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BACKGROUND: The kidney plays an important role in maintaining normal blood pH. Metabolic acidosis (MA) upregulates the pathway that mitochondria in the proximal tubule (PT) use to produce ammonia and bicarbonate from glutamine, and is associated with AKI. However, the extent to which MA causes AKI, and thus whether treating MA would be beneficial, is unclear. METHODS: Gavage with ammonium chloride induced acute MA. Multiphoton imaging of mitochondria (NADH/membrane potential) and transport function (dextran/albumin uptake), oxygen consumption rate (OCR) measurements in isolated tubules, histologic analysis, and electron microscopy in fixed tissue, and urinary biomarkers (KIM-1/clara cell 16) assessed tubular cell structure and function in mouse kidney cortex. RESULTS: MA induces an acute change in NAD redox state (toward oxidation) in PT mitochondria, without changing the mitochondrial energization state. This change is associated with a switch toward complex I activity and decreased maximal OCR, and a major alteration in normal lipid metabolism, resulting in marked lipid accumulation in PTs and the formation of large multilamellar bodies. These changes, in turn, lead to acute tubular damage and a severe defect in solute uptake. Increasing blood pH with intravenous bicarbonate substantially improves tubular function, whereas preinjection with the NAD precursor nicotinamide (NAM) is highly protective. CONCLUSIONS. MA induces AKI via changes in PT NAD and lipid metabolism, which can be reversed or prevented by treatment strategies that are viable in humans. These findings might also help to explain why MA accelerates decline in function in CKD.

Ca2+ is an important second messenger that translates extracellular stimuli into intracellular responses. Although there has been significant progress in understanding Ca2+ dynamics in organs such as the brain, the nature of Ca2+ signals in the kidney is still poorly understood. Here, we show that by using a genetically expressed highly sensitive reporter (GCaMP6s), it is possible to perform imaging of Ca2+ signals at high resolution in the mouse kidney in vivo. Moreover, by applying machine learning-based automated analysis using a Ca2+-independent signal, quantitative data can be extracted in an unbiased manner. By projecting the resulting data onto the structure of the kidney, we show that different tubular segments display highly distinct spatiotemporal patterns of Ca2+ signals. Furthermore, we provide evidence that Ca2+ activity in the proximal tubule decreases with increasing distance from the glomerulus. Finally, we demonstrate that substantial changes in intracellular Ca2+ can be detected in proximal tubules in a cisplatin model of acute kidney injury, which can be linked to alterations in cell structure and transport function. In summary, we describe a powerful new tool to investigate how single cell behavior is integrated with whole organ structure and function and how it is altered in disease states relevant to humans.

O Estágio Profissional (EP) é uma etapa fundamental do percurso académico, pois é quando acontece a passagem da vida de estudante para o mundo profissional. Este é um processo de partilha com os colegas estagiários, com o professor cooperante (PC), o orientador e toda a comunidade educativa da instituição. O EP fez com que todos os conhecimentos que foram adquiridos na licenciatura e no 1º ano de Mestrado, fossem aplicados na prática, onde tive a possibilidade de exercer a profissão e perceber as verdadeiras situações práticas de uma escola. Este EP teve as suas dificuldades, principalmente, o segundo período que me obrigou a um ensino à distância. Este documento está dividido em cinco grandes capítulos: a Introdução, o Enquadramento Pessoal, o Enquadramento Institucional, o Enquadramento Operacional e as Considerações Finais. Ao longo do ano letivo, surgiram diversos problemas em relação ao planeamento, à realização e avaliação. Neste pressuposto, e de um ponto de vista pessoal, este percurso efetuado ao longo do EP foi crucial para a minha evolução e mudou a minha forma de estar na profissão. Ainda assim, é de salientar que os alunos foram elementos cruciais para este processo, o que me levou a redescobrir a Educação Física (EF), ajudando a que me tornasse mais autónoma e determinada como docente. Ao longo deste EP, a motivação, a comunicação e a instrução foram aspetos fundamentais neste processo. Assim, o papel da professora estagiária foi sempre motivar os alunos para a prática, tendo por base as metodologias e estratégias inovadoras. Por fim, neste documento foi feita uma reflexão que prioriza o valor educacional de toda a experiência e o impacto que teve para o meu desenvolvimento, seja ao nível pessoal, como profissional. The Professional Internship (PE) is a key stage of the academic journey, as it is when the transition from student life to the professional world takes place. This is a process of sharing with fellow trainees, the cooperating teacher (CP), the supervisor and the entire educational community of the institution. The EP made all the knowledge that was acquired in the undergraduate degree and in the first year of the Master's degree, be applied in practice, where I had the opportunity to practice the profession and understand the real practical situations of a school. This PE had its difficulties, mainly the second period that forced me to do distance learning. This document is divided into five main chapters: Introduction, Personal Framework, Institutional Framework, Operational Framework, and Final Considerations. Throughout the school year, several problems arose in relation to planning, implementation and evaluation. In this sense, and from a personal point of view, this journey made throughout the PE was crucial to my evolution and changed my way of being in the profession. Still, it should be noted that the students were crucial elements for this process, which led me to rediscover Physical Education, helping me to become more autonomous and determined as a teacher. Throughout this PE, motivation, communication and instruction were fundamental aspects in this process. Thus, the role of the trainee teacher was always to motivate students to practice, based on innovative methodologies and strategies. Finally, in this paper a reflection was made that prioritizes the educational value of the whole experience and the impact it had for my development, both on a personal and professional level.

The development of intravital imaging with multiphoton microscopy has had a major impact on kidney research. It provides the unique opportunity to visualize dynamic behavior of cells and organelles in their native environment and to relate this to the complex 3-dimensional structure of the organ. Moreover, changes in cell/organelle function can be followed in real time in response to physiological interventions or disease-causing insults. However, realizing the enormous potential of this exciting approach has necessitated overcoming several substantial practical hurdles. In this article, we outline the nature of these challenges and how a variety of technical advances have provided effective solutions. In particular, improvements in laser/microscope technology, fluorescent probes, transgenic animals, and abdominal windows are collectively making previously opaque processes visible. Meanwhile, the rise of machine learning-based image analysis is facilitating the rapid generation of large amounts of quantitative data, amenable to deeper statistical interrogation. Taken together, the increased capabilities of multiphoton imaging are opening up huge new possibilities to study structure-function relationships in the kidney in unprecedented detail. In addition, they are yielding important new insights into cellular mechanisms of tissue damage, repair, and adaptive remodeling during disease states. Thus, intravital microscopy is truly entering an exciting new era in translational kidney research.

Alpha intercalated cells (αICs) in the kidney collecting duct (CD) belong to a family of mitochondria rich cells (MRCs) and have a crucial role in acidifying the urine via apical V-ATPase pumps. The nature of metabolism in αICs and its relationship to transport was not well-understood. Here, using multiphoton live cell imaging in mouse kidney tissue, FIB-SEM, and other complementary techniques, we provide new insights into mitochondrial structure and function in αICs. We show that αIC mitochondria have a rounded structure and are not located in close proximity to V-ATPase containing vesicles. They display a bright NAD(P)H fluorescence signal and low uptake of voltage-dependent dyes, but are energized by a pH gradient. However, expression of complex V (ATP synthase) is relatively low in αICs, even when stimulated by metabolic acidosis. In contrast, anaerobic glycolytic capacity is surprisingly high, and sufficient to maintain intracellular calcium homeostasis in the presence of complete aerobic inhibition. Moreover, glycolysis is essential for V-ATPase-mediated proton pumping. Key findings were replicated in narrow/clear cells in the epididymis, also part of the MRC family. In summary, using a range of cutting-edge techniques to investigate αIC metabolism in situ, we have discovered that these mitochondria dense cells have a high glycolytic capacity.

A pesquisa tem como objetivo estudar o perfil, comportamento e atitudes do consumidor da farmácia. Pretende-se perceber se as mudanças no comportamento do consumidor face à saúde se refletem na farmácia, no farmacêutico e na forma como este é visto pelo cliente. Para responder à questão de partida, implementou-se um modelo de pesquisa descritiva que teve por base a aplicação de um questionário estruturado e de uma entrevista semiestruturada, os quais foram analisados com base no cruzamento das faixas etárias e através de uma análise de conteúdo, respetivamente. Na revisão de literatura, abordam-se temas fulcrais para o desenvolvimento do projeto como comportamento do consumidor, marketing de serviços, marketing farmacêutico e marketing relacional, que permitem consolidar conhecimentos de modo a permitir uma análise mais completa. O estudo conclui que existe uma diferença no comportamento consoante a faixa etária do consumidor, por exemplo, as faixas mais novas adotam comportamentos de compra pouco planeados e as faixas mais velhas planeiam a sua compra, apesar de valorizarem mais a presença do farmacêutico. The research aims to study the profile, behavior and attitudes of the pharmacy consumer. It is intended to understand whether the changes in consumer behavior in relation to health are reflected in the pharmacy, the pharmacist and the way this is seen by the client. To answer the question of departure, a descriptive research model was implemented, based on the application of a structured questionnaire and a semi-structured interview, which were analyzed based on the intersection of age groups and through content analysis, respectively. In the literature review, we discuss key themes for the development of the project, such as consumer behavior, service marketing, pharmaceutical marketing and relational marketing, which allow us to consolidate knowledge in order to make a better analysis. The study concludes that there is a difference in behavior depending on the age range of the consumer, for example, the newer track users adopt poorly planned purchasing behaviors and the users of older tracks plan their purchase, even though they value more the presence of the pharmacist.

Several lines of evidence support the existence of a renin-angiotensin system (RAS) in the retina that is separated from the blood stream by the retinal pigment epithelium (RPE). Under physiological conditions, increased activity of intraretinal RAS regulates neuronal activity of the retina but patho-physiologically participates in retinal degeneration such as hypertensive or diabetic retinopathy. Interestingly, the RPE appears to be a modulator of intraretinal RAS in response to changes in systemic RAS. As increased systemic RAS activity is associated with increased sympathetic tonus, we investigated whether systemic β-adrenergic stimulation of the RPE also modulates renin expression in the RPE. In vivo, the mouse RPE expresses the β-adrenergic receptor subtypes 1 and 2. Staining of retina sagittal sections showed tyrosine hydroxylase positive nerve endings in the choroid indicating adrenaline/noradrenaline production sites in close proximity to the RPE. Systemic infusion of isoproterenol increased renin expression in the RPE but not in the retina. This increase was sensitive to concomitant systemic application of the angiotensin-2 receptor-type-1 blocker losartan. In vitro analysis of renin gene expression using polarized porcine RPE showed that the activity of the renin promoter can be increased by cAMP stimulation (IBMX/forskolin) but was not influenced by angiotensin-2. Thus, with the identification of the β-adrenergic system we added a new regulator of the retinal RAS with relevance for retinal function and pathology. Furthermore, it appears that the RPE is not only a close interaction partner of the photoreceptors but also a regulator or retinal activity in general.

Background A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1’s role and the mechanism regulating its function in the DCT. Methods We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor–1 (I1), mediates cAMP’s effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Results Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)–dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro , and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Conclusions Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

Each year more people are diagnosed with skin cancer all over the world. The large incidence in populations is causing a huge concern to the scientific community, which leads the development of multiple studies related to diagnose this type of cancer. Therefore computer-aided systems are becoming more important in this field due to the challenging task of discriminate benign from malignant skin lesions. These systems can process several images and are intended to make a decision based on the diagnosis achieved by the processing of the images which will reduce the dependency on the experience of the dermatologist and the time consumed in the visual interpretation of each lesion. The main goal of this thesis is the study of the evolution of pigmented skin lesions. Starting from two images of the same lesion at different moments of evaluation, that is the identification of changes that may lead to the intervention of the specialist. These possible alterations may be evidenced through image processing techniques implemented using MATLAB which may help the physician to make a decision. This work addresses three main steps in image processing namely pre-processing, segmentation and feature extraction and aims to obtain results based on the temporal analysis of the lesion.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure. Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations. Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATMaggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death. Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Dissertação de mestrado em Crime, Diferença e Desigualdade, A reclusão representa um período de afastamento dos indivíduos face ao “exterior” sendo necessário garantir, aquando do seu regresso, a possibilidade de reintegração. Neste sentido, esta dissertação estudou de que forma pode ser preparado o regresso à sociedade através do papel que a reinserção assume durante a reclusão pela ação dos Técnicos Superiores de Reeducação (TSR) e Guardas Prisionais. Tendo em conta a escassez de estudos, em Portugal, que olhem para o fenómeno da reclusão a partir das representações dos funcionários prisionais e constatando, a partir dos seus discursos, que a reinserção social apresenta condicionalismos, pretende-se, com base em 24 entrevistas realizadas em duas prisões portuguesas (Estabelecimento Prisional do Porto e Estabelecimento Prisional de Lisboa), a estes dois grupos de profissionais, analisar as suas perspetivas acerca do papel da reinserção social na vida dos reclusos, conhecer as ferramentas de que dispõem para os auxiliar neste processo, bem como constatar as dificuldades e as possíveis melhorias que podem ser introduzidas em meio prisional. Concluímos que estes profissionais consideram que aquele que deveria ser o papel da prisão, a reinserção dos indivíduos, se encontra sobreposto por um cariz punitivo. Avançam que a prisão se tem demonstrado uma resposta ineficaz para combater as taxas de reincidência e, como tal, consideram que o trabalho desenvolvido no sentido da reinserção assume um papel de extrema importância. Defendem que o seu trabalho deve ser iniciado no momento de entrada dos reclusos na prisão e que os ganhos obtidos pela intervenção devem ser reforçados após a reclusão. Acreditam que a criação de um ambiente o mais próximo possível ao exterior vai de encontro às pretensões de reinserção, bem como a inserção dos reclusos em atividades e programas que lhes permitam adquirir novas competências. Acrescentam em relação ao desempenho das suas funções, diversos obstáculos e apresentam algumas soluções para ultrapassar esses condicionalismos. Defendem que seria importante que a formação os preparasse para a realidade que acabam por vivenciar no quotidiano prisional. Afirmam que seria necessário mais apoio institucional, adequado às exigências da profissão, bem como um maior investimento em infraestruturas (e.g., escola e oficinas) para garantir a ocupação dos reclusos. Estas propostas, entre outras, permitirão potenciar o papel dos profissionais tornando-o mais próximo e interventivo., Incarceration represents a period of remoteness from the individual to the "outside" and it is necessary to ensure, upon his return, the possibility of reintegration. In this sense, this dissertation studied how the return to society can be prepared through the role that reinsertion takes during the seclusion by the action of the Superior Technicians of Reeducation and Prison Guards. Taking into account the lack of studies in Portugal that look at the phenomenon of imprisonment based on the representations of prison officials, and based on their speeches that social reintegration presents constraints, it is intended, based on 24 interviews carried out in two Portuguese prisons (Prision Establishment of Porto and Prison Establishment of Lisbon), to these two groups of professionals, to analyze their perspectives on the role of social reintegration in the life of prisoners, to know the tools they have to assist them in this process, as well as to identify the difficulties and possible improvements that can be introduced in prisons. We conclude that these professionals consider that what should be the role of prison, the reintegration of individuals, is superimposed by a punitive nature. They point out that the imprisonment has shown an ineffective response to combat recidivism rates and, as such, consider that the work carried out towards reintegration plays an extremely important role. They argue that their work should be initiated when inmates enter prison, and that the gains from intervention should be strengthened after incarceration. They believe that the creation of an environment as close as possible to the exterior meets the pretensions of reintegration, as well as the insertion of inmates into activities and programs that allow them to acquire new skills. They also add a number of obstacles to the performance of their duties and offer some solutions to overcome these constraints. They argue that it would be important for the training to prepare them for the reality they end up experiencing in prison daily life. They also added that more institutional support would be needed, adequate to the demands of the profession, as well as greater investment in infrastructure (e.g., school and workshops) to ensure the occupation of prisoners. These proposals, among others, will make it possible to enhance the role of professionals by making them closer and more participatory.

Endogenous Ca(2+)-activated Cl(-) currents (CaCCs) are abundant and present in very different cell types. Very good evidence has been provided that endogenous CaCC is produced by anoctamin 1 (Ano1) and Ano2. Insight into the physiological role of anoctamins has been provided for Ano1, Ano2 and Ano6; however, the physiological role of the other seven members of the anoctamin family remains obscure. Anoctamins 1 and 2 may operate as individual Ca(2+)-sensitive channel proteins or may require accessory subunits for complete function. We find that overexpressed Ano1 has properties resembling all those of endogenous CaCCs, although with some noticeable biophysical and regulatory differences when compared with endogenous channels. Apart from Ano1 and Ano2, expression of Ano6 also produces a Cl(-) conductance. Depending on the cellular background, Ano6 currents may have variable properties. Anoctamins 1 and 6 are frequent in epithelial cells, often coexpressed together with Ano8, Ano9 and Ano10. Most available data on anoctamins were obtained from mouse tissues and from cultured cells, which may not be representative of native human tissues.

Endogenous Ca(2+)-activated Cl(-) channels (CaCC) demonstrate biophysical and pharmacological properties that are well represented in cells overexpressing anoctamin 1 (Ano 1, TMEM16A), a protein that has been identified recently as CaCC. Proteins of the anoctamin family (anoctamin 1-10, TMEM16A-K) are widely expressed. The number of reports demonstrating their physiological and clinical relevance is quickly rising. Anoctamins gain additional interest through their potential role in cell volume regulation and malignancy. Available data suggest that Ano 1 forms stable dimers and probably liaise with accessory proteins such as calmodulin or other anoctamins. In order to understand how anoctamins produce Ca(2+)-activated Cl(-) currents, it will be necessary to obtain better insight into their molecular structure, interactions with partner proteins, and mode of activation.

Molecular identification of the Ca(2+)-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca(2+)-dependent Cl(-) secretion in epithelia. TMEM16A is an intrinsic constituent of Ca(2+)-dependent Cl(-) channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl(-) secretion in native epithelia activated by Ca(2+)-dependent agonists is missing in mice lacking expression of TMEM16A. Ca(2+)-dependent Cl(-) transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A(-/-) animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A(-/-) mice. These results clearly demonstrate the broad physiological role of TMEM16A(-/-) for Ca(2+)-dependent Cl(-) secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.

The collecting duct (CD) is the final segment of the kidney involved in the fine regulation of osmotic and ionic balance. During dehydration, arginine vasopressin (AVP) stimulates the expression and trafficking of aquaporin 2 (AQP2) to the apical membrane of CD principal cells, thereby allowing water reabsorption from the primary urine. Conversely, when the secretion of AVP is lowered, as for instance upon water ingestion or as a consequence of diabetes insipidus, the CD remains water impermeable leading to enhanced diuresis and urine dilution. In addition, an AVP-independent mechanism of urine dilution is also at play when fasting. Piezo1/2 are recently discovered essential components of the non-selective mechanically activated cationic channels. Using quantitative PCR analysis and taking advantage of a β-galactosidase reporter mouse, we demonstrate that Piezo1 is preferentially expressed in CD principal cells of the inner medulla at the adult stage, unlike Piezo2. Remarkably, siRNAs knock-down or conditional genetic deletion of Piezo1 specifically in renal cells fully suppresses activity of the stretch-activated non-selective cationic channels (SACs). Piezo1 in CD cells is dispensable for urine concentration upon dehydration. However, urinary dilution and decrease in urea concentration following rehydration are both significantly delayed in the absence of Piezo1. Moreover, decreases in urine osmolarity and urea concentration associated with fasting are fully impaired upon Piezo1 deletion in CD cells. Altogether, these findings indicate that Piezo1 is critically required for SAC activity in CD principal cells and is implicated in urinary osmoregulation.

Piezo1 and Piezo2 are critically required for nonselective cationic mechanosensitive channels in mammalian cells. Within the last 5 years, tremendous progress has been made in understanding the function of Piezo1/2 in embryonic development, physiology, and associated disease states. A recent breakthrough was the discovery of a chemical opener for Piezo1, indicating that mechanosensitive ion channels can be opened independently of mechanical stress. We will review these new exciting findings, which might pave the road for the identification of novel therapeutic strategies.

O presente projeto aborda uma temática bastante atual da nossa sociedade e é realizado no âmbito do Mestrado em Intervenção Psicossocial com Crianças e Jovens em Risco. A pobreza e a exclusão social são problemas que caracterizam a nossa sociedade, sendo que uma das políticas sociais que mais relevância apresenta neste âmbito se refere ao Rendimento Social de Inserção (RSI). Com o objetivo de compreender evolução no processamento de RSI entre 2004 e 2013 em número de beneficiários e rendimento/beneficiário, em Portugal, tendo em conta também variáveis sociodemográficas, foi utilizada uma base de dados que está disponível no site da Segurança Social. Neste sentido a amostra, que é constituída pelos beneficiários do RSI em Portugal Continental e Ilhas, de 2004 a 2013, é coincidente com a população. Com a análise dos resultados verificou-se que o ano de 2004 foi igualmente o ano onde se verificou o menor número de beneficiários de RSI bem como de rendimento/beneficiário, em todos os centros distritais; que entre os anos 2004 e 2013 o número de beneficiários de RSI foi sempre superior para o género feminino ainda que com padrão evolutivo semelhante em ambos os géneros. This project addresses a current thematic of our society and is included in the master’s degree in Psychosocial Intervention with Children and Youngsters at Risk. Poverty and social exclusion are problems that characterize our society. One of the most important social policies in this context is Social Inclusion Income (RSI). In order to understand the evolution of this social policy, between 2004 and 2013, in number of beneficiaries and income/beneficiary, in Portugal, taking also into account socio-demographic variables, we used a database which is available on the website of the Social Assistance. In this sense, the sample, which is constituted by the RSI beneficiaries from Continental Portugal and Islands, from 2004 to 2013, is coincident with the population. It was found that the year of 2004 registered the smallest number of RSI beneficiaries as well as income/beneficiary in all district centers; and that between 2004 and 2013 the number of RSI beneficiaries was always higher in female gender even though evolutionary pattern was similar in both genders.

Piezo1 and Piezo2 are critically required for nonselective cationic mechanosensitive channels in mammalian cells. Within the last 5 years, tremendous progress has been made in understanding the function of Piezo1/2 in embryonic development, physiology, and associated disease states. A recent breakthrough was the discovery of a chemical opener for Piezo1, indicating that mechanosensitive ion channels can be opened independently of mechanical stress. We will review these new exciting findings, which might pave the road for the identification of novel therapeutic strategies.

Dissertação de Mestrado em Psicologia Clínica e da Saúde. Objetivo: Avaliar a eficácia de longo prazo dos antidepressivos no tratamento da perturbação depressiva major e/ou perturbação distímica. Métodos de Pesquisa: Realização de pesquisa eletrónica via EBSCO nas seguintes bases de dados com restrição de língua: Academic Search Complete; CINAHL Plus; MedicLatina; MedLine; PsycArticles; Psychology and Behavioral Sciences Collection; PsycINFO; SocINDEX; e SportDiscus. Critérios de Seleção: Todos os estudos randomizados e com grupo de controlo (RCTs) de período igual ou superior a um ano após a randomização, entre antidepressivos versus placebo, em pacientes diagnosticados com perturbação depressiva major e/ou perturbação distímica, na ausência de episódios de mania e/ou hipomania e outras comorbilidades. Recolha e Análise dos Dados: Os estudos incluídos foram inspecionados por dois autores. Foi avaliada a qualidade metodológica do risco de viés de diferentes parâmetros do estudo, tendo por base as diretrizes da Cochrane Collaboration. Os resultados avaliados foram: mortalidade, estado global, estado mental e efeitos secundários. Resultados: Num total de 4225 resultados obtidos, 10 preencheram os critérios de inclusão. Todos os estudos demonstraram a capacidade de prevenção de recaídas/recorrências dos antidepressivos, quando comparados com o placebo. A diferença da taxa média entre antidepressivos versus placebo: por recaída/recorrência foi de 23%; por abandono do tratamento por outras razões que não por recaída/recorrência foi de 3%; e a proporção de participantes que não completaram o estudo foi de 24%. Todos os estudos incluídos apresentam lacunas metodológicas nos diferentes parâmetros analisados (e.g. inclusão apenas dos participantes que manifestaram benefício clínico do tratamento antidepressivo, num momento antecedente ao estudo; presença de conflito de interesses). Conclusão: Perante os resultados obtidos na atual revisão, considera-se que ainda não existem evidências claras e confiáveis sobre a eficácia a longo prazo do tratamento antidepressivo na depressão. Aims: To evaluate the long-term efficacy of antidepressants in the treatment of major depressive disorder and/or dysthymic disorder. Search Methods: Conducting electronic survey via the following EBSCO databases with constraint language: Academic Search Complete; CINAHL Plus; MedicLatina; MedLine; PsycArticles; Psychology and Behavioral Sciences Collection; PsycINFO; SocINDEX; and SportDiscus. Selection Criteria: All randomized studies with control group (RCTs) of period equal to or more than 1 year after randomization between antidepressants versus placebo in patients with major depressive disorder and/or dysthymic disorder, in the absence of episodes of mania and/or hypomania and other comorbidities. Collection and Analysis Data: The included studies were inspected by two authors. It was assessed the methodological quality of the risk of bias of different parameters of the study, based on the guidelines of the Cochrane Collaboration. The results assessed were: mortality, global state, mental state, and side effects. Main Results: A total of 4225 results, 10 met the inclusion criteria. All studies demonstrated the ability to prevent relapse/recurrence of antidepressants compared to placebo. The average rate of the difference between antidepressants versus placebo: by relapse/recurrence was 23%; by dropout treatment for other reasons than a relapse/recurrence was 3%; and the proportion of participants who did not complete the study was 24%. All the included studies show methodological weaknesses in the differents the parameters analyzed (e.g. including only participants who expressed clinical benefit of antidepressant treatment, a time preceding the study; presence of conflict of interest). Conclusion: Given the results obtained in the current revision, it is considered that there are still no clear and reliable evidence about the long-term efficacy of antidepressant treatment in depression. Orientação: Professor Doutor Gil Nata.

International audience; Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non-selective stretch-activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin-2 (PC2) or, to a greater extent its pathogenic mutant PC2-740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N-terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.

Airways consist of a heterogeneous population of cells, comprising ciliated cells, Clara cells and goblet cells. Electrolyte secretion by the airways is necessary to produce the airway surface liquid that allows for mucociliary clearance of the lungs. Secretion is driven by opening of Cl− selective ion channels in the apical membrane of airway epithelial cells, through either receptor mediated increase in intracellular cAMP or cytosolic Ca2+. Traditionally cAMP-dependent and Ca2+-dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca2+ activated Cl− channel TMEM16A (anoctamin 1) and with detailed knowledge of the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR), it has become possible to look more closely into this relationship.

Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca(2+) activated Cl(-) channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4'000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca(2+) activated Cl(-) currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca(2+) signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.

Outwardly rectifying chloride channels (ORCC, ICOR) of intermediate single-channel conductance of around 50 pS, are ubiquitously expressed, but have remained a mystery since their description more than 25 y ago. These channels have been shown to be activated on membrane excision and depolarization of the membrane voltage and by cAMP in the presence of the cystic fibrosis transmembrane conductance regulator. We show that anoctamin 6 (Ano6), a member of the recently identified family of putative Cl − channels, is the crucial component of ORCC single-channel and whole-cell currents in airway epithelial cells and Jurkat T lymphocytes. Cystic fibrosis transmembrane conductance regulator augmented ORCC produced by Ano6 in A549 airway epithelial cells. Ano6 is activated during membrane depolarization or apoptosis of Jurkat T lymphocytes and epithelial cells, and is inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid, 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid, or AO1. Ano6 belongs to the basic equipment of any cell type, including colonic surface epithelial cells. It forms the essential component of ORCC and seems to have a role for cell shrinkage and programmed cell death.

Endogenous Ca(2+)-activated Cl(-) currents (CaCCs) are abundant and present in very different cell types. Very good evidence has been provided that endogenous CaCC is produced by anoctamin 1 (Ano1) and Ano2. Insight into the physiological role of anoctamins has been provided for Ano1, Ano2 and Ano6; however, the physiological role of the other seven members of the anoctamin family remains obscure. Anoctamins 1 and 2 may operate as individual Ca(2+)-sensitive channel proteins or may require accessory subunits for complete function. We find that overexpressed Ano1 has properties resembling all those of endogenous CaCCs, although with some noticeable biophysical and regulatory differences when compared with endogenous channels. Apart from Ano1 and Ano2, expression of Ano6 also produces a Cl(-) conductance. Depending on the cellular background, Ano6 currents may have variable properties. Anoctamins 1 and 6 are frequent in epithelial cells, often coexpressed together with Ano8, Ano9 and Ano10. Most available data on anoctamins were obtained from mouse tissues and from cultured cells, which may not be representative of native human tissues.

Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca2+-activated Cl- currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca2+-activated Cl- channel as well as a regulator of Ca2+ signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl- channels by controlling intracellular Ca2+ levels. Best1 interacts with important Ca2+-signaling proteins such as STIM1 and can interact directly with other Ca2+-activated Cl- channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca2+-activated Cl- channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca2+-dependent Cl- channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca2+-activated Cl- currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains.

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1-ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca(2+)-activated Cl(-) conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl(-) conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca(2+) sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca(2+)-dependent Cl(-) channels.

Bestrophin 1 (Best1) controls intracellular Ca 2+ concentration, induces Ca 2+ -activated Cl - conductance, and increases proliferation of colon carcinoma cells. Here, we show that expression of Best1 in mouse renal collecting duct (CD) cells causes i ) an increase in cell proliferation, ii ) a loss of amiloride-sensitive Na + absorption, iii ) induction of Ca 2+ -dependent Cl - conductance (CaCC), and iv ) epithelial-to-mesenchymal transition. During conditions of high proliferation or when we exposed CD cells to serum or TGF–β1, we observed upregulation of Best1, increased CaCC, redistribution of the epithelial-to-mesenchymal transition marker β-catenin, and upregulation of vimentin. In contrast, suppression of Best1 by RNAi inhibited proliferation, reduced CaCC, and downregulated markers of EMT. CaCC and expression of Best1 were independent of the cell cycle but clearly correlated to cell proliferation and cell density. During renal inflammation in LPS-treated mice or after unilateral ureteral obstruction, we observed transient upregulation of Best1. These data indicate that repression of cell proliferation, CaCC, and expression of Best1 occurs during mesenchymal-to-epithelial transition once CD cells polarize and terminally differentiate. These results may suggest a role for Best1 in renal fibrosis and tissue repair.

Ion channels like voltage-gated ether-á-go-go (Eag1) K(+) channels or Ca(2+)-activated Cl(-) channels have been shown to support cell proliferation. Bestrophin 1 (Best1) has been proposed to form Ca(2+)-activated Cl(-) channels in epithelial cells. Here we show that original T(84) colonic carcinoma cells grow slowly (T(84)-slow) and express low amounts of Eag1 and Best1, whereas spontaneously transformed T(84) cells grow fast (T(84)-fast) and express high levels of both proteins. Both Eag1 and Best1 currents are up-regulated in T(84)-fast cells. Eag1 currents were cell cycle-dependent with up-regulation during G(1)/S transition. T(84)-slow, but not T(84)-fast, cells formed tight monolayers when grown on permeable supports. RNA interference inhibition of Eag1 and Best1 reduced proliferation of T(84)-fast cells, whereas overexpression of Best1 turned T(84)-slow into fast-growing cells. Eag1 and Best1 improve intracellular Ca(2+) signaling and cell volume regulation. These results establish a novel role for bestrophins in cell proliferation.