Caveolae — from ultrastructure to molecular mechanisms

Almost 50 years after the first sighting of small pits that covered the surface of mammalian cells, investigators are now getting to grips with the detailed workings of these enigmatic structures that we now know as caveolae.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Genetic disruption of the growth hormone receptor does not influence motoneuron survival in the developing mouse

In the rodent central nervous system (CNS) during the five days prior to birth, both growth hormone (GH) and its receptor (GHR) undergo transient increases in expression to levels considerably higher than those found postnatally. This increase in expression coincides with the period of neuronal programmed cell death (PCD) in the developing CNS. To evaluate the involvement of growth hormone in the process of PCD, we have quantified the number of motoneurons in the spinal cord and brain stem of wild type and littermate GHR-deficient mice at the beginning and end of the neuronal PCD period. We found no change in motoneuron survival in either the brachial or lumbar lateral motor columns of the spinal cord or in the trochlear, trigeminal, facial or hypoglossal nuclei in the brain stem. We also found no significant differences in spinal cord volume, muscle fiber diameter, or body weight of GHR-deficient fetal mice when compared to their littermate controls. Therefore, despite considerable in vitro evidence for GH action on neurons and glia, genetic disruption of GHR signalling has no effect on prenatal motoneuron number in the mouse, under normal physiological conditions. This may be a result of compensation by the signalling of other neurotrophic cytokines.

Characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells - The initial fate of unbound E-cadherin

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

Experiências formativas no início da docência mediadas pelo PIBID educação física da Universidade Estadual de Feira de Santana, Bahia

O presente trabalho objetiva compreender como o Coordenador, os Supervisores e os Bolsistas do PIBID Educação Física da Universidade Estadual de Feira de Santana (UEFS) no município de Feira de Santana – Bahia percebem os processos de iniciação à docência na formação inicial de professores de Educação Física no PIBID Educação Física – UEFS. Por meio da Pesquisa Narrativa, numa perspectiva de pesquisa e de formação, oportunizamos aos sujeitos da pesquisa entrevistas narrativas compostas pela reflexividade crítica necessária na constituição da docência. Analisamos a produção acadêmica sobre a temática da iniciação à docência no tempo da formação inicial nos bancos de dados da Associação Nacional de Pós-Graduação e Pesquisa em Educação (ANPED) e do Congresso Brasileiro de Ciências do Esporte (CONBRACE) e discutimos com Antonio Nóvoa, Carlos Marcelo e Molina Neto sobre as concepções de formação e as experiências vividas no início da docência e buscamos em Walter Benjamin e Jorge Larrosa ancoragens para o entendimento do conceito de experiência. Os resultados apontam para o reconhecimento do PIBID como um programa que aproxima do campo de atuação interferindo na escolha pela docência. Sinalizam que o programa possibilita a articulação entre a universidade e a escola fortalecendo a perspectiva de formação que acontece coletivamente. Confirmam que o PIBID permite a constatação dos desafios presentes na escola e da necessidade de transformação desta realidade com a articulação dos saberes acadêmicos e os escolares. Neste sentido, constatamos a afirmação da entrada na carreira, como uma experiência formativa que intensifica o desenvolvimento profissional docente e, portanto, deve ser concebida como política nacional institucionalizada de iniciação à docência.

Política de segurança pública no Brasil contemporâneo : entre a segurança cidadã e a continuidade autoritária

O objeto de estudo dessa pesquisa é a política de segurança pública brasileira visando compreender o seu percurso ideológico e político no contexto de retomada e da consolidação da democracia no Brasil, após 21 anos de ditadura militar. Considerando o contexto no qual se verifica a existência de disputa política em torno da concepção de segurança pública, o objetivo geral deste trabalho é compreender a matriz estruturante da política de segurança pública no Brasil contemporâneo. Seu intuito visa responder à pergunta inicial e condutora do interesse que estrutura este trabalho, aqui apresentada nos seguintes termos: a política de segurança pública no Brasil após o restabelecimento das eleições diretas para a Presidência da República está em vias de transição, tendendo a assumir caráter democrático ou a força da tradição autoritária na cultura política brasileira tem-se garantido a sua continuidade neste campo da intervenção estatal? Ancoramos a nossa reflexão nas categorias teóricas de dominação, coerção e consenso no pensamento clássico de Hobbes, Marx, Weber e Gramsci, extraindo deles os elementos que nos auxiliam no entendimento da política de segurança pública brasileira. Para o estudo dessa política foi fundamental operarmos uma profunda revisão bibliográfica, especialmente para entender como a manutenção da ordem foi se desenhando no contexto brasileiro e como historicamente tem prevalecido um modelo de segurança pública marcado pelo autoritarismo. Entretanto, a partir da redemocratização brasileira há a emergência de outro paradigma para a política de segurança pública, a segurança cidadã, propondo, entre outras coisas, a reforma das instituições de segurança pública e a formação em direitos humanos nas instituições policiais. Para a análise do paradigma emergente de segurança, buscamos apoio no Programa Nacional dos Direitos Humanos e no Plano Nacional de Segurança Pública, documentos federais que representam a construção de uma nova intencionalidade para a segurança pública no Brasil. Finalmente, reconhecemos que, embora haja significativas reformas na segurança pública, tal política, diante da prevalência de um paradigma de segurança tradicional com fortes componentes autoritários, se encontra entre a segurança cidadã e a continuidade autoritária.

Os Media Participativos e a importância do Cidadão e da Instituição enquanto Marcas mobilizadoras na área da Oncologia

Present paper aims at surveying the relevance that brands and identities may have in participatory platforms in the context of oncology. Despite the fact that these platforms provide the same tools to citizens and institutions alike, their capacity for mobilization is distinct. In general, institutions seem to be more successful, although this is not always the case. Discussing the concept of brand and identity, as well as understanding how the differences between citizens and institutions may blur within the context of new media, may contribute to answering the above question. We anchor our analysis on the study of social network Facebook, oncological institutions and citizen support groups, and various health studies. The ultimate goal will be the clarification of the importance of brands and identity as a contributive factor towards new solutions in participatory media

Kuailous ou Po tau ya : imagens no feminino sobre os portugueses em Macau

Tendo Macau por contexto, quinze anos após a transferência da sua administração para a República Popular da China e cinco séculos de presença portuguesa, o estudo efetua uma abordagem às representações e estereótipos sociais dos portugueses, a partir de um inquérito a cento e setenta e cinco mulheres chinesas desta Região Administrativa Especial (RAEM). Tendo como marcos conceptuais a Teoria das Representações Sociais, de Moscovici, e a Teoria da Identidade Social, de Tajfel, o estudo procura explicitar os saberes sociais construídos por esse grupo de referência, determinantes na forma como se processa a comunicação e a interação grupal com portugueses e âncora para a construção de estereótipos com os quais esses processos são simplificados, mas também enviesados. Antevendo-se, num horizonte não muito longínquo, decisões quanto à manutenção do princípio “um país, dois sistemas”, que viabiliza a preservação de marcas portuguesas (como a língua) e uma abertura especial à presença de portugueses em Macau, a compreensão das imagens e metáforas sobre esta comunidade constitui estratégia relevante para a perspetivação, no presente como no futuro, das relações intergrupais que a envolvem. O estudo conclui que as representações sobre os portugueses são, no essencial, de pendor positivo e exibem uma abertura à proximidade social e à participação desta comunidade nas dinâmicas sociais da RAEM. Mas assinala também áreas de potencial maior conflitualidade na defesa, pelo endogrupo, de áreas vitais de afirmação dessa nova ordem social pós- transferência.

Protein-ligand docking study: diterpenes from Juniperus brevifolia as anticancer and antimicrobial agentes

REDCAT: Natural Products and related Redox Catalysts: Basic Research and Applications in Medicine and Agriculture, Aveiro, 25-27 Novembro de 2012.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Estudo do comportamento sísmico de barragens de gravidade com ancoragens passivas

Dissertação de Natureza Científica para obtenção do grau de Mestre em Engenharia Civil na Área de Especialização de Estruturas

O papel do pivot na produção da notícia: estudo de caso da SIC

Relatório de estágio apresentado à Escola Superior de Comunicação Social como parte dos requisitos para obtenção de grau de mestre em Jornalismo.

A Review of antenas for indoor positioning systems

This paper provides a review of antennas applied for indoor positioning or localization systems. The desired requirements of those antennas when integrated in anchor nodes (reference nodes) are discussed, according to different localization techniques and their performance. The described antennas will be subdivided into the following sections according to the nature of measurements: received signal strength (RSS), time of flight (ToF), and direction of arrival (DoA). This paper intends to provide a useful guide for antenna designers who are interested in developing suitable antennas for indoor localization systems.