Modulação de proteínas regulatórias e juncionais da barreira hematotesticular em células de Sertoli humanas (HSec) expostas ao Monobutil ftalato (MBP) e ao Bisfenl A (BPA)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Criptorquidia e exposição in utero ao di(n-butil)-ftalato e à acrilamida: avaliação morfológica do dano testicular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dielectrophoresis (dep) and electrowetting (ewod) as an anti-fouling process for antibacterial surfaces

Nowadays the medical field is struggling to decrease bacteria biofilm formation which leads to infection. Biomedical devices sterilization has not changed over a long period of time. This results in high costs for hospitals healthcare managements. The objective of this project is to investigate electric field effects and surface energy manipulation as solutions for preventing bacteria biofilm for future devices. Based on electrokinectic environments 2 different methods were tested: feasibility of electric gradient through mediums (DEP) reinforced by numerical simulations; and EWOD by the fabrication of golden interdigitated electrodes on silicon glass substrates, standard ~480 nm Teflon (PTFE) layer and polymeric gasket to contain the bacteria medium. In the first experiment quantitative analysis was carried out to achieve forces required to reject bacteria without considering dielectric environment limitations as bacteria and medium frequency dependence. In the second experiment applied voltages was characterized by droplets contact angle measurements and put to the live bacteria tests. The project resulted on promising results for DEP application due to its wide range of frequency that can be used to make a “general” bacteria rejecting; but in terms of practicality, EWOD probably have higher potential for success but more experiments are needed to verify if can prevent biofilm adhesion besides the Teflon non-adhesive properties (including limitations as Teflon breakthrough, layer sensitivity) at incubation times larger than 24 hours.

Rôles de la protéine tyrosine phosphatase DEP-1 dans l'angiogenèse, la perméabilité vasculaire et la progression tumorale

L’angiogenèse et l’augmentation de la perméabilité vasculaire sont des éléments clés pour la croissance et la progression tumorale. Par conséquent, de nombreux efforts sont déployés à comprendre les mécanismes moléculaires impliqués dans la formation et le remodelage des vaisseaux sanguins de manière à identifier de nouvelles cibles thérapeutiques potentielles. De cette optique, les travaux de cette thèse se sont concentrés sur la protéine tyrosine phosphatase DEP-1, initialement identifiée comme un régulateur négatif de la prolifération et de la phosphorylation du VEGFR2 lorsque fortement exprimée dans les cellules endothéliales. Toutefois, en utilisant une approche d’ARNi, il a été démontré que via sa capacité à déphosphoryler la tyrosine inhibitrice de Src (Y529), DEP-1 était également un régulateur positif de l’activation de Src dans les cellules endothéliales stimulées au VEGF. Puisque Src joue un rôle central dans la promotion de l’angiogenèse et la perméabilité vasculaire, nous avons en plus démontré que DEP-1 était un promoteur de ces fonctions in vitro et que la tyrosine phosphorylation de sa queue C-terminale, permettant l’interaction et l’activation de Src, était requise. Les travaux de recherche présentés dans cette thèse démontrent dans un premier temps à partir d’une souris Dep1 KO, dont le développement ne présente aucun phénotype apparent, que la perte de l’expression de DEP-1 se traduit en une inhibition de l’activation de Src et de l’un de ses substrats, la VE-Cadherine, en réponse au VEGF chez la souris adulte. Nos résultats démontrent donc, pour la première fois, le rôle primordial de DEP-1 dans l’induction de la perméabilité vasculaire et de la formation de capillaires in vivo. Conséquemment, la croissance tumorale et la formation de métastases aux poumons sont réduites due à une inhibition de leur vascularisation ce qui se traduit par une diminution de la prolifération et une augmentation de l’apoptose des cellules cancéreuses. De façon intéressante, l’expression élevée de DEP-1 dans les vaisseaux sanguins tumoraux de patientes atteintes du cancer du sein corrèle avec une vascularisation accrue de la tumeur. En plus du rôle de DEP-1 dans la réponse angiogénqiue à l’âge adulte, nos travaux ont également démontré le rôle important de DEP-1 lors de la vascularisation de la rétine, un modèle in vivo d’angiogenèse développementale. Dans ce contexte, DEP-1 inhibe la prolifération des cellules endothéliales et limite leur bourgeonnement et la complexification du réseau vasculaire rétinien en permettant l’expression adéquate du Dll4, un régulateur crucial de l’organisation de la vascularisation développementale. Cette expression du Dll4 découlerait de la stabilisation de la β-caténine par l’inactivation de la GSK3β, un régulateur important de la dégradation de la β-caténine, en réponse au VEGF selon la voie de signalisation VEGFR2-Src-PI3K-Akt-GSK3β. Ainsi, ces travaux identifient DEP-1 comme un régulateur important de l’organisation vasculaire rétinienne. Les rôles positifs de DEP-1 dans les cellules endothéliales découlent principalement de sa capacité à lier et activer la kinase Src. En plus de contribuer à la réponse angiogénique, Src est également un oncogène bien caractérisé notamment pour sa contribution au programme invasif des cellules cancéreuses mammaires. Les travaux de cette thèse illustrent que DEP-1 est préférentiellement exprimée dans les cellules cancéreuses mammaires invasives et qu’il régule l’activation de Src, de voies de signalisation invasives et, par le fait même, de l’invasivité de ces cellules in vitro et in vivo. De façon intéressante, ces observations corrèlent avec des données cliniques où l’expression modérée de DEP-1 est associée à un mauvais pronostic de survie et de rechute. Ces résultats démontrent donc, pour la première fois, le rôle positif de DEP-1 dans l’activation de Src au niveau des cellules endothéliales et des cellules cancéreuses mammaires ce qui permet la régulation du bourgeonnement endothélial, de la perméabilité vasculaire, de l’angiogenèse normale et pathologique en plus de l’invasion tumorale.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

DEP-derived density from two snow trenches from Kohnen Station, Dronning Maud Land, Antarctica from the 2012/13 field season

The density of firn is an important property for monitoring and modeling the ice sheet as well as to model the pore close-off and thus to interpret ice core-based greenhouse gas records. One feature, which is still in debate, is the potential existence of an annual cycle of firn density in low-accumulation regions. Several studies describe or assume seasonally successive density layers, horizontally evenly distributed, as seen in radar data. On the other hand, high-resolution density measurements on firn cores in Antarctica and Greenland showed no clear seasonal cycle in the top few meters. A major caveat of most existing snow-pit and firn-core based studies is that they represent one vertical profile from a laterally heterogeneous density field. To overcome this, we created an extensive dataset of horizontal and vertical density data at Kohnen Station, Dronning Maud Land on the East Antarctic Plateau. We drilled and analyzed three 90 m long firn cores as well as 160 one meter long vertical profiles from two elongated snow trenches to obtain a two dimensional view of the density variations. The analysis of the 45 m wide and 1 m deep density fields reveals a seasonal cycle in density. However, the seasonality is overprinted by strong stratigraphic noise, making it invisible when analyzing single firn cores. Our density dataset extends the view from the local ice-core perspective to a hundred meter scale and thus supports linking spatially integrating methods such as radar and seismic studies to ice and firn cores.

Estágio na direção de apoio à venda - Dep. de Operações do Turismo de Portugal

Atualmente, com a mudança de paradigma que se verifica no perfil de turista, sendo este cada vez mais informado e exigente no planeamento das suas viagens, cada vez mais as empresas turísticas necessitam de se diferenciar para conseguirem criar vantagem competitiva e manter as suas quotas de mercado. Ora, como solução a esta realidade, o marketing e a promoção turísticas surgem como forma de gestão da experiência do turista na sua viagem para que se garantam a satisfação das suas necessidades e se exceda as suas expetativas. Os destinos turísticos, hoje em dia, também se encontram influenciados pela realidade supracitada e arranjaram formas de se promover e manter competitivos. Assim, surgem os organismos oficiais como elementos com o papel de promover um destino, seja ele um país, região ou até mesmo alguns produtos/segmentos. Desta forma, em Portugal, existe o Turismo de Portugal, I.P. - Autoridade Turística Nacional – com responsabilidades a nível da promoção, valorização e sustentabilidade da atividade turística. Dentro deste, existe a Direção de Apoio à Venda, onde foi realizado o estágio, e com funções principais de coordenar as atividades das Equipas de Turismo no estrangeiro, assistir as agências de promoção estrangeiras na promoção de Portugal, construir e manter uma base de dados do trade internacional atualizada e garantir o apoio à oferta nacional. Com a aposta no apoio ao trade, em detrimento das campanhas de promoção tradicionais, a estratégia do Turismo de Portugal passa a cooperar com operadores turísticos, imprensa internacional e outros canais de distribuição estrangeiros em como vender o destino Portugal aliado a um maior esforço nos meios online. Com base nesta premissa, denotou-se a necessidade de providenciar ao trade internacional um portal onde estes pudessem instruir-se sobre o destino Portugal e as suas regiões, produtos e até mesmo outras informações úteis. Assim, surgiu o projeto VisitPortugal-Trade que se comporta como um complemento ao portal VisitPortugal no seu apoio ao trade. Desta forma, este projeto surge como uma solução de apoio à operação turística organizada, sejam eles pertencentes aos mercados principais emissores de turistas, ou mercados ainda não consolidados, através da formação – através de um curso E-learning – e informação – por via de conteúdo promocional. Paralelamente, a oferta turística nacional, irá beneficiar de um acesso mais facilitado a contactos de operadores estrangeiros, devidamente tipificado, e servindo assim como um forte apoio à internacionalização do seu negócio.