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Centro de Investigación en Sanidad Animal (CISA), Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry., M. Monteiro, A. S. Lavrador, R. A. Santos, and F. Rangel were supported by grants SFRH/BD/114995/2016, ZEBRALGRE_BM_2019-003, SFRH/BD/131069/2017, and SFRH/BD/138375/2018, respectively, from FCT — Foundation for Science and Technology, under the POCI program. A. Couto C. Serra and P. Enes have a scientific employment contract supported by national funds through FCT. E. da Costa and M. R. Domingues are financially supported by FCT/MCTES (Portugal) through CESAM (UIDB/50017/2020 + UIDP/50017/2020), QOPNA (FCT UID/QUI/00062/2019), LAQV/REQUIMTE (UIDB/50006/2020), and RNEM (LISBOA-01–0145-FEDER-402–022125). This research was partially supported by the Strategic Funding to UID/Multi/04423/2019 (POCI-01–0145-FEDER-007621) through national funds provided by FCT under the project PTDC/CVT-WEL/5207/2014.

Off mainland Portugal, the common dolphin (Delphinus delphis) is the most sighted cetacean, although information on this species is limited. The Atlantic coast of Southern Portugal is characterized by an intense wind-driven upwelling, creating ideal conditions for common dolphins. Using data collected aboard whale-watching boats (1929 sightings and 4548 h effort during 2010–2014), this study aims to understand the relationships between abundance rates (AR) of dolphins of different age classes (adults, juveniles, calves and newborns) and oceanographic [chlorophyll a (Chl-a) and sea surface temperature (SST)] variables. Over 70% of the groups contained immature animals. The AR of adults was negatively related with Chl-a, but not related to SST values. The AR of juveniles was positively related with SST. For calves and newborns, although the relationship between SST and AR is similar to that observed for juveniles, the effect could not be distinguished from zero. There was no relationship between Chl-a levels and the AR of juveniles, calves and newborns. These results corroborate previous findings that common dolphins tend to occur in highly productive areas demonstrating linkages between their abundance and oceanographic variables, and that this region may be a potential nursery ground.

Pentedrone and methylone can express stereoselectivity in toxicokinetic and toxicodynamic processes. Similarly, their chiral discrimination in metabolism, which was not yet evaluated, can result in different metabolic profiles and subsequent hepatotoxic effects. Therefore, the aim of this work was to assess, for the first time, both the hepatic cytotoxic and metabolic profile of pentedrone and methylone enantiomers using physiologically relevant in vitro models. The hepatotoxicity of these compounds was observed in a concentration-dependent manner in human stem-cell-derived hepatocyte-like cells (HLCs) cultured under 3D (3D-HLCs) and 2D (2D-HLCs) conditions. Enantioselectivity, on the other hand, was only shown for pentedrone (1 mM) in 3D-HLCs, being R-(−)-pentedrone the most cytotoxic. Furthermore, the metabolic profile was initially evaluated in human liver microsomes (HLM) and further demonstrated in 3D-HLCs and 2D-HLCs applying a gas chromatography coupled to a mass spectrometer (GC–MS) technique. Methylone and pentedrone showed distinct and preferential metabolic routes for their enantiomers, resulting in the production of differentiated metabolites; R-(+)-methylone and R-(−)-pentedrone are the most metabolized enantiomers. In conclusion, the results demonstrated enantioselectivity for pentedrone and methylone in the metabolic processes, with enantioselectivity in cytotoxicity for pentedrone.

Portuguese network for SARS-CoV-2 genomics (Consortium): Agostinho José S Lira, Aida M Sousa Fernandes, Alexandra Estrada, Alexandra Nunes, Alfredo Rodrigues, Ana Caldas, Ana Constança, Ana Margarida Henriques, Ana Miguel Matos, Ana Oliveira, Ana Paula Dias, Ana Pelerito, Ana Rita Couto, Anabela Vilares, António Albuquerque, Baltazar Nunes, Bruna R Gouveia, Carina de Fátima Rodrigues, Carla Feliciano, Carla Roque, Carlos Cardoso, Carlos Sousa, Cathy Paulino, Célia Rodrigues Bettencourt, Claudia C Branco, Cláudia Nunes Dos Santos, Conceição Godinho, Constantino P Caetano, Cristina Correia, Cristina Toscano, Cristina Veríssimo, Daniela Silva, Diana Patrícia Pinto da Silva, Eliana Costa, Elizabeth Pádua, Fátima Martins, Fátima Vale, Fernanda Vilarinho, Fernando Branca, Filomena Caldeira, Filomena Lacerda, Francisca Rocha, Graça Andrade, Helena Ribeiro, Helena Rodrigues, Herberto Jesus, Hugo Sousa, Idalina Ferreira, Inês Baldaque, Inês Costa, Inês Gomes, Inna Slobidnyk, Isabel Albergaria, Isabel Dias, Isabel Fernandes, Isabel Lopes de Carvalho, Ivone Água-Doce, Jácome Bruges Armas, Joana Ramos, João Carlos Sousa, João Costa, João Dias, João Rodrigues, João Sobral, Jorge Machado, Jorge Meneses, José Alves, José Vicente Constantino, Laura Brum, Leonor Silveira, Líbia Zé-Zé, Lidia Santos, Ludivina Freitas, Luís Silva, Luisa Mota-Vieira, Lurdes Lopes, Lurdes Monteiro, Márcia Faria, Margarida Farinha, Margarida Vaz, Maria Alice Pinto, Maria Ana Pessanha, Maria Beatriz Tomaz, Maria Calle Vellés, Maria da Graça Maciel de Soveral, Maria Helena Ramos, Maria Isabel Veiga, Maria João Gargate, Maria João Peres, Maria José Borrego, Maria Matos Figueiredo, Mariana Martins, Mariana Viana, Maurício Melim, Miguel Babarro Jorreto, Miguel Fevereiro, Miguel Pinheiro, Mónica Oleastro, Nair Seixas, Nelson Ventura, Nuno Verdasca, Olga Costa, Patrícia Barros, Patricia Fonseca, Patricia Miguel, Paula Bajanca-Lavado, Paula Branquinho, Paula Palminha, Paula Soares, Paula Valente, Paulo Leandro, Paulo Pereira, Pedro Cardoso, Pedro Pechirra, Pedro Ramos, Raquel Neves, Raquel Rocha, Raquel Rodrigues, Raquel Sabino, Regina Sá, Ricardo Filipe Romão Ferreira, Ricardo Rodrigues, Rita C Veloso, Rita Cordeiro, Rita Côrte-Real, Rita de Sousa, Rita Gralha, Rita Macedo, Rita Matos, Rita Rodrigues, Sandra Paulo, Sara Sousa, Sílvia Lopo, Sónia Marta Santos Magalhães, Sónia Rodrigues, Sónia Silva, Susana Ladeiro, Susana Martins, Susana Silva, Teresa Salvado, Tiago Luís, Valquíria Alves, Vera Manageiro Background: Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods: By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARS-CoV-2 introductions and early dissemination in Portugal. Results: We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions: Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance. Plain language summary: Analysing SARS-CoV-2 genetic material and how it changes over time can help us understand how the virus spreads between countries and determine the impact of control measures. In this study, we investigated SARS-CoV-2 transmission and evolution in the early stages of the COVID-19 pandemic in Portugal. In particular, we reconstructed the routes and timeliness of viral introductions into the country and assessed the relative contribution of each introduction in terms of how the epidemic evolved over time. We detected at least 277 independent introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. This study reflects an unprecedented effort in the field of the infectious diseases in Portugal, highlighting the need for systematic and geographically-representative surveillance to aid public health efforts to control the virus. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Por tugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

14 Pág. Centro de Investigación en Sanidad Animal (CISA), The search for natural immunotherapeutic agents has become increasingly important for fish disease treatment and prophylaxis. The present research explored the effects of macro- and microalgal extracts on zebrafish juvenile growth and immune status. A plant-based diet was used as a control (diet C), and seven other diets were prepared similar to diet C, supplemented with 10 g Kg−1 of each algal extract (Fucus vesiculosus, F; Nannochloropsis gaditana, N; and Ulva rigida, U), or a combination of extracts (diets FN, FU, NU and FNU). A fishmeal-based diet was included as a positive control (diet C+). Diet C+ outperformed the remaining dietary treatments, growth-wise. None of the extracts improved diet C negative effects. However, the inclusion of U. rigida and N. gaditana extracts promoted an immunomodulatory action after 1 week of trial, by upregulating cytokine expression. This effect subsided after 30 days, suggesting tolerance may be developed over time. In contrast, effects on growth-related genes were still observed after 5 weeks of feeding F, N, U and FN diets. Algal extract dietary inclusion did not compromise intestinal integrity. These findings support the potential of algal extracts as functional feed additives during short periods (, Fincanced by the Foundation for Science and Technology (grants number: SFRH/BD/114995/2016,EBRALGRE_ BM_2019-003 UID/Multi/04423/2019 POCI-01-0145-FEDER-007621 PTDC/CVT-WEL/5207/2014).

19 Pág. Centro de Investigación en Sanidad Animal (CISA), Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare., This research was funded by U. Porto and SOJA DE PORTUGAL, grant number PP-IJUP2019-SOJA DE PORTUGAL-14 This research was also partially supported by the FCT, grant numbers POCI-01-0145-FEDER-007621 and PTDC/CVT-WEL/5207/2014.

REPLYING TO H. Murua et al. Nature https://doi.org/10.1038/s41586-021-03396-4 (2021) Our previously published paper1 provided global fine-scale spatiotemporal estimates (1° × 1°; monthly) of overlap and fishing exposure risk (FEI) between satellite-tracked shark space use and automatic identification system (AIS) longline fishing effort. We did not assess shark mortality directly, but in addition to replying to the Comment by Murua et al.2, we confirm—using regression analysis of spatially matched data—that fishing-induced pelagic shark mortality (catch per unit effort (CPUE)) is greater where FEI is higher. We focused on assessing shark horizontal spatiotemporal overlap and exposure risk with fisheries because spatial overlap is a major driver of fishing capture susceptibility and previous shark ecological risk assessments (ERAs) assumed a homogenous shark density within species-range distributions3,4,5 or used coarse-scale modelled occurrence data, rather than more ecologically realistic risk estimates in heterogeneous habitats that were selected by sharks over time. Furthermore, our shark spatial exposure risk implicitly accounts for other susceptibility factors with equal or similar probabilities to those commonly used in shark ERAs3,5.

The interest in using marine organisms as a source of novel bioactive compounds has increased recently, reinforcing the need to find efficient and cost-effective methods to extract these biocompounds. Thus, the present study has focused on evaluating four solvent systems and three extraction methods to obtain extracts of three macroalgae (Gracilaria sp., Fucus vesiculosus, and Ulva rigida) and two microalgae (Chlorella sp. and Nannochloropsis gaditana). The extracts were evaluated regarding yield, phenolic content, and potential antioxidant activity. Results showed that a lower organic solvent:water ratio increased macroalgae, but not microalgae, biomass extraction efficiency. Levels of total phenolics, ortho-diphenols and flavonoids were highly influenced by algae material and solvent system applied. Strong 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity was obtained with ethanol:water, 80:20 (E80:20) microalgal extracts, while no visible trend was detected for macroalgae extracts. However, methanolic extracts were the most effective in scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In macroalgae, a high positive correlation was observed between phenolic content and antioxidant capacity while in microalgae, the opposite was verified, suggesting that in microalgae, phenolic compounds are not the main contributors to the observed antioxidant capacity.

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αβ or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αβ and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bassDicentrarchus labrax,to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRβ, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.

29 pages, 9 figures, 4 tables.-- This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credite, Climate-driven expansions of ocean hypoxic zones are predicted to concentrate pelagic fish in oxygenated surface layers, but how expanding hypoxia and fisheries will interact to affect threatened pelagic sharks remains unknown. Here, analysis of satellite-tracked blue sharks and environmental modelling in the eastern tropical Atlantic oxygen minimum zone (OMZ) shows shark maximum dive depths decreased due to combined effects of decreasing dissolved oxygen (DO) at depth, high sea surface temperatures, and increased surface-layer net primary production. Multiple factors associated with climate-driven deoxygenation contributed to blue shark vertical habitat compression, potentially increasing their vulnerability to surface fisheries. Greater intensity of longline fishing effort occurred above the OMZ compared to adjacent waters. Higher shark catches were associated with strong DO gradients, suggesting potential aggregation along suitable DO gradients contributed to habitat compression and higher fishing-induced mortality. Fisheries controls to counteract deoxygenation effects on shark catches will be needed as oceans continue warming, Funding was provided by the UK Natural Environment Research Council (NERC) (NE/R00997/X/1), European Research Council (ERC-AdG-2019 883583 OCEAN DEOXYFISH), NERC Oceans 2025 Strategic Programme (all to DWS), the Save Our Seas Foundation (DWS, NQ), Fundacao para a Ciencia e a Tecnologia (FCT) under PTDC/BIA/28855/2017 and COMPETE POCI-01–0145-FEDER-028855 (NQ, DWS), and MARINFO–NORTE-01–0145-FEDER-000031 (funded by Norte Portugal Regional Research article Ecology Operational Program [NORTE2020], under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund-RDF), and Xunta de Galicia Spain under the Isabel Barreto Program 2009–2012 (GM). FCT supported NQ (CEECIND/02857/2018), MV (PTDC/BIA-COM/28855/2017), GM (PTDC/MAR-BIO/4458/2012), FV (CEECIND/03469/2017), and JF (through the strategic project UID/MAR/04292/2013 to MARE). Direccao Regional de Cieˆ ncia e Tecnologia (DRCT) also supported JF (M3.1a/F/062/2016). DWS was supported by a Marine Biological Association Senior Research Fellowship

REPLYING TO A. V. Harry & J. M. Braccini Nature https://doi.org/10.1038/s41586-021-03463-w (2021) Our global analysis1 estimated the overlap and fishing exposure risk (FEI) using the space use of satellite-tracked sharks and longline fishing effort monitored by the automatic identification system (AIS). In the accompanying Comment, Harry and Braccini2 draw attention to two localized shark–longline vessel overlap hotspots in Australian waters, stating that 47 fishing vessels were misclassified as longline and purse seine vessels in the Global Fishing Watch (GFW)3 2012–2016 AIS fishing effort data product that we used. This, they propose2, results in misidentifications that highlight fishing exposure hotspots that are subject to an unexpected level of sensitivity in the analysis and they suggest that misidentifications could broadly affect the calculations of fishing exposure and the central conclusions of our study1. We acknowledged in our previously published paper1 that gear reclassifications were likely to occur for a small percentage of the more than 70,000 vessels studied, however, here we demonstrate that even using much larger numbers of vessel reclassifications than those proposed by Harry and Braccini2, the central results and conclusions of our paper1 do not change.

Genomic surveillance of SARS-CoV-2 was rapidly implemented in Portugal by the National Institute of Health in collaboration with a nationwide consortium of >50 hospitals/laboratories. Here, we track the geo-temporal spread of a SARS-CoV-2 variant with a mutation (D839Y) in a potential host-interacting region involving the Spike fusion peptide, which is a target motif of anti-viral drugs that plays a key role in SARS-CoV-2 infectivity. The Spike Y839 variant was most likely imported from Italy in mid-late February and massively disseminated in Portugal during the early epidemic, becoming prevalent in the Northern and Central regions of Portugal where it represented 22% and 59% of the sampled genomes, respectively, by 30 April. Based on our high sequencing sampling during the early epidemics [15.5% (1275/8251) and 6.0% (1500/24987) of all confirmed cases until the end of March and April, respectively], we estimate that between 14 March and 9 April (covering the epidemic exponential phase) the relative frequency of the Spike Y839 variant increased at a rate of 12.1% (6.1%–18.2%, CI 95%) every three days, being potentially associated with 24.8% (20.8–29.7%, CI 95%; 3177–4542 cases, CI 95%) of all COVID-19 cases in Portugal during this period. Our data support population/epidemiological (founder) effects contributing to the Y839 variant super spread. The potential existence of selective advantage is also discussed, although experimental validation is required. Despite huge differences in genome sampling worldwide, SARS-CoV-2 Spike D839Y has been detected in 13 countries in four continents, supporting the need for close surveillance and functional assays of Spike variants. This study is co-funded by Fundação para a Ciência e a Tecnologia and Agência de Investigação Clínica e Inovação Biomédica [grant number 234_596874175] on behalf of the Research 4 COVID-19 call. This work is also a result of the GenomePT project [grant number POCI-01-0145- FEDER-022184], supported by COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

Dissertação de Mestrado Integrado em Psicologia apresentada à Faculdade de Psicologia e de Ciências da Educação O sono constituiu umas das principais necessidades do ser humano com impacto significativo na qualidade de vida e nos indicadores de bem-estar físico, emocional e psicológico. No caso da doença renal crónica a existência de queixas ao nível do sono é comum, associada a um risco acrescido para o desenvolvimento de distúrbios do sono através de vários mecanismos fisiopatológicos. A doença renal crónica compreende diferentes estádios de gravidade, sendo a partir de um determinado momento necessário o recurso a um tratamento de substituição renal como a hemodiálise, ao qual se pode seguir o transplante renal. O número de doentes renais crónicos continua a aumentar e, como resultado disso, é provável que a prevalência de distúrbios do sono também aumente. Neste sentido, este estudo teve como principal objetivo identificar e caraterizar dificuldades e padrões de sono numa amostra da população clínica com doença renal crónica (em hemodiálise ou transplantada) por comparação com pessoas sem doença renal, para que seja possível objetivar com maior precisão o potencial impacto do tratamento de substituição renal na qualidade de sono destes doentes. Mais especificamente, pretendemos comparar doentes em tratamento de hemodiálise com um grupo emparelhado sem doença renal; comparar doentes renais em hemodiálise com doentes já transplantados, assim como comparar transplantados com pessoas sem doença renal. Para além disso, tendo em conta o sono enquanto preditor de qualidade de vida são incluídos neste estudo indicadores de qualidade de vida e variáveis psicológicas como a ansiedade e depressão. Este estudo foi conduzido a partir de uma amostra total constituída por 223 participantes divididos por três grupos: 92 doentes renais em hemodiálise (55% do sexo masculino), com uma idade média de 69 anos (DP=13,67); 40 transplantados renais (50% do sexo feminino), com uma idade média de 42 anos (DP= 9,59); e 91 não insuficientes renais (56% do sexo masculino), com uma média de idade de 69 anos (DP= 14,02). O processo de recolha decorreu através da distribuição de protocolos de autorresposta impressos (compreendendo PSQI, ISI, ESS e Stop-Bang, para autoavaliação do sono; a HADS e o WHOQOL-Bref), para o grupo em hemodiálise num centro diálise da cidade de Coimbra e para não insuficientes renais em Centros Recreativos Séniores, tendo em vista recrutar participantes com características sociodemográficas comparáveis às dos doentes em hemodiálise. Por último, no que respeita ao grupo de transplantados renais procedeu-se à distribuição de protocolos disponibilizados online. A divulgação incidiu em Associações Portuguesas de Transplante Renal, grupos online de apoio e no Grupo Desportivo de Transplantados Renais em Portugal (GDTP). Os resultados do presente estudo sugerem que os problemas de sono são consideravelmente superiores em doentes renais crónicos submetidos a ambas as terapêuticas de substituição renal em comparação com sujeitos saudáveis ao nível da função renal. A realização de transplante renal sugere melhorias ao nível da qualidade de sono, nos indicadores de qualidade de vida e na redução de sintomatologia ansiosa e depressiva em doentes renais crónicos em comparação com o tratamento de hemodiálise. Apesar disso, a realização de transplante comporta um conjunto de novos desafios o que pode contribuir, a par de motivos orgânicos inerentes à realização de transplante, para a presença de queixas relacionadas com o sono. Neste estudo, os transplantados renais apresentam indicadores sugestivos de insónia e sonolência diurna superiores quando comparados com sujeitos em hemodiálise. Sleep constitutes a basic need with significant impact on the quality of life, physical, emotional, and psychological well-being of human beings. In the case of chronic kidney disease, sleep complaints are commonly present, and are associated with an increased risk for the development of sleep disorders through various pathophysiological mechanisms. Chronic kidney disease comprises different stages of severity. End-stage kidney disease requires renal replacement treatment such as hemodialysis or subsequent kidney transplant. The number of people with this chronic condition continues to increase and, as a result, the prevalence of sleep disorders and complaints is also likely to increase. In this sense, the main purpose of this study was to examine sleep patterns and difficulties of the clinical population with chronic kidney disease compared to a matched sample not displaying the disease, by studying the impact of renal replacement treatment on the quality of sleep of these patients. In addition, considering sleep as a predictor of quality of life, indicators of quality of life and psychological variables such as anxiety and depression were considered. This research project was conducted from a total sample of 223 participants divided into three main groups: 92 kidney patients on hemodialysis (55% male), with an average age of 69 years-old (SD= 13,67); 40 kidney transplants (50% female), with an average age of 42 years-old (SD= 9,59); and 91 subjects without renal failure (56% male), with an average age of 69 years-old (SD =14,02). The research protocol comprised measures of sleep (PSQI; ISI; ESS; Stop-Bang), anxiety, depression (HADS), and quality of life (WHOQOL-Bref). The collection process took place through the distribution of printed self-response protocols, for the hemodialysis group in one of existing Coimbra city dialysis center, and participants without renal failure recruited in Senior Recreational Centers (in order to recruit participants displaying sociodemographic similarities in comparison to the ones composing the clinical sample). Finally, for the kidney transplant group, protocols were distributed online, and the dissemination was focused on Portuguese Renal Transplant Associations, online support groups and the Portuguese Renal Transplant Sports Group (PRTS). The results of the present study suggest that the presence of sleep problems is considerably higher in chronic kidney patients undergoing both renal replacement therapies compared to healthy participants in terms of kidney function. The kidney transplantation is associated with improvements in the quality of sleep, in the quality of life indicators and in the reduction of anxious and depression symptoms in comparison with the hemodialysis patients. Despite this, undergoing and living with renal transplant involves a set of new challenges that may in addition to organic reasons inherent to the transplant, contribute to the presence of complaints related to sleep. In this study, kidney transplant recipients have indicators suggestive of higher insomnia and daytime sleepiness when compared with subjects on hemodialysis.

This study aimed to evaluate the effect of diets including 10%, 20%, and 30% of black soldier fly larvae (Hermetia illucens) meal (HM) (diets HM10, HM20, and HM30, respectively), replacing 17%, 35%, and 52% of fishmeal (FM), on growth performance, whole-body composition and fatty acid (FA) profile, plasma biochemistry, and intermediary metabolism of meagre (Argyrosomus regius) juveniles. For that purpose, triplicate groups of 18.0 ± 0.02 g fish were fed a control diet (CTR) without HM, and the experimental diets for 9 weeks. Growth, feed efficiency, protein efficiency ratio, nitrogen and energy retentions linearly decreased with the increase of dietary HM level. With the exception of ash, whole-body composition was not linearly affected by dietary HM inclusion. Regarding whole-body FA profile, total saturated fatty acids (SFA), SFA: polyunsaturated fatty acids (PUFA) ratio, and total n-6PUFA linearly increased with increasing levels of dietary HM, while total n-3PUFA, n-3 long chain PUFA (LC-PUFA), n-3:n-6 ratio, and unsaturation index linearly decreased with increasing levels of HM in the diets. Docosahexaenoic acid (DHA) whole-body retention linearly decreased while lauric acid (12:0) retention increased with the increase of HM in the diet. Plasma glucose linearly increased, while plasma triglycerides and total lipids decreased with the dietary level of HM. Hepatic amino acid catabolic (alanine aminotransferease and aspartate aminotransferease), and lipogenic (glucose-6-phosphate dehydrogenase and malic enzyme) enzymes activities were unaffected by diet composition, whereas lipid catabolic (3-hydroxyacyl-CoA dehydrogenase) enzyme activity linearly decreased with dietary HM level. Glutamate dehydrogenase, an amino acid catabolic enzyme, was lower in fish fed the HM20 diet than the other HM-including diets. Overall, 10% of HM, corresponding to 17% of FM replacement, might be included in meagre diets without major adverse effects on growth, feed utilization, whole-body composition and FA profile.

Funding: The authors thank all the patients who participated in this research and made it possible. We also thank Isa Dutra (MSc), João Paulo Pinheiro (BsC) and Raquel Meneses (BsC) from Hospital Santo Espírito da Ilha Terceira, EPE, Epidemiology and Molecular Biology Service (SEEBMO), Angra do Heroísmo, Portugal, who performed the DNA extractions and Vânia Machado (MSc), who participated in the elaboration of the pedigrees. BP wassupported by “Fundo Regional para a Ciência e Tecnologia (FRCT)” (M3.1.2/F/023/2011) Objectives: DISH/CC is a poorly understood phenotype characterised by peripheral and axial enthesopathic calcifications, frequently fulfilling the radiological criteria for Diffuse Idiopathic Skeletal Hyperostosis (DISH, MIM 106400), and in some cases associated with Calcium Pyrophosphate Dihydrate (CPPD) Chondrocalcinosis (CC). The concurrence of DISH and CC suggests a shared pathogenic mechanism. In order to identify genetic variants for susceptibility we performed whole exome sequencing in four patients showing this phenotype. Materials and methods: Exome data were filtered in order to find a variant or a group of variants that could be associated with the DISH/CC phenotype. Variants of interest were subsequently confirmed by Sanger sequencing. Selected variants were screened in a cohort of 65 DISH/CC patients vs 118 controls from Azores. The statistical analysis was performed using PLINK V1.07. Results:We identified 21 genetic variants in 17 genes that were directly or indirectly related to mineralization, several are predicted to have a strong effect at a protein level. Phylogenetic analysis of altered amino acids indicates that these are either highly conserved in vertebrates or conserved in mammals. In case-control analyses, variant rs34473884 in PPP2R2D was significantly associated with the DISH/CC phenotype (p=0.028; OR=1.789, 95% CI= 1.060-3.021). Conclusion: The results of the present and preceding studies with the DISH/CC families suggests that the phenotype has a polygenic basis. The PPP2R2D gene could be involved in this phenotype in an as yet unknown way. publishersversion published

Senegalese sole Solea senegalensis is currently farmed in recirculation aquaculture systems that often involve water re-oxygenation, which in turn may cause acute or prolonged hyperoxia exposures. In order to understand the impact of acute hyperoxia on the fish immune system and peripheral tissues such as gills and gut, Senegalese sole juveniles (30.05 ± 1.72 g) were exposed to normoxia (100% O2sat) as control and two hyperoxic conditions (150 and 200% O2sat) and sampled at 4 and 24 h. Fish haematological profile, total and differential blood cell counts and plasma immune parameters were analysed. Histomorphology and immunofluorescence analyses of gills and intestine were performed, respectively, whereas head-kidney samples were used for assessing the expression of immune-related genes. Results indicate that acute hyperoxia exposure may reduce fish erythrocyte and haemoglobin levels. Moreover, decreases in total leucocytes numbers, circulating lymphocytes, monocytes, alternative complement pathway activity and expression of cyclooxygenase-2 were observed in fish exposed to hyperoxia. In contrast, hyperoxia did not induce major effects on gill histomorphology nor in the protein content of ion and glucose cotransporters as well as a macrophage marker (V-ATPase) in the intestine. Although the activation of humoral mechanisms and immune-related genes were not dramatically affected by acute hyperoxia, the compromised immune cell status and the reduction of some inflammatory indicators are issues to consider under acute hyperoxia conditions.

There is a general consensus that many shark species are declining in numbers. However, effective management measures often depend on knowing how trends in abundance and distribution are influenced by environmental conditions. Several efforts to describe the occurrence and distribution of basking sharks Cetorhinus maximus have been made in northern Europe, particularly around the UK, but nothing is known regarding their occurrence in southern areas, such as the south of Portugal. Using 2 decades of observational data collected in the south of Portugal, we show that the occurrence of basking sharks in the area was highly seasonal, with individuals being observed mainly during spring. Based on in situ and satellite-derived environmental variables and climate indices, we also demonstrate that temporal trends were associated with the beginning of the upwelling season and that the inter-annual changes were related to lower values of sea surface temperature, North Atlantic Oscillation index, upwelling index, 2-mo lagged chlorophyll a and 3-mo lagged Atlantic Multidecadal Oscillation index, and higher values of 2-mo lagged upwelling index. These findings suggest that basking sharks are associated with the expansion of cold waters following upwelling events in the region, probably due to the aggregation and increase of zooplankton. Although the temperature recorded during our study years ranged from 14 to 24 degrees C, sharks were mainly observed when temperatures were lower than 20 degrees C, corroborating their preference for colder water. This study provides the first knowledge on the habitat use of basking sharks in southern European Atlantic areas. PROMAR Project [31-03-05-FEP-0037] Portuguese Foundation for Science and Technology (FCT)

Effective ocean management and the conservation of highly migratory species depend on resolving the overlap between animal movements and distributions, and fishing effort. However, this information is lacking at a global scale. Here we show, using a big-data approach that combines satellite-tracked movements of pelagic sharks and global fishing fleets, that 24% of the mean monthly space used by sharks falls under the footprint of pelagic longline fisheries. Space-use hotspots of commercially valuable sharks and of internationally protected species had the highest overlap with longlines (up to 76% and 64%, respectively), and were also associated with significant increases in fishing effort. We conclude that pelagic sharks have limited spatial refuge from current levels of fishing effort in marine areas beyond national jurisdictions (the high seas). Our results demonstrate an urgent need for conservation and management measures at high-seas hotspots of shark space use, and highlight the potential of simultaneous satellite surveillance of megafauna and fishers as a tool for near-real-time, dynamic management., Data analysis was funded in part by the Marine Biological Association (MBA) and the UK Natural Environment Research Council (NERC) (NE/R00997X/1) (to D.W.S.) with additional research support from the Save Our Seas Foundation and the NERC Oceans 2025 Strategic Research Programme, in which D.W.S. was a principal investigator. D.W.S. was supported by an MBA Senior Research Fellowship, N.Q. by European Regional Development Fund (FEDER) via the Programa Operacional Competitividade e Internacionalização (COMPETE), National Funds via Fundação para a Ciência e a Tecnologia (FCT) under PTDC/MAR/100345/2008 and COMPETE FCOMP-01-0124-FEDER-010580 (to N.Q. and D.W.S.), and Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF) in project MarInfo (NORTE-01-0145-FEDER-000031). Additional support was provided by an FCT Investigator Fellowship IF/01611/2013 (N.Q.), FCT Doctoral Fellowship PD/BD/52603/2014 (M.V.), PTDC/MAR-BIO/4458/2012, Xunta de Galicia - Isabel Barreto Program 2009-2012 (G.M.), Australian Research Council (ARC) grant DE170100841 and operational funds from the Australian Institute for Marine Science (AIMS) (both to A.M.M.S.).

This study aimed to evaluate the effect of including 10%, 20% and 30% of mealworm (Tenebrio molitor, TM) larvae meal (diets TM10, TM20 and TM30, respectively), replacing 25%, 50% and 75% of fishmeal (FM) in a isoproteic (50%) and isolipidic (19%) FM-based diet (CTR) for meagre (Argyrosomus regius). The diets were fed for 9 weeks to triplicate groups of fish with 18.0 g initial body weight and results were evaluated for growth performance; whole-body composition; amino acid (AA) catabolism and digestive enzymes activity; nutrients and chitin digestibility. A digestibility trial was also simultaneously conducted for 48 days. Growth and feed utilization parameters decreased linearly as dietary TM inclusion level increased, while mortality, hepatosomatic and visceral indexes, and AA catabolism enzymes activity were unaffected by the dietary treatments. Whole-body protein was reduced in fish fed TM20 and TM30 diets, with the latter group also having lower energy content than fish fed the CTR diet. The apparent digestibility coefficients (ADC) of dry matter, energy, protein, and AA (except leucine, valine and phenylalanine), decreased as dietary TM inclusion level increased. The lower ADC of protein was related to a linear decrease in trypsin activity. Lipase and total alkaline protease activities were also lower in fish fed the TM30 diet than in the other groups. A quadratic response of total alkaline protease with the increase of dietary TM meal was similar to that observed in the intensity of zymograms bands corresponding to proteases other than trypsin or chymotrypsin-like. Chitin ADC was null and no chitinolytic activity was found in the intestine. Overall, meagre presented a limited capacity to utilize TM, with a 10% TM dietary inclusion already resulting in significant impairment of fish digestive capacity and growth performance, while higher TM dietary levels further negatively affecting fish whole-body composition.

The ATP-binding cassette transporter 6 (ABCC6) gene encodes a cellular transmembrane protein transporter (MRP6) that is involved in the regulation of tissue calcification in mammals. Mutations in ABCC6 are associated with human ectopic calcification disorders. To gain insight into its evolution and involvement in tissue calcification we conducted a comparative analysis of the ABCC6 gene and the related gene ABCC1 from invertebrates to vertebrates where a bony endoskeleton first evolved. Taking into consideration the role of ABCC6 in ectopic calcification of human skin we analysed the involvement of both genes in the regeneration of scales, mineralized structures that develop in fish skin. The ABCC6 gene was only found in bony vertebrate genomes and was absent from Elasmobranchs, Agnatha and from invertebrates. In teleost fish the abcc6 gene duplicated but the two genes persisted only in some teleost genomes. Six disease causing amino acid mutations in human MRP6 are a normal feature of abcc6 in fish, suggesting they do not have a deleterious effect on the protein. After scale removal the abcc6 (5 and 10 days) and abcc1 (10 days) gene expression was up-regulated relative to the intact control skin and this coincided with a time of intense scale mineralization.

Objectives Ankylosing spondylitis (AS) is the most prevalent form of spondyloarthritis, with a known genetic association with the HLA-B27 molecule. The aim of this study was to assess the contribution of the HLA-G, HLA-E and HLA-F to AS susceptibility/protection in Portuguese patients with HLA-B27 AS and HLA-B27 unaffected controls. Methods High-resolution typing of HLA-G, HLA-E and HLA-F was performed in 228 patients with HLA-B27 AS and 244 HLA-B27 unaffected controls. Allelic, genotypic and haplotypic frequencies were compared between cohorts. To replicate the results, single nucleotide polymorphisms (SNPs) in HLA-E and HLA-F genes were typed in Australian cohorts. For further confirmation, a group of European-descent patients with AS and unaffected controls were genotyped for Major Histocompatibility Complex SNPs using the Illumina microarray. Results In the Portuguese population, no significant differences were found in HLA-G. For HLA-E, a significant difference was detected for the genotype HLA-E*01:01:01/01:03:01 (p=0.009; pc=0.009; OR=0.51), with a protection effect. For HLA-F, significant differences were detected in the allele HLA-F*01:01:02 (p=0.0049; pc=0.0098; OR=0.60) and corresponding SNP rs2075682 (p=0.0004; pc=0.0008; OR=0.53), suggesting protection and in the genotype HLA-F*01:01:01/01:03:01 (p=0.011; pc=0.043; OR=2.00), suggesting a susceptibility effect. Three G-E-F haplotypes with significant differences were detected but occur in a very small number of individuals. The only significant differences detected in the replication studies were for HLA-E rs1059510 in the Australians and for HLA-F rs1736924 in the European-descent cohorts. Conclusion Our results reveal suggestive AS protective and susceptibility effects from both HLA-E and HLA-F loci, however with population differences. To our knowledge, this is the first study showing association of HLA-F with AS.

The effect of a dietary incorporation of the seaweedsUlva lactucaandChondrus crispusas functional ingredients was evaluatedin gilthead seabream (Sparus aurata) juvenile growth, feed efficiency, muscle lipid composition, intermediary metabolism,oxidative status and gut histomorphology. Fish (15 g initial body weight) were fed with isoproteic (46%) and isolipidic (18%)diets with fish meal (FM) and plant feedstuffs (PF) (27:73 protein from FM:PF) as main protein sources (control diet). Three otherdiets were formulated similar to the control but including 5%U. lactuca,5%C. crispusor 2.5% of both algae (dietsUlva,Chondrusand Mix, respectively). Dietary incorporation ofUlvadid not affect growth performance (P> 0.05) while it leads tohigher muscleΣSFA (saturated fatty acids); and liver lipid peroxidation (LPO). Moreover, lower muscleΣPUFA (polyunsatu-rated fatty acids) and plasma glucose was observed. Dietary incorporation ofChondrusleads to lower growth; whole-body drymatter and lipid content; muscleΣPUFA andω3 fatty acids; and plasma glucose. In addition, higher muscleΣMUFA (mono-unsaturated fatty acids); and liver LPO was observed. Dietary incorporation of both algae (Mix) led to lower growth; whole-bodylipid content; muscleΣPUFA andω3 fatty acids; plasma glucose; and hepatic PK (pyruvate kinase) and HOAD (3-hydroxyacyl-CoA dehydrogenase). Furthermore, it leads to higher muscleΣSFA andΣMUFA; and liver LPO. Overall, no beneficial effects ofincluding the tested seaweeds in the diets was observed, while oxidative status was negatively affected in all dietary treatments., This work was supported by the StructuredR&D&I Project INNOVMAR (ref. NORTE-01-0145-FEDER-000035)within the research line INSEAFOOD, founded by the NorthernRegional Operational Programme (NORTE2020) through the EuropeanRegional Development Fund (ERDF). The Fundação para a Ciência e aTecnologia (FCT) and the FEDER, under programme PT2020 (ProjectUID/QUI/50006/2013) are also acknowledged for the financial funding.I.G., R.M. and A.C. were supported by FCT grants (SFRH/BPD/114959/2016, SFRH/BD/115870/2016 and SFRH/BPD/101354/2014, respec-tively). F.C. was a recipient of a grant within the Project ZEBRALGRE(PTDC/CVT-WEL/5207/2014).

For an increased incorporation of plant ingredients in aquafeeds at the expense of fish meal (FM) and fish oil (FO), more knowledge is needed on the effects at the intestine level of dietary vegetable oils (VO) and carbohydrates (CH), and of possible interactions. For that purpose, in this study, the activities of digestive pancreatic enzymes (amylase, lipase, total alkaline proteases), gut microbiota, and histomorphology were assessed in gilthead sea bream (IBW 71.0 ± 1.5 g) fed four diets differing in lipid source (FO or a blend of VO) and carbohydrate content (0% or 20% gelatinized starch) for 81 days. No major changes in digestive enzyme activities were noticed in fish fed the experimental diets. Dietary VO, but not CH content, modified intestinal microbial profile, by increasing the similarity of bacterial communities. Especially when combined with CH, dietary VO promoted abnormal enterocyte architecture. Liver histology was also accessed, and an increased cytoplasmic vacuolization of hepatocytes was related with dietary CH inclusion, being only significantly different in fish fed FO-based diets. Overall, nutritional interactions between dietary lipid source and carbohydrate content were not observed on digestive enzyme activities and microbial profile. However, the intestine histological modifications observed in fish fed the VOCH+ diet suggest a negative interaction between dietary VO and CH. This requires a more in depth assessment in future studies as it can have negative consequences at a functional level.

Summary This study confirms for Madeira Island (Portugal) population the Type 1 Diabetes (T1D) susceptible and protective Human leucocyte antigens (HLA) markers previously reported in other populations and adds some local specificities. Among the strongest T1D HLA associations, stands out, as susceptible, the alleles DRB1*04:05 (OR = 7.3), DQB1*03:02 (OR = 6.1) and DQA1*03:03 (OR = 4.5), as well as the haplotypes DRB1*04:05-DQA1*03:03-DQB1*03:02 (OR = 100.9) and DRB1*04:04-DQA1*03:01-DQB1*03:02 (OR = 22.1), and DQB1*06:02 (OR = 0.07) and DRB1*15:01-DQA1*01:02-DQB1*06:02 (OR = 0.04) as protective. HLA-DQA1 positive for Arginine at position 52 (Arg52) (OR = 15.2) and HLA-DQB1 negative for Aspartic acid at the position 57 (Asp57) (OR = 9.0) alleles appear to be important genetic markers for T1D susceptibility, with higher odds ratio values than any single allele and than most of the haplotypes. Genotypes generated by the association of markers Arg52 DQA1 positive and Asp57 DQB1 negative increase T1D susceptibility much more than one would expected by a simple additive effect of those markers separately (OR = 26.9). This study also confirms an increased risk for DRB1*04/DRB1*03 heterozygote genotypes (OR = 16.8) and also a DRB1*04-DQA1*03:01-DQB1*03:02 haplotype susceptibility dependent on the DRB1*04 allele (DRB1*04:01, OR = 7.9; DRB1*04:02, OR = 3.2; DRB1*04:04, OR = 22.1).

Submitted by António Freitas (amsf@uma.pt) on 2018-11-12T14:15:40Z No. of bitstreams: 1 HLA‐DQA1 and HLA‐DQB1 allele diversity and its extended haplotypes in Madeira Island.pdf: 74411 bytes, checksum: 26a2d2d7e46087fd3accf0ced0cc0d6f (MD5) Made available in DSpace on 2018-11-12T14:15:40Z (GMT). No. of bitstreams: 1 HLA‐DQA1 and HLA‐DQB1 allele diversity and its extended haplotypes in Madeira Island.pdf: 74411 bytes, checksum: 26a2d2d7e46087fd3accf0ced0cc0d6f (MD5) Previous issue date: 2017 info:eu-repo/semantics/publishedVersion

Em colaboração com a Rede Portuguesa de Laboratóros para o Diagnóstico da Gripe Introduction: Immune profile for influenza viruses is highly changeable over time. Serological studies can assess the prevalence of influenza, estimate the risk of infection, highlight asymptomatic infection rate and can also provide data on vaccine coverage. The aims of the study were to evaluate pre-existing cross-protection against influenza A(H3) drift viruses and to assess influenza immunity in the Portuguese population. Materials and methods: We developed a cross-sectional study based on a convenience sample of 626 sera collected during June 2014, covering all age groups, both gender and all administrative health regions of Portugal. Sera antibody titers for seasonal and new A(H3) drift influenza virus were evaluated by hemagglutination inhibition assay (HI). Seroprevalence to each seasonal influenza vaccine strain virus and to the new A(H3) drift circulating strain was estimated by age group, gender and region and compared with seasonal influenza-like illness (ILI) incidence rates before and after the study period. Results: Our findings suggest that seroprevalences of influenza A(H3) (39.9%; 95% CI: 36.2–43.8) and A(H1)pdm09 (29.7%; 95% CI: 26.3–33.4) antibodies were higher than for influenza B, in line with high ILI incidence rates for A(H3) followed by A(H1)pdm09, during 2013/2014 season. Low pre-existing crossprotection against new A(H3) drift viruses were observed in A(H3) seropositive individuals (46%). Both against influenza A(H1)pdm09 and A(H3) seroprotection was highest in younger than 14-years old. Protective antibodies against influenza B were highest in those older than 65 years old, especially for B/Yamagata lineage, 33.3% (95% CI: 25.7–41.9). Women showed a high seroprevalence to influenza, although without statistical significance, when compared to men. A significant decreasing trend in seroprotection from north to south regions of Portugal mainland was observed. Conclusions: Our results emphasize that low seroprotection increases the risk of influenza infection in the following winter season. Seroepidemiological studies can inform policy makers on the need for vaccination and additional preventive measures. This work was supported by the National Institute of Health Doutor Ricardo Jorge, IP Lisbon, Portugal. info:eu-repo/semantics/acceptedVersion

Objectives. To investigate the efficacy of infliximab in the treatment of severe calcium pyrophosphate deposition diseases (CPPD).Methods. Two patients with severe CPPD and diffuse idiopathic skeletal hyperostosis- (DISH-) like phenotype are described. Both patients were resistant to therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). Both patients were treated with infliximab, a TNF-αreceptor antagonist, for nine years.Results. Treatment with infliximab resulted in major clinical and laboratory improvements without relevant side effects.Conclusions. These results suggest that infliximab may be an effective treatment of severe CPDD.

Submitted by Isabel Gomes (itg@lisboa.ucp.pt) on 2017-03-07T11:25:24Z No. of bitstreams: 1 Tese_AnaAreias.pdf: 2488096 bytes, checksum: 6de6b5948e32640872f493b3b8945a78 (MD5) Approved for entry into archive by Isabel Gomes (itg@lisboa.ucp.pt) on 2017-03-07T11:25:48Z (GMT) No. of bitstreams: 1 Tese_AnaAreias.pdf: 2488096 bytes, checksum: 6de6b5948e32640872f493b3b8945a78 (MD5) Made available in DSpace on 2017-03-07T11:25:48Z (GMT). No. of bitstreams: 1 Tese_AnaAreias.pdf: 2488096 bytes, checksum: 6de6b5948e32640872f493b3b8945a78 (MD5) Previous issue date: 2016-08-31

Over the last decade, ocean sunfish movements have been monitored worldwide using various satellite tracking methods. This study reports the near-real time monitoring of fine-scale (< 10 m) behaviour of sunfish. The study was conducted in southern Portugal in May 2014 and involved satellite tags and underwater and surface robotic vehicles to measure both the movements and the contextual environment of the fish. A total of four individuals were tracked using custom-made GPS satellite tags providing geolocation estimates of fine-scale resolution. These accurate positions further informed sunfish areas of restricted search (ARS), which were directly correlated to steep thermal frontal zones. Simultaneously, and for two different occasions, an Autonomous Underwater Vehicle (AUV) video-recorded the path of the tracked fish and detected buoyant particles in the water column. Importantly, the densities of these particles were also directly correlated to steep thermal gradients. Thus, both sunfish foraging behaviour (ARS) and possibly prey densities, were found to be influenced by analogous environmental conditions. In addition, the dynamic structure of the water transited by the tracked individuals was described by a Lagrangian modelling approach. The model informed the distribution of zooplankton in the region, both horizontally and in the water column, and the resultant simulated densities positively correlated with sunfish ARS behaviour estimator (rs = 0.184, p

This report describes a 32-year-old woman presenting since childhood with progressive calcium pyrophosphate disease (CPPD), characterized by severe arthropathy and chondrocalcinosis involving multiple peripheral joints and intervertebral disks. Because ANKH mutations have been previously described in familial CPPD, the proband’s DNA was assessed at this locus by direct sequencing of promoter and coding regions and revealed 3 sequence variants in ANKH. Sequences of exon 1 revealed a novel isolated nonsynonymous mutation (c.13 C>T), altering amino acid in codon 5 from proline to serine (CCG>TCG). Sequencing of parental DNA revealed an identical mutation in the proband’s father but not the mother. Subsequent clinical evaluation demonstrated extensive chondrocalcinosis and degenerative arthropathy in the proband’s father. In summary, we report a novel mutation, not previously described, in ANKH exon 1, wherein serine replaces proline, in a case of early-onset severe CPPD associated with metabolic abnormalities, with similar findings in the proband’s father.

Summary HLA class I diversity (loci A, B and C) was analysed in four populations, two from North Cameroon (Podokwo and Uldeme) and two from South Cameroon (Ewondo and Bamileke). Northern and southern Cameroon populations show a substantial genetic diversity in terms of haplotype sharing and genetic distances, even despite the low percentage of variance due to differences among populations evidenced by analysis of molecular variance. The signals of differentiation among populations are consistent with their linguistic affiliation, and support previous evidence, based on autosomal microsatellites and protein loci, which has shown that the complex pattern of genetic variation of Cameroon can in part be described by contrasting the northern and southern part of the country. Looking at our results in the more general framework of HLA diversity in sub-Saharan Africa, it turns out that the Podokwo and Uldeme show some genetic links to populations of the southern western branch of the Sahel corridor, while their high frequency of A*02 and C*04 alleles is congruent with previously hypothesised introgression of non-sub-Saharan alleles. On the other hand, signals of shared ancestry between the Bamileke and Ewondo and the Bantu speakers from central and southern Africa were detected.

Background: Atherosclerosis and thrombosis are the major manifestations underlying cardiovascular diseases (CVD), which are the leading cause of mortality and morbidity worldwide. Both result from an interaction between genetic and environmental risk factors. The goal of our study was to evaluate several polymorphisms identified as predisposing factors to atherosclerosis and thrombosis. Material and Methods: A series of 155 healthy unrelated individuals of Azorean origin were analyzed using the CVD StripAssay (ViennaLab Diagnostics, Austria) for the most established polymorphisms involved in blood coagulation (F2, F5, F13A1, FGB), fibrinolitic system (SERPINE1), platelet adhesion (ITGB3), homocysteine metabolism (MTHFR), reninangio-tensin system (ACE) and lipid metabolism (APOE). Results: No significant differences were observed in allelic frequencies when comparing our data to mainland Portugal. Group stratification according to the number of “increased” risk alleles, demonstrated that 116/155 (75%) individuals belong to the moderate risk group (5 - 10 risk alleles). Conclusions: Although acknowledging the fact that the allelic states at the analysed loci lack predictive value, the fact that a high frequency of individuals presents at least 5 risk alleles (124/155; 80%) is important for the establishment of the appropriate preventive measures in the Azorean population.

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.

BACKGROUND: The association between susceptibility to multiple sclerosis (MS) and HLA-DRB1*15 has been reported in various European populations. OBJECTIVE: To investigate the relationship between MS, HLA-DRB1*15 and other DRB1 alleles in a Portuguese population and their association with clinical course of MS. METHODS: The HLA-DRB1 alleles were analyzed by PCR-SSP in 248 MS patients and 282 healthy controls. In order to relate HLA-DRB1 alleles to disease aggressiveness, patients with relapsing remitting MS and secondary progressive MS were subdivided into 3 groups: 'benign' MS patients who maintain an Extended Disability Status Scale (EDSS) score of 3 after the same period and 'aggressive' MS those with EDSS>or=6 within 15 years of disease onset. RESULTS: As expected, a higher frequency of HLA-DRB1*15 was found in MS patients (29.8% vs 19.9%, odds ratio (OR)=1.72, 95% CI=1.15-2.56, p=0.008). The HLA-DRB1*03 allele was positively associated with MS in the overall patient population (22.6% vs 15.6%, OR=1.58, 95% CI=1.02-2.45). Concerning disease aggressiveness, HLA-DRB1*15 occurred more frequently in the group with benign disease (42.6% vs 19.9%, OR=2.99, 95% CI=1.56-5.72) and in the group with non-benign disease (34.1% vs 19.9%, OR=2.09, 95% CI=1.05-4.16) compared with controls. When time to reach an EDSS=3 or EDSS=6 was considered as end point, HLA-DRB1*15 negative patients were found to have a worse prognosis. CONCLUSIONS: In this population of Portuguese MS patients, the HLA-DRB1*15 allele is established as a genetic marker for susceptibility to MS and is also associated with a better outcome.

Gitelman syndrome (GS) is a rare autossomal recessive inherited tubulopathy, characterized by defective tubular reabsorption of magnesium and potassium, mostly caused by mutations in the SLC12A3 gene. The association of GS with chondrocalcinosis (CC) has been described in the literature as a typical example of hypomagnesemiainduced crystal deposition disease. We are presenting one case where the genetic cause for GS was identified in a proband with secondary early onset CC. A 60 years-old male patient with CC, hypomagnesemia and hypokalemia was identified in our hospital as a result of clinical and laboratory assessments. The clinical diagnosis of GS was performed and SLC12A3 gene was screened in the proband; variants detected were further searched in family members. The proband was homozygous for the S615L mutation; additionally, only one from the seven family members which were heterozygous presents CC. The presence of CC in two other individuals is most likely sporadic, in agreement with their advanced age.

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Objective. Twelve families that were multiply affected with diffuse idiopathic skeletal hyperostosis (DISH) and/or chondrocalcinosis, were identified on the island of Terceira, The Azores, potentially supporting the hypothesis that the 2 disorders share common etiopathogenic factors. The present study was undertaken to investigate this hypothesis. Methods. One hundred three individuals from 12 unrelated families were assessed. Probands were identified from patients attending the Rheumatic Diseases Clinic, Hospital de Santo Espirito, in The Azores. Family members were assessed by rheumatologists and radiologists. Radiographs of all family members were obtained, including radiographs of the dorsolumbar spine, pelvis, knees, elbows, and wrists, and all cases were screened for known features of chondrocalcinosis. Results. Ectopic calcifications were identified in 70 patients. The most frequent symptoms or findings were as follows: axial pain, elbow, knee and metacarpophalangeal (MCP) joint pain, swelling, and/or deformity, and radiographic enthesopathic changes. Elbow and MCP joint periarticular calcifications were observed in 35 and 5 patients, respectively, and chondrocalcinosis was identified in 12 patients. Fifteen patients had sacroiliac disease (ankylosis or sclerosis) on computed tomography scans. Fifty-two patients could be classified as having definite (17%), probable (26%), or possible (31%) DISH. Concomitant DISH and chondrocalcinosis was diagnosed in 12 patients. Pyrophosphate crystals were identified from knee effusions in 13 patients. The pattern of disease transmission was compatible with an autosomal-dominant monogenic disease. The mean age at which symptoms developed was 38 years. Conclusion. These families may represent a familial type of pyrophosphate arthropathy with a phenotype that includes peripheral and axial enthesopathic calcifications. The concurrence of DISH and chondrocalcinosis suggests a shared pathogenic mechanism in the 2 conditions.