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A tensão entre dois tempos distintos como o passado e o futuro é algo complicado de entender e percecionar. Esse tempo intermédio que chamamos de presente é efémero e muito acelerado. Quando damos por nós a pensar no que se passa no momento, já este é passado e o tempo em que vivemos faz já parte do futuro. Com isto, em que momento este futuro e passado podem viver em sintonia, numa simbiose de tempos que faz esta tensão entre ambos ser equilibrada? É necessário entender que influência tem um no outro. Claramente, o passado tem uma grande influência no futuro e, no caso da arquitetura e na sua evolução ao longo dos anos, o passado está sempre presente, podendo dizer-se que este está na base do futuro. "A Tradição é um desafio à inovação".1 No caso concreto da Casa, como dispositivo do dia a dia do ser humano, a sua mudança é inevitável, acompanhando os novos hábitos que criamos. É, deste modo, necessário entender o que vai ser este futuro, próximo ou não, e que alterações tem de sofrer a casa, na sua disposição e composição entre espaços interiores e exteriores, e de que forma, mesmo tendo por base uma estrutura antiga, podemos chamá-la Casa do Futuro. Assim, e pegando no caso concreto de uma casa de família, pretende-se abordá-la e conseguir chegar a esse limite entre os dois tempos, para que ambos vivam em harmonia e se adaptem às necessidades pretendidas, passando, primeiramente, por uma fase de caraterização da casa e, posteriormente, a um projeto concreto de intervenção. Mas como nos podemos agarrar a essa estrutura antiga da Casa e transpô-la para o futuro? O que é uma Casa do Futuro? Este pensamento traz outras questões essenciais que são: Quem habita a casa? Qual o modo de vida de quem a habita? E que casa é essa na qual queremos intervir? Perceber um modo de intervenção invoca a importância que o tempo tem e o que está entre o que permanece e o que se transforma, uma nova dimensão entre as Coisas e as Pessoas. Cria-se, assim, uma tensão, um jogo de contrários, de opostos e roturas, em que é necessário encontrar no que está, o que precisa de ser feito. É preciso 'tratar com respeito, mas sem receio'. Para além disto, a influência dos arquitetos Alison e Peter Smithson é preponderante para o desenvolvimento do projeto, já que refletem sobre este assunto no Movimento Moderno, através do seu projeto Casa do Futuro. The tension between two different times such as the past and the future is rather complicated to realize and to perceive. That split time, which we call the present is ephemeral and very fast. Moreover, before we know it, the present is a fleeting moment, it is in the past already and the time we live in is within the future. Herewith, how can this future and this past be tuned, in a symbiosis of times that balances this tension? It is necessary to grasp how they influence each other. Clearly, the past has a great influence in the future; regarding architecture and how it has evolved throughout the years, the past is always present, and it is true to say that this is a basis for the future. "Tradition is a challenge to innovation".2 In the specific case of the House, as a day-by-day mechanism of the human being, its change is inevitable, as it follows the new ways we create. It is, therefore, necessary to understand what this future will be (near future or not) and what changes the house must endure, as far as indoor and outdoor spaces are made up and displayed, and in what way, even after being based in an old structure, it can be referred to as House of the Future. Accordingly, and taking the specific case of a family house, the objective is to approach it and to be able to reach that split time between those two times, so that both can adapt to the intended needs, the first of the steps being the profiling of the house and, subsequently, the developing of a concrete intervention project. However, how can one hold on to that House's old structure and transpose it to the future? What is a House of the Future? This notion raises other important issues, namely: Who inhabits the house? What is the lifestyle of its inhabitants? And what is that house on which we want to take action? Understanding a means of intervention refers to the importance of time and what remains between what continues and what is transformed, a new dimension between Things and People. It makes for a tension, a game of contraries, of opposites and ruptures, where it is necessary to find out what needs to be done within that that exists already. It is necessary to 'treat with respect, but with no fear'. Furthermore, the influence by architects Alison and Peter Smithson takes a leading role as regards the project's development since they address this matter in the Modern Movement, in their project House of the Future.

Study question Could endometriosis-associated infertility be mitigated by metformin? Summary answer Metformin treatment restored fertility to control rates in endometriosis-induced mice that present lower fertility. No effect was observed on sham-operated mice. What is known already Endometriosis is a gynaecological disorder characterized by ectopic vascularized endometrial tissue growth, mainly in pelvic cavity, which provokes pain and infertility. Corroborating observations in women, endometriosis decreases oocyte quality and pregnancy success in rodents, without affecting the number of ovulations, resorption rate and fetal weight. Thus, animal models of endometriosis constitute a valuable tool to elucidate the pathophysiology of the disease and putative pharmacological therapies. Metformin is widely used for diabetes type–2 treatment, reducing glucose, oxidative stress and inflammation. Due to its antioxidant properties, metformin has shown to induce regression of endometrial implants in a rat model of endometriosis. Study design, size, duration B6CBA/F1 female mice were randomly divided in groups and subjected to treatment: 1-Endometriosis (n = 20); 2-Sham-operated (n = 12); 3-Endometriosis with metformin (n = 20); 4-Sham-operated with metformin (n = 20). Endometriosis was surgically induced by heterologous transplantation of endometrium from one donor in receptors from the same strain mice. Implants were confirmed and monitored by ultrasound. 50mg/kg/day of metformin was orally administrated during 3 months to Groups 3 and 4. Half of mice in each group were mated to fertility study. Participants/materials, setting, methods Endometriomas were monitored at 3 timepoints during the experiments. Biometric parameters of mice and number of implantation sites, fetuses and fetal weight were recorded. Fertility rates were assessed by the average number of fetuses in each group. Histological characterization of ovary, uterus, endometriomas, and peritoneal tissue at the implant site was performed by Hemathoxylin & Eosin (H&E) staining. Statistical study among groups was carried out and significant differences were considered for student t-test < 0.05. Main results and the role of chance A decrease of 30% of fertility rate was verified in mice with endometriosis (p = 0,01); treatment with metformin was able to revert this decrease (p = 0,04). Interestingly, no differences in fertility were found in sham-operated mice under metformin treatment relatively with those of group 2 (p = 0,16). Although the number of absorptions observed in mice of the endometriosis group was higher, no statistical difference was reached comparatively with other groups. No biometrical differences were found between mice with endometriosis receiving metformin and those that do not receive the drug. Regarding H&E staining we verified that endometriomas showed histologically resemblances to uterus. Moreover, endometriomas from mice without treatment with metformin were dark brown, recalling for human endometriomas called “chocolate” cysts, while the endometriomas from mice who were treated with metformin were visually clearly. We postulate that these findings owes to a metformin-mediated decrease of oxidative imbalance and inflammatory response, induction of regression of endometriomas and regulation of oestrogen secretion. Limitations, reasons for caution Extrapolation of data from animal models to human needs caution, considering that endometriosis pattern differs between species. Also, further investigation, focused in identification of molecular targets of metformin and molecular pathways activated in endometriosis, is needed and in course. Wider implications of the findings: With these results, we indicate metformin as a novel and safe strategy to mitigate endometriosis-related oxidative stress and indeed could be used as a valid pharmacological approach to ameliorate endometriosis-associated infertility. Trial registration number Not applicable

Dissertação de Mestrado em Política Social O acolhimento familiar é uma medida de promoção dos direitos e de proteção das crianças e jovens em perigo, consagrada na Lei de Promoção e Proteção de Crianças e Jovens em Perigo, que consiste na retirada de uma criança ou jovem à sua família de origem, quando as necessidades essenciais ao desenvolvimento não foram satisfeitas. Apesar de a lei mencionar o acolhimento familiar como medida de colocação preferencial, em detrimento do acolhimento residencial, o número de crianças institucionalizadas é elevado e mantém a tendência para aumentar (CASA, 2019). Este estudo procura compreender os fatores da pouca aplicação da medida, através das perceções de três conjuntos de sujeitos, com diferentes papéis, funções sociais e acesso à tomada de decisão: profissionais na área do acolhimento (n=11); especialistas com conhecimento científico acerca da medida (n=12); representantes dos grupos parlamentares (n=6), com legitimidade política. Os sujeitos (N=29) foram selecionados por amostragem por conveniência e entrevistados identificando obstáculos à efetiva aplicação da medida; fornecendo sugestões de melhoria da medida; e situando-a enquanto medida promotora de direitos das crianças. Os resultados sugerem consenso em relação à ideia de que o acolhimento familiar proporciona às crianças o usufruto do seu direito a uma família; revelam perceções complementares sobre as dificuldades na aplicação da medida: falta de informação; predomínio de uma cultura de institucionalização, que se traduz numa preferência efetiva por medidas de colocação; dificuldades na conciliação entre a vida profissional, pessoal e familiar, e falta de benefícios fiscais e laborais. As sugestões recolhidas incluem a realização de uma campanha de divulgação da medida; melhoria na conciliação entre vida profissional, pessoal e familiar e mais vontade política para que a medida cumpra os objetivos previstos na lei. Foster care is a measure of social policy aimed to promote the rights and protect children and young people in danger, enshrined in the Law of Protection of Children and Young People at Risk, which consists of the removal of a child or young person from its family, when the needs essential to its development, have not been fulfilled. Despite the mention in the law to foster care as preferential to residential care, the number of institutionalized children is higher than that of children in foster care, with an upward tendency (CASA, 2019). This study seeks to deeper understand the main factors for this to be a measure rarely used, through the analysis of perceptions of three sets of subjects with different social roles and professional functions and access to decision making: professionals in the field (n=11); experts (n=12); representatives of political parties in Parliament (n=6). Study participants (N=29), intentionally selected, has been interviewed to identify difficulties towards an effective application of the measure; suggesting how to improve the measure; and defining the measure as effective in children's rights promotion. Results suggest consensus around the benefit for children of foster care; reveal complementary perceptions on the effective measure application obstacles, including lack of information; dominance of an institutionalization culture; hard balance on professional, personal and family life and lack of tax and labour benefits. The suggestions made include a campaign publicizing the measure; work-family life improvement and political will increasing the measure potential to fulfil the objectives stated in the law. N/A

White adipose tissue (WAT) browning is a potent mechanism to dissipate energy as heat and, thus, its activation constitutes a promise therapeutic approach to obesity. We previously reported the melanocortin α-melanocyte stimulating hormone (α-MSH) ability to increase the number of beige cells in subcutaneous inguinal WAT (ingWAT) in high fat diet (HFD)-fed mice. The current study examined the browning effect of intraperitoneally administered α-MSH on diverse fat depots from mice fed with HFD or standard rodent diet (SD). For this, mRNA expression of browning hallmark genes was quantified concomitantly with histological examination of the adipose tissue samples (epidydimal (eWAT), mesenteric (mWAT), retroperitoneal (rpWAT) or ingWAT). As well, α-MSH impact on body weight, serum profile, WAT mass and lipolytic rates were evaluated. In the visceral depots mWAT, eWAT and rpWAT from HFD-fed mice, α-MSH was not able to induce a browning mechanism. Surprisingly, in SD-fed mice, α-MSH decreased the expression of several beige-specific genes in rpWAT and promoted an increase of the size of lipid droplets. No browning effect was observed in ingWAT from SD-fed mice. We also verified that HFD ingestion per se stimulated the browning mechanisms in rpWAT, but not in mWAT and eWAT. In conclusion, the fat depots from diverse anatomical locations respond differently to α-MSH treatment when exposed to different diets.

Background/objectives The browning of white adipose tissue (WAT) has been in the spotlight during the last years, becoming an attractive approach to combat obesity. Melanocortin neuropeptides, such as -melanocyte-stimulating hormone (alpha-MSH), are well-known regulators of appetite at the central nervous system, but its role in adipocyte metabolism is poorly elucidated. This study sought to verify if alpha-MSH can induce transdifferentiation of white to brown/beige adipocytes and to determine whether it can ameliorate the obesity phenotype. Methods The browning effect of alpha-MSH was determined in isolated adipocytes using the 3T3-L1 cell line and in inguinal subcutaneous adipose tissue (ingWAT) of diet-induced obese (DIO) mice by quantifying the expression of browning hallmark genes, oxygen consumption, and mitochondrial biogenesis. alpha-MSH protection from diet-induced obesity was evaluated by analyzing mice body weight, fat mass, and lipid and glucose serum profiles. Results Here, we report that alpha-MSH activates a thermogenic gene program and increases the mitochondrial respiratory rate in 3T3-L1 adipocytes and ingWAT of DIO mice. Without affecting food intake, peripheral administration of alpha-MSH decreases body weight and ingWAT mass, promoting a significant rise in the number of smaller adipocytes, whereas it lowered the larger ones. Additionally, there was an increase in the mass of brown adipose tissue. Browning activation occurs concomitantly with improvement on serum lipid profile, insulin resistance, and glucose homeostasis. Conclusions This study highlights the anti-obesity properties of melanocortins by promoting ingWAT browning and provides new perspectives for future designing of more effective therapeutic strategies.

// Diana Castro 1 , Marcia Moreira 1 , Alexandra Monteiro Gouveia 1, 2, 3 , Daniel Humberto Pozza 1, 3 and Ramon Andrade De Mello 4, 5, 6 1 Department of Experimental Biology, Faculty of Medicine, University of Porto, Porto, Portugal 2 Institute for Cellular and Molecular Biology (IBMC), Institute for Health Innovation, University of Porto, Porto, Portugal 3 Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal 4 Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal 5 Department of Medicine, Faculty of Medicine, University of Porto, Porto, Portugal 6 Cearense School of Oncology, Ceara Cancer Institute, Fortaleza, Brazil Correspondence to: Ramon Andrade De Mello, email: ramondemello@doctors.org.uk Keywords: microRNAs, lung cancer, inflammation, epithelial mesenchymal transition, interleukin 1 Received: April 12, 2017 Accepted: August 26, 2017 Published: September 16, 2017 ABSTRACT Lung cancer (LC) is a serious public health problem responsible for the majority of cancer deaths and comorbidities in developed countries. Tobacco smoking is considered the main risk factor for LC; however, only a few smokers will be affected by this cancer. Current screening methods are focused on identifying the early stages of this malignancy. Thus, new data concerning the roles of microRNA alterations in inflammation, epithelial-mesenchymal transition and lung disease have increased hope about LC pathogenesis, diagnosis, treatment and prognosis. MicroRNA mechanisms include angiogenesis promotion, cell cycle regulation by modulating cellular proliferation and apoptosis, and migration and invasion inhibition. In this context, this manuscript reviews the current information about many important microRNAs as they relate to the initiation and progression of LC.

The Melanocortin 5 Receptor (MC5R) is a cell surface receptor that belongs to the class of G-protein coupled receptors (GPCRs), which comprises an intracellular carboxylic domain, seven transmembrane helices and an extracellular amino terminal. Over the last few years, MC5R has been implicated in the regulation of lipid metabolism in exocrine glands, muscle and even in adipose tissue and its function is quite dependent on its correct cell membrane targeting. In this context, the purpose of this work was to study the role of MC5R N-terminus in the receptor trafficking from the endoplasmic reticulum (ER) through the Golgi complex to the plasma membrane. Analysis of N-terminal deleted forms of MC5R revealed that the first 21 amino acids contain the information responsible for the receptor cell surface expression and the removal of further amino acids interfere with the receptor synthesis. In this setting, several mutant forms of the receptor were created by site directed mutagenesis of the MC5R first 21 amino acids and their presence at the plasma membrane was assessed. We have found that two small motifs, constituted by residues Ser4/Ser5 and Ser17/Glu18, are clearly involved in the correct targeting of MC5R to the cell surface. Fluorescence microscopy analysis has revealed that MC5R constructs with mutations in those residues are mainly retained at the ER/Golgi complex. Furthermore, the homodimerization ability of the receptor is maintained in these mutant forms, suggesting that other mechanisms are involved in the regulation of the anterograde transport of MC5R by those N-terminal domains.

As empresas tiveram desde cedo o objetivo de usar os sistemas de informação para tornar os seus processos de negócio mais eficazes e aumentar a competitividade. Assim, as ferramentas de gestão de processos de negócio têm-se tornado cada vez mais comuns nas organizações porque as auxiliam a alcançar os seus objetivos. No entanto, ainda existem ferramentas como a Plataforma de Processos Operacionais que por razões como a obsolescência da solução, não cumprem com esses objetivos, podendo reduzir/impossibilitar o desenvolvimento do negócio. Assim sendo, há a necessidade de ser implementada uma nova solução. Este trabalho, fundamentado primeiramente numa pesquisa bibliográfica, e posteriormente, na realização da prova de conceito, apresenta a avaliação da ferramenta da Red Hat confrontando as suas funcionalidades com os requisitos definidos. A avaliação, realizada requisito a requisito, começou pelos requisitos de desenvolvimento/implementação e terminou nos requisitos não-funcionais. Além de demonstrar a aderência dos requisitos, os representantes da empresa também responderam às questões apresentadas pelos representantes da Caixa Geral de Depósitos. Através dos resultados pude concluir que, apesar do produto ter atendido à maioria dos requisitos definidos, a solução não será utilizada para um piloto dada a pouca disponibilidade de recursos por parte do fornecedor. Assim, atendendo à necessidade urgente de avançar com a migração tecnológica da Plataforma de Processos Operacionais e estando a decorrer o processo de upgrade da ferramenta processual atualmente utilizada na plataforma de crédito, a Caixa Geral de Depósitos pretende adotar igualmente o Iprocess como motor de workflow para a Plataforma de Processos Operacionais. Companies have had from an early age the goal of using information systems to make their business processes more efficient in order to increase competitiveness. In this sense, business process management tools have become increasingly common in organizations because they help them achieve their goals. However, there are still tools such as the Operational Process Platform that for reasons such as the obsolescence of the solution, do not fulfill these objectives, which may reduce/preclude the development of the business. Therefore, there is a need to implement a new solution. This work based firstly on a bibliographical research, and later, on the proof of concept, presents the evaluation of the tool of Red Hat confronting its functionalities with the defined requirements. The evaluation, carried out from requirement to requirement, began with the requirements of development/implementation and ended with non-functional requirements. In addition to demonstrating compliance with the requirements, the company also answered the questions presented by representatives of Caixa Geral de Depósitos. From the results I was able to conclude that although the product has met most of the defined requirements, the solution will not be used for a pilot given the poor availability of resources by the supplier. Therefore, given the urgent need to move forward with the technological migration of the Operational Processes Platform and given the process of upgrading the process tool currently used in the credit platform is in progress, Caixa Geral de Depósitos intends to adopt Iprocess as a workflow engine for the Operational Process Platform.

O presente trabalho teve como objetivo analisar os Espaços de Jogo e Recreio (EJR) públicos do distrito de Coimbra, em particular os Parques Infantis. Apresentamos as funções e finalidades dos EJR, normas e regulamentos, assim como as suas potencialidades e objetivos na divulgação e desenvolvimento de uma vida saudável, na importância do livre brincar e, naturalmente, a relevância do movimento e atividade física. Interpreta-se a evolução dos EJR, conferindo especial realce à atual hipermodernidade e às mudanças evidentes das últimas décadas. Foram observados 169 EJR, usando-se para o efeito o instrumento FOPIN. Os dados obtidos permitem concluir pela tendência significativa para a estandardização dos EJR, quer no que concerne ao tipo de piso, quer aos equipamentos fabricados. As condições de segurança são genericamente boas, mas constata-se o funcionamento de EJR com irregularidades graves, que de acordo com as normas legislativas conduziriam ao seu liminar encerramento pela entidade fiscalizadora. As condições de limpeza e higiene de muitos EJR são baixas e a maioria não possui água potável. Predominam equipamentos monoestruturais e artificiais, que na maioria não proporcionam oportunidades de prática para todos os tipos de habilidades motoras fundamentais na infância.

Cardiovascular disease risk factors (CVDRF), especially diabetes mellitus (DM), disrupt oxidative stress response. This condition underlies endothelial dysfunction, early manifested in men as erectile dysfunction. The current study is aimed at elucidating the impact of CVDRF in the oxidation responsive AMPK/SIRT1-PGC-1α-SIRT3 pathway and related miRNAs in the human corpus cavernosum. Human penile tissue fragments from individuals submitted to programmed urological surgeries (n=27), aged 43-63 years, were clustered depending on the presence of CVDRF; the control group included samples from patients without CVDRF, and groups A and B included samples from patients with DM and additional CVDRF, totalizing ≤2 CVDRF (group A) and ≥3 CVDRF (group B). Dual-immunolabelling of SIRT3, SOD2, or GPX1 with α-actin in tissue sections was carried out. The assessment of expression levels of NOX1, phospho-AMPKα, total AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPX1 was performed by western blotting and of miR-200a, miR-34a, miR-421, and miR-206 by real-time PCR. Phospho-AMPKα and SIRT3 expression was found significantly increased in group B relative to other groups, suggesting a marked influence of CVDRF, additional to DM, in the regulation of these enzymes. NOX1 was also increased in group B relative to controls. Only an increasing tendency was observed in the phospho-AMPKα/total AMPKα ratio, SIRT1, and PGC-1α expression in groups A and B when compared with controls. Concerning antioxidant enzymes, GPX1 expression was found incremented in group A, but SOD2 expression was decreased in groups A and B, comparative with controls. Group B presented significantly diminished levels of miR-421 and miR-200a, but only a decreasing trend on miR-34 and miR-206 expression was observed. Taken together, our findings demonstrated that besides DM, additional CVDRF presented a cumulative effect in the cellular response to oxidative unbalance, contributing to AMPK/SIRT1-PGC-1α-SIRT3 pathway activation. SOD2, a major mitochondrial antioxidant defence, did not follow the same variation.

Resumo O Sistema Único de Saúde tem passado por constante evolução e ampliação desde a publicação da Constituição Federal de 1988. O Distrito Federal apresentou contribuições no campo da Política de Atenção Primária à Saúde, sobretudo entre os anos 2016 e 2018, ao definir a Estratégia Saúde da Família como forma definitiva de organização dos serviços na APS e estabelecer um conjunto de ações orientadas para a conversão ao modelo. Esse artigo descreve e analisa os principais processos dessa mudança. Utilizou-se o método de análise documental de portarias, resoluções, relatórios e outros documentos, bem como dados disponíveis nas bases oficiais. Destacam-se a elaboração de um arcabouço normativo que inclui a implantação das equipes, os processos de trabalho dos profissionais, mudanças nas especialidades médicas e reordenamento dos profissionais de saúde que já atuavam na APS. A experiência demonstra a viabilidade de mudanças incrementais nas políticas de saúde em prol da ampliação de acesso da população aos serviços de saúde, por meio da definição de prioridades, melhor gestão da força de trabalho, capacitação e planejamento descentralizado, repercutindo na elevação da cobertura populacional de Saúde da Família dos iniciais 28% para 69%, alcançados em um período de dois anos. Abstract Conclusion The Unified Health System has undergone constant evolution and expansion since the publication of Brazil’s 1988 Federal Constitution. The Federal District has provided contributions to the field of Primary Health Care Policy, especially between 2016 and 2018, by defining the Family Health Strategy as the definitive way of organizing Primary Health Care (PHC) services and by establishing a set of actions aimed at conversion of existing PHC services to this model. This article describes and analyzes the key processes of this change. The method used was analysis of ordinances, resolutions, reports and other documents, as well as assessment of data from official databases. We emphasize the development of a normative structure that includes the teams’ implementation, the professionals’ work processes, changes in medical specialties and reorganization of the health professionals who already worked in PHC. with an effect of increasing the population’s Family Health Strategy coverage (in a population of approximately 3 million inhabitants) from 28% to 69% during a two-year period, the experience demonstrates the feasibility of incremental changes in health policies such as defining priorities, better management of the workforce, training and decentralized planning to increase access to health services.

Transforming growth factor beta (TGF-ß) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-ß and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-ß, TGF-ß receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-ß and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-ß and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-ß mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-ß receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-ß expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

The job market works under asymmetric information, making it hard for firms to know the real capabilities of the workers they hire. Workers can help overcome this asymmetry by signaling their productivity with investments in education. However, it’s fair to assume that workers sometimes miscalculate their own abilities, which disturbs the functioning of the signal. This study intends to analyze the effects of overconfidence amongst workers on the various parameters of the game by conducting an experiment. The results from the experiment suggest that the profits of both workers and firms will be greatly impaired as a consequence.

The Unified Health System has undergone constant evolution and expansion since the publication of Brazil's 1988 Federal Constitution. The Federal District has provided contributions to the field of Primary Health Care Policy, especially between 2016 and 2018, by defining the Family Health Strategy as the definitive way of organizing Primary Health Care (PHC) services and by establishing a set of actions aimed at conversion of existing PHC services to this model. This article describes and analyzes the key processes of this change. The method used was analysis of ordinances, resolutions, reports and other documents, as well as assessment of data from official databases. We emphasize the development of a normative structure that includes the teams' implementation, the professionals' work processes, changes in medical specialties and reorganization of the health professionals who already worked in PHC. with an effect of increasing the population's Family Health Strategy coverage (in a population of approximately 3 million inhabitants) from 28% to 69% during a two-year period, the experience demonstrates the feasibility of incremental changes in health policies such as defining priorities, better management of the workforce, training and decentralized planning to increase access to health services.O Sistema Único de Saúde tem passado por constante evolução e ampliação desde a publicação da Constituição Federal de 1988. O Distrito Federal apresentou contribuições no campo da Política de Atenção Primária à Saúde, sobretudo entre os anos 2016 e 2018, ao definir a Estratégia Saúde da Família como forma definitiva de organização dos serviços na APS e estabelecer um conjunto de ações orientadas para a conversão ao modelo. Esse artigo descreve e analisa os principais processos dessa mudança. Utilizou-se o método de análise documental de portarias, resoluções, relatórios e outros documentos, bem como dados disponíveis nas bases oficiais. Destacam-se a elaboração de um arcabouço normativo que inclui a implantação das equipes, os processos de trabalho dos profissionais, mudanças nas especialidades médicas e reordenamento dos profissionais de saúde que já atuavam na APS. A experiência demonstra a viabilidade de mudanças incrementais nas políticas de saúde em prol da ampliação de acesso da população aos serviços de saúde, por meio da definição de prioridades, melhor gestão da força de trabalho, capacitação e planejamento descentralizado, repercutindo na elevação da cobertura populacional de Saúde da Família dos iniciais 28% para 69%, alcançados em um período de dois anos.

Introduction Sirtuin (SIRT)1 was recently identified in human corpus cavernosum (CC). We hypothesized that other sirtuins could also be expressed in the CC. Expression of these enzymes in tissues is affected by aging, the main independent risk factor for erectile dysfunction besides other cardiovascular disease risk factors (CVDRF), such as diabetes or obesity. Aim The aim of this study was to characterize the expression of SIRT1-3 and SIRT5–7 in human CC relatively to age and CVDRF. Methods Samples of CC collected from patients submitted to programmed surgeries or organ donors were divided in three groups according to age and presence of CVDRF. Expression of SIRT1–3 and SIRT5–7 mRNAs was assessed by real-time polymerase chain reaction. Cellular localization and semi-quantification of sirtuins proteins were performed by immunofluorescence and Western blotting (WB), respectively. Nuclear factor kappa B (NFkB)-p65, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) levels were also assayed by WB. Main Outcome Measures The main outcome measure was to characterize the expression of SIRT1–3 and SIRT5–7 in human CC. Results SIRT1–3 and SIRT5–7 mRNAs were detected in all individuals, without statistical differences among groups, excepting SIRT7 that decreased four times in aged groups relatively to young (P = 0.013). WB analysis demonstrated that aged individuals with CVDRF presented higher levels of SIRT7 protein relatively to young (P = 0.0495) and lower levels of SIRT3 protein relatively to healthy aged (P = 0.0077). Expression of NFkB-p65 and iNOS were higher in aged than in young individuals (P = 0.0185; P = 0.004, respectively). No differences in other sirtuins or total eNOS were seen among groups although phospho eNOS Ser1177 levels decreased in groups of aged men relatively to young (P = 0.0043; P = 0.0099). Conclusions Our results demonstrate for the first time expression of SIRT2–3 and SIRT5–7 in the human CC. Aged individuals with CVDRF presented an increase in SIRT7 protein levels and a decrease in mitochondrial SIRT3. This finding suggests that CVDRF induces the loss of antioxidant defense mechanisms leading to endothelial injury.

Background Endothelial dysfunction underlies cardiovascular disease that frequently affects aged individuals. Characterized by local decrease in nitric oxide, it results from down-regulation of endothelial nitric oxide synthase (eNOS) expression/activity. Aiming to elucidate the molecular mechanisms involved in age-related endothelial dysfunction and to unveil potential therapeutic targets, we tested how diet pattern, exercise and atorvastatin modulate the expression of eNOS, inducible NOS (iNOS), endothelin-1, sirtuins (SIRT) and microRNA-155 in the erectile tissue of high-fat fed aged rats. Methods Sprague-Dawley male rats fed with high-fat diet until they completed 12 months were grouped and subjected to energy restriction (ER), ER and atorvastatin, or, ER, atorvastatin and physical exercise. Controls were fed with standard rodent chow. The blood pressure was measured using the tail-cuff method before sacrifice at 18 months. Glucose, total cholesterol, HDL, triglyceride and CRP were assessed in blood and eNOS, endothelin-1, iNOS and sirtuins were detected by immunofluorescence in the penis sections; eNOS, endothelin-1, iNOS, SIRT2–4 and SIRT6–7 were semi-quantified by western blotting in tissue homogenates. MicroRNA-155 was quantified using RT-PCR in formalin-fixed paraffin embedded sections. To compare the studied variables, two-tail student t test was used. Results Atorvastatin promotes eNOS expression and is more efficient than ER or exercise in the control of hyperlipidemia and inflammation. Among the studied sirtuins, detected for the first time in the erectile tissue of the aged rat, SIRT2 aligns with eNOS expression. Both proteins exhibit over-expression in animals with combined exercise, atorvastatin and ER. Analysis of microRNA-155 expression also suggests its intervention in the regulation of eNOS expression. ER, particularly when combined with atorvastatin, was able to reverse the increase of iNOS and endothelin-1 in high-fat fed rats. Conclusions The present results indicate that the association of ER, atorvastatin and exercise is more efficient than isolated interventions in the prevention of endothelial dysfunction. Electronic supplementary material The online version of this article (10.1186/s12986-018-0265-z) contains supplementary material, which is available to authorized users.

The aging process is characterized by progressive accumulation of damaged biomolecules in the endoplasmic reticulum, as result of increased oxidative stress accompanying cellular senescence. In agreement, we hypothesized that WI-38 human cellular models of replicative senescence and stress-induced premature senescence (SIPS) induced by hydrogen peroxide (H2O2-SIPS) or copper sulfate (CuSO4-SIPS) would present endoplasmic reticulum chaperoning mechanisms impairment and unfolded protein response activation. Results show that in replicative senescence and CuSO4-SIPS, immunoglobulin binding protein, calnexin, protein disulfide isomerase, and ER oxireductin-1 levels adjust to restore proteostasis and inositol-requiring enzyme-1 (IRE1)-, activating transcription factor 6 (ATF6)-, and pancreatic ER kinase (PERK)-mediated unfolded protein response are activated. However, H2O2-SIPS does not exhibit IRE1 and ATF6 pathways activation but a PERK-mediated upregulation of CCAAT/enhancer-binding protein homologous protein, showing that CuSO4-SIPS mimics better the endoplasmic reticulum molecular events of replicative senescence than H2O2-SIPS. Moreover, unfolded protein response activation is required for both SIPS models induction, because PERK and IRE1 inhibitors decreased senescence-associated beta-galactosidase appearance. In CuSO4-SIPS, the decrease in senescence levels is associated with PERK-driven, but IRE1 independent, cell cycle arrest while in H2O2-SIPS cell proliferation is PERK independent. These results add a step further on the molecular mechanisms that regulate senescence induction; moreover, they validate CuSO4-SIPS model as a useful tool to study cellular stress responses during aging, hoping to postpone age-related health decline.

Over recent years, hair has become the ideal matrix for retrospective investigation of chronic abuse, including for tramadol. However, in order to exclude the possibility of external contamination, it is also important to quantify simultaneously its main metabolite, O-desmethyltramadol (M1), which presence in hair reflects systemic exposure. In the present study a methodology aimed at the simultaneous quantification of tramadol and M1 in human hair was developed and validated for the first time. After decontamination of hair samples (60 mg), tramadol and M1 were extracted with methanol in an ultrasonic bath (~5 h). Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. Subsequently to derivatization, analysis was performed by gas chromatography-electron impact/mass spectrometry (GC-EI/MS). The method proved to be selective. The regression analysis for both analytes was shown to be linear in the range of 0.1-20.0 ng/mg with correlation coefficients of 0.9995 and 0.9997 for tramadol and M1, respectively. The coefficients of variation oscillated between 3.85 and 13.24%. The limits of detection were 0.03 and 0.02 ng/mg, and the lower limits of quantification were 0.08 and 0.06 ng/mg for tramadol and M1, respectively. The proof of applicability was performed in hair samples from six patients undergoing tramadol therapy. All samples were positive for tramadol and M1.

Copper sulfate-induced premature senescence (CuSO

Copper sulfate-induced premature senescence (CuSO4-SIPS) consistently mimetized molecular mechanisms of replicative senescence, particularly at the endoplasmic reticulum proteostasis level. In fact, disruption of protein homeostasis has been associated to age-related cell/tissue dysfunction and human disorders susceptibility. Resveratrol is a polyphenolic compound with proved antiaging properties under particular conditions. In this setting, we aimed to evaluate resveratrol ability to attenuate cellular senescence induction and to unravel related molecular mechanisms. Using CuSO4-SIPS WI-38 fibroblasts, resveratrol is shown to attenuate typical senescence alterations on cell morphology, senescence-associated beta-galactosidase activity, and cell proliferation. The mechanisms implicated in this antisenescence effect seem to be independent of senescence-associated genes and proteins regulation but are reliant on cellular proteostasis improvement. In fact, resveratrol supplementation restores copper-induced increased protein content, attenuates BiP level, and reduces carbonylated and polyubiquitinated proteins by autophagy induction. Our data provide compelling evidence for the beneficial effects of resveratrol by mitigating CuSO4-SIPS stressful consequences by the modulation of protein quality control systems. These findings highlight the importance of a balanced cellular proteostasis and add further knowledge on molecular mechanisms mediating resveratrol antisenescence effects. Moreover, they contribute to identifying specific molecular targets whose modulation will prevent age-associated cell dysfunction and improve human healthspan.

Purpose During ovarian follicle growth, local cellular interactions are essential for oocyte quality acquisition and successful fertilization. While cumulus cells (CCs) nurture oocytes, they also deliver oocyte-secreted factors (OSFs) that activate receptors on CCs. We hypothesized that disturbance of those interactions contributes to age-related lower reproductive success in women submitted to assisted reproductive technology treatments. Methods Women aged 27-48, without recognized personal reproductive disorder, were enrolled in the study and divided in < 35- and >= 35-year-old groups. CCs collected upon follicle aspiration were processed for immunocytochemistry and RNA extraction. The expression patterns of OSF receptors BMPR2, ALK 4, ALK5, and activin receptor-like kinase (ALK6) were studied. Results Independently of age, receptors were found mostly in the cell periphery. The quantitative assay revealed that in older women, BMPR2, ALK 4, and ALK6 were all significantly decreased, whereas ALK5 was slightly increased. Conclusions Female age imparts an effect on the expression of OSF receptors in CCs. The findings indicate that reproductive aging affects the local regulation of signaling pathways mediated by BMPR2, ALK6, and ALK4 receptor activation, suggesting their joint involvement.

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15 min, a G i -protein driven, β-arrestins 1/2 independent process, and a late sustained activation that is regulated by β-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.

The development of analytical techniques that enable the use of hair as an alternative matrix for the analysis of drugs of abuse is useful for confirming the exposure in a larger time window (weeks to months, depending on the length of the hair shaft). In the present study a methodology aimed at the simultaneous quantification of cocaine and morphine in human hair was developed and validated. After decontamination, hair samples (20 mg) were incubated with a mixture of methanol/hydrochloric acid (2:1) at 65 °C overnight (~16 h) in order to extract the drugs of the matrix. Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. After derivatization with N-methyl-N-(trimethylsilyl) trifluoroacetamide, blank, standards and samples were analyzed by gas chromatography/electron impact–mass spectrometry (GC-EI/MS). The method proved to be selective, as there were no interferences of endogenous compounds with the same retention time as cocaine, morphine and ethylmorphine (internal standard). The regression analysis for both analytes showed linearity in the range 0.25–10.00 ng/mg with correlation coefficients ranging from 0.9989 to 0.9991. The coefficients of variation oscillated between 0.83 and 14.60%. The limits of detection were 0.01 and 0.02 ng/mg, and the limits of quantification were 0.03 and 0.06 ng/mg for cocaine and morphine, respectively. The proposed GC-EI/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of cocaine and morphine in hair samples. The proof of applicability was performed in hair samples obtained from drug addicts enrolled in a Regional Detoxification Treatment Center. The importance of hair samples is highlighted, since positives results were obtained when urine immunoassay analyses were negative. Copyright © 2012 John Wiley & Sons, Ltd.

Aims Exercise-stimulated myokine secretion into circulation may be related with browning in white adipose tissue (WAT), representing a positive metabolic effect on whole-body fat mass. However, limited information is yet available regarding the impact of exercise on myokine-related modulation of adipocyte phenotype in WAT from obese rats. Main methods Sprague-Dawley rats (n = 60) were divided into sedentary and voluntary physical activity (VPA) groups and fed with standard (35 kcal% fat) or high-fat (HFD, 71 kcal% fat)-isoenergetic diets. The VPA-groups had unrestricted access to wheel running throughout the protocol. After-9 weeks, half of sedentary standard (SS) and sedentary HFD (HS)-fed animals were exercised on treadmill (endurance training, ET) for 8-weeks while maintaining the dietary treatments. Key findings The adipocyte hypertrophy induced by HFD were attenuated by VPA and ET. HFD decreased 5′ AMP-activated protein kinase (AMPK) activity in muscle as well as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and uncoupling protein 1 (UCP1) proteins in eWAT, while not affecting circulating irisin. VPA increased eWAT Tmem26 mRNA levels in the standard diet-fed group, whereas ET increased AMPK, interleukin 6 (IL-6) and fibronectin type III domain-containing protein 5 (FNDC5) protein expression in muscle, but had no impact on circulating irisin protein content. In eWAT, ET increased bone morphogenetic protein 7 (Bmp7), Cidea and PGC-1α in both diet-fed animals, whereas BMP7, Prdm16, UCP1 and FNDC5 only in standard diet-fed group. Significance Data suggest that ET-induced myokine production seems to contribute, at least in part, to the “brown-like” phenotype in WAT from rats fed a HFD.

Normal pubertal development in humans involves two distinct processes: maturation of adrenal androgen secretion (adrenarche) and activation of the hypothalamic–pituitary–gonadal axis (gonadarche). One factor thought to contribute to the adrenarche in man is increased adrenal 17-hydroxylase (CYP17) activity. In the rat, there is evidence for adrenal involvement in the initiation of puberty, but the adrenal glands of this species are generally thought to express CYP17 only very poorly at best. To further examine the nature of postnatal adrenal development in rat, plasma samples and adrenal tissues were taken from animals aged 2–90 days, circulating adrenal steroids assayed, and adrenal zones assessed quantitatively. A relative increase in zona reticularis, and peaks of circulating cortisol, androstenedione, and 17-OH-progesterone were observed around postnatal days 16–20, clearly before the development of the gonads, which begins at 30–35 days. Quantitative reverse transcriptase PCR confirmed a peak in mRNA coding for CYP17 in adrenal tissue from rats of similar age. The results suggest that the rat adrenal has the capacity to secrete steroids arising from 17-hydroxylation, and that this may contribute to a process similar to human adrenarche.

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro peroxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

The melanocortin system is composed by the agonists adrenocorticotropic hormone and alpha, beta and gamma-melanocyte-stimulating hormone, and two naturally occurring antagonists, agouti and agouti-related protein. These ligands act by interaction with a family of five melanocortin receptors (MCRs), assisted by MCRs accessory proteins (MRAPs). MCRs stimulation activates different signaling pathways that mediate a diverse array of physiological processes, including pigmentation, energy metabolism, inflammation and exocrine secretion. This review focuses on the regulatory mechanisms of MCRs signaling, highlighting the differences among the five receptors. MCRs signal through G-dependent and independent mechanisms and their functional coupling to agonists at the cell surface is regulated by interacting proteins, namely MRAPs and beta-arrestins. The knowledge of the distinct modulation pattern of MCRs signaling and function may be helpful for the future design of novel drugs able to combine specificity, safety and effectiveness in the course of their therapeutic use.

O objetivo deste estudo foi estudar o papel dos hemisférios cerebrais no reconhecimento visual de objetos. Participaram nesta investigação 19 sujeitos saudáveis que foram submetidos a uma tarefa de lateralização de objetos vs não-objetos com um paradigma de memória de reconhecimento. Os registos de potenciais evocados foram recolhidos através de 12 canais ligados a amplificadores BrainProducts. Foram analisados o P300 e o componente positivo tardio (LPC) na fase de teste. Os resultados mostram vantagens no processamento de não-objetos pelo hemisfério direito e de objetos pelo esquerdo, bem como uma maior amplitude P300 e LPC nas regiões parietais. Estes resultados sugerem que não-objetos são identificados mais eficazmente quando processados inicialmente pelo hemisfério direito, o que vai de acordo com as suas capacidades visuais. O mesmo resultado foi verificado para os objetos quando processados inicialmente pelo hemisfério esquerdo, o que é congruente com as suas capacidades semânticas., The aim of this investigation was to study the role of left and right hemispheres in visual object recognition. 19 healthy participants were exposed to a recognition memory paradigm with lateralized study phase of objects vs. non-objects. ERP data was collected using 12 channels connected to standard BrainProducts amplifiers. The P300 and the Late Positive Component (LPC) were analyzed at central test phase. The results showed advantages in stimuli processing for non-objects biased to the right hemisphere and for objects biased to the left hemisphere, along with larger P300 and LPC amplitudes over parietal sites. These data suggest that non-objects are encoded more effectively when processed initially by the right hemisphere, which concur with right hemisphere visual abilities semantic abilities. The same is true for objects processed initially by the left hemisphere, which supports the previous findings about the left hemisphere semantic abilities., Orientação: Jorge Oliveira

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the alpha-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of alpha-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in alpha-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed. In 3T3-L1 adipocytes, alpha-MSH-activated MC5R signals through the cAMP/PKA and MAPK/ERK1/2 pathways. PICA was fundamental for HSL and PLIN1 activation and lipolysis regulation. ERK1/2 inhibition strongly interfered with the release of NEFAs but not glycerol. In addition, the intracellular TG levels, which were decreased after MC5R activation, were restored after ERK1/2 inhibition, indicating that these kinases are involved in NEFA re-esterification rather than lipolysis regulation. This notion is also supported by the observation that the alpha-MSH-mediated activation of phosphoenolpyruvate carboxykinase (PEPCK) was abolished in the presence of ERK1/2 inhibitors. Altogether, these results indicate that alpha-MSH-activated MC5R regulates two tightly coupled pathways in adipocytes: lipolysis and re-esterification. The global effect is a decrease in adipocyte fat mass, which is important for strategies to ameliorate obesity.

Over recent years, hair has become the ideal matrix for retrospective investigation of chronic abuse, including for tramadol. However, in order to exclude the possibility of external contamination, it is also important to quantify simultaneously its main metabolite, O-desmethyltramadol (M1), which presence in hair reflects systemic exposure. In the present study a methodology aimed at the simultaneous quantification of tramadol and M1 in human hair was developed and validated for the first time. After decontamination of hair samples (60 mg), tramadol and M1 were extracted with methanol in an ultrasonic bath (~5 h). Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. Subsequently to derivatization, analysis was performed by gas chromatography-electron impact/mass spectrometry (GC-EI/MS). The method proved to be selective. The regression analysis for both analytes was shown to be linear in the range of 0.1-20.0 ng/mg with correlation coefficients of 0.9995 and 0.9997 for tramadol and M1, respectively. The coefficients of variation oscillated between 3.85 and 13.24%. The limits of detection were 0.03 and 0.02 ng/mg, and the lower limits of quantification were 0.08 and 0.06 ng/mg for tramadol and M1, respectively. The proof of applicability was performed in hair samples from six patients undergoing tramadol therapy. All samples were positive for tramadol and M1.

Melanocortin receptors (MCRs) belong to the G-protein-coupled receptor family of transmembrane proteins. They recognize specific ligands named melanocortins that are mainly produced in the pituitary and hypothalamus. Newly synthesized MCRs at the endoplasmic reticulum are subjected to quality control mechanisms that screen for the correct structure, folding or processing, essential for their proper cell surface expression. Some motifs, located at the N- or C-terminus or even on transmembrane and in loop regions, have been implicated in these biological processes. This article reviews these specific domains and the role of accessory proteins and post-translation modifications in MCRs' targeting to cell surface. Additionally, promising approaches involving pharmacological stabilization of misfolded and misrouted mutant MCRs, which improve their forward transport, are reported. Understanding the MCRs' structural determinants fundamental for their proper cell surface integration is essential for correcting abnormalities found in some diseases.

The development of analytical techniques that enable the use of hair as an alternative matrix for the analysis of drugs of abuse is useful for confirming the exposure in a larger time window (weeks to months, depending on the length of the hair shaft). In the present study a methodology aimed at the simultaneous quantification of cocaine and morphine in human hair was developed and validated. After decontamination, hair samples (20 mg) were incubated with a mixture of methanol/hydrochloric acid (2:1) at 65 °C overnight (~16 h) in order to extract the drugs of the matrix. Purification was performed by solid-phase extraction using mixed-mode extraction cartridges. After derivatization with N-methyl-N-(trimethylsilyl) trifluoroacetamide, blank, standards and samples were analyzed by gas chromatography/electron impact-mass spectrometry (GC-EI/MS). The method proved to be selective, as there were no interferences of endogenous compounds with the same retention time as cocaine, morphine and ethylmorphine (internal standard). The regression analysis for both analytes showed linearity in the range 0.25-10.00 ng/mg with correlation coefficients ranging from 0.9989 to 0.9991. The coefficients of variation oscillated between 0.83 and 14.60%. The limits of detection were 0.01 and 0.02 ng/mg, and the limits of quantification were 0.03 and 0.06 ng/mg for cocaine and morphine, respectively. The proposed GC-EI/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of cocaine and morphine in hair samples. The proof of applicability was performed in hair samples obtained from drug addicts enrolled in a Regional Detoxification Treatment Center. The importance of hair samples is highlighted, since positives results were obtained when urine immunoassay analyses were negative.

Dissertação apresentada ao Instituto Politécnico de Castelo Branco para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Educação Especial - Domínio Cognitivo e Motor. Nesta dissertação apresentam-se os resultados de uma investigação no que concerne à importância da utilização do conto, da imagem e da leitura, no processo de ensino-aprendizagem, no que respeita aos conteúdos da disciplina de História e Geografia de Portugal (HGP) aplicada a alunos portadores de Dificuldades Intelectuais e Desenvolvimentais (DID). Orientada pelos efeitos pedagógicos do conto e pelo pressuposto de que a leitura e a imagem têm impacto na aquisição de competências, a investigação desenvolveu-se em torno de um grupo de alunos portadores de DID, de um Agrupamento de Escolas do centro do país. Os resultados apurados da aplicação do conto Estórias da História de Portugal e análise das fichas de autoavaliação permitiram constatar o elevado grau de importância do conto, da imagem e da leitura como ferramentas didáticas e lúdicas eficazes na abordagem da História de Portugal, tendo-se registado resultados positivos na aquisição de competências naqueles alunos com DID.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated -galactosidase (SA -gal) activity, altered cell morphology and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favour of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA -gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), Fibronectin, Transforming growth factor -1 (TGF 1), Insulin growth factor binding protein 3 (IGFBP3) and Heme oxygenase 1 (HO-1). Western blotting results confirmed enhanced intracellular p21, ApoJ and TGF 1 in copper treated cells. Thus, similarly to other SIPS inducing agents, HDFs exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper associated senescence and lead to a better understanding of copper-related disorders establishment and progression. Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated beta-galactosidase (SA beta-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA beta-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor beta-1 (TGF beta 1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF beta 1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Introduction. Aging is a recognized risk factor for erectile dysfunction (ED), contributing independently to vascular damage of penile tissue. Vascular maintenance depends on angiogenic balance in tissues. Vascular endothelial growth factor (VEGF) is a modulator of endothelial cells functions, after engagement to specific receptor kinase domain region (KDR). Other factors, such as angiopoietins, cross talk with VEGF, modulating its effects. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) compete for binding to Tie-2 and, while Ang1 promotes vascular stabilization, Ang2 acts as a partial agonist or antagonist of Ang1 signaling, depending on VEGF bioavailability.Aims. To quantify the expression of Ang1, Ang2, Tie-2, VEGF, and KDR by real-time polymerase chain reaction (PCR) in human corpus cavernosum (CC) from young and aged healthy individuals.Methods. Human CC fragments were obtained from organ donors without known risk factors to ED and divided in two groups: young (16-35 years) and aged (59-74 years). RNA was extracted and converted to cDNA. Real-time PCR reactions employed appropriate primers. KDR, Tie-2, Akt, and phospho-Akt protein levels were also assessed by Western blotting (WB). Computer-assisted evaluation of vascular areas was performed.Main Outcome Measures. Study of angiopoietins-Tie-2 and VEGF-KDR systems in human CC during aging by real-time PCR and WB. The ratios Ang1/Tie-2 and VEGF/KDR and Akt levels were also determined.Results. Real-time PCR results showed a sixfold significant reduction in the Ang1/Tie-2 ratio during aging. Ang2, VEGF, and KDR expression results were highly variable. Nevertheless, the ratio VEGF/KDR was significantly higher in the aged individuals. Akt and phospho-Akt levels were similar in both groups. Immunohistological evaluation revealed a significant decrease in vascular areas and endothelial surface in CC with aging, despite no differences found in vessel number.Conclusions. The obtained results suggest an aging-associated downregulation of angiopoietins/Tie-2 system and an apparent compensatory upregulation of the VEGF/KDR system. Introduction. Aging is a recognized risk factor for erectile dysfunction (ED), contributing independently to vascular damage of penile tissue. Vascular maintenance depends on angiogenic balance in tissues. Vascular endothelial growth factor (VEGF) is a modulator of endothelial cells functions, after engagement to specific receptor kinase domain region (KDR). Other factors, such as angiopoietins, cross talk with VEGF, modulating its effects. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) compete for binding to Tie-2 and, while Ang1 promotes vascular stabilization, Ang2 acts as a partial agonist or antagonist of Ang1 signaling, depending on VEGF bioavailability.Aims. To quantify the expression of Ang1, Ang2, Tie-2, VEGF, and KDR by real-time polymerase chain reaction (PCR) in human corpus cavernosum (CC) from young and aged healthy individuals.Methods. Human CC fragments were obtained from organ donors without known risk factors to ED and divided in two groups: young (16-35 years) and aged (59-74 years). RNA was extracted and converted to cDNA. Real-time PCR reactions employed appropriate primers. KDR, Tie-2, Akt, and phospho-Akt protein levels were also assessed by Western blotting (WB). Computer-assisted evaluation of vascular areas was performed.Main Outcome Measures. Study of angiopoietins-Tie-2 and VEGF-KDR systems in human CC during aging by real-time PCR and WB. The ratios Ang1/Tie-2 and VEGF/KDR and Akt levels were also determined.Results. Real-time PCR results showed a sixfold significant reduction in the Ang1/Tie-2 ratio during aging. Ang2, VEGF, and KDR expression results were highly variable. Nevertheless, the ratio VEGF/KDR was significantly higher in the aged individuals. Akt and phospho-Akt levels were similar in both groups. Immunohistological evaluation revealed a significant decrease in vascular areas and endothelial surface in CC with aging, despite no differences found in vessel number.Conclusions. The obtained results suggest an aging-associated downregulation of angiopoietins/Tie-2 system and an apparent compensatory upregulation of the VEGF/KDR system.

Melanocortin 5 receptor (MC5R) is a G protein coupled receptor (GPCR) with high affinity for alpha-melanocyte-stimulating hormone (alpha-MSH). To unravel some of the downstream cell-signaling pathways activated by this receptor, HEK293 cells were transiently and stably transfected with a vector encoding green fluorescent protein (GFP)-tagged MC5R. In these cells the receptor was correctly addressed to the cell surface and was functional, as shown by the MC5R-induced formation of intracellular cAMP. In fact, the MC5R agonist alpha-MSH induced a 10- or 16-fold increase (transient or stable cells, respectively) above the cAMP levels found in unstimulated cells. Moreover, in cells stably expressing MC5R-GFP, alpha-MSH promoted ERK1/2 phosphorylation in a dose-dependent manner (EC50=7.3 nM) with the maximal effect occurring after 5 min of agonist incubation. The signaling pathway conveyed through ERK1/2 is not linked to cAMP, since the phosphorylation of these kinases is unchanged by the inhibition of adenylyl cyclase. Also, ERK1/2 activation is not significantly affected by protein kinase A (PKA), protein kinase C (PKC) and protein kinase B or Akt (Akt/PKB) specific inhibitors. However, alpha-MSH-induced ERK1/2 activation is abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Altogether, these findings demonstrate that MC5R signals through a PI3K-regulated Akt-independent pathway leading to downstream activation of ERK1/2. The involvement of these MAPK suggests that MC5R could be implicated in cellular proliferation or differentiation mechanisms.

Dissertação de Mestrado em Estudos Euro-Asiáticos apresentada à Universidade Aberta O objectivo deste estudo é fazermos uma abordagem dos acontecimentos mais importantes, ocorridos em Timor-Leste, de modo a conseguirmos dar resposta à grande questão deste trabalho: Timor-Leste pode ser considerado como um Estado falhado? Assim, começamos por focar o propósito da colonização portuguesa neste território. Os portugueses chegaram a Timor, em meados de 1514, com intuitos mercantis e de difusão da fé cristã, permanecendo nesta ilha durante quase 500 anos. A convivência foi, quase sempre, pacifica visto Portugal não interferir nos usos e costumes locais. A forma de estar, ser e viver do povo timorense manteve-se inalterada ao longo dos séculos seguintes. A revolução do 25 de Abril de 1974 em Portugal, reflectiu-se em Timor-Leste, com o surgimento de partidos políticos que pretendiam alcançar a independência. Deu-se uma luta interna pela conquista do poder levando à guerra civil, à saída da administração portuguesa e à posterior invasão indonésia. Esta invasão foi feita com a conivência de poderosas potências internacionais, nomeadamente a Austrália e os Estados Unidos da América (EUA), permitindo que em Julho de 1976, a Indonésia declarasse Timor-Leste como a sua 27ª província. Mas será que esta anexação foi aceite pelos timorenses? E pela Organização das Nações Unidas (ONU)? O que nos permite afirmar é que num espaço de 30 anos, o país foi destruído pela segunda vez (a primeira tinha sido durante a Segunda Guerra Mundial aquando da invasão do Japão), levando o povo timorense a um desgaste efectivo. Após vários anos de lutas internas e externas, em 1999, foi dado ao povo timorense a possibilidade de escolher o seu futuro. A 30 de Agosto de 1999 realizou-se um referendo, cujo resultado foi esmagadoramente a favor da independência. A Indonésia abandonou Timor-Leste, mas será que aceitou de bom grado a decisão do povo timorense? Perante as noticias, de terror e destruição, que chegavam, toda a sociedade portuguesa se uniu em defesa daquele povo distante e desconhecido. Aquando da reentrada de portugueses em Timor-Leste, estes não foram vistos como antigos colonizadores mas como defensores e apoiantes dos timorenses. Finalmente em Maio de 2002 Timor-Leste tornou-se um Estado independente, escolhendo como língua nacional o Português e o Tétum. No entanto, será que esta escolha agradou aos países vizinhos que tinham eventuais interesses em dominar Timor? Desde sempre têm existido interesses internos e externos que pretendem que Timor-Leste não exerça efectivamente os seus poderes, criando vários problemas, com o intuito de levar à instabilidade interna. Esta instabilidade levou a que, em Fevereiro de 2006, se instalasse uma crise político-militar em Timor-Leste. Mais uma vez, foi solicitado o apoio a Portugal, para os ajudar a Timor-Leste: o emergir de um Estado Página 6 resolver este problema, bem como os apoiar e preparar para as eleições que se aproximavam. Este apoio foi concedido, visando a preparação dos militares e do povo timorense para o processo eleitoral. Assim, num clima pacífico, em 2007 realizaram-se as eleições presidenciais e legislativas, cujos resultados foram obtidos com sucesso. Todavia, em Fevereiro de 2008, houve dois atentados contra o Presidente da República e o Primeiro-Ministro, revelando a insatisfação de um grupo de ex-militares perante a situação do país. Eventualmente, será que todos estes conflitos internos eram e são apoiados por países vizinhos, com interesses económicos na área? Estes países pretendem considerar Timor-Leste, à luz da comunidade internacional, um Estado falhado? Em 2008, as necessidades deste povo continuam a ser variadas, verificando-se uma falta de estratégia na identificação de prioridades que possibilitem acções concretas e eficazes em prol do desenvolvimento deste país. The purpose of this study is to address the most important events that occurred in Timor-Leste, so that we can address the core issue of this work: Can Timor-Leste be regarded as a failed state? So we began by focusing on the purpose of the Portuguese colonization in this territory. The Portuguese arrived in Timor in mid-1514, for commercial reasons and for the dissemination of the Christian faith, staying on this island for almost five hundred years. The living was almost always peaceful since Portugal did not interfere in local traditions and customs. The way of living of the people of East Timor has remained unchanged over the centuries. The revolution of 25th of April 1974, in Portugal, was reflected in Timor-Leste, with the emergence of political parties wishing to achieve independence. There was an internal struggle for power that led to civil war, to the withdrawal of the ex-Portuguese administration and to the subsequent Indonesian invasion. This invasion was done with the connivance of powerful international powers, including Australia and the United States of America, allowing that, in July 1976, Indonesia declared East Timor as its 27th province. But was this annexation accepted by the East Timorese? And by the UN? What we can affirm is that in the following thirty years the country was destroyed for the second time (the first was during World War II during the invasion of Japan), making the Timorese people truly suffer the consequences. After several years of internal and external strife, in 1999, the Timorese people were given the opportunity to choose their future. On the 30th of August 1999 a referendum was held, and the result was overwhelmingly in favour of independence. Indonesia left East Timor but did it gladly accept the decision of the Timorese people? Given the news of terror and destruction that came, all of the Portuguese society was united in defence of the people far and unknown. Upon re-entry of the Portuguese in Timor-Leste, they were not seen as former colonizers, but as advocates and supporters of East Timorese. Finally in May 2002 Timor-Leste became an independent state, choosing Portuguese and Tetum as the national languages. However, did this choice please the neighbours who had interests in dominating Timor? Historically, there are internal and external interests that claim that East Timor does not exercise its powers effectively, creating several problems, in order to lead to internal instability. This instability led to, in February 2006, a political and military crisis in Timor-Leste. Again, support of Portugal was sought, to help the Timorese people to solve this problem as well as to support and prepare for the elections approaching. This support was given, preparing the military and the Timorese people to the electoral process. Thus, peacefully, in Timor-Leste: o emergir de um Estado Página 8 2007 there were the presidential and legislative elections, whose results were obtained successfully. However, in February 2008, there were two attacks against the President and the Prime Minister, revealing the dissatisfaction of a group of former soldiers about the situation of the country. Were and are all these internal conflicts supported by neighbouring countries with economic interests in the area? Do these countries want to consider East Timor, in the light of the international community, a failed state? In 2008, the needs of the people continue to be varied and there is a lack of strategy to identify priorities that would allow concrete and effective action in support of development of this country.

The melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) with a typical seventransmembrane-domain structure. It is assumed that all GPCRs undergo several post-transcriptional modifications during synthesis and folding until finally target to the cell membrane. The available information about the specific domains responsible for the correct targeting of the melanocortin receptors to the cell surface is scarce. Regarding MC5R, it was shown that the first 20 aminoacids of the human receptor can be deleted without affecting the ligand binding affinity, but further deletions of the N terminus resulted in total loss of binding. These results were not directly correlated with the cell surface expression levels of the receptor. Thus, we aim to define the specific MC5R domains important to its correct trafficking and signaling, and preliminary results are here presented.