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- ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in ArabidopsisPublication . Rodrigues, Américo; Adamo, Mattia; Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rabissi, Agnese; Lumbreras, Victoria; González-Guzmán, Miguel; Antoni, Regina; Rodriguez, Pedro L.; Baena-González, ElenaPlant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways.
- Avaliação de parâmetros de cultivo de Lysmata debelius Bruce, 1983 ao longo do desenvolvimento larvar e juvenilPublication . Salgado, Daniel Francisco; Leandro, Sérgio Miguel Franco Martins; Marques, Sónia Cristina FerreiraA presente dissertação teve como principal objetivo completar o ciclo de vida do camarão-de-fogo, de Lysmata debelius Bruce, 1983, em condições controladas de cativeiro, contribuindo para a sua domesticação e para o avanço da aquacultura ornamental. Para tal, foram definidos três eixos centrais de investigação: determinar o período até à obtenção de indivíduos com tamanho comercial, avaliar a influência da densidade de cultivo e da luz LED azul e branco (azul: 450-470nm e branco: 450-700nm) na sobrevivência e no assentamento larvar. Estes fatores são considerados determinantes para a definição de protocolos de cultivo eficientes e sustentáveis. Os resultados demonstraram que a sobrevivência larvar de Lysmata debelius foi fortemente influenciada pela densidade de cultivo, com melhores valores registados nas densidades mais baixas (90–140 larvas L-1. A taxa de assentamento não revelou diferenças estatisticamente significativas entre tratamentos. O tempo até ao primeiro assentamento foi mais curto nos ensaios sem luz (81,67 ± 2,31 dias), em comparação com aqueles conduzidos sob luz LED azul e branco (89,67–93,33 dias), embora sem diferenças estatisticamente significativas. Por outro lado, o tempo médio total até ao assentamento mostrou diferenças estatisticamente significativas na ausência de luz, situando-se entre 86,67 e 91,18 dias, face a 93,50–96,82 dias em presença de luz, sem efeito relevante da densidade. Finalmente, ao integrar as fases larvar e juvenil, estimou-se que o período total desde a desova até às dimensões do tamanho comercial (3 cm), variou entre 194 e 202 dias. Estes resultados, fornecem uma base sólida para a compreensão da biologia larvar de L. debelius, fundamental para a otimização dos protocolos de cultivo desta espécie e reforçam a necessidade de considerar múltiplos fatores ambientais e de manejo no desenho de sistemas produtivos. Do ponto de vista prático, esta investigação confirmou a viabilidade da produção sustentável desta espécie, abrindo caminho para reduzir a pressão sobre populações selvagens e responder à crescente procura do mercado. A consolidação de protocolos larvares e reprodutivos representa um passo essencial para a expansão da aquacultura ornamental, promovendo práticas mais responsáveis e ambientalmente sustentáveis, resultando numa menor dependência de exemplares capturados em populações naturais. Em forma conclusiva, este trabalho estabelece as bases para o cultivo controlado de Lysmata debelius, representando um contributo científico e aplicado relevante para a sua domesticação e para a consolidação da aquacultura ornamental como setor sustentável e inovador.
- Characterization of Selected Wild Mediterranean Fruits and Comparative Efficacy as Inhibitors of Oxidative Reactions in Emulsified Raw Pork Burger PattiesPublication . Ganhão, Rui; Estévez, Mario; Kylli, Petri; Heinonen, Marina; Morcuende, DavidIn the present study, water, ethanolic, and methanolic extracts from seven selected wild fruits originally from the Mediterranean area, namely, strawberry tree (Arbutus unedo L., AU), azarole (Crataegus azarolus L., CA), common hawthorn (Crataegus monogyna L., CM), blackthorn (Prunus spinosa L., PS), dog rose (Rosa canina L., RC), elm-leaf blackberry (Rubus ulmifolius Schott, RU), and rowan (Sorbus aucuparia L., SA), were analyzed for the total amount and profile of phenolic compounds and for the in vitro antioxidant activity against the DPPH and ABTS radicals (study 1). The seven fruits showed different chemical compositions, which consequently led to different antioxidant potentials. Among the seven fruits initially analyzed, AU, CM, RC, and RU had the highest amount of phenolic compounds and displayed the greatest antioxidant activity in vitro. Extracts from these four fruits were tested as inhibitors of lipid oxidation in raw pork burger patties subjected to refrigerated storage at 2 °C for 12 days (study 2). The quantitative measurements of thiobarbituric acid reactive substances (TBA-RS), hexanal content, and color stability were used as indicators of oxidative reactions. The four selected fruits displayed intense antioxidant activity against lipid oxidation, which highlights the potential usage of these fruits as ingredients for the manufacture of healthy meat products. Among them, RC and AU were particularly efficient as their protective effect against lipid oxidation was more intense than that displayed by quercetin (230 mg/kg of burger patty).
- Co-Occurrence of ACSSuT and Cephalosporin Resistance Phenotypes Is Mediated by int1- Associated Elements in Nontyphoidal Salmonella enterica from Human Infections in SpainPublication . Campos, Maria Jorge; Palomo, Gonzalo; Hormeño, Lorena; Ugarte, María; Porrero, María Concepción; Herrera-León, Silvia; Vadillo, Santiago; Píriz, Segundo; Quesada, AlbertoA screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004-2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. β-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum β-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance.
- Diel vertical behavior of Copepoda community (naupliar, copepodites and adults) at the boundary of a temperate estuary and coastal watersPublication . Gonçalves, A.M.M.; Pardal, M.A.; Marques, S.C.; S. Mendes; M.J. Fernández-Gómez; M.P. Galindo-Villardón; U.M. AzeiteiroDespite a growing interest in diel vertical migration as a research topic, there are few studies in southern European marine coastal systems. This study determined the main structuring hydrological and physical factors at different temporal scales in copepod assemblage distribution patterns. Seasonal, tidal, lunar and diel vertical migrations accomplished by horizontal movements were examined on the main copepod fraction of the Mondego estuary, Portugal. Seasonal samples were conducted hourly at the mouth of the estuary, during diel cycles (25h), both over neap and spring tides, at the bottom and surface, using a 63μm and 335μm mesh size nets. Simultaneously, four sites inside the estuary were sampled during flood tide to evaluate and compare copepods species' distribution along the estuary. Species life cycles were also categorized. Spring-spring tide best expresses the stable part of copepod-environment dynamics. Acartia tonsa and Oithona nana were distributed mainly at the bottom during ebb tides. A clear resident estuarine performance was noticeable in O.nana proving the estuarine preferences of the species. Neritic species showed preferences by saline waters, whereas the resident species were found mainly at estuarine areas. Copepodites stages showed a similar distribution pattern as estuarine species, avoiding leaving the estuary. In contrast nauplii and Oithona plumifera showed higher densities at surface flood tides. Indeed, vertical migrations accomplished by horizontal movements were mainly influenced by depth and tidal cycles, whereas day and night were not ecologically significant.
- Dissemination of Antimicrobial-Resistant Clones of Salmonella enterica Among Domestic Animals, Wild Animals, and HumansPublication . Palomo, Gonzalo; Campos, Maria Jorge; Ugarte, María; Porrero, María Concepción; Alonso, Juan Manuel; Borge, Carmen; Vadillo, Santiago; Domínguez, Lucas; Quesada, Alberto; Píriz, SegundoNon-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. This work focuses on the identification of Salmonella enterica clonal strains which, presenting a wide distribution potential, express resistance determinants that compromise effectiveness of the antimicrobial therapy. The screening was performed on 506 Salmonella enterica isolates from animals and humans, which were characterized by serovar and phage typing, genome macrorestriction and pulsed-field gel electrophoresis, and detection of phenotypic and genotypic traits for antimicrobial resistance. A Salmonella Enteritidis strain with strong quinolone resistance is spread on three host environments carrying one of the four variants found for the GyrA protein: (1) Asp87Tyr, the major polymorphism found in 39 Salmonella isolates from human origin and six from poultry; (2) Ser83Phe, with four isolates from human origin and one from white stork (Ciconia ciconia); and (3) Asp87Asn or (4) Asp87Gly, with two isolates each from human origins. Several Salmonella Typhimurium strains that presented int1 elements and the classically associated pentaresistance (ACSSuT) phenotype were found distributed between two host environments: domestic animals and humans, domestics and wild animals, or wild fauna plus humans. This study points out the importance of monitoring gut microbiota and its antimicrobial resistance from wildlife, in parallel to livestock animals and humans, especially for animal species that are in close contact with people.
- An empirical comparison of Canonical Correspondence Analysis and STATICO in the identification of spatio-temporal ecological relationshipsPublication . Mendes, Susana; Fernández-Gómez, M. José; Pereira, Mário Jorge; Azeiteiro, Ulisses Miranda; Galindo-Villardón, M. PurificaciónThe wide-ranging and rapidly evolving nature of ecological studies mean that it is not possible to cover all existing and emerging techniques for analyzing multivariate data. However, two important methods enticed many followers: the Canonical Correspondence Analysis (CCA) and the STATICO analysis. Despite the particular characteristics of each, they have similarities and differences, which when analyzed properly, can, together, provide important complementary results to those that are usually exploited by researchers. If on one hand, the use of CCA is completely generalized and implemented, solving many problems formulated by ecologists, on the other hand, this method has some weaknesses mainly caused by the imposition of the number of variables that is required to be applied (much higher in comparison with samples). Also, the STATICO method has no such restrictions, but requires that the number of variables (species or environment) is the same in each time or space.Yet, the STATICO method presents information that can be more detailed since it allows visualizing the variability within groups (either in time or space). In this study, the data needed for implementing these methods are sketched, as well as the comparison is made showing the advantages and disadvantages of each method. The treated ecological data are a sequence of pairs of ecological tables, where species abundances and environmental variables are measured at different, specified locations, over the course of time.
- Estudo comparativo do perfil químico e das bioatividades de três macroalgas marinhas em diferentes contextos ecológicosPublication . Matias, Margarida Henriques; Mouga, Teresa Margarida Lopes da Silva; Afonso, Clélia Paulete Correia NevesEsta dissertação teve como principal objetivo comparar três espécies de macroalgas – Gracilaria gracilis, Sargassum muticum e Ulva sp. – colhidas em épocas distintas do ano e em locais distintos da costa portuguesa, através da caraterização do perfil bioquímico e das bioatividades (antimicrobianas, antioxidantes e bioestimulantes). A caraterização ecológica dos locais demonstrou condições ambientais contrastantes: a Figueira da Foz apresenta forte influência marítima e alguma fluvial, com águas relativamente frias; a Lagoa de Óbidos é marcada por águas calmas e elevada variabilidade físico-química; e Peniche carateriza-se pelas praias rochosas, sujeitas a forte agitação marítima. Estas diferenças refletem-se sobretudo na salinidade e no oxigénio dissolvido, condicionando a fisiologia e a composição bioquímica das algas. Em diferentes análises observaram-se diferenças significativas entre espécies e também entre locais, enquanto a sazonalidade mostrou menor impacto nos resultados. Os resultados mostraram que o teor de humidade é superior nas amostras colhidas em setembro/novembro, indicando um efeito sazonal. No teor de cinzas, G. gracilis possui os teores mais baixos, sem diferenças significativas entre épocas e locais. Quanto ao conteúdo mineral, destaca-se o cálcio, variando entre 1,91 e 17,91 mg/g, enquanto o arsénio excede os limites regulamentados, em todas as amostras. Além disso, a G. gracilis apresentou o maior teor de hidratos de carbono, enquanto o teor proteico variou entre espécies, com a Ulva sp. de Buarcos de março (UBM) a obter o valor máximo (4,78%). Relativamente à matéria gorda total, a Ulva sp., registou o valor mais baixo (0,52%), enquanto o S. muticum apresentou o valor máximo (3,23%), exibindo predominantemente ácido palmítico (C16:0) e ácido oleico (C18:1 n9 cis). O rácio ómega-6/ómega-3, por sua vez, é superior a 1 nas amostras de S. muticum (entre 1,9 e 2,73) e maioritariamente inferior a 1 na Ulva sp., exceto na UBM. Na análise dos pigmentos fotossintéticos, a clorofila a é o pigmento mais abundante, atingindo 8,45 mg/g em S. muticum. Adicionalmente, a fucoxantina variou entre 0,77 e 1,87 mg/g em S. muticum e a ficoeritrina-R oscilou entre 0,047 e 0,549 mg/g em G. gracilis. Também se verificou que a abundância de pigmentos é fortemente influenciada pelo local de recolha. Os extratos hidroetanólicos não exibiram atividade antimicrobiana relevante, mas demonstraram forte atividade antioxidante, destacando-se a UBM (93,85% de inibição no DPPH e 564,16 μmol Fe/g no FRAP). No QTP, por sua vez, destacou-se o S. muticum de Óbidos em março, com uma concentração de 38,42 mg GAE/g. As frações residuais dos extratos revelaram um elevado potencial bioestimulante nas sementes de alface e tomate, sendo que, relativamente ao crescimento, destaca-se a Ulva sp.. Estas descobertas evidenciam que a composição bioquímica e as bioatividades das espécies em estudo são influenciadas pela espécie, pelo local e pela sazonalidade, realçando o seu potencial como recursos de valor acrescentado na área nutricional, agronómico e biotecnológico.
- Feeding habits of Solea senegalensis in earthen ponds in Sado estuaryPublication . Castelo Branco, Maria Ana; Arruda, Marco A.; Gamito, SofiaThe senegale sole, Solea senegalensis Kaup, 1858, is a commercially valuable fish and it is beginning to be cultured in a semi-intensive way in fish farms in southern European countries. The present study was initiated to investigate the diet of S. senegalensis under semi-extensive conditions. The feeding habits of sole were studied in two earthen ponds of a fish farm in the Sado estuary. In one earthen pond, artificial fish ration was given. S. senegalensis feeds on few prey items, its diet is mainly composed of insect larvae (Chironomus salinarus) and polychaeta (Hediste diversicolor). The diet composition of this species suggests feeding specialization, by consuming mainly annelids and insect larvae and by avoiding other items, extremely abundant in the environment, such as gastropods. In the water reservoir where ration was given, some fish consumed simultaneously benthic organism together with ration. However, benthic organisms seem to be the most important component of S. senegalensis diet.
- Immobilized heterologous Rhizopus oryzae lipase: A feasible biocatalyst for the production of human milk fat substitutesPublication . Tecelão, Carla; Guillén, Marina; Valero, Francisco; Ferreira-Dias, SuzanaThis work aims at evaluating the potentialities of a heterologous Rhizopus oryzae lipase (rROL) as a feasible biocatalyst, to be used as an alternative to commercial lipases, for the synthesis of Human Milk Fat Substitutes (HMFS). This enzyme was immobilized in three different supports (Accurel® MP 1000, Eupergit® C and Lewatit® VP OC 1600) and tested as catalysts for the acidolysis between tripalmitin and oleic acid, batchwise, at 60 ◦C, in solvent-free medium. The highest molar incorporations of oleic acid in the triacylglycerols were achieved with rROL in Lewatit® VP OC 1600 and Accurel® MP 1000, although the highest immobilization yields were observed with Eupergit® C. Quasi-equilibrium was attained after 6 h or 12 h reaction, with about 30 mol.% and 22 mol.% of oleic acid incorporation, when rROL immobilized in Lewatit and Accurel were used, respectively. Also, 23 h batch operational stability tests were performed: half-life times (t1/2) of 34.5 h and 64.0 h were achieved for rROL immobilized in Accurel® MP 1000 and in Lewatit® VP OC 1600, respectively. An important increase in the operational stability was observed for rROL immobilized in Lewatit after rehydration at the end of each batch (t1/2 = 202 h). Thus, the loss of activity may be explained by a progressive dehydration occurring along the reaction.