Orientador: Maria José Soares Mendes Giannini / Banca: Christiane Pienna Soares / Banca: Paulo Inácio da Costa / Banca: Márcia de Souza Carvalho Melhem / Banca: Gil Bernard / Resumo: Neste trabalho foi realizado um estudo comparativo entre isolados de Pb18 e Pb265, que têm capacidades distintas de adesão às células Hela e Vero, sob influência da temperatura e diversos fatores químicos. Foram também avaliados eventos em células de epitélio pulmonar expostas à gp 43 e células de Pb18, antes e após inoculação animal, representando, respectivamente, isolados menos e mais virulentos. Os resultados mostram que as adesinas fúngicas são termo-lábeis, sugerindo sua natureza protéica. Os açúcares aminados glucosamina e galactosamina foram os mais eficientes para inibir a adesão celular, em relação à manose, glicose e galactose, indicando que esta atividade envolve mecanismos específicos tipo lectina. O estudo com componente da matriz extracelular mostrou que a laminina, assim como seus derivados sintéticos, inibiu a adesão do isolado Pb18, fato não verificado com Pb265, tanto com células Hela, quanto com a linhagem Vero, mas variável sob influência de fatores químicos. As distintas vias de sinalização foram demonstradas pelos eventos Ras-Raf, Rho e AKT verificados, sendo que o isolado menos virulento causou menos estresse citotóxico, comprovado por menor sinal Ras- Raf e AKT de proliferação. Os sinais intensos de proliferação observados nos experimentos com o isolado mais virulento e exposição à gp 43 poderiam ser associados à maior sobrevida do fungo que se internaliza mais facilmente nas células do hospedeiro, dificultando o seu reconhecimento por células do sistema monocítico-fagocitário, antes de causar apoptose, evadir e disseminar-se. A ativação das vias de sobrevida, associada a sinais citosólicos quando o Pb entra na célula epitelial, foi demonstrada de modo inédito neste trabalho. / Abstract: In this work it was accomplished a comparative study between isolates Pb18 and Pb265, which have distinct capacities of adhesion to the cells Hela and Vero under influence of temperature and several chemical factors. Events in pulmonary epithelial cells exposed to gp 43 and in Pb18 cells, before and after animal inoculation, representing, respectively, isolates less and more virulent were evaluated as well. The results show that the fungal adhesins are term-labels, suggesting its proteical nature. The aminated sugars glucosamine and galactosamine were the most efficient, in comparison with manose, glycose and galactose, in inhibiting the cellular adhesion, indicating that this activity involves specific mechanisms lectin-type. The study with extracellular matrix component indicated that the laminin, as well as its synthetic derivatives, inhibited the adhesion of the isolate Pb18 to the cells Vero and Hela, what did not occur with Pb265, but it was variable under influence of chemical factors. The distinct signaling pathways were demonstrated by the Ras-Raf, Rho and AKT events, considering that the less virulent isolate caused minor cytotoxic stress, proved by minor Ras-Raf and AKT proliferation signals. The intense proliferation signals observed in the experiments with more virulent isolate and exposition to gp 43 could be associated to larger time of survival of the fungus that more easily goes inside the host cells, making it difficult to the monocyte-macrophage system cells to recognize it before the apoptosis, the evasion and the dissemination. The Ras-Raf and AKT pathways acted synergetically with the effects of the cell survival. The decrease of the AKT event implied partial loss of the survival signal that intensified, following and in a opposite way, with Ras-Raf decrease. The activation of the survival pathways, associated to citosolic signals when Pb goes inside the epithelial cell, was for the first time demonstrated in this work. / Doutor

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T19:53:30Z (GMT). No. of bitstreams: 1 Santos_AlineMarados_D.pdf: 7023687 bytes, checksum: ccd063ea57be631b6b555f86e24af597 (MD5) Previous issue date: 2011 / Resumo: A tirosino-quinase de adesão focal (FAK) tem papel crítico na mediação da migração, sobrevivência e proliferação celular. Estudos anteriores de nosso laboratório demonstraram que a FAK é ativada pelo estresse mecânico em miócitos cardíacos e que ela se coimunoprecipita com a miosina sarcomérica. No presente trabalho foi demonstrado que o domínio FERM da FAK medeia à interação com a miosina sarcomérica, sendo que esta interação leva a inibição da autofosforilação da FAK, enquanto que a ativação prévia da FAK reduz sua interação com a miosina in vitro. Ensaios de cross linking acoplado a espectrometria de massas e espalhamento de raios X a baixos ângulos demonstraram que a miosina interage em uma fenda localizada entre os subdomínios do domínio FERM. Experimentos de microscopia confocal demonstraram que estas proteínas estão colocalizadas em miócitos cardíacos de ratos neonatos e adultos. Ensaios de imunoprecipitação revelaram que aproximadamente 40% da FAK está basalmente associada à miosina sarcomérica enquanto que, após o estiramento celular esta associação reduziu paralelamente à ativação da FAK. A porcentagem de FAK associada à miosina não se alterou com a ativação da FAK após tratamento com fenilefrina, diferente da ativação pelo estresse mecânico. A interferência na interação FAK/miosina pelo silenciamento gênico da miosina culminou com a ativação da FAK e o tratamento dos miócitos cardíacos com o peptídeo FP-1, derivado do subdomínio F2 do domínio FERM, levou a uma diminuição na interação FAK/miosina e ao aumento na fosforilação/ativação da FAK. O tratamento prolongado com FP-1 resultou em hipertrofia dos miócitos cardíacos de ratos neonatos, efeito concomitante à ativação da via de sinalização Akt, TSC2 e S6Kinase. Tanto o silenciamento da FAK quanto o tratamento com rapamicina bloquearam a hipertrofia decorrente do tratamento com FP-1. Os dados deste trabalho indicam que a interação da FAK com a miosina sarcomérica é sensível ao estresse mecânico e que possui papel regulatório na manutenção da quiescência basal da FAK e no controle das vias de sinalização mediadas por esta quinase, como a via de crescimento celular AKT/mTOR/S6Kinase, em miócitos cardíacos em cultura / Abstract: The Focal Adhesion Kinase (FAK) plays a critical role in mediating the migration, survival and cell proliferation. Previous studies from our laboratory demonstrated that FAK is activated by mechanical stress in cardiac myocytes and it co-immunoprecipitate with sarcomeric myosin. Here, we demonstrated that the FAK FERM domain mediates the interaction with sarcomeric myosin, and that this interaction leads to inhibition of FAK autophosphorylation in vitro, whereas the previous activation of FAK reduces its affinity to myosin. A model based on small angle X-ray scattering analyses and crosslinking technology coupled with mass spectrometry indicated that a cleft in FERM domain is critical to the interaction of FAK to myosin. Confocal microscopy experiments showed that these proteins are colocalized in cardiomyocytes of neonatal and adult rats. Immunoprecipitation assays revealed that approximately 40% of FAK is basally associated with sarcomeric myosin while cardiomyocyte stretching reduced this association in parallel with FAK activation. The percentage of FAK associated with myosin was not change in response to FAK activation by treatment with phenylephrine, unlike in response to FAK by mechanical stress. The interference in the FAK/Myosin interaction by myosin silencing approach culminated with the activation of FAK. The treatment of cells with the FP-1 peptide, derived from the FAK FERM domain, lead to a decrease in the interaction with sarcomeric myosin and an increase in FAK activation. Prolonged treatment with FP-1 resulted in morphological hypertrophy of neonatal rat cardiomyocytes which was an effect concomitant with activation of the Akt, TSC2 and S6Kinase signaling pathway. Both FAK silencing and the rapamycin treatment blocked the morphological hypertrophy resulting of the treatment with FP-1. This study indicated that the interaction of FAK with sarcomeric myosin is sensitive to mechanical stress and it has regulatory role in maintaining of FAK quiescence and control of signaling pathways mediated by this kinase, such as cell growth via AKT/mTOR/S6Kinase in cardiac myocytes in culture / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

No description available.

Résumé Les systèmes microbiologiques naturels (colonne d’eau), semi-naturels (station d’épuration), mais surtout industriels ou de laboratoire (bioréacteurs) sont communément représentés par des modèles mathématiques destinés à l’étude, à la compréhension des phénomènes ou au contrôle des processus (de production, par exemple). Dans l’énorme majorité des cas, lorsque les cellules (procaryotes ou eucaryotes) mises en jeu dans ces systèmes sont en suspension, le formalisme de ces modèles non structurés traite le système comme s’il était homogène. Or, en toute rigueur, il est clair que cette approche n’est qu’une approximation et que nous avons à faire à des phénomènes hétérogènes, formés de plusieurs phases (solide, liquide, gazeuse) intimement mélangées. Nous désignons ces systèmes comme « polyphasiques dispersés » (SPD). Ce sont des systèmes thermodynami-quement instables, (presque) toujours ouverts. La démarche que nous avons entreprise consiste à examiner si le fait de considérer des systèmes dits « homogènes » comme des systèmes hétérogènes (ce qu’ils sont en réalité) apporte, malgré une complication du traitement mathématique, un complément d’information significatif et pertinent. La démarche s’est faite en deux temps : · Une étape purement théorique, destinée à établir de manière rigoureuse et générale les bilans de matière pour chaque composé du système dans chacune de ces phases. · Une étape appliquée, visant à démontrer, au travers d’exemples concrets, la validité du concept et de la démarche. Pour l’étude des applications, pour diverses raisons, nous avons choisi d’étudier un bioréacteur ouvert « simple », le chémostat. Les bilans généraux dérivés à la première étape ont donc été appliqués à ce réacteur et plusieurs exemples, tirés de la littérature, pour la plupart, ont été traités dans le cadre des SPD. Les principaux résultats exposés dans le travail concernent : - sur le plan général, la pertinence d’une partition des systèmes en plusieurs phases, ce qui fait apparaître à la fois des flux d’échange interphasiques (qui n’apparaissent pas dans les systèmes dits monophasiques) et la possibilité de représenter le système à plusieurs niveaux de description. - quant aux applications, outre quelques petits exemples simples, nous proposons 1) un nouveau mécanisme pour représenter la dissipation de l’énergie cellulaire (un domaine encore très controversé), grâce à une approche implicite (c’est-à-dire, sans hypothèses particulières sur la forme des cinétiques intracellulaires) et 2) un modèle simple, original et innovant pour expliquer les signaux chimiques intercellulaires, les phénomènes de seuil et le branchement métabolique respiro-fermentatif en général et chez Saccharomyces cerevisiae en particulier, un mécanisme d’intérêt fondamental et industriel (levuristes et fermentations alcooliques). Abstract. Natural microbiological systems (rivers, seas, …), semi-natural (wastewater treatment plants), but especially industrial or lab-scale systems (bioreactors) are commonly represented by mathematical models intended for the study, the understanding of phenomena or for the control of processes (production, for example). In almost in every case, when the cells (prokaryotic or eukaryotic) concerned in these systems are in suspension, the formalism of these unstructured models treats the system as if it were homogeneous. However, in any rigor, this approach is clearly only an approximation and we have to deal with heterogeneous phenomena, formed of several phases (solid, liquid, gas) closely mixed. We refer to these systems as “polyphasic dispersed systems” (PDS). They are thermodynamically unstable systems, and are (practically) always open. The approach we undertook consists in examining if treating apparent «homogeneous» systems as heterogeneous systems (what they actually are) brings, in spite of some mathematical complications, further significant and relevant information’s. We proceeded in two steps: · A purely theoretical stage, intended to establish in a rigorous and general way the mass balances for each compound in each phases of the system. · A applied stage, aiming at showing, through concrete examples, the soundness of the concept and of the method. Concerning the applications, for several reasons, we chose to study a “simple” open bioreactor: the chemostat. The general balances previously derived in a general way were hence applied to this reactor and a number of examples, mainly obtained from the literature, were treated within the PDS framework. The principal results presented in this work concern: - on the general level, the importance of partitioning the system in different phases, enlightening at the same time interphasic exchange flows (which do not appear in the systems known as monophasic) and the possibility of representing the system on several levels of description. - concerning the applications, in addition to some small simple examples, we propose 1) a new mechanism representing the cellular energy dissipation (a still very controversial field), using an implicit approach (i.e., without particular assumptions about the form of the intracellular kinetics) and 2) a simple, original and inventive model explaining cellular chemical signaling, threshold phenomena and a general metabolic switch occurring during respirofermentative transition. The latter was especially tested on Saccharomyces cerevisiae data to interpret the Crabtree effect in yeast, a mechanism of fundamental and industrial importance (in connection with baker’s yeast production and alcoholic fermentations).

Grâce aux nombreuses études sur le sujet, nous savons qu’une stimulation inflammatoire vasculaire excessive entraîne un débalancement des fonctions homéostatiques de l’endothélium. Ce débalancement est à l’origine d’une dysfonction endothéliale définie comme étant l’étape clé contribuant au développement de l’athérosclérose. Le Toll-like receptor-2 (TLR2) est impliqué dans l’activation cellulaire via la transcription des gènes liés à l’inflammation. Il reconnaît des molécules microbiennes mais également des facteurs endogènes non-infectieux tels que sécrétés par les tissus endommagés provenant de la dysfonction endothéliale. Ainsi, l’activation et la signalisation du TLR2 sont en étroite relation avec le développement de l’athérosclérose. Les études épidémiologiques ont confirmé le rôle athéroprotecteur de l’œstrogène via de nombreux mécanismes d’action. Ainsi, nous avons cherché à identifier de nouvelles cibles moléculaires permettant de mieux interpréter les bénéfices potentiels de l’œstrogène sur le système cardiaque. Pour la première fois chez les cellules endothéliales (CE) vasculaires de souris, nos travaux ont confirmé l’effet anti-inflammatoire de l’œstrogène via la diminution de l’expression et de l’activité du TLR2. Nous avons également déterminé l’influence de l’œstrogène sur le profil de la réponse inflammatoire de ce récepteur en mesurant les potentiels endothéliaux de migration et d’adhésion. De plus, nous avons caractérisé les voies de signalisation impliquées en démontrant l’influence négative de l’œstrogène sur la phosphorylation des kinases activées par le TLR2; illustrant l’interaction entre l’œstrogène et la signalisation de ce récepteur. Nos travaux amènent ainsi de nouvelles connaissances sur la régulation endothéliale du TLR2 et mettent en lumière les effets anti-inflammatoires et vasculaires rapides de l’œstrogène. / Evidence supports the contribution of immune responses in atherosclerosis development in part by alterations in the endothelium activation status and by the recruitment of inflammatory cells triggered by cardiovascular risk factors. These alterations are the principal cause of endothelial dysfunction defined as the key step contributing to the development of atherosclerosis. Via the transcription of genes related to inflammation, the Toll-like receptor-2 (TLR2) is involved in endothelial cell activation. It generally recognizes microbial molecules but also non-infectious endogenous factors such as those secreted by damaged tissues from the endothelial dysfunction. Thus, activation and signalization of the TLR2 are closely linked with the development of atherosclerosis. Epidemiological studies have confirmed the atheroprotective role of estrogen through multiple mechanisms of action. Thus, to better interpret the potential benefits of estrogen on the cardiovascular system, we sought to identify new molecular targets such as TLR2 regulation. For the first time in mouse vascular endothelial cells (EC), our results have confirmed the anti-inflammatory effect of estrogen via the decreased expression and activity of TLR2. We also determined the influence of estrogen on the profile of the inflammatory response triggered through this receptor by measuring endothelial migration and adhesion potentials. Furthermore, we demonstrated the interaction between estrogen and TLR2 signalling pathways with a negative influence of estrogen on the phosphorylation level of kinases activated by this receptor. Thus, our study brings new insights into the endothelial regulation of TLR2 and highlights rapid anti-inflammatory and cardioprotective effects from estrogen.

Grâce aux nombreuses études sur le sujet, nous savons qu’une stimulation inflammatoire vasculaire excessive entraîne un débalancement des fonctions homéostatiques de l’endothélium. Ce débalancement est à l’origine d’une dysfonction endothéliale définie comme étant l’étape clé contribuant au développement de l’athérosclérose. Le Toll-like receptor-2 (TLR2) est impliqué dans l’activation cellulaire via la transcription des gènes liés à l’inflammation. Il reconnaît des molécules microbiennes mais également des facteurs endogènes non-infectieux tels que sécrétés par les tissus endommagés provenant de la dysfonction endothéliale. Ainsi, l’activation et la signalisation du TLR2 sont en étroite relation avec le développement de l’athérosclérose. Les études épidémiologiques ont confirmé le rôle athéroprotecteur de l’œstrogène via de nombreux mécanismes d’action. Ainsi, nous avons cherché à identifier de nouvelles cibles moléculaires permettant de mieux interpréter les bénéfices potentiels de l’œstrogène sur le système cardiaque. Pour la première fois chez les cellules endothéliales (CE) vasculaires de souris, nos travaux ont confirmé l’effet anti-inflammatoire de l’œstrogène via la diminution de l’expression et de l’activité du TLR2. Nous avons également déterminé l’influence de l’œstrogène sur le profil de la réponse inflammatoire de ce récepteur en mesurant les potentiels endothéliaux de migration et d’adhésion. De plus, nous avons caractérisé les voies de signalisation impliquées en démontrant l’influence négative de l’œstrogène sur la phosphorylation des kinases activées par le TLR2; illustrant l’interaction entre l’œstrogène et la signalisation de ce récepteur. Nos travaux amènent ainsi de nouvelles connaissances sur la régulation endothéliale du TLR2 et mettent en lumière les effets anti-inflammatoires et vasculaires rapides de l’œstrogène. / Evidence supports the contribution of immune responses in atherosclerosis development in part by alterations in the endothelium activation status and by the recruitment of inflammatory cells triggered by cardiovascular risk factors. These alterations are the principal cause of endothelial dysfunction defined as the key step contributing to the development of atherosclerosis. Via the transcription of genes related to inflammation, the Toll-like receptor-2 (TLR2) is involved in endothelial cell activation. It generally recognizes microbial molecules but also non-infectious endogenous factors such as those secreted by damaged tissues from the endothelial dysfunction. Thus, activation and signalization of the TLR2 are closely linked with the development of atherosclerosis. Epidemiological studies have confirmed the atheroprotective role of estrogen through multiple mechanisms of action. Thus, to better interpret the potential benefits of estrogen on the cardiovascular system, we sought to identify new molecular targets such as TLR2 regulation. For the first time in mouse vascular endothelial cells (EC), our results have confirmed the anti-inflammatory effect of estrogen via the decreased expression and activity of TLR2. We also determined the influence of estrogen on the profile of the inflammatory response triggered through this receptor by measuring endothelial migration and adhesion potentials. Furthermore, we demonstrated the interaction between estrogen and TLR2 signalling pathways with a negative influence of estrogen on the phosphorylation level of kinases activated by this receptor. Thus, our study brings new insights into the endothelial regulation of TLR2 and highlights rapid anti-inflammatory and cardioprotective effects from estrogen.

Les systèmes microbiologiques naturels (colonne d’eau), semi-naturels (station d’épuration), mais surtout industriels ou de laboratoire (bioréacteurs) sont communément représentés par des modèles mathématiques destinés à l’étude, à la compréhension des phénomènes ou au contrôle des processus (de production, par exemple).<p><p>Dans l’énorme majorité des cas, lorsque les cellules (procaryotes ou eucaryotes) mises en jeu dans ces systèmes sont en suspension, le formalisme de ces modèles non structurés traite le système comme s’il était homogène. Or, en toute rigueur, il est clair que cette approche n’est qu’une approximation et que nous avons à faire à des phénomènes hétérogènes, formés de plusieurs phases (solide, liquide, gazeuse) intimement mélangées. Nous désignons ces systèmes comme « polyphasiques dispersés » (SPD). Ce sont des systèmes thermodynami-quement instables, (presque) toujours ouverts.<p><p>La démarche que nous avons entreprise consiste à examiner si le fait de considérer des systèmes dits « homogènes » comme des systèmes hétérogènes (ce qu’ils sont en réalité) apporte, malgré une complication du traitement mathématique, un complément d’information significatif et pertinent. <p><p>La démarche s’est faite en deux temps :<p>·\ / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished