Influxo de Ca2+ mediado por canais TRP em células secretoras de insulina

Dissertação de mestrado em Bioquímica apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A célula-β está equipada com um mecanismo de influxo de Ca2+ distinto do mediado por canais CaV. Pretendeu-se neste trabalho identificar este mecanismo, recorrendo a técnicas de microscopia quantitativa de fluorescência para monitorizar a [Ca2+]i em ilhéus únicos de ratinho. A estimulação do influxo de Ca2+ foi efectuada, na maior parte dos casos, aumentando a [Ca2+]o para 7 mM (doravante designada como „pulso de Ca2+‟). A aplicação de um pulso de Ca2+ na presença de 3 mM glicose aumentou a [Ca2+]i. O efeito não foi afectado por hiperpolarização com o agonista de canais KATP diazóxido (100 μM), mas foi muito potenciado pelo bloqueador de canais CaV1.0 nifedipina (10 μM). Nifedipina aumentou a [Ca2+]i e acelerou o quenching da fluorescência de FURA-2 por Mn2+ no ponto isosbéstico do indicador. Os efeitos dos pulsos de Ca2+ na presença de nifedipina foram bloqueados por dois fenamatos, difenilamina-2-carboxilato (DPC, 500 μM) e ácido mefenâmico (3-80 μM; IC50=10.3 μM). O ácido mefenâmico é um inibidor aparentemente selectivo de canais TRPM3, uma sub-classe de canais TRP activada pelo esteróide neuroactivo pregnenolona. DPC aumentou a [Ca2+]i acentuadamente na ausência de nifedipina, pondo em evidência acções colaterais indesejáveis noutros transportadores de Ca2+; o efeito correspondente de ácido mefenâmico foi negligenciável. O ácido mefenâmico não afectou os aumentos da [Ca2+]i induzidos por pulsos de Ca2+ na ausência de nifedipina, indicando que os canais subjacentes são diferentes dos canais TRPM3. Também foram avaliados os efeitos de pulsos de Ca2+ utilizando células BRIN-BD11 únicas. Os pulsos de Ca2+ induziram respostas compostas por vários aumentos rápidos e transitórios, com latências variáveis, um padrão que era notoriamente diferente do registado em ilhéus. Esta observação sugere que, em células BRIN-BD11, o mecanismo subjacente ao efeito dos pulsos de Ca2+ é manifestamente diferente do que ocorre em ilhéus. Conclui-se que as células-β de ratinho estão equipadas com canais TRPM3 sensíveis a pregnenolona, nifedipina e ácido mefenâmico. Sendo estes canais permeáveis a outros catiões divalentes, é provável que sejam responsáveis por IV recapturar iões Zn2+, que são necessários para a estabilização da insulina nos grânulos da célula-β.There is evidence for the operation of a Ca2+ influx pathway distinct from CaV channels in pancreatic beta cells. I aimed at identifying this pathway, using quantitative FURA-2 fluorescence microscopy to monitor global [Ca2+]i from single mouse islets. Stimulation of Ca2+ influx was effected mostly by raising [Ca2+]o to 7 mM (henceforth designated as a „Ca2+ pulse‟). Applying a Ca2+ pulse in presence of 3 mM glucose raised the [Ca2+]i. The effect was insensitive to further hyperpolarization with the KATP channel opener diazoxide (100 μM) but was greatly enhanced by the CaV1.0 blocker nifedipine (10 μM). Nifedipine itself raised the [Ca2+]i and accelerated Mn2+ quenching of FURA-2 fluorescence at the dye‟s isosbestic point. The effects of Ca2+ pulses in presence of nifedipine were blocked by two fenamates, diphenylamine-2-carboxylate (DPC, 500 μM) and mefenamic acid (3-80 μM; IC50: 10.3 μM). Mefenamic acid is a seemingly selective inhibitor of TRPM3 channels, a TRP channel sub-class activated by the neuroactive steroid pregnenolone. DPC raised the [Ca2+]i markedly in absence of nifedipine, highlighting unwanted actions of the drug on Ca2+ transport mechanisms unrelated to TRPM3 channels; the corresponding effect of mefenamic acid was negligible. Mefenamic acid did not affect the [Ca2+]i rises evoked by Ca2+ pulses in absence of nifedipine, indicating that the underlying channels are distinct from TRPM3 channels. I have also assessed the effects of Ca2+ pulses using single insulin-secreting BRIN-BD11 cells. Ca2+ pulses elicited multi-spike responses with variable lag-times, a pattern that contrasted markedly with that in islets, suggesting that the underlying mechanism is different. It is concluded that mouse beta-cells are equipped with pregnenolone-, nifedipine- and mefenamic acid-sensitive TRPM3 channels. Since these channels are permeant to other divalent cations, they may provide the re-uptake of Zn2+ ions, required to stabilize insulin in the beta-cell granules

Building a Portuguese Coalition for Biodiversity Genomics

The diverse physiography of the Portuguese land and marine territory, spanning from continental Europe to the Atlantic archipelagos, has made it an important repository of biodiversity throughout the Pleistocene glacial cycles, leading to a remarkable diversity of species and ecosystems. This rich biodiversity is under threat from anthropogenic drivers, such as climate change, invasive species, land use changes, overexploitation or pathogen (re)emergence. The inventory, characterization and study of biodiversity at inter- and intra-specific levels using genomics is crucial to promote its preservation and recovery by informing biodiversity conservation policies, management measures and research. The participation of researchers from Portuguese institutions in the European Reference Genome Atlas (ERGA) initiative, and its pilot effort to generate reference genomes for European biodiversity, has reinforced the establishment of Biogenome Portugal. This nascent institutional network will connect the national community of researchers in genomics. Here, we describe the Portuguese contribution to ERGA’s pilot effort, which will generate high-quality reference genomes of six species from Portugal that are endemic, iconic and/or endangered, and include plants, insects and vertebrates (fish, birds and mammals) from mainland Portugal or the Azores islands. In addition, we outline the objectives of Biogenome Portugal, which aims to (i) promote scientific collaboration, (ii) contribute to advanced training, (iii) stimulate the participation of institutions and researchers based in Portugal in international biodiversity genomics initiatives, and (iv) contribute to the transfer of knowledge to stakeholders and engaging the public to preserve biodiversity. This initiative will strengthen biodiversity genomics research in Portugal and fuel the genomic inventory of Portuguese eukaryotic species. Such efforts will be critical to the conservation of the country’s rich biodiversity and will contribute to ERGA’s goal of generating reference genomes for European species.info:eu-repo/semantics/publishedVersio

Acesso a Tratamento Endovascular para Acidente Vascular Cerebral Isquémico em Portugal

Introduction: Since the publication of endovascular treatment trials and European Stroke Guidelines, Portugal has re-organized stroke healthcare. The nine centers performing endovascular treatment are not equally distributed within the country, which may lead to differential access to endovascular treatment. Our main aim was to perform a descriptive analysis of the main treatment metrics regarding endovascular treatment in mainland Portugal and its administrative districts. Material and Methods: A retrospective national multicentric cohort study was conducted, including all ischemic stroke patients treated with endovascular treatment in mainland Portugal over two years (July 2015 to June 2017). All endovascular treatment centers contributed to an anonymized database. Demographic, stroke-related and procedure-related variables were collected. Crude endovascular treatment rates were calculated per 100 000 inhabitants for mainland Portugal, and each district and endovascular treatment standardized ratios (indirect age-sex standardization) were also calculated. Patient time metrics were computed as the median time between stroke onset, first-door, and puncture. Results: A total of 1625 endovascular treatment procedures were registered. The endovascular treatment rate was 8.27/100 000 inhabitants/year. We found regional heterogeneity in endovascular treatment rates (1.58 to 16.53/100 000/year), with higher rates in districts closer to endovascular treatment centers. When analyzed by district, the median time from stroke onset to puncture ranged from 212 to 432 minutes, reflecting regional heterogeneity. Conclusion: The overall national rate of EVT in the first two years after the organization of EVT-capable centers is one of the highest among European countries, however, significant regional disparities were documented. Moreover, stroke-onset-to-first-door times and in-hospital procedural times in the EVT centers were comparable to those reported in the randomized controlled trials performed in high-volume tertiary hospitals.Introdução: A aprovação do tratamento endovascular para o acidente vascular cerebral isquémico obrigou à reorganização dos cuidados de saúde em Portugal. Os nove centros que realizam tratamento endovascular não estão distribuídos equitativamente pelo território, o que poderá causar acesso diferencial a tratamento. O principal objetivo deste estudo é realizar uma análise descritiva da frequência e métricas temporais do tratamento endovascular em Portugal continental e seus distritos. Material e Métodos: Estudo de coorte nacional multicêntrico, incluindo todos os doentes com acidente vascular cerebral isquémico submetidos a tratamento endovascular em Portugal continental durante um período de dois anos (julho 2015 a junho 2017). Foram colhidos dados demográficos, relacionados com o acidente vascular cerebral e variáveis do procedimento. Taxas de tratamento endovascular brutas e ajustadas (ajuste indireto a idade e sexo) foram calculadas por 100 000 habitantes/ano para Portugal continental e cada distrito. Métricas de procedimento como tempo entre instalação, primeira porta e punção foram também analisadas. Resultados: Foram registados 1625 tratamentos endovasculares, indicando uma taxa bruta nacional de tratamento endovascular de 8,27/100 000 habitantes/ano. As taxas de tratamento endovascular entre distritos variaram entre 1,58 e 16,53/100 000/ano, com taxas mais elevadas nos distritos próximos a hospitais com tratamento endovascular. O tempo entre sintomas e punção femural entre distritos variou entre 212 e 432 minutos. Conclusão: Portugal continental apresenta uma taxa nacional de tratamento endovascular elevada, apresentando, contudo, assimetrias regionais no acesso. As métricas temporais foram comparáveis com as observadas nos ensaios clínicos piloto

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

An estimate of the number of tropical tree species

The high species richness of tropical forests has long been recognized, yet there remains substantial uncertainty regarding the actual number of tropical tree species. Using a pantropical tree inventory database from closed canopy forests, consisting of 657,630 trees belonging to 11,371 species, we use a fitted value of Fisher’s alpha and an approximate pantropical stem total to estimate the minimum number of tropical forest tree species to fall between ∼40,000 and ∼53,000, i.e. at the high end of previous estimates. Contrary to common assumption, the Indo-Pacific region was found to be as species-rich as the Neotropics, with both regions having a minimum of ∼19,000–25,000 tree species. Continental Africa is relatively depauperate with a minimum of ∼4,500–6,000 tree species. Very few species are shared among the African, American, and the Indo-Pacific regions. We provide a methodological framework for estimating species richness in trees that may help refine species richness estimates of tree-dependent taxa

A search for ultra-high-energy photons at the Pierre Auger Observatory exploiting air-shower universality

The Pierre Auger Observatory is the most sensitive detector to primary photons with energies above ∼0.2 EeV. It measures extensive air showers using a hybrid technique that combines a fluorescence detector (FD) with a ground array of particle detectors (SD). The signatures of a photon-induced air shower are a larger atmospheric depth at the shower maximum (X$_{max}$) and a steeper lateral distribution function, along with a lower number of muons with respect to the bulk of hadron-induced background. Using observables measured by the FD and SD, three photon searches in different energy bands are performed. In particular, between threshold energies of 1-10 EeV, a new analysis technique has been developed by combining the FD-based measurement of X$_{max}$ with the SD signal through a parameter related to its muon content, derived from the universality of the air showers. This technique has led to a better photon/hadron separation and, consequently, to a higher search sensitivity, resulting in a tighter upper limit than before. The outcome of this new analysis is presented here, along with previous results in the energy ranges below 1 EeV and above 10 EeV. From the data collected by the Pierre Auger Observatory in about 15 years of operation, the most stringent constraints on the fraction of photons in the cosmic flux are set over almost three decades in energy

Study on multi-ELVES in the Pierre Auger Observatory

Since 2013, the four sites of the Fluorescence Detector (FD) of the Pierre Auger Observatory record ELVES with a dedicated trigger. These UV light emissions are correlated to distant lightning strikes. The length of recorded traces has been increased from 100 μs (2013), to 300 μs (2014-16), to 900 μs (2017-present), to progressively extend the observation of the light emission towards the vertical of the causative lightning and beyond. A large fraction of the observed events shows double ELVES within the time window, and, in some cases, even more complex structures are observed. The nature of the multi-ELVES is not completely understood but may be related to the different types of lightning in which they are originated. For example, it is known that Narrow Bipolar Events can produce double ELVES, and Energetic In-cloud Pulses, occurring between the main negative and upper positive charge layer of clouds, can induce double and even quadruple ELVES in the ionosphere. This report shows the seasonal and daily dependence of the time gap, amplitude ratio, and correlation between the pulse widths of the peaks in a sample of 1000+ multi-ELVES events recorded during the period 2014-20. The events have been compared with data from other satellite and ground-based sensing devices to study the correlation of their properties with lightning observables such as altitude and polarity