05F e L38-05D, foram obtidas 100 progênies F2:3, as quais foram retrocruzadas com cada um dos parentais, resultando em 200 progênies de retrocruzamento. Estas progênies foram avaliadas em dez ambientes, com duas repetições por ambiente, em Piracicaba SP, utilizando o delineamento ?-látice. A avaliação do stay green foi feita em dez plantas competitivas por parcela, 120 dias após a semeadura, utilizando uma escala de notas variando de 1 a 5, em que 1 correspondeu às plantas com todas as folhas acima e pelo menos duas folhas verdes abaixo da espiga e 5 à planta com todas as folhas secas. O caráter florescimento feminino foi avaliado e utilizado como covariável para ajuste das variações de maturação das progênies, e as médias das parcelas foram utilizadas para as análises. A estimativa da variância aditiva foi muito superior à de dominância e a variância da interação aditiva por ambiente apresentou maior magnitude que a variância aditiva. O tipo de ação gênica foi de dominância parcial, mas devido ao desequilíbrio de ligação da população, este pode estar superestimado. O coeficiente de herdabilidade em nível de plantas apresentou baixa magnitude (~25%) enquanto que em nível de médias a magnitude foi elevada (~70%). Estes resultados indicam que os efeitos aditivos são mais importantes que os dominantes no controle do caráter stay green e que os efeitos aditivos não são consistentes nos diversos ambientes de avaliação. Assim, o nível de heterose deve apresentar baixa magnitude, e a seleção fenotípica não pode ser realizada em nível de plantas, mas baseada em médias de experimentos com repetições, conduzidas em diversos ambientes para minimizar os efeitos destes e suas interações com os genótipos. A linhagem L08-05F apresentou maior nível de stay green que a linhagem L38-05D e, portanto, pode ser utilizada como fonte de alelos favoráveis para serem transferidos para outras linhagens visando aumentar o nível deste caráter em linhagens que apresentarem baixos níveis de senescência retardada. / Several researches have reported that the increase in maize productivity could be attributed to the tolerance to abiotic stresses rather than to an increase on a plant productivity per se. Moisture stress is one of the most important stresses that reduce the maize productivity, but because of the difficulties for the direct selection for tolerance to moisture stress secondary traits as delayed senescence, also known as stay green trait, has been employed for this purpose. However, there is limited information reported on the inheritance of this trait. Thus, the objective of this research was to study the inheritance of the stay green trait in maize using the design III (Comstock and Robinson, 1952). One hundred F2:3 progenies were developed from a population derived from the cross between the inbred lines L08-05F and L38-05D; and these progenies were backcrossed to the inbred parents giving rise to 200 backcrossed progenies. These progenies were evaluated in ten environments, with two replications per environment, in Piracicaba City, São Paulo State, using the experimental design ?-lattice. The stay green trait was recorded in ten competitive plants per plot 120 days after sowing, using a scale with scores from 1 to 5, where score 1 was assigned to the plants with all leaves above the ear and at least two leaves below the ear green, and score 5 was assigned to plants with all leaves senescent. The trait days to silking was also recorded and used as covariate to adjust the maturation variation of the backcrossed progenies, and the mean of the plots was used for analysis. The estimate of the additive variance was quite larger than the dominance variance, the additive x environment variance was quite larger than the additive variance, and the average level of dominance was partial dominance, but since the population was in linkage disequilibrium the level of dominance may be overestimated. The heritability coefficient on a plant level was of low magnitude (~25%) while that one on a mean level presented high magnitude (~70%). These results indicated that the additive effects were more important than the dominance effects for the control of the stay green trait, and that the additive effects are not consistent across the environments. Therefore, the level of heterosis should present low magnitude, and the phenotypic selection should not be conducted on a plant level; instead selection should be based on the means of replicated experiments assessed in several environments to minimize their effects and the genotype by environment interactions. The inbred line L08-05F presented higher level of stay green than the inbred L38-05D and thus the former inbred could be used as a source of favorable alleles to be transferred to others inbreds to increase the level of this trait in inbreds that have low levels of delayed senescence.

Introdução: o processo fisiológico de envelhecimento traduz-se em diversas alterações estruturais do sistema musculoesquelético. Estas, por sua vez, acarretam em modificações funcionais que se repercutem na dependência do senescente, determinando a diminuição da sua qualidade de vida. Das estratégias existentes para atenuar os efeitos da senescência o treinamento de potência tem sido apontado como preferido. Existem, contudo, indícios de que um treinamento baseado na potencialização da ação do ciclo alongamento-encurtamento seja uma melhor escolha. Hipóteses do estudo: pelos resultados obtidos em intervenções similares, hipotetiza-se que as participantes do protocolo de intervenção proposto apresentarão um aumento da densidade mineral óssea, do volume muscular, da capacidade funcional e melhora de alguns parâmetros biomecânicos da marcha, bem como da sua qualidade de vida. Objetivos: o objetivo geral do presente trabalho é, portanto, propor um modelo de treinamento fundamentado na potencialização da ação do ciclo alongamento-encurtamento e averiguar os seus efeitos em parâmetros selecionados da morfologia osteomuscular, capacidade funcional e qualidade de vida de idosas. Materiais e Métodos: 21 idosas sedentárias (66,9±4,2 anos) executaram o protocolo proposto durante 20 semanas, tendo os efeitos na densidade mineral óssea de fêmur, coluna, tíbia e rádio; efeitos na composição corporal, na força, no equilíbrio, na marcha, na flexibilidade e na qualidade de vida comparados aos efeitos obtidos pelo grupo controle (N=17, 65,0±3,4 anos), que não alterou o seu nível de atividade física. O protocolo de intervenção, composto por onze exercícios de força realizados com o intuito de potencializar a ação do ciclo alongamento-encurtamento, que apresentava duas modalidades de salto (salto vertical com contramovimento e drop jump), exigia a realização da fase concêntrica na maior velocidade possível. Resultados: quando comparado com o grupo controle, o grupo experimental apresentou alterações significantes (p<0,05) na densidade mineral óssea de coluna (g=1,06) e sua microarquitetura (g=0,80), na microarquitetura da tíbia (g=0,82), na força máxima (g=2,39) e potência (g=1,38) de extensores de joelho, na velocidade máxima de marcha (g=0,96), na flexibilidade de membros inferiores (g=1,05) e superiores (g=0,86) e no domínio Atividades passadas, presentes e futuras da qualidade de vida (g=1,08). Conclusão: os resultados apontam para a eficácia da proposta de intervenção, apresentando-se como uma nova estratégia para atenuar e até mesmo reverter algumas perdas estruturais e funcionais impostas pelo processo de envelhecimento / Introduction: the physiological aging process induces several structural changes in the musculoskeletal system. These, in turn, result in functional changes that are reflected in the senescent dependency, determining the reduction in their quality of life. Power training has been identified as ideal to mitigate the effects of aging. However, there are indications that an intervention based on the potentiation of the stretch-shortening cycle action is a better choice. Study hypotheses: the participants of the proposed intervention will increase their bone mineral density, muscle volume, functional capacity and will show some improvement in their gait, as well as in their quality of life. Purposes: the main objective of this study was to propose a training model based in the potentiation of the stretch-shortening cycle action and assess its effects on selected parameters of musculoskeletal morphology, functional capacity and quality of life of elderly women. Methods: 21 sendentary elderly women (66.9 ± 4.2 years) performed the proposed intervention protocol for 20 weeks and the effects on bone mineral density of the femur, spine, tibia and radio; effects on body composition, strength, balance, gait, flexibility and quality of life were compared to the effects obtained by the control group (N = 17, 65.0 ± 3.4 years) that did not change their level of physical activity. The program was composed by eleven strength exercises performed in order to enhance stretch-shortening cycle action, had two jump exercises (vertical jump with countermovement and drop jump) and had the concentric phase of the movements performed as fast as possible. Results: when compared to the control group the experimental group showed significant changes (p <0.05) in bone mineral density of the spine (g = 1.06) and its microarchitecture (g = 0.80), the microarchitecture of the tibia (g = 0.82), the knee extensors maximum force (g = 2.39) and power (g = 1.38), the maximum walking speed (g = 0.96), the lower (g = 1.05) and upper (g = 0.86) limbs flexibility and in the domain past, present and future activities of the quality of life (g = 1.08). Conclusion: the results point to the effectiveness of the proposed intervention, suggesting it as a new strategy to slow down and even reverse some structural and functional losses imposed by the aging process

FGFs compreendem um grande família de 24 proteínas, participando de processos chaves nos mais variados tecidos, tendo funções parácrina, autócrina e intrácrina, regulando mitogênese, diferenciação celular, morfogênese e cicatrização. Mas, a relação estrutura-função dos FGFs é pobremente entendida. O membro protótipo desta família é o FGF-2, que apresenta quatro isoformas moleculares incluindo a forma de 18 kDa que é secretada e se liga aos receptores específicos (FGFRs) e dispara uma complexa sinalização. As outras isoformas, de alto peso molecular (21, 22 e 22,5 kDa) são expressas por códons alternativos (CUG) e permanecem no interior da célula interagindo com parceiros moleculares desconhecidos. Para antecipar mecanismos e parceiros do FGF-2 HMW foi realizada modelagem molecular desta isoforma que mostrou: uma estrutura do N-terminal da proteína com motivo &#946;&#8594;&#945;&#8594&#946; e manutenção do barril &#946;. A busca por parceiros intracelulares, foi realizada através da técnica do duplo hibrido de levedura, usando um biblioteca de cDNA de cérebro de rato. Foram encontrados 4 possíveis parceiros: BRD2, UBE2I, BRPF1, PC4. Todas essas interações foram confirmadas através do crescimento da levedura em meio sem histidina, produção de &#946;-galactosidase e ensaios de \"pull-down\" com GST. Analises por FACS confirmam que FGF2 não causa apoptose em células adrenais tumorais Y1 de camundongo, mas promovem um acumulo de células na fase S com bloqueio do ciclo celular e da proliferação, configurando uma forma de senescência. Resultados com as células humanas HEK-ER:Ras permitem fazer a seguinte generalização: FGF2 induz senescência em células malignas transformadas pelos oncogenes raso A superexpressão da proteína de fusão FGF-2(18kDa):protA, mas não a da FGF-2(22,5 kDa):protA, protege a célula Y1 da senescência induzida por FGF-2. Por outro lado, a superexpressão destas mesmas isoformas de FGF-2 fusionadas à proteína A em células imortalizadas Balb3T3 não causou transformação celular e nem alterou a resposta mitogênica destas células ao FGF-2 recombinante adicionado ao meio de cultura. Células Y1 quando tratadas com FGF-2 recombinante produz ROS intracelular e libera anions superóxido no meio extracelular. Além disso, o anti-oxidante NAC protege estas células da indução de senescência induzida por FGF-2, sugerindo que ROS pode ser intermediário no disparo de senescência por FGF-2. / FGFs comprise a large fami1y of 24 proteins that play key roles in a number of tissues as local paracrine, autocrine and intracrine regulators of mitogenesis, cellular differentiation, organ morphogenesis and tissue repair. Structure-function relationship among FGFs is still poorly understood. FGF-2, the fami1y prototype member, exists as four molecular species. The 18 kDa form is released to the extracellular milieu and binds to specific receptors (FGFR), initiating a complex array of signals. Other isoforms of higher molecular weights (21, 22 and 22,5 kDa) are translated from alternative codons (CUG) and remain inside of the cell interacting with unknown partners. Aiming to anticipate mechanisms and partners, we modeled the FGF2-HMW molecule, showing that the protein displays &#946;&#8594;&#945;&#8594&#946; motif in the N-terminal region and maintains the &#946;-barrel structure common to ali FGFs. By the yeast two-hybrid method, using a cDNA rat brain library, we found four possible partners for FGF2-HMW: BRD2, UBE2I, BRP1 and PC4. Ali partners were confirmed by yeast growth without histidine, production of &#946;-galactosidase and \"pull-down\" assays with GST. FACS analyses confirmed that FGF2 does not cause apoptosis in mouse Y1 adrenal tumor cells. But, FGF2 inhibited S phase progression blocking cell cycle and proliferation, characterizing a form of senescence. In addition, results obtained with the human HEK-ER:Ras cells support the following general statement: FGF2 triggers senescence in malignant cells transformed by ras oncogenes. Ectopic expression of the fusion protein FGF-2(18 kDa):protA, but not of FGF-2(22,s kDa):protA, protected Y1 cells senescence induced by FGF-2. On the other hand, ectopic expression of FGF-2 isoforms fusioned to protA in Balb3T3 immortalized cells did not cause transformation and neither modified the mitogenic response of this cell to recombinant FGF2. Recombinant FGF-2 stimules Y1 cells to produce intracellular ROS and to release superoxide anions into intracellular medium. Moreover, the ROS scavenger NAC protect Y1 cells from senescence induced by FGF-2, suggesting that ROS may be mediate senescence triggering induced by FGF-2.

Entre todos os cânceres de pele, o melanoma está entre os menos comuns, mas é responsável pela maior parte das mortes. No caso da doença metastática, não há um tratamento satisfatório capaz de prolongar a vida do paciente. Isso leva à necessidade de novas estratégias e de novos tratamentos que possam reverter a quimiorresistência do tumor. Entre as proteínas que têm seu perfil de expressão modificado no melanoma está a proibitina, cuja expressão aumenta durante a progressão tumoral. Proibitina é uma chaperona mitocondrial pertencente a uma família de proteínas que possuem um resíduo hidrofóbico SPFH, que confere a ela uma capacidade de ancoragem e de organização de espaços em membranas. Além disso, no compartimento nuclear, é um inibidor da família de fatores de transcrição E2F, juntamente com a proteína retinoblastoma (Rb). Em melanomas, proibitina localiza-se no citoplasma, associada à mitocôndria, e no núcleo. No citoplasma, proibitina faz parte da resposta a diversas drogas, como cisplatina, dacarbazina, temozolamida, vimblastina e tunicamicina pode estar relacionado com o aumento de espécies reativas de oxigênio (ROS), já que essas drogas podem de alguma forma induzir ROS intracelular. O aumento de expressão de proibitina, nesse contexto, poderia fazer parte de uma resposta protetora da mitocôndria, o que em última análise protegeria a célula contra a morte celular, já que a inibição de proibitina sensibiliza a célula ao tratamento com cisplatina ou tunicamicina. Além disso, o estresse provocado pela privação de soro fetal bovino em linhagens de melanoma leva ao aumento de expressão de proibitina e é acompanhado pela indução de ROS. No núcleo, proibitina esta colocalizada com MCM5 e MCM7, mas não MCM2. A inibição de proibitina leva ao aumento de expressão de metaloproteinases de matriz extracelular, não só em melanomas, mas também em linhagens de câncer de mama e de câncer de pulmão. Ainda, proibitina parece estar relacionada com o fenômeno da transição epitélio mesênquima, já que a inibição de proibitina leva ao aumento de expressão de marcadores mesenquimais como N-caderina e vimentina e a perda de expressão de marcadores epiteliais, como a E-caderina. Outras funções controladas por E2F1 que proibitina pode estar modulando são a capacidade de E2F1 induzir reparo de DNA devido a lesões causadas por radiação UVB e a indução de senescência. A inibição de proibitina em linhagem de câncer de pulmão protegeu a célula contra o dano genotóxico causado pela radiação UVB, pelo aumento da proteína de reparo de DNA Gadd45a, que é induzida por E2F1. Ainda, a inibição de proibitina diminuiu a quantidade de células senescência induzida por adriamicina em linhagens de melanoma. Ainda, a expressão de proibitina responde a fatores do microambiente tumoral como TGF?, IL4 e LPS juntamente com INF? e, além disso, têm sua expressão diminuída durante a maturação de macrófagos. Esses resultados mostram que proibitina pode atuar protegendo o tumor ou bloqueando vias importantes para seu desenvolvimento, dependendo da sua compartimentalização subcelular / Among all skin cancers, melanoma is the least common, but is responsible for most deaths. In metastatic disease, no satisfactory treatment can prolong the patient\'s life. This leads to the need for new strategies and new treatments that may reverse tumor chemoresistance. Among proteins that have their expression profile altered in melanoma is prohibitin whose expression increases during tumor progression. Prohibitin is a mitochondrial chaperone belonging to a family of proteins which possess a hydrophobic residue SPFH, which gives it a capacity for anchorage and organization in membrane regions. Furthermore, in the nuclear compartment, prohibitin is an inhibitor of the E2F transcription factor family, together with the retinoblastoma protein (Rb). In melanomas, prohibitin is located in the cytoplasm, associated to the mitochondria, and inside the nucleus. In the cytoplasm, prohibitin is part of the response to various drugs such as cisplatin, dacarbazine, temozolomide, vinblastine and tunicamycin and may be associated with increased reactive oxygen species (ROS), since these drugs can somehow induce intracellular ROS. Prohibitin overxpression in this context could be part of a protective response of the mitochondria, which ultimately protect cells against death, as prohibitin inhibition sensitizes cells to cisplatin or tunicamycin treatment. Moreover, the stress caused by deprivation of fetal bovine serum in melanoma cell lines leads to prohibitin overexpression and is accompanied by ROS induction. In the nucleus, prohibitin is colocalized to MCM5 and MCM7, but not to MCM2. Inhibition of prohibitin leads to increased expression of matrix metalloproteinases, not only on melanomas but also on breast cancer and lung cancer cell lines. Further, prohibitin appears to be related to the phenomenon of epithelial mesenchymal transition, since prohibitin inhibition leads to increased expression of mesenchymal markers such as N-cadherin and vimentin and loss of expression of epithelial markers, such as E-cadherin. Other functions controlled by E2F1 that are modulated by prohibitin include be the ability of E2F1 to induce DNA repair against UVB radiation and the induction of cellular senescence. Inhibition of prohibitin in lung cancer cell line protected against genotoxic damage caused by UVB radiation, due to DNA repair protein Gadd45a overexpression, which is induced by E2F1. Also, prohibitin inhibition decreased the amount of cell senescence induced by adriamycin in melanoma cell line. Further, prohibitin expression is triggered by tumor microenvironmental factors such as TGF?, IL4, and LPS together with INF? and, in addition, prohibitin expression decreases during macrophages maturation. These results show that prohibitin may act protecting the tumor or blocking pathways important for its development, depending on its subcellular compartment distribution

A informação sobre o tipo de herança de um caráter considerado para fins de seleção é de extrema importância para o sucesso dos programas de melhoramento. O caráter senescência retardada, usualmente chamado de stay-green, tem sido relacionado em diversas culturas à tolerância a estresses abióticos, principalmente ao estresse devido à seca. Embora a maioria dos híbridos de milho comerciais sejam stay-green, as informações sobre o seu tipo de herança são muito limitadas. Assim, este trabalho teve como objetivo estudar a herança do caráter stay-green em milho tropical. O material genético utilizado incluiu 55 linhagens de diversas origens, a fim de representar a variabilidade genética em milho tropical. Foram realizados cruzamentos dialélicos parciais, onde 50 linhagens foram cruzadas com outras 5 linhagens utilizadas como testadoras, originando 250 cruzamentos. Os 250 cruzamentos e seis híbridos comerciais foram avaliados em 8 ambientes no delineamento de látice simples 16x16 com duas repetições. O caráter stay-green foi avaliado em cinco plantas competitivas por parcela, 120 dias após a semeadura, através de uma escala de notas visual de 1 a 5, onde a nota 1 se referia às plantas verdes e a nota 5 às plantas secas. Foi necessário tomar dados de florescimento feminino para utilizá-los como covariável nas análises estatísticas e corrigir as diferenças de maturação entre os cruzamentos. A análise de variância dialélica foi realizada de acordo com o método 4 do modelo 1 de Griffing (1956), adaptado para dialelos parciais em múltiplos ambientes. A capacidade geral de combinação (CGC), tanto para as linhagens como para os testadores, e a capacidade específica de combinação (CEC) foram altamente significativas )01,0(<=P, mostrando que tanto a CGC como a CEC contribuíram significativamente para a expressão do caráter. Porém a contribuição da CGC foi de 69,06% e a da CEC foi de 30,94% para a variação entre cruzamentos, indicando que os efeitos aditivos, relacionados à CGC, são mais importantes que os efeitos não aditivos (dominância e epistasia), que são relacionados à CEC, na variação dos cruzamentos. Tanto a CGC como a CEC interagiram significativamente com o ambiente, evidenciando que estes parâmetros não são consistentes nos diversos ambientes. Então, a seleção para o caráter stay-green deve ser baseada em médias de experimentos avaliados com repetições em diversos ambientes. / Information on the inheritance of traits to be selected is of paramount importance for the success of breeding programs. The trait delayed senescence, usually named \"stay-green\" trait, has been related to tolerance to abiotic stresses, mainly drought stress, in several crop species. Although the majority of commercial maize hybrids are \"stay-green\", limited information are available on its inheritance. Thus, this research was conducted to study the inheritance of the stay-green trait in tropical maize. The genetic material included 55 inbred lines from several sources to represent the genetic variation of tropical maize. Fifty inbred lines were crossed to 05 inbreds as testers following the partial diallel cross design, giving rise to 250 single crosses. The crosses and six commercial hybrids, 256 entries, were evaluated at eight environments using a 16 x 16 lattice design with two replications per environment. The stay-green trait was recorded 120 days after sowing, in five competitive plants per plot, following a visual note scale, i.e., from 1 to 5, where 1 refers to green plants and 5 to no-green plants. Also, the trait days to mid-silking was recorded and used as covariate to correct for differences of maturing among crosses. The analysis of variance of the diallel crosses was computed following the method 4 model 1 of Griffing (1956) extended to multiple environments. The general combining ability (GCA) for both the inbreds and the testers, and the specific combining ability (SCA) were all highly significant (P<=0.01), showing that GCA as well as SCA contribute significantly for the expression of the trait. However, the contribution of the GCA was 69.06% and of the SCA was 30.94% for the variation among the crosses, indicating that the additive effects, which are related to GCA, are more important than the non-additive effects (dominance and epistasis), which are related to SCA, for the variation of the crosses. Both GCA and SCA interacted significantly with the environments, showing that these parameters were not consistent across the environments. Thus, selection for the stay-green trait should be based on the means of experiments evaluated in several environments.

Os trabalhos foram conduzidos no Laboratório de Fisiologia Pós-Colheita do Departamento de Ciências Biológicas da ESALQ-USP, no período de agosto de 2001 a agosto de 2002. Verificaram-se os efeitos isolados do regulador vegetal ácido giberélico, 1-metilciclopropeno e aminoetoxivinilglicina, e também nas combinações de 1-MCP com GA3 e AVG com GA3, em pós-colheita de tangor ‘Murcote’ e de lima ácida ‘Tahiti’. A escolha das variedades ocorreu em função do seu potencial, tanto para o mercado interno, quanto para a exportação de frutas frescas. As concentrações utilizadas foram: 20 mg L -1 de GA3; 0,1, 0,5 e 1,0 mL L -1 de 1-MCP; 10, 50 e 100 mg L -1 de AVG e as combinações de 0,5 mL L -1 de 1-MCP com 20 mg L -1 de GA3 e 50 mg L -1 de AVG com 20 mg L -1 de GA3, além do controle. Aplicou-se o 1-MCP através da exposição dos frutos ao gás durante 12 h em caixas herméticas a 20 o C. O AVG e o GA3 foram aplicados submergindo os frutos em solução aquosa contendo as devidas concentrações, durante um minuto. Nas combinações, o GA3 foi aplicado após os tratamentos com 1-MCP ou AVG. O delineamento experimental adotado foi inteiramente casualizado, em esquema fatorial para verificar a ação do 1-MCP e do AVG em tangor ‘Murcote’e na lima ‘Tahiti’. Para as combinações de 1-MCP+GA3 e do AVG+GA3, utilizou-se o delineamento experimental inteiramente casualizado com parcelas subdivididas no tempo. Determinou-se a evolução da coloração na casca (L, C* e h o ), os níveis de sólidos solúveis totais ( o Brix), acidez titulável total (%), vitamina C, perda de massa (%), quantidade de suco (%) e taxa respiratória dos frutos sob refrigeração e após 3 dias a 25 o C. Os resultados foram submetidos à análise de regressão. O 1-MCP não tem efeito sobre a coloração de casca, em aplicações tardias em tangor ‘Murcote’. A concentração de 0,5 mL L -1 foi suficiente para reduzir a taxa respiratória e o consumo de vitamina C. As características físico-químicas mantiveram-se adequadas para o consumo durante 45 dias de armazenamento refrigerado. O AVG aplicado em pós-colheita realçou a intensidade da cor na casca de tangor ‘Murcote’, diminuiu o consumo de sólidos solúveis totais, a taxa respiratória e não afetou a acidez titulável. As combinações de 1-MCP e AVG com GA3 não tiveram efeitos na coloração da casca do tangor ‘Murcote’, após estágio avançado de pigmentação na casca. As combinações também não afetaram o nível de acidez titulável, vitamina C, a perda de massa e a taxa respiratória do tangor ‘Murcote’. Com relação à lima ácida ‘Tahiti’, o tratamento com 1-MCP a 0,5 ou 1,0 mL L -1 atrasou mudanças na coloração da casca (croma e ângulo de cor), promoveu maior teor de sólidos solúveis totais e diminuiu a amplitude de variação do ratio. O AVG não impediu a mudança na cor da casca, porém diminuiu o consumo de SST e de ATT; não afetou a taxa respiratória e o nível de vitamina C. A combinação do 1-MCP+GA3 retardou a evolução na mudança da cor de casca durante 60 dias. O nível de vitamina C manteve-se elevado. Não houve alterações na taxa respiratória e na quantidade de suco nos frutos da lima ácida ‘Tahiti’. / The experiments were conducted from August 2001 to August 2002 in the post-harvest Physiology Laboratory, Department of Biology, ESALQ-USP. It was checked the isolated effect of gibberellic acid, 1-methylcyclopropene and aminoethoxyvinilglycine, and also the combinations of 1-MCP with GA3 and AVG with GA3, on ‘Murcott’ tangor and ‘Tahiti’ lime post-harvest. The cultivars were chosen because of their potential, both for the internal market as for fresh fruit exportation. The following concentrations were applied: GA3 at 20 mg L -1 ; 1-MCP at 0.1, 0.5 and 1.0 mL L -1 ; AVG at 10, 50 and 100 mg L -1 and the combinations of 1-MCP at 0.5 mL L -1 with GA3 at 20 mg L -1 and AVG at 50 mg L -1 with GA3 at 20 mg L -1 , and the control. The fruits were exposed to 1-MCP gas during 12 h in hermetic boxes at 20 o C. AVG and GA3 were applied dipping the fruits, for a minute, in solutions with the mentioned concentrations. In combinations, GA3 was applied after the treatments with 1-MCP or AVG. The experimental design was totally randomized factorial to check the action of 1-MCP and AVG on ‘Murcott’ tangor and ‘Tahiti’ lime. For the combinations of 1- MCP+GA3 and AVG+GA3, totally randomized design was used with time subdivided parcels. It was determined the evolution in the peel color (L, C* e h o ), the levels of total soluble solids ( o Brix), total titriable acidity (%), vitamin C, mass loss (%), juice content (%) and respiratory rate of the fruits under cold storage and after 3 days at 25 o C. The results were submitted to regression analysis. 1-MCP doesn’t have any effect on the peel color, in late application, on tangor ‘Murcott’. The 1-MCP at 0.5 mL L -1 was enough to reduce the respiratory rate and the waste of C vitamin. The physic-chemical characteristics were kept suitable for the consumption during 45 days of cold storage. The AVG, in post-harvest application, enhanced the peel color intensity of ‘Murcott’ tangor, decreased the waste of total soluble solids, the respiratory rate and didn’t affect the total titrable acidity. The combinations of 1-MCP and AVG with GA3 didn’t have any effect on the peel color of ‘Murcott’ tangor, after an advanced stage of peel coloration. The combinations didn’t affect the levels of total titrable acidity, C vitamin, mass loss and respiratory rate of ‘Murcott’ tangor. In relation to ‘Tahiti’ lime, the treatment with 1-MCP at 0.5 or 1.0 mL L -1 delayed changes in the peel color (color croma and angle), promoted higher concentration of total soluble solids and decreased the range of ratio variation. The AVG didn’t prevent peel color change, however it decreased the waste of TSS and of TTA; it didn’t affect the respiratory rate and the level of C vitamin. The combination of 1-MCP+GA3 delayed the evolution in the change of peel color during 60 days. The level of C vitamin was kept high. There weren’t changes in the respiratory rate and in juice content ‘Tahiti’ lime.