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

Scaling relations of branching pulsatile flow

Apparently complex flow structures obey to scaling relations that enable to make it viable the study of their configuration and flow dynamics. This is the case of flow structures that exhibit several branching levels and are thought to perform optimally. Here we present scaling relations of diameters and lengths of branching cylindrical channels with pulsatile flows, and compare them with other relations published in the literature. It is shown that, under constant global volume of the flow tree, and for zero pulse frequency these scaling relations reduce to Murrays's law of consecutive diameters. Optimal scaling depends on pulse frequency, distensibility of the channel walls, and asymmetry of the daughter vessels. In case that in addition to global volume of the flow tree, the pressure head is also kept constant, a similar scaling law of channel lengths emerges that holds together with the law of diameter scaling. The effect of channel distensibility is shown to be somehow important, such that for achieving optimal performance (lowest impedance) channels with lower relative distensibility must have their diameter increased. Results are compared with those of other models for the case of some arteries

Heart rate, arterial distensibility, and optimal performance of the arterial tree

In this study we explore the ability of a previously developed model of pulsatile flow for explaining the observed reduction of arterial distensibility with heart rate. The parameters relevant for the analysis are arterial wall distensibility together with permeability and reflection coefficients of the end capillaries. A non-specific artery and the ensemble of tissues supplied by that artery were considered in the model. The blood current within that artery was equalized to the sum of all micro currents in the tissues supplied by that artery. A formula emerged that relates changes in arterial distensibility with heart rate, and also with some particular aspects of microcirculation. Then, that formula was tested with data of distensibilities of the radial and carotid arteries observed at the heart rates of 63, 90, and 110 b.p.m. The formula correctly predicted the trend of decreased distensibility with heart rate for both arteries. Moreover, due to the fact that the carotid artery supplies the brain, and because the Blood–Brain barrier is highly restrictive to colloids in the blood, for the carotid artery the formula predicted a less marked decrease in distensibility than in the case of the radial artery feeding muscle tissue, which has a greater permeability to colloids, a trend that was confirmed by data. It was found that reduction of arterial distensibility with heart rate was greater in arteries that supply end capillaries with high permeability and low reflection coefficients

Beyond public transports: The portuguese environmental disclosure in the public transports sector

Even though throughout the years there has been a growth in the society’s mindset concerning environmental and social issues, it is still a field with several gaps and which severely lacks legislation. Several studies have already showed that by increasing legislation, there may be an increase on the amount of the companies’ environmental disclosures. Therefore, in order to regulate and increase the environmental disclosures in Europe, the European Union issued a Recommendation for its Member-States which was transposed to Portugal as an accounting standard – DC29 (Environmental Matters) - which should’ve been followed by the companies. The purpose of this research was to analyse how the environmental reporting practices have evolved in the Portuguese Public Transports sector, and whether it was influenced, or not, by the issuing of the DC29 Accounting Standard (And from 2010 onwards, the NCRF26). The public transports sector is currently one of the most pollutant sectors of the European Union. This thesis was based on content analysis of the Annual Accounts and Sustainability Reports of four of the biggest companies of the Portuguese Public Transports sector. Findings seemed to indicate that, even though the companies are not disclosing accordingly to the standard, the quantity of disclosure did increase, and the quality also evolved into more comparable, and more objective information.Apesar de ao longo dos últimos anos ter existido uma clara mudança de pensamento relacionada com aspectos ambientais e sociais, este é ainda um tema com várias falhas, sendo de realçar a falta de legislação. Vários estudos académicos mostraram uma relação entre o aumento da legislação existente e o da divulgação ambiental das empresas. Assim, de modo a regular e aumentar a divulgação ambiental na Europa, a União Europeia emitiu uma recomendação para os seus Estados-membros que foi transposta para Portugal na forma de Directriz Contabilística (DC 29 - Matérias Ambientais), que deveria ter sido seguida pelas empresas nas suas Demonstrações Financeiras. O objectivo desta tese é o de analisar o desenvolvimento das práticas de divulgação ambiental no sector dos transportes públicos, e se este foi ou não, influenciado pela publicação da DC 29, sendo que este é, neste momento, um dos sectores mais poluentes da União Europeia. Esta tese foi elaborada com base na análise de conteúdo dos Relatórios Anuais, de Contas e de Sustentabilidade, de quatro das maiores empresas portuguesas de Transportes Públicos. Os resultados revelaram que, apesar de as empresas não estarem a divulgar as suas matérias ambientais de acordo com o que está explícito na DC 29 (e a partir de 2010 na NCRF 26), a quantidade da divulgação de facto aumentou, a sua qualidade evoluiu positivamente e as partes interessadas passaram a ter acesso a uma informação mais comparável e mais objectiva

Exploring Memorable Sacred Tourism Experiences and Place Attachment

Ever since people have travelled to sacred places, religious and sacred sites have become some of the most visited destinations globally (Griftiths, 2011; Nyaupane et al., 2015), providing fascinating forms of travel (Griffin & Raj, 2017). However, despite the global importance and popularity of religious tourism and sacred places, few studies measure the tourists’ experiences in those destinations (Albayrak et al., 2018). Moreover, the standardisation of tourism products, including sacred ones, and the competitive market, requires that tourism destinations distinguish themselves by creating unique experiences. Currently, tourists seek appealing, unique, and memorable experiences that enhance their emotional link with tourism destinations. Previous research studies revealed that tourism experiences and place attachment were related. However, research in this area is still scarce, especially in sacred places. Within this context and based on the tourism experience and place attachment scales, the present study explores the relationship between tourist experience and place attachment in a sacred destination. Insights of an empirical study of 329 tourists who visited the sacred Portuguese destination of the Shrine of Fatima allowed us to conclude that memorable experience have the following characteristics: 1) meaningfulness and refreshment, 2) hedonism, 3) novelty, and 4) involvement. Place attachment also exhibits four dimensions: 1) place identity, 2) place dependence, 3) place familiarity, and 4) place symbolism. The present research contributes to the theoretical development of memorable tourism experiences and place attachment in the literature by addressing tourism experience and pace attachment dimensions in relation to sacred destinations. Also, the results could have potential practical implications for religious and holy destination planning, marketing, and management, promoting the differentiating qualities that attract tourists and involving them with these settings in a unique and memorable tourism experience

Ultrasound-assisted biosynthesis of novel methotrexate-conjugates

New methotrexate-acylglycerols and methotrexate-cyclodextrins (, and -CD) conjugates were obtained via esterification or transesterification reactions. All reactions were catalysed by esterases namely immobilized Lipase from Candida antarctica B and Lipase from Thermomyces lanuginosus. The use of ultrasound to assist the reactions revealed to be a key factor to obtain high conversion yields on both MTX conjugates. Transesterification reactions including long chain triacylglycerols were only successful when ultrasound was applied. In cyclodextrins esterification a higher number of MTX molecules was also linked to cyclodextrins when ultrasound was used. All the conjugates were characterized by MALDI-TOF and NMR spectroscopy.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Jennifer Noro also thanks to FCT – Fundação para a Ciência e a Tecnologia for funding her scholarship (SFRH/BD/121673/2016). This work has also received funding from the European Union Horizon 2020 research and innovation programme under grant agreement NMP-06-2015-683356 FOLSMART.info:eu-repo/semantics/publishedVersio

Para uma leitura do texto-mundo: educação literária e expressão e educação dramática/teatro - O conto é teu, o conto é nosso

Nos últimos anos, a nossa experiência docente/investigativa em contexto académico tem-nos levado a concluir/intuir que a formação literária e artística, em geral, dos nossos alunos da Licenciatura em Educação Básica, futuros profissionais de educação/mediadores de leitura, é, mesmo numa fase que se pode considerar já avançada dos estudos, manifestamente débil, evidenciando níveis de competência literária muito distintos/diferenciados, em alguns casos, até, constrangedores quanto, por exemplo, ao “intertexto leitor”. O projeto em implementação e do qual pretendemos dar conta nesta comunicação reveste-se de uma dimensão interdisciplinar, procurando não apenas acentuar a transversalidade da língua portuguesa, materializada em textos literários (do acervo tradicional à literatura canónica), mas também da própria cultura artística. Trata-se de um projeto desenhado com alunos dos 1º e 2º anos da Licenciatura em Educação Básica, do Instituto de Educação da Universidade do Minho. Neste âmbito e numa perspetiva interdisciplinar, pretende-se implementar um conjunto de atividades de mediação literária e artística, no decurso da formação inicial de professores do 1º ciclo do ensino básico e educadores de infância. Optando por uma metodologia de investigação-ação, numa primeira fase, propomo-nos conhecer a cultura literária dos alunos, para, posteriormente e designadamente no contexto de UC como Literatura para a Infância e a Juventude e Expressão Dramática, entre outras, alargar o contacto com modos literários cuja receção é aparentemente menos generalizada (poético e dramático/teatral); desenvolver dimensões fonológicas e de interpretação inerentes a contextos de dizer e de oralidade (narração, formas poético-líricas e texto dramático); perceber como a leitura poderá contribuir para a melhoria da capacidade de desconstrução de textos e, consequentemente, para a formação de futuros cidadãos ativos na leitura do texto-mundo.CIEC - Centro de Investigação em Estudos da Criança, IE, UMinho (UI 317 da FCT), Portugal, Fundos Nacionais através da FCT (Fundação para a Ciência e a Tecnologia) e cofinanciado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) com a referência POCI-01-0145-FEDER-007562info:eu-repo/semantics/publishedVersio

Cascading Crypthecodinium cohnii biorefinery: global warming potential and techno-economic assessment

ABSTRACT: Prior to the commissioning of a new industrial biorefinery it is deemed necessary to evaluate if the new project will be beneficial or detrimental to climate change, one of the main drivers for the sustainable development goals (SDG) of the United Nations. In particular, how SDG 7, Clean and Efficient Energy, SDG 3, Good Health and Well Being, SDG 9, Industry Innovation and Infrastructure, and SDG 12, Responsible Production and Consumption, would engage in a new biorefinery design, beneficial to climate change, i.e., fostering SDG 13, Climate Action. This study uses life cycle assessment methodology (LCA) to delve in detail into the Global Warming Impact category, project scenario GHG savings, using a conventional and a dynamic emission flux approach until 2060 (30-year lifetime). Water, heat and electricity circularity are in place by using a water recirculation process and a combined heat and power unit (CHP). A new historical approach to derive low and higher-end commodity prices (chemicals, electricity, heat, jet/maritime fuel, DHA, N-fertilizer) is used for the calculation of the economic indicators: Return of investment (ROI) and inflation-adjusted return (IAR), based upon the consumer price index (CPI). Main conclusions are: supercritical fluid extraction is the hotspot of energy consumption; C. cohnii bio-oil without DHA has higher sulfur concentration than crude oil based jet fuel requiring desulfurization, however the sulfur levels are compatible with maritime fuels; starting its operation in 2030, by 2100 an overall GHG savings of 73% (conventional LCA approach) or 85% (dynamic LCA approach) is projected; economic feasibility for oil productivity and content of 0.14 g/L/h and 27% (w/w) oil content, respectively (of which 31% is DHA), occurs for DHA-cost 100 times higher than reference fish oil based DHA; however future genetic engineering achieving 0.4 g/L/h and 70% (w/w) oil content (of which 31% is DHA), reduces the threshold to 20 times higher cost than reference fish oil based DHA; N-fertilizer, district heating and jet fuel may have similar values then their fossil counterparts.info:eu-repo/semantics/publishedVersio

Impact of brewery wastewater inhibitors in pure and mixed cultures of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07

ABSTRACT: Brewery wastewater (BWW) is an appealing low-cost substrate for the production of single cell oils by oleaginous microorganisms. However, it may contain inhibitor compounds that may affect the microbial metabolism. This work investigated, for the first time, the presence of potential inhibitor compounds in primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) for the pure and mixed cultivation of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07. Three organic acids (OrgAc) were identified in the brewery effluents (acetic, propionic and butyric acids). Yeast and microalga pure and mixed cultivations were performed in PBWW and SBWW in order to understand the behaviour of the microorganisms, individually and together. Flow cytometry (FC) was used to monitor each microbial population during the mixed cultivations, and to study the yeast and microalga cell viability throughout all cultivations. The yeast cells in pure cultures grown in both effluents were severely affected by the OrgAc presence confirmed by the cell stress results obtained by FC. However, in the mixed cultures, the yeast cells were able to develop, and the levels of stress conditions were considerably lower. Only in microalga pure and mixed cultures efficient OrgAc removal was observed.info:eu-repo/semantics/publishedVersio

New dual-stage pH control fed-batch cultivation strategy for the improvement of lipids and carotenoids production by the red yeast Rhodosporidium toruloides NCYC 921

The optimal medium pH to produce biomass and fatty acids by the red yeast Rhodosporidium toruloides NCYC 921 is 4.0, and to produce carotenoids is 5.0. Based on this difference, a dual-stage pH control fed-batch cultivation strategy for the enhancement of lipids and carotenoids production by this yeast was studied. The results showed that when the yeast growth phase was conducted at pH 4.0, and the products accumulation phase was conducted at pH 5.0, biomass, total fatty acid and total carotenoid productivities were significantly improved comparing with the yeast fed batch cultivations carried out at fixed medium pH (4 or 5). Under dual-stage pH control conditions, the biomass, carotenoids and lipids productivities attained 2.35 g/L h, 0.29 g/L h and 0.40 g/L h, respectively. It was also observed that the oxygen played a major role in the yeast carotenoid production