Diversos atletas e praticantes de atividades físicas incorporam em suas rotinas de treinamento, exercícios aeróbios e de força motora simultaneamente. Contudo, essa combinação conhecida como treinamento concorrente (TC) tem demonstrado uma atenuação da resposta adaptativa da força e hipertrofia muscular. O presente estudo analisou se alguns genes e proteínas envolvidos na resposta hipertrófica e na biogênse mitocondrial do músculo esquelético poderiam explicar a atenuação da resposta adaptativa com o TC. Trinta e sete sujeitos foram divididos nos grupos: controle (C), aeróbio (TA), força (TF) e concorrente (TC) e submetidos a oito semanas de treinamento. Os resultados significantes foram: aumento na força dinâmica máxima de 270,3 (±45,5) para 320,3 (±57,0) Kg para o TF e de 268,4 (±47,6) para 315,7 (±63,5) para o TC; área de secção transversa do quadríceps de 8332,4 (±817,5) mm2 para 8849,5 (±893,3) mm2 para o TF e de 8340,8 (±1000,0) mm2 para 8996,8 (±919,5 )mm2 para o TC; o gene da mTOR demonstrou aumento significante de 1,01 (±0,10) U.A para 1,44 (±0,17) U.A no TF e redução de 1,01 (±0,15) para 0,536 (± 0,25) U.A da p70S6K1 no TC; a expressão total da proteína p70S6K1 demonstrou aumentou no grupo TC em relação ao C (1,1 (±0,2) U.A vs 0,8 (±0,3) U.A), a fosforilação da Akt no resíduo ser473 e da p70S6K1no resíduo thr389 aumentou somente no TF em relação ao C (1,3 (±0,2) U.A vs 0,9 (±0,1) U.A e 1,3 (±0,4) vs 0,8 (±0,3) U.A, respectivamente). O grupo TF e TC demonstraram adaptações similares nas variáveis de força e hipertrofia muscular apesar de algumas diferenças na resposta molecular. Esses achados indicam que na fase inicial do TC as diferenças na adaptação molecular não refletem em alterações na força e hipertrofia muscular quando comparadas ao TF / Many athletes and individuals involved in physical training perform strength and endurance exercises in the same training unit. However, this combination, referred as concurrent training (CT), has shown to blunt strength and skeletal muscle growth responses. This study investigated whether some genes and proteins associated with muscle growth and mitochondrial biogenesis may explain the decreased adaptive response to CT. Thirty seven participants were divided into four groups: control (C), endurance (TA), strength (TF) and concurrent (TC) and submitted to eight weeks of training. Significant results were found in the following variables from pre to post training: maximum dynamic strength - TF from 270,3 (±45,5) to 320,3 (±57,0) Kg and TC from 268,4 (±47,6) to 315,7 (±63,5); quadriceps cross sectional area (CSA) - TF from 8332,4 (±817,5) mm2 to 8849,5 (±893,3) mm2, TC from 8340,8 (±1000,0) mm2 to 8996,8 (±919,5)mm2; mTOR gene expression increased significantly post-training only for the TF (1,01 (±0,10) A.U to 1,44 (±0,17) A.U) and p70S6K1 was significantly reduced post-training (1,01 (±0,15) to 0,536 (± 0,25) A.U) for the TC; p70S6K1 total protein content was significantly greater after TC when compared with C (1,1 (±0,2) U.A vs 0,8 (±0,3)) and phosphorylation of both Akt at ser473 and p70S6K1 at thr389 increased only after TF compared with C (1,3 (±0,2) U.A vs 0,9 (±0,1) U.A and 1,3 (±0,4) vs 0,8 (±0,3) U.A, respectively). TF and TC groups had similar improvements in muscle strength and hypertrophy, besides some differences in the molecular responses. These differences at the molecular level in early phases of the TC do not blunt muscle strength and hypertrophy adaptations compared with the TF

Doenças da córnea estão entre as principais causas de cegueira e os mecanismos de lesão e reparação estão em grande parte concentradas no epitélio, porém os mediadores e alvos para possíveis intervenções terapêuticas são pouco conhecidos.Nas agressões ao epitélio da córnea, a ativação dos receptores vanilóides de potencial transitório 1 (TRPV1) leva a resposta inflamatória e cicatricial. O objetivo desse trabalho é identificar os mecanismos de sinalização e resposta do TRPV1 na córnea de animais experimentais. O cultivo de células epiteliais de córnea de ratos, a identificação de mediadores por western blot, medida do influxo de cálcio, resposta de citocinas medidas por ensaio quantitativo de PCR em tempo real (RNAm) e ELISA (proteína) após estímulos nocioceptivos ou inflamatórios; e a investigação histológica e imunohistoquimica em modelos animais, sejam ratos com diabetes mellitus (DM) por 8 semanas ou expostos a cloreto de benzalcônio a 0,2% (BAC) por 7 dias. Camundongos TRPV1-/- comparados aos controles C57 foram comparados sem estimulo, ou sob estimulo único com CAP a 1µM, BAC 0,2 % por 7 dias ou após queimadura alcalina com NaOH a 1M. Os resultados mostraram a presença de TRPV1 em epitélio de córnea em cultura primária, mostraram que essas células respondem com influxo de cálcio ao estímulo com o agonista Capsaicina a 1µM e aumento de TNF? e IL-1? (sendo máximo nas combinações CAP+CPZ e Win+CAP, respectivamente). Em ratos com DM e BAC ocorre a redução da expressão do RNAm de IL-1? (p=0,0269) na córnea, entre outras alterações. Os camundongos TRPV1-/- tem fenótipo físico e ocular normal, porém reduzida sensibilidade a CAP 1µM. O tratamento com BAC leva a diminuição da secreção lacrimal (p=0,0011) e de IL-1? e TNF? na glândula lacrimal (GL) de TRPV1-/- machos (p=0,0177, p=0,0245, respectivamente). A queimadura alcalina com NaOH 1M resultou em melhor cicatrização e maior espessura epitelial no grupo TRPV1-/-. Em ix conclusão, o TRPV1 está presente na córnea e atua em resposta a agressões externas. Situações adquiridas como DM e toxicidade por BAC, reconhecidas como neuropáticas diminuem a expressão de mediadores inflamatórios. A ausência genética e TRPV1 não altera o fenótipo, mas apresenta menor sensibilidade e melhor restauração da superfície ocular após queimadura alcalina / Corneal diseases are among the leading causes of blindness.Damage and repair mechanisms are largely concentrated in the epithelium, but the mediators and targets for possible therapeutic interventions are poorly understood. The aggressions to the corneal epithelium, the activation of vanilloid receptors transient potential 1 (TRPV1) leads to inflammatory and wound healing. The aim of this study is to identify the signaling mechanisms and TRPV1 response in the cornea of experimental animals.The culture of epithelial cells from rat cornea, to identify mediators by western blot analysis, measurement of calcium influx, cytokine response measured by PCR real time (mRNA) and ELISA (protein) nocioceptivos or after inflammatory stimuli; and the histological and immunohistochemical investigation in animal models are mice with diabetes mellitus (DM) for 8 weeks, and exposed to benzalkonium chloride at 0.2% (BAC) for 7 days. Mice TRPV1-/- compared to control C57 were compared with no stimulus, or under single stimulation with 1 µM Capsaicine (CAP), BAC 0.2% for 7 days or after the alkali burn with NaOH 1 M.The results showed the presence of TRPV1 in corneal epithelium in primary culture, showed that these cells respond to calcium influx the stimulus with capsaicin agonist 1?M and increase of TNF? and IL-1? (being maximum at the CAP + CPZ combinations and Win + CAP, respectively). In rats with DM and BAC occurs reducing the expression of IL-1? mRNA (p = 0.0269) in the cornea, among other changes. The TRPV1-/- mice have physical phenotype and normal ocular but low sensitivity to CAP 1?M. Treatment with BAC leads to decreased tear secretion (p = 0.0011) and IL-1? and TNF? in the lacrimal gland (GL) of TRPV1-/- mice (p = 0.0177, p = 0.0245, respectively) . The alkaline burn with NaOH 1M resulted in better healing and greater epithelial thickness in TRPV1-/- group .In conclusion, TRPV1 is present in the cornea and operates in response to external aggressions. Situations existing as DM and xi toxicity BAC recognized as neuropathic decrease the expression of inflammatory mediators. Genetic absence and TRPV1 does not alter the phenotype, but has lower sensitivity and better restoration of the ocular surface after alkaline burn

Les complexes du groupe Polycomb (PcG) sont des répresseurs transcriptionnels capables de maintenir un état inactivé de la chromatine au niveau de leurs gènes cibles via des modifications post-traductionnelles des histones. Historiquement définis comme des répresseurs des gènes homéotiques, les protéines du PcG sont maintenant reconnues comme des répresseurs de gènes contrôlant le cycle cellulaire. Dans cette étude nous avons appréhendé l'implication des gènes du PcG E(z), Su(z)12, Pc, ph, Sce, Scm et Psc-Su(z)2 dans le contrôle de l'état prolifératif des cellules épithéliales des disques imaginaux in vivo. En utilisant des mutations nulles de ces gènes, nous avons étudié l'implication de ces protéines dans des processus biologiques tels que la prolifération, la croissance cellulaire, la différentiation et l'apoptose dont la dérégulation est associée à la tumorigénèse. Au cours de ce travail, nous avons constaté que les complexes du PcG ne se comportent absolument pas comme attendu : non seulement les différentes protéines composants le complexe PRC1 n'assument pas les mêmes fonctions, mais, par ailleurs, les complexes PRC1 et PRC2 ne collaborent pas et présentent même des effets antagonistes. Ainsi, nous avons répartit ces protéines dans deux sous-groupes : Le premier contient PH et Psc-Su(z)2 et agit comme suppresseur de tumeurs. Le deuxième groupe contient E(z), Su(z)12 et PC, des protéines qui favorisent la prolifération cellulaire plutôt que de l'inhiber. Enfin, nous avons recherché les cibles dont la dérégulation pourrait corréler avec les phénotypes associés à ces mutants. Nous avons identifié que certaines voies de signalisations impliquées dans le développement de l'œil de la Drosophile sont régulées de façon opposés par les protéines de ces deux sous-groupes. / Polycomb group (PcG) proteins are transcriptional repressors that were historically identified as regulators of homeotic genes. However, PcG proteins are now recognized as repressors of genes controlling the cell cycle. In this study we analyzed the role of the PcG genes E(z), Su(z)12, Pc, ph, Sce, Scm, and Psc-Su(z)2 in control of proliferation of epithelial cells in imaginal discs in vivo. Using null mutations of these genes, we investigated the involvement of these proteins in growth, differentiation and cell polarity. Surprisingly, we found that mutation of specific PcG proteins induce differential effects on the overall growth of the eye-antennal imaginal disc. In particular, we investigated the involvement of these proteins in biological processes such as proliferation, cell growth, differentiation and apoptosis, whose deregulation is associated with tumorigenesis. In this work, we found that PcG complexes do not behave as expected: different PRC1 proteins components do not assume the same functions, and PRC1 and PRC2 complexes may actually induce antagonistic effects. Thus, we have divided these proteins into two subgroups: The first contains PH and Psc-Su(z)2 and acts as tumor suppressors. The second group contains E(z), Su(z)12 and PC, and these proteins appear to favor cell proliferation. Finally, we looked for targets whose deregulation may correlate with the mutant phenotypes. We have identified several signaling pathways involved in Drosophila eye development that are regulated in an opposing manner in mutants of these two subgroups.

O fungo filamentoso Trichoderma reesei é uma espécie de grande importância biotecnológica no que tange a degradação de biomassa lignocelulósica para a produção de bioetanol em larga escala. Seu sistema de enzimas celulolíticas é muito eficiente, apesar de ser possuir poucas celulases, sugerindo que o controle dessas enzimas vai além da regulação transcricional. Assim, neste trabalho nós obtivemos o perfil fosfoproteômico de T. reesei cultivado em glicose e bagaço de cana-de-açúcar a partir de análise fosfoproteômica LC-MS/MS por spectral counting. A comparação entre os perfis de fosfoproteínas obtidos das linhagens parental QM6a de T. reesei e dos mutantes knockout para TMK1 e TMK2 permitiu a demonstração de que essas MAPK agem de maneira interconectada com outras vias de transdução de sinal na célula, especialmente a via de TOR e de AMPc-PKA, para regulação da produção de celulases. Além disso, também demonstramos a regulação da resposta a estresse celular por TMK2, e o papel da fosforilação no controle direto de enzimas CAZy. Nossos resultados mostram que a fosforilação desempenha papel importante na regulação dessas enzimas e de outras funções celulares no fungo após a transcrição de seus respectivos genes. O agrupamento desses dados permite melhor entendimento da via de sinalização mediada pelas MAPK TMK1 e TMK2 de T. reesei, e como as modificações pós-traducionais promovidas por elas afetam no sensing de nutrientes celulares e, por consequência, na produção de enzimas celulolíticas, de forma direta ou indireta. / The filamentous fungus Trichoderma reesei has great biotechnological importance in regards to the lignocellulosic biomass degradation for large-scale production of bioethanol. Its cellulolytic system is very efficient, despite being composed by only a few cellulases, which suggests that the control of these enzymes goes beyond their transcriptional regulation. Thus, in this study, we performed a spectral counting LC-MS/MS analysis and achieved the phosphoproteomic profile of T. reesei grown either in glucose or sugarcane bagasse as sole carbon source. The comparison between the phosphoproteins profiles obtained from the parental strain QM6a and the knockout mutants for TMK1 and TMK2 allowed us to demonstrate that these MAPK act in an interconnected manner with other signaling transduction pathways, especially the TOR and cAMP-PKA pathways, in order to regulate the cellulases production. Furthermore, we were also able to determine the regulation of cellular stress response by TMK2, and the role of phosphorylation in the direct control of CAZymes. Our results show that phosphorylation plays an important role on the control of these enzymes and other cellular functions in T. reesei after the transcription of their respective genes. Taken together, this data allows better comprehension of the signaling pathways mediated by TMK1 and TMK2 in T. reesei, and how the post-translational modification promoted by these MAPK might affect the nutrient sensing and, therefore, the production of the cellulolytic enzymes, either directly or indirectly.

L'autophagie est un processus d'autodigestion qui se produit dans toutes les cellules eucaryotes et conduit à la dégradation d'éléments du cytoplasme (organites, macromolécules) par le lysosome. Ce mécanisme, qui se produit de manière basale, permet le renouvellement du contenu cytoplasmique mais également la survie cellulaire lorsqu'il est induit par différents stress (carence nutritionnelle, hypoxie…). L'autophagie est alors impliquée dans diverses pathologies comme les maladies neurodégénératives et le cancer car sa dérégulation peut grandement perturber l'homéostasie cellulaire. Le but de ma thèse est de déterminer le rôle de la protéine nucléaire et cytoplasmique BAT3 dans l'autophagie et d'étudier son mécanisme de régulation. Cette protéine de 150 kDa, également appelée BAG6 ou Scythe, est composée de nombreux domaines protéiques (UBL, Prolin-rich, NLS, BAG) qui lui permettent d'interagir avec de multiples partenaires. Sa fonction majeure réside dans le contrôle qualité du cytoplasme mais BAT3 est aussi impliquée dans l'immunité ou l'apoptose. Ce travail identifie la protéine BAT3 comme essentiel pour l'autophagie basale et induite. Nous montrons que son mécanisme d'action passe par la régulation de la localisation de l'acétyltransférase p300 et l'acétylation de ces substrats : p53 et une protéine de la machinerie de l'autophagie : ATG7. En effet, BAT3 (i) limite la présence de p300 dans le cytosol en (ii) maintenant un faible et régulable niveau d'acétylation d'ATG7 et (iii) permet l'acétylation de p53 dans le noyau au cours de la carence nutritionnelle, événement indispensable à l'induction de l'autophagie. / Autophagy, literally meaning self-eating, is a highly evolutionary conserved process in eukaryotes in which parts of the cytoplasm (organelles, macromolecules) are degraded by lysosomes. Basal autophagy is a quality control mechanism allowing the renewal of the cytoplasm but autophagy is also induced by cellular stress (starvation, hypoxia…) to improve cell survival. Autophagy has been implicated in several physiopathologies such as cancer or neurodegenerative diseases. Deregulations of autophagy may profoundly affect homeostasis.The purpose of my thesis is to explore the role of the nucleo-cytoplasmic shuttling protein BAT3 in autophagy and the mechanism of BAT3-dependent autophagy.Also known as BAG6 or Scythe, this 150 kDa protein is composed of various domain (UBL, Prolin-Rich, NLS, BAG) by which BAT3 interacts with multiple partners. The major of role BAT3 seems to be the protein quality control but BAT3 is also implicated in immunity and apoptosis. Our work demonstrates that the protein BAT3 is essential for basal and starvation-induced autophagy. We show that BAT3 regulation of autophagy is mediated by the modulation of p300 acetyltransferase intracellular localization and acetylation of two subtrates: p53 and the autophagy-related protein ATG7. Indeed, Bat3 allows: (i) the limitation of p300 into cytosol resulting in (ii) the maintenance of a low level of ATG7 acetylation and (iii) the increase of the starvation-induced p53 autophagy leading to the induction of autophagy.

Estudos anteriores demonstraram que a crotoxina (CTX), o principal componente do veneno de Crotalus durissus terrifucus, apresenta ação anti-inflamatória, inibindo a migração celular e a atividade fagocítica de neutrófilos peritoneais. Esses efeitos inibitórios são prolongados, uma vez que podem ser observados até 14 dias após a administração de uma única dose dessa toxina. Considerando-se a vida média curta dos neutrófilos, é difícil explicar como a ação inibitória da CTX sobre os neutrófilos circulantes e peritoneais persiste por períodos prolongados após a administração de uma única dose da toxina. Dessa forma, o objetivo desse estudo foi avaliar o efeito in vitro e in vivo da CTX sobre a atividade funcional dos neutrófilos da medula óssea de camundongos e alguns dos mecanismos moleculares envolvidos na ação inibitória da CTX sobre as funções avaliadas. Para os ensaios in vitro, os neutrófilos foram incubados com a CTX (0,08 μg/mL), por 1 ou 24 horas. Para os ensaios in vivo, os animais foram pré-tratados com uma única administração de CTX (44 mg/kg), 1 dia antes do isolamento das células. Uma vez obtidos os neutrófilos, os seguintes parâmetros funcionais foram avaliados: quimiotaxia, adesão à fibronectina, fagocitose, produção de espécies reativas do oxigênio e desgranulação. Os resultados obtidos demonstraram que a CTX, in vitro e in vivo, inibiu os processos de quimiotaxia, adesão à fibronectina e fagocitose de partículas opsonizadas, entretanto não alterou a produção de espécies reativas do oxigênio ou a desgranulação em neutrófilos da medula óssea. Esses resultados demonstram que a CTX induz efeito inibitório sobre a funcionalidade dos neutrófilos da medula óssea, particularmente sobre funções associadas à polimerização de actina e consequente reorganização do citoesqueleto. Ainda, com o objetivo de elucidar os possíveis mecanismos envolvidos neste efeito inibitório, foram realizados ensaios para a análise da expressão do receptor CR3, bem como para a avaliação da expressão total e da atividade de proteínas de sinalização intracelular envolvidas na polimerização de actina nos neutrófilos. Os resultados obtidos mostraram que a CTX inibiu a expressão de ambas as subunidades (CD11b e CD18) do receptor CR3, bem como inibiu a atividade de Syk, Vav1, Cdc42, Rac1 e RhoA e a expressão da subunidade 1B do complexo Arp2/3. Em conjunto, os resultados desse estudo mostraram que a CTX inibe a funcionalidade dos neutrófilos da medula óssea e que essa ação está associada à inibição do receptor CR3, bem como à inibição da atividade de Syk e de suas proteínas downstream, o que resulta na redução da formação de filamentos de F-actina. Os resultados desse estudo comprovam a hipótese de que ação inibitória prolongada da CTX sobre a atividade dos neutrófilos circulantes e peritoneais está associada a alterações funcionais dos neutrófilos da medula óssea. Ainda, considerando-se a participação central dessas células na resposta inflamatória aguda, esse estudo contribui para a elucidação do efeito anti-inflamatório prolongado da CTX / Previous studies demonstrated that crotoxin (CTX), the main component of Crotalus durissus terrificus venom, presents anti-inflammatory properties, inhibiting cell migration and the phagocytic activity of peritoneal neutrophils. These inhibitory effects are long-lasting, since it can be observed up to 14 days after a single administration of this toxin. Considering the short half-life of neutrophils, it is difficult to explain how the inhibitory effect of CTX on circulating and peritoneal neutrophils persists for long periods after a single injection of this toxin. Thus, the aim of this study was to evaluate the in vitro and in vivo effect of CTX on the functionality of bone marrow neutrophils from mice and some of the molecular mechanisms involved on the inhibitory effect of CTX on these functions. For in vitro assays, neutrophils were incubated with CTX (0,08 μg/mL), for 1 or 24 hours. For in vivo assays, the animals were pretreated with a single administration of CTX (44 mg/kg), 1 day before the isolation of cells. Once obtained the neutrophils, the following functional parameters were evaluated: chemotaxis, adhesion to fibronectin, phagocytosis, reactive oxygen species production and degranulation. The results demonstrated that CTX, in vitro and in vivo, inhibited the processes of chemotaxis, adhesion to fibronection and phagocytosis of opsonized particles, however, it did not alter the reactive oxygen species production and degranulation. These results showed that CTX induces an inhibitory effect on the functionality of bone marrow neutrophils, particularly on functions that depend on actin polymerization and cytoskeleton rearrangement. Furthermore, to elucidate some possible mechanisms involved on this inhibitory effect, assays to analyze the expression of the receptor CR3, as well as, assays to analyze the total expression and activity of signaling proteins involved on actin polymerization were done. The results showed that CTX inhibited both subunits of CR3 (CD11b and CD18) and the activity of Syk, Vav1, Cdc42, Rac1 and RhoA and the expression of the subunit 1B from Arp2/3. Together, the results presented herein demonstrate that CTX inhibits the functionally of bone marrow neutrophils and that this effect is associated to the inhibition of CR3 receptor and inhibition of the activity of Syk and its downstream signaling proteins, which results in the decrease of F-actin. The results prove the hypothesis that the long-lasting inhibitory effect of CTX on the activity of circulating and peritoneal neutrophils is associated to functional modifications of bone marrow neutrophils. Besides, considering the central role of these cells on the inflammatory response, this study contributes to the better understanding of the long-lasting anti-inflammatory effect of CTX

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas β3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores β3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade βγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas. / During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of β3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of β3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through βγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

Model checking is a formal verification technique which is widely used in different areas for automated verification and analysis. In this study, we applied a Model Checking method to a biological system. Firstly we constructed a single-cell, Boolean network model for the signaling pathways of apoptosis (programmed cell death) in lung cancers by combining the intrinsic and extrinsic Apoptosis pathways, p53 signaling pathway and p53 - DAP Kinase pathway in Lung cancers. We translated this model to the NuSMV input language. Then we converted known experimental results to CTL properties and checked the conformance of our model with respect to biological experimental results. We examined the dynamics of the apoptosis in lung cancer using NuSMV symbolic model checker and identified the relationship between apoptosis and lung cancer. Finally we generalized the whole process by introducing translation rules and CTL property patterns for biological queries so that model checking any signaling pathway can be automated.

Cell signaling is a list of chemical reactions that are used for intercellular and intracellular communication. Signaling pathways denote these chemical reactions in a systematic manner. Today, many signaling pathways are constructed by several experimental methods. However there are still many communication skills of cells that are needed to be discovered. RNAi system allows us to see the phenotypes when some genes are removed from living cells. By observing these phenotypes, we can build signaling pathways. However it is costly in terms of time and space complexity. Furthermore, there are some interactions RNAi data cannot distinguish that results in many different signaling pathways all of which are consistent with the RNAi data. In this thesis, we combine genetic algorithms with some greedy approaches to find the topologies that fit the Boolean single knock-down RNAi experiments. Our algorithm finds nearly all of the results for small inputs in a few minutes. It can also find a significant number of results for larger inputs. Then we eliminate isomorphic topologies from the output set of this algorithm. This process fairly reduces the number of topologies. Afterwards we offer a simple scheme for suggesting new wet-lab RNAi experiments which is necessary to have a complete approach to find the actual network. Also we describe a new activation and deactivation model for pathways when the activation of the phenotype after RNAi knock-downs are given as weighted variables. We adapt the first genetic algorithm approach to this model for directly finding the most possible network.

Τα ουδετερόφιλα κύτταρα του ανθρώπινου περιφερικού αίματος, ανήκουν σε μία κατηγορία κυττάρων, τα οποία ονομάζονται επαγγελματίες φαγοκύτταρα και τα οποία είναι υπεύθυνα για την πρόσληψη και θανάτωση των παθογόνων μικροοργανισμών (Bessler et al.2002). Από παλαιότερες έρευνες της ερευνητικής μας ομάδας, έχει δειχθεί η συμμετοχή των σηματοδοτικών μορίων FAK, Elk-1 και MAP κινασών στη ρύθμιση της κυτταροφαγίας από τα εξειδικευμένα φαγοκύτταρα (πλασματοκύτταρα), που βρίσκονται στην αιμολέμφο της μύγας της Μεσογείου, Ceratitis capitata. (Lamprou et al. 2007, Mamali et al. 2008). Είναι επίσης γνωστό ότι κατά την κυτταροφαγία παράγονται διάφορες μορφές δραστικού οξυγόνου (ROS) και αζώτου (RNS), με κύριες το Η2Ο2 και ΝΟ αντίστοιχα. Οι παραγόμενες ROS και RNS συμμετέχουν ως μόρια μεταγωγής σήματος σε διάφορα ενδοκυτταρικά σηματοδοτικά μονοπάτια που ρυθμίζουν την κυτταροφαγία από τα λευκοκύτταρα. Όμως τα βιβλιογραφικά δεδομένα στο πεδίο αυτό είναι λιγοστά. Στην παρούσα εργασία, μελετήθηκε η συμμετοχή των FAK, Elk-1, MAP κινασών και Η2Ο2 στην κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου καθώς και η αλληλεπίδραση μεταξύ των FAK, Elk-1, MAP κινασών και του Η2Ο2 που παράγεται από τα κύτταρα λόγω της παρουσίας των βακτηρίων E.coli και S.aureus. Με κυτταρομετρία ροής και τη χρήση του εξειδικευμένου φθοροχρώματος διυδροροδαμίνη (DHR), πιστοποιήθηκε η παραγωγή Η2Ο2 από τα ουδετερόφιλα κύτταρα του αίματος, παρουσία E.coli και S.aureus. Η ενεργός συμμετοχή του παραγόμενου Η2Ο2 αποδείχτηκε όταν η μείωση της παραγωγής υπεροξεικών ανιόντων με τη χρήση του εξειδικευμένου αναστολέα Ν-εθυλεν-μαλεϊμίδιο (NEM), μείωσε την πρόσληψη βακτηρίων από τα κύτταρα. Η συμμετοχή των FAK και Elk-1 στην κυτταροφαγία ελέγχθηκε με καταστολή της γονιδιακής τους έκφρασης με τη χρήση μορίων siRNA, ενώ αντίστοιχα για τις MAP κινάσες χρησιμοποιήθηκαν αναστολείς της φωσφορυλίωσης τους, οι οποίοι καταστέλλουν την ενεργότητα τους. Με τη χρήση των ίδιων μεθόδων αναστολής για τα μόρια FAK, Elk-1 και MAPKs, μελετήθηκε και ο ρόλος τους στην παραγωγή Η2Ο2 κατά την κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου. Τέλος, ερευνήθηκε και ο ρόλος του Η2Ο2, σαν σηματοδοτικό μόριο κατά τη διαδικασία της κυτταροφαγίας, μελετώντας με ανάλυση κατά western, την επίδραση της αναστολής της παραγωγής του Η2Ο2, στην φωσφορυλίωση (ενεργοποίηση) των MAP κινασών. / Neutrophil cells from human peripheral blood belong in a group of cells, that are called professional phagocytes and are responsible for the uptake and killing of pathogen microorganisms (Bessler et al.2002). Research data from our lab, has shown that the signaling molecules FAK, Elk-1 and MAP kinases participate in the regulation of phagocytosis from the phagocytes of the Mediterranean fly, C.capitata (Lamprou et al. 2007, Mamali et al. 2008). It is also known, that during phagocytosis phagocytes produce different forms of reactive oxygen species (ROS) and nitrogen species (RNS), mainly Η2Ο2 and ΝΟ, respectively. Produced ROS and RNS, act as signaling molecules in different intracellular signaling pathways that regulate phagocytosis in leykocytes. . In the present work, we examined the participation of FAK, Elk-1, MAPKs and Η2Ο2 in the phagocytosis of bacteria E.coli and S.aureus and also the interaction between FAK, Elk- 1, MAPKs and Η2Ο2 that is produced from the cells, due to the presence of the bacteria E.coli and S.aureus. Using flow cytometry and the specialized fluorescent dye Dihydrorodamine-123 (DHR), we certified the production of Η2Ο2 from the human neutrophil cells, in the presence of E.coli and S.aureus. The participation of the produced Η2Ο2 was shown, when the reduction of the produced Η2Ο2 with the use of the inhibitor Ν-etheylen-maleimide (NEM), reduced the uptake of bacteria from the cells. The participation of FAK and Elk-1 in phagocytosis was examined by suppressing their expression with the use of siRNA molecules, while for the MAP kinases we used specific activation inhibitors. By using the same inhibitng methods for the molecules FAK, Elk-1 and MAPKs we studied their role in the production of Η2Ο2 during the phagocytosis of bacteria E.coli and S.aureus from human neutrophil cells. Finally, by reducing the production of superoxide anions, thereby reducing the production of H2O2, by using the specific inhibitor Ν-etheylen-maleimide (NEM), we examined the expression of the phosphorylated forms of MAPKs, by western analysis, in human neutrophils in the presence of the bacteria E.coli and S.aureus.