P2 purinoceptors signaling in fibroblasts of rat subcutaneous tissue

Mestrado em Biologia Molecular e CelularO tecido conjuntivo parece estar envolvido na génese de diversas condições patológicas. O aumento da rigidez do tecido conjuntivo, resultante da fibrose, pode constituir um factor importante no mecanismo patogénico da dor crónica resistente a fármacos (Langevin & Sherman, 2007). Por outro lado, os nucleótidos extracelulares parecem estar envolvidos na fisiopatologia da dor crónica (Burnstock, 2001). Assim, este estudo teve como objectivo averiguar o efeito dos nucleótidos de adenina e uridina na proliferação e síntese de colagénio tipo I de fibroblastos do tecido subcutâneo de rato em cultura. Os resultados obtidos mostram que a incubação com UTP (0.3-100 M, n=5) induz um aumento da proliferação e da produção de colagénio tipo I, o qual é dependente da concentração. Contrariamente, o agonista selectivo dos receptores P2Y2, o MRS 2768 (10 μM, n=3), não teve qualquer efeito no que se refere à proliferação, mas diminuiu significativamente (P<0.05) a síntese de colagénio tipo I. Uma vez que o aumento da produção de colagénio induzida pelo UTP (100 μM) foi proporcional ao aumento do número de células (proliferação celular),podemos especular que este aumento se deve ao aumento do número de células per si do que a uma maior actividade sintética de cada célula. Assim, ao normalizar os valores do colagénio tipo I em relação aos valores obtidos do MTT para os mesmos momentos/dias, deixamos de observar diferenças estatisticamente significativas entre o controlo e as células expostas ao UTP. Uma vez que os receptores P2Y2 não parecem estar envolvidos nesta resposta do UTP (100 μM), esta poderá estar a ser mediada pela activação dos receptores P2Y4 e/ou P2Y6. Considerando que o RB-2 (10 μM, n=5), um antagonista não selectivo que actua preferencialmente no subtipo de receptores P2Y4, não foi capaz de modificar a resposta induzida pelo UTP (100 μM), os receptores P2Y4 parecem também não estar envolvidos. Por outro lado, o MRS 2578 (100 nM), um antagonista selectivo dos receptores P2Y6, atenuou de forma significativa o aumento induzido pelo UTP (100 μM). A corroborar os nossos resultados, uma análise imunocitoquímica mostrou uma imunorreactividade positiva contra os receptores P2Y2 e P2Y6, mostrando um padrão de marcação citoplasmático/membranar, o qual é típico para este tipo de receptores, ao contrário do padrão nuclear exibido pelo anticorpo contra os receptores P2Y4. Relativamente ao envolvimento dos receptores sensíveis ao ADP, os resultados obtidos mostraram que o ADPβS (10-100 μM, n=3-6), um análogo estável do ADP, não parece induzir efeitos significativamente diferentes (P>0.05) na proliferação celular. Contudo, a sua incubação continuada aumentou a produção de colagénio tipo I de forma dependente da concentração (P<0.05). De modo a identificar os receptores purinérgicos envolvidos neste efeito, testamos o ADPβS (100 μM) na presença do MRS 2179 (0.3 μM), do AR-C 66096 (0.1 μM), e do MRS 2211 (10 μM), os quais antagonizam selectivamente os receptores P2Y1, P2Y12 e P2Y13, respectivamente. O efeito facilitatório induzido pelo ADPβS (100 μM) foi atenuado de forma significativa na presença do antagonista dos receptores P2Y1, o MRS 2179 (0.3 μM, n=3), sem ser afectado pelo antagonista dos receptores P2Y12, o AR- C 66096 (0.1 μM, n=3). Pelo contrário, o MRS 2211 (10 μM, n=2) potenciou o aumento da produção de colagénio induzida pelo ADPβS (100 μM), indicando assim que a síntese de colagénio tipo I induzida pelo receptor P2Y1 pode estar a ser parcialmente influenciada por uma activação síncrona do receptor inibitório P2Y13. Por último, uma análise por imunocitoquímica mostrou que estas células apresentam imunorreactividade positiva para os receptores P2Y1 e P2Y13, exibindo um padrão citoplasmático/membranar, contrariamente ao padrão nuclear dos receptores ostentado pelo anticorpo contra os receptores P2Y12. Concluindo, a remodelação da fáscia superficial induzida pelos fibroblastos parece ser regulada por um balanço entre a activação dos receptores P2Y2 e P2Y6, assim como dos receptores P2Y13 e P2Y1. Clarificar as vias que conduzem ao processo de fibrose pode representar uma oportunidade para esclarecer o seu envolvimento na patogénese da dor crónica musculo-esquelética, bem como ser útil no desenvolvimento de novas estratégias terapêuticas.Connective tissue may be involved in the pathogenesis of a wide variety of disease conditions. Increased connective tissue stiffness due to fibrosis may be an important link to the pathogenic mechanism leading to drug-resistant chronic pain (Langevin & Sherman, 2007). In addition, extracellular nucleotides seem to be involved in the pathophysiology of chronic pain (Burnstock, 2001). Therefore, we aimed at investigating the effect of adenine and uridine nucleotides on the proliferation and synthesis of type I collagen by rat fibroblasts from subcutaneous connective tissue. The results showed that continuous incubation of UTP (0.3-100 M, n=5) concentration-dependently increased fibroblasts proliferation, as also increased the synthesis of type I collagen above the control levels. Conversely, the selective P2Y2 agonist, MRS 2768 (10 μM, n=3), was devoid of effect in what concerns proliferation, but significantly (P<0.05) decreased type I collagen synthesis. Since the increase in type I collagen synthesis induced by UTP (100 μM) was proportional to the increase in the amount of cells in the culture (fibroblasts proliferation), we speculated that such an increase could be related to the increase in the cell number rather than a higher synthetic activity. Thus, we performed a more detailed data analysis, in which we normalized type I collagen production taking into consideration the MTT values obtained at the same time points, and we observed no longer significant differences between control and UTP-exposed cells. Discounting the contribution of MRS 2768-sensitive P2Y2 receptors, UTP (100 μM)-induced increase in cells proliferation could be due to P2Y4 and/or P2Y6 receptor activation. Since RB-2 (10 μM, n=5), a non-selective antagonist that acts preferentially on the P2Y4 subtype, did not modify the effect of UTP (100 μM), P2Y4 does not seem to be involved. In turn, MRS 2578 (100 nM), which is a selective P2Y6 antagonist, significantly attenuated UTP (100 μM)-induced increase. To corroborate our results, an immunocytochemistry analysis showed a positive immunoreactivity against the P2Y2 and P2Y6 receptors exhibiting a cytoplasmic/membrane labeling pattern, which is typical for those receptors in many different cells, conversely to the nuclear labeling pattern exhibited by the antibody against the P2Y4. To investigate the involvement of ADP-sensitive P2 receptors on cell proliferation and extracellular matrix production, fibroblast cultures were continuously incubated with the stable ADP analogue, ADPβS (10-100 μM). Results obtained with ADPβS (10-100 μM, n=3-6) showed no significant (P>0.05) differences in fibroblast cells proliferation. However, a continuous incubation with ADPβS (10-100 μM, n=2-5) concentration-dependently increased type I collagen production by fibroblasts (P<0.05). In order to identify which purinoceptor(s) that could be mediating this effect, we tested ADPβS (100 μM) in the presence of MRS 2179 (0.3 μM), AR-C 66096 (0.1 μM), and MRS 2211 (10 μM), which antagonize selectively ADP-sensitive P2Y1, P2Y12 and P2Y13 receptors, respectively. The facilitatory effect of ADPβS (100 μM) was significantly attenuated in the presence of the P2Y1 antagonist, MRS 2179 (0.3 μM, n=3), without being affected by the P2Y12 antagonist, AR- C 66096 (0.1 μM, n=3). In contrast, MRS 2211 (10 μM, n=2) potentiated the effect of ADPβS (100 μM) on type I collagen synthesis, thus indicating that the P2Y1-receptor-induction of type I collagen synthesis may be partially counteracted by synchronous activation of the inhibitory P2Y13 receptor. Finally, an immunocytochemistry analysis showed that these cells exhibit immunoreactivity to P2Y1 and P2Y13 receptors with a cytoplasmic/membrane staining pattern, conversely to the nuclear pattern of P2Y12. Concluding, a delicate balance between the activation of P2Y2 and P2Y6, as well as P2Y13 and P2Y1 purinoceptors, might regulate fibroblast’s induced superficial fascia remodeling. Targeting the pathways leading to fibrosis may represent an opportunity to clarify its involvement in the pathogenesis of musculoskeletal chronic pain and it may be useful for designing novel therapeutic strategies to overcome this disease

Tráfico de droga nos aeroportos: análise de uma realidade pouco explorada

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de licenciada em CriminologiaO tráfico de droga é um tema recorrente na sociedade contemporânea que, na sua extensão, engloba todos os cidadãos. Pode-se afirmar que é um tema já com bastantes anos de história e, ainda hoje, desperta um particular interesse em diversos autores. O tema foi escolhido por ser diversificado, interessante, e também relativamente ligado à área da Criminologia. Os aeroportos são um local onde transitam diariamente milhões de pessoas, associada a uma vasta complexidade no seu funcionamento. Com isto, a ideia de fazer um projeto cujo tema é o tráfico aeroportuário surgiu por não haver muitos artigos que relacionem as duas designações diretamente, e por isso surge na tentativa de interligar as mesmas projetando um trabalho coeso, aliciante e elucidativo.Drug trafficking is a recurring theme in contemporary society, in its extent, covers all citizens. One can say that it is s topic with many years of history and still today arouses a particular arouses a particular interest in several authors. The theme was chosen for being diverse, interesting, and also relatively connected to the field of Criminology. The airports are a place where passing through daily million people, associated with a vast complexity in its operation. With this, the idea of making a project whose theme is the airport traffic came about because there was no many articles that link the two names directly, and so arises in trying to connect the same designing a cohesive work, attractive and informative.N/

Structure and adaptation of arteries to pulsatile flow - The case of the ascending aorta

PURPOSE: The objectives are: (i) assess the development of the impedance of some arteries during the first decades of life; (ii) determine the influence of pulse rate in arterial impedance; (iii) compare the structure of some arterial segments with optimized structures with respect to blood flow; and (iv) explain the elongation of the ascending aorta throughout life in healthy subjects. METHODS: A model of the arterial network previously developed by the authors, together with data of lengths, diameters, and distensibilities of arterial segments reported in the literature were used. The impedances of the aorta and carotid artery were calculated based on that model. Similarly, the impedances of various arteries corresponding to heart rates of 65 bpm and 120 bpm were calculated. Values observed in arterial segments were compared with the respective optimal values from the viewpoint of hemodynamic performance. This allowed drawing conclusions on the arterial segments that might be critical with regard to hemodynamics. RESULTS: It was found that in healthy people impedances of the aorta and the carotid artery decrease markedly with age especially during body growth. It was also found that impedances of the main arteries do not significantly change with heart rate, even if sharp changes in arterial distensibility are observed. With respect to optimal flow performance, it was found that scaling between diameters of branching arteries is generally close to optimality, while the corresponding length scaling is far from optimality. It was also found that the ascending aorta and aortic arch are among those arterial segments whose lengths are much smaller than the optimum values. An explanation is offered for the age associated elongation of the aorta in healthy people. CONCLUSIONS: In healthy subjects, the human arterial system continues to optimize its performance at least until the age of 60

O impacto do estágio na exploração vocacional e na adaptabilidade de carreira em estudantes do ensino profissional

Dissertação de mest., Psicologia (Psicologia da Educação), Faculdade de Ciências Humanas e Sociais, Univ. do Algarve, 2013A aprendizagem em contexto real de trabalho é um elemento essencial da formação dos estudantes dos cursos Profissionais. No campo da literatura vocacional, o modelo de desenvolvimento da carreira de Donald Super, a perspectiva desenvolvimentista-contextualista de Vondraceck, Lerner e Schulenberg e a teoria construtivista da adaptabilidade da carreira de Mark Savickas sustentam que a experiência de trabalho pode exercer impacto no desenvolvimento vocacional dos jovens. Apesar disso, os estudos empíricos nem sempre são consonantes relativamente a esta expectativa. É neste contexto que surge a presente investigação, num formato longitudinal (pré e pós estágio), com o objetivo de estudar a relação entre as qualidades do estágio curricular e o desenvolvimento vocacional numa amostra de 60 estudantes dos cursos Profissionais. Os resultados obtidos destacam o papel da autonomia, da supervisão, do suporte social, da diversidade de tarefas e das oportunidades de aprendizagem (dimensões da qualidade da experiência de estágio) no desenvolvimento vocacional dos estudantes, nomeadamente ao nível da atividade exploratória e da adaptabilidade da carreira. Tais fatores poderão servir de base para orientar o acompanhamento do estágio, pelo que importa valorizar a articulação de esforços de todos os agentes implicados no processo de formação dos estudantes (os professores orientadores de estágio, os supervisores das entidades acolhedoras, ou até os colegas), tendo em vista o desenvolvimento da adaptabilidade de carreira e a manutenção da qualidade das experiências de estágio, de modo a facilitar a transição do aluno para o mundo do trabalho. No final da dissertação, são igualmente apresentadas as limitações e apontadas futuras linhas de investigação neste âmbito

Comparison of Canopy Openness in Different Cocoa (Theobroma cacao) Production Systems in Alto Beni, Bolivia

Cocoa (Theobroma cacao L.) grows naturally as an understory tree in tropical forests and produces well under shaded and non-shaded conditions. It is cultivated by small scale farmers in South America under various conditions, ranging from monocultures to different kinds of agroforestry systems. While in monocultures it is exposed to direct sunlight, one or various tree species shade the cocoa in agroforestry systems. Also organic cocoa cultivation is becoming more and more popular due to premium prices and increasing ecological consciousness. In Alto Beni, Bolivia, the Research Institute of Organic Agriculture (FiBL) and local partners have established a long-term field trial to compare cocoa production systems. The bi-factorial randomised block design includes management and biodiversity factors combined to the following five cocoa treatments: monoculture and agroforestry systems both under organic and conventional management, and successional agroforestry system (high plant species diversity) under organic management and for further comparison fallow plots of same age as the cocoa plots. Research is done in all fields of agronomic, economic and environmental interest. This study focuses on the comparison of the canopy openness of the different cocoa production systems and fallow plots. Knowledge about the canopy openness enables the estimation of light entering the production system, especially on the cocoa layer (photosynthesis relevant) and also on the soil as canopy openness influences the microclimate in the plantation. Another aspect of the canopy is the impact on the throughfall within the plot. Over the time, variations in the canopy structure indicate the production of biomass, of nutrient enrichment by throughfall (rain-wash and nutrient leaf leaching in the canopy) and may indicate pruning necessities when the plant cover above the cocoa exceeds critical values. To estimate the canopy openness, in the years 2012 and 2013 hemispherical photography was taken with fisheye lenses in the different cocoa production systems and in the fallow plots. The photos were analysed with the programme Gap Light Analyser. First results of canopy openness between the cocoa systems will be shown and discussed for leave area index and potential microclimate differences

Stratford Centre : reimagined playground

The following article is a collection of images looking at how youth counter cultural groups of urban dancers, skateboarders and roller skaters reappropriate a commercial space in East London and reimagine a pluralist playground. The narratives of trespass and occupation of Stratford Centre—a "private-public" space used both as a shopping centre and a 24-hour thoroughfare—are visually explored through photographs depicting moments of happiness, sharing, friendship and creative collaboration between young people. This work challenges the concepts of public space and common right to the city by unveiling the marginalisation and exclusion of these young alternative cultures and the lack of formal urban spaces for (young) people to engage in different activities—more spontaneous and recreational than the ones of work and consumption. Through subversive practices, the young people gathering at night in the underused space of the shopping centre reclaim and take back their right to socialise, express themselves and play. They suggest alternative and sensory rich ways of experiencing urban space and create possibilities of action that are not offered by the capitalist city. Driven by the desire to positively represent this vibrant and inclusive place, this body of work attempts to convey something of the atmosphere of an open space characterised by great social cohesion and performed by a community of young talented people.peer-reviewe

Activities as a research resource

Activities play a guiding central role in occupational therapy, articulating to its utilization, the understanding and assessment of its capabilities in numerous and different interventions arising from this field. In this study, we aim to present the use of activities as a research methodology resource. Considering the pathways studied in a master’s research followed by a PhD work, some applications of this resource are reported as a powerful strategy of investigation for the occupational therapy researcher. It is about structuring, systematizing, and analyzing the activities proposed in professional practice by the theoretical and methodological rigor used in the research works. It emphasizes how the use of these activities can be considered important data, records, instruments and sources for different research methods, especially for qualitative analyses. Workshop activities were offered in the above mentioned studies; they were used as communication instruments and expressions of personal and collective experiences, supplying data to understand the actions of subjects and collectives. Furthermore, this strategy was applied as spaces of experimentation, learning and expression, where each participant was conceived as an active being of the process, in a way that the proposal could result in democratic experiences that reflected greater interest and participation, and a more complex presentation of the research data. It is worth mentioning that other methodological procedures, which substantiated the analyses and interpretation of the data collected, were also used. It was possible to conclude that activities, mainly as a qualitative research resource, constituted materialities in different languages and expressions that enriched the analyses arising from micro-realities, producing a repertoire of information that supported the interpretations required for the investigative processes

The impact of sitting volleyball participation on the lives of players with impairments

Forbidden to stand aims to provide a comprehensive account of how participation in sitting volleyball (SV) has impacted upon the lives of players with impairments. To achieve this aim, this study uses capabilities approach, a theoretical and methodological framework unexplored in sport contexts but widely appraised in political philosophy as one of the most comprehensive approaches to well-being and quality of life. One of the implications of the use of capabilities approach was the compulsory need to pay attention not only to personal capabilities per se, but also to the contextual elements of the individuals experience in SV. As such, whilst identifying, describing and assessing the main personal capabilities in which participation in SV had a significant impact, the present study presents simultaneously an anthropological account of the SV field in the United Kingdom (UK) as it developed. In connecting capabilities approach and disability sport for the first time, this study contributes to our understanding of the impact of sport on the whole lives of people and to the development of a holistic tool to measure personal development, helping to address an acknowledged omission of such instruments in the academic field of adapted physical activity. In order to respect the pluralism and complexity of capabilities approach, an ethnographic methodological design was used due to its flexibility in combining a plurality of theoretical insights; data sources and perspectives. During the study the researcher performed different roles within the SV community facilitating empirical data collection using the ethnographic tool kit. A key development in this process was the definition of an analytical thematic framework which directed the extensive analysis of the whole data set. A set of ten relevant capabilities were then identified as the most relevant for SV players with impairments, and SV impact on those capabilities described. This study reveals that while the potential to enact and promote capabilities is present in SV context in the UK, it is very dependent upon influential factors operating at a personal, cultural and environmental levels. At a personal level, the enjoyment and expansion of capabilities in players with impairments was very much influenced by the possession of substantial financial resources and previous sporting capital; thus the players who have expanded their capabilities the most were individuals who already possessed a good level of capabilities enjoyment. At the cultural level, while SV field detains important qualities to promote capabilities enjoyment such as an equalisation of the social worth between people with and without impairments, these were often overridden by the political and cultural dominance of an able-bodied volleyball ethos. At an environmental level, the overdependence of Volleyball institutions from the funding allocated by national sport agencies such as UK Sport, as well as the incipient development of SV grassroots stream clearly placed SV in a vulnerable position in relation to external political forces. The most important outcomes of the present study is the identification of life dimensions that are significantly affected by participation in SV as well as the identification of the most important factors mediating such impact. Beyond the fields of disability sport and adapted physical activity, a theoretical/methodological symbiotic relation between capabilities approach and social sciences of sport would encourage those involved in sport to refocus their mission on people and human development instead of on economic and institutional benefits

The epididymis as a target of endocrine disruption: effect on calcium homeostasis

The epididymis is a central player in male fertility. Hormone regulation, mainly in the caput region, establishes the biochemical composition (water, ions, organic molecules) of the epididymal fluid, and it is during the transit through the epididymis, and by exposure to this unique environment, that sperm undergo the transformations required to become fully functional. Studies in knockout animals for calcium (Ca2+) channels demonstrated the importance of regulating epididymal Ca2+ levels for spermatozoa function, as these animals displayed impaired sperm motility, viability, and fertilization capacity. In recent decades, concern was raised about endocrine disruption and male fertility. Endocrine disruptors (EDs) can perturb the normal hormone signalling disrupting spermatogenesis. However, the effect of EDs in the epididymis function is largely unknown. Vinclozolin (VNZ), an endocrine disruptor (ED) with antiandrogenic activity, has been shown to affect male reproduction. The present thesis investigated the effect of VNZ on rat epididymal cells survival and function, Ca2+ homeostasis and antioxidant defences. Caput sections were cultured ex vivo in the presence or absence of VNZ 500 µM. Ca2+ levels, the expression of Ca2+-sensitive receptor (CaSR) and Ca2+ transient receptor potential vanilloid subfamily member 6 (TRPV6), the activity of glutathione peroxidase (GPX) and superoxide dismutase (SOD) and the activity of caspase-3 were assessed. CaSR and TRPV6 expression decreased in the presence of VNZ, together with an increase in the concentrations of extracellular Ca2+. Also, the activity of the antioxidant enzymes GPX and SOD and the activity of caspase-3 decreased in the VNZ-treated group. The obtained results demonstrated for the first time that VNZ dysregulates Ca2+ homeostasis and decreases the antioxidant defences in the epididymis, which indicates that the exposure to VNZ may affect sperm quality and male fertility.O epidídimo é uma estrutura fundamental para a fertilidade masculina. A regulação hormonal, principalmente na região do caput, estabelece a composição bioquímica (água, iões, moléculas orgânicas) do fluido do epidídimo, e é durante a exposição a esse ambiente único que os espermatozoides sofrem as transformações necessárias para se tornarem totalmente funcionais. Estudos em animais knockout para canais de cálcio (Ca2+) demonstraram a importância da regulação dos níveis de Ca2+ no epidídimo para a função dos espermatozóides, uma vez que esses animais apresentaram motilidade e viabilidade espermática reduzidas, assim como, capacidade de fertilização prejudicada. Nas últimas décadas, tem surgido uma preocupação crescente relativamente ao efeito dos chamados desreguladores endócrinos (DEs) na fertilidade masculina. Sabe-se que os DEs podem perturbar a normal sinalização hormonal, desregulando a espermatogénese, no entanto, o efeito dos DEs na função do epidídimo é ainda amplamente desconhecido. Foi descrito que a vinclozolina (VNZ), um DE com atividade antiandrogénica, afeta a reprodução masculina. Assim, a presente tese investigou o efeito da VNZ na sobrevivência e função celular, homeostase do Ca2+ e defesas antioxidantes no epidídimo de rato. Foram realizadas culturas ex vivo de seções da região do caput na presença ou ausência de VNZ 500 µM. Posteriormente, foram avaliados os níveis de Ca2+, a expressão do receptor sensível ao Ca2+ (CaSR), do receptor transiente de potencial membro da subfamília 6 (TRPV6), a atividade da glutationa peroxidase (GPX) e superóxido dismutase (SOD) e a atividade da caspase-3. Observou-se uma diminuição da expressão do CaSR e do TRPV6 na presença de VNZ, juntamente com um aumento das concentrações de Ca2+ extracelular. Além disso, a atividade das enzimas antioxidantes GPX e SOD e a atividade da caspase-3 diminuíram no grupo tratado com VNZ. Os presentes resultados demonstraram pela primeira vez as ações da VNZ na desregulação da homeostase do Ca2+ e redução das defesas antioxidantes no epidídimo, o que indica que a exposição a este DE pode afetar a qualidade do esperma e a fertilidade masculina