Lipid signalling in grapevine resistance against fungal pathogens

Grapevine (Vitis vinifera L) is one of the most economically important crops worldwide, mostly due to its uses for wine and table grape production. However, it is prone to several diseases. Downy and powdery mildews and grey mold, caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively, are among the most devastating ones. Disease control strategies include phytochemical applications every growing season, jeopardizing the sustainability of viticulture. Understanding the molecular processes behind disease resistance or susceptibility is vital to define alternative control strategies and select new disease resistance traits for breeding programs. The identification of molecular markers that allow discriminating tolerant and susceptible grapevine genotypes to their pathogens is an important step to help breeders select genotypes for crossings to produce hybrids with good winemaking and disease tolerance traits. Lipids and lipid-derived metabolites are not only major structural and metabolic constituents of the cell, but they also function as modulators of a multitude of signal transduction pathways evoked by biotic stresses. It has been proposed that specific fatty acids (FA) may be involved in plant resistance against pathogens with different colonization strategies (biotroph, hemibiotroph and necrotroph). Previous results indicate that the content of several FA suffers alterations at early time-points after grapevine inoculation with the biotrophic oomycete Plasmopara viticola. These alterations are linked with reactive oxygen species and Jasmonic acid (JA) associated signalling. Moreover, lipid molecules and their derivatives, including JA, when applied externally, can cause a modulation of the lipid and FA signalling mechanisms in a similar manner to the pathogen challenge. Plants that are exposed to these elicitor molecules show a quicker and more intense defence response upon contact with a pathogen. The extracellular matrix (ie apoplast) is the first battlefield where pathogen recognition occurs and secretion of both defence molecules and pathogen effectors take place. Therefore, the apoplast is one of the most important cell compartments in plant-pathogen interaction. Nonetheless, despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Because apoplastic fluid extraction from woody plants is a challenging task, studies regarding grapevine apoplast are still scarce to this day. There are two published studies on the grapevine leaf apoplast proteome and none on its metabolome. In this work, the problems raised above were addressed. The analysis of the constitutive lipid and FA composition of tolerant and susceptible grapevine genotypes to P. viticola was carried out, along with the expression analysis of FA desaturase (FAD) genes. These studies allowed to identify lipids and FA as potential biomarkers for tolerance or susceptibility to P. viticola. The saturated FA, mainly in monogalactosyldiacylglycerol and phosphatidyl choline are candidate tolerance biomarkers and the polyunsaturated linoleic acid (C18:2) as well as the plastidial lipids are candidate susceptibility biomarkers. Moreover, the higher expression levels of FAD4, FAD6 and FAD8 in susceptible genotypes suggest that they might also be considered as candidate biomarkers for susceptibility. The analysis of the total leaf FA composition revealed corroborating results in terms of FA saturation degree and FAD expression, and it is a more rapid and less costly approach (discussed in the chapters II and III). Due to the relevance of the JA mediated lipid signalling in the grapevine-P. viticola interaction, another question that raised was whether this mechanism would be conserved in the interaction with other pathogens with different invasion and/or lifestyles. Therefore, the FA modulation events, crucial for JA synthesis and signalling, were also addressed in the grapevine interaction with E. necator (biotroph, invading the plant leaf in the adaxial page from wound apertures) and B. cinerea (necrotroph). While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection (discussed in chapter IV). Since lipid molecules and JA showed previously to play important roles in the grapevine defence responses to P. viticola, the potential role of this molecule as a FA signalling trigger was studied. In fact, FA modulation after JA elicitation is similar to that described previously after P. viticola inoculation even in a susceptible cultivar, highlighting the potential of this molecule as an alternative to prevent grapevine diseases (discussed in chapter V). To uncover the lipid signalling events of the first moments of plant pathogen interaction, a thorough analysis of the apoplastic fluid is necessary. A new methodological approach to isolate grapevine leaf apoplast compatible with proteomic and lipidomic based studies was defined. The constitutive metabolome was assessed by FTICR-MS, which allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes. Among them, lipids are the most abundant molecular class. This methodology represents an optimization to the existing protocols and opens the way to study the lipid signalling events in the first battlefield of the grapevine-pathogen interaction (discussed in chapter VI). This work allowed to bring us a few steps closer to the complete disclosure of the grapevine lipid mediated defence mechanisms highlighting also candidate molecules to be used in future breeding programs for disease tolerance

relatório de estágio

Mestrado, Enfermagem Comunitária, 2014, Escola Superior de Enfermagem de LisboaA vacinação é considerada, de entre todas as medidas de Saúde Pública, a que melhor relação custo-efetividade tem evidenciado. É um dos mais bem-sucedidos programas de saúde de baixo custo nas intervenções de saúde pública. A terceira revolução em saúde está associada à necessidade de redução de custos face às mudanças na etiologia da morbilidade e mortalidade. Embora a racionalização dos custos seja inevitável, espera-se que se traduza em medidas de capacitação e coresponsabilização do cidadão de modo a que lhe seja proporcionado o melhor serviço com vista à promoção e proteção da sua saúde. Este projeto foi implementado numa UCSP do ACES Lisboa Central. A intervenção dirige-se especificamente às crianças nascidas em 2010, 2005 e 1998 com vacinas em atraso. A amostra é de 52 crianças. O objetivo geral é promover a imunização individual e de grupo (famílias e comunidade onde se inserem), segundo o atual PNV. É desejável que a taxa de cobertura vacinal nacional seja, em média, igual ou superior a 95% para cada vacina do PNV. “A não vacinação assume uma variedade de formas que se encontram insuficientemente estudadas e comparadas em diferentes planos, nacional e internacional.” (Cunha & Durand, 2008, p. 372). O projeto tem como referencial teórico de enfermagem, o Modelo da Promoção de Saúde de Nola Pender e segue a metodologia do Processo de Planeamento em Saúde. O instrumento de colheita de dados utilizado foi um questionário dirigido aos pais e como método de priorização utilizou-se a grelha de análise. Foram identificados conhecimentos diminuídos sobre doenças evitáveis através do PNV e imunização deficiente. Os resultados mostram que 53% dos pais procuram informação sobre vacinas na internet, 60% dos inquiridos pedem informações sobre doenças ao médico e apenas 7% dos pais recorrem ao enfermeiro. As estratégias selecionadas foram adequadas aos resultados, recorrendo a novas tecnologias de informação

Impacto psicológico da crise financeira

Tese de doutoramento, Psicologia, Faculdade de Ciências Humanas e Sociais, Universidade do Algarve, 2014Alguns países Europeus, incluindo Portugal, sofreram recentemente uma crise económica. Stressores económicos, como dificuldade económica, ameaça financeira e bem-estar financeiro, podem contribuir negativamente para a saúde psicológica dos indivíduos. Esta investigação teve como objectivo explorar a influência das variáveis dificuldade económica, ameaça financeira e bem-estar financeiro (stressores económicos) sobre as variáveis stresse, ansiedade e depressão (indicadores de saúde mental), bem como testar o efeito moderador do coping e do suporte social na relação acima mencionada. Um total de 729 participantes portugueses responderam a um questionário que é composto por instrumentos que avaliam os stressores económicos e os indicadores de saúde mental. Os resultados encontrados permitem verificar que, de uma forma geral, os stressores económicos têm um impacto significativo nos indicadores de saúde mental. Em tempos de crise económica, a dificuldade económica e a ameaça financeira são preditores significativos de ansiedade, depressão e stresse e o bem-estar financeiro está significativamente associado à ansiedade e à depressão. A influência dos stressores económicos sobre o stresse, ansiedade e depressão podem ser moderados pelo coping e pelo suporte social. Observa-se ainda que o coping e o suporte social em conjunto moderam as reacções aos stressores económicos reduzindo consideravelmente as experiências de stresse, ansiedade e depressão

Sintase do óxido nítrico de Leishmania infantum: da sequência à estrutura

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2015O óxido nítrico (NO) tem um grande impacto nos seres vivos. No Homem, o NO participa em vários processos fisiológicos, como a regulação da pressão sanguínea, a neurotransmissão e a resposta imune. Nestes processos, o NO funciona como uma molécula sinalizadora. No entanto, em níveis mais elevados, este radical pode ter efeitos tóxicos nas células, fazendo parte da resposta de defesa contra mcirorganismos patogénicos. De facto, há um consenso de que, nas infeções por Leishmania e Trypanosoma cruzi, o NO produzido pelos macrófagos infetados medeia a morte dos parasitas. Em parasitas do género Leishmania, o NO é ainda importante na interação entre o parasita e o hospedeiro. Para além disso, sabe-se atualmente que a concentração de NO produzido pelos parasitas varia ao longo do seu ciclo de vida, sendo tanto maior quanto maior é a sua infetividade. Assim, o NO assume um papel importante no estabelecimento da infeção por Leishmania. O enzima sintase do óxido nítrico (NOS, EC: 1.14.13.39), que catalisa a reação de formação de NO a partir de L-arginina, é, portanto essencial para o parasita. Atualmente, sabe-se que não existe homologia de sequência entre o NOS de Leishmania infantum (LiNOS) e de humano. Neste sentido, o LiNOS surge como um potencial alvo terapêutico para o combate da leishmaniose visceral, doença pela qual o parasita é responsável. Neste projeto foi demonstrado, pela primeira vez, que o LiNOS expresso nativamente forma um homotetrâmero. Foi ainda possível concluir que o tetrâmero de LiNOS é estabilizado na presença de heme e de tetrahidrobiopterina. Através da análise da sequência da proteína em estudo, foi prevista a existência de uma região desordenada entre os resíduos 210 e 270 e de um segmento transmembranar entre os resíduos 424 e 446. Na análise das regiões desordenadas do LiNOS, foi também identificado um local de ligação a montante e/ou a jusante desta região, sendo esta ligação potencialmente estabelecida com a caveolina. A estrutura tridimensional do monómero de LiNOS foi ainda prevista por homologia, tendo sido possível prevê-la para a maioria da sequência (resíduos 95-584). Os resultados obtidos permitiram gerar informação crucial para dirigir os próximos passos experimentais no sentido da sua elucidação total, assim como de outros aspetos relacionados com a função.Nitric oxide (NO) exerts a great impact on living beings. On human cells, NO participates in several physiological processes, such as blood pressure regulation, neurotransmission and immune response. In these processes, NO works as a signaling molecule. However, when in higher levels, this radical can exert toxic effects on cells as part of the immune response against pathogenic microorganisms. In fact, that is a consensus that, in infections by Leishmania and Trypanosoma cruzi, NO produced by infected macrophages mediates parasite death. In Leishmania genus parasites, NO is also important for the parasite-host interaction. Besides, it is now known that the concentration of NO produced by the parasites varies during its life cycle and it increases as the parasite’s infectivity increases. Hence, NO plays an important role on the establishment of the infection by Leishmania. The enzyme nitric oxide synthase (NOS, EC: 1.14.13.39), wich catalizes the synthesis of NO from L-arginine, is essential for the parasite. At this time, it is known that Leishmania infanum NOS (LiNOS) has no sequence homology with human NOS. Therefore, LiNOS arises as a potential therapeutic target for the treatment of visceral leishmaniasis, the desiese for wich the parasite is responsible. It was established during this project, for the first time, that natively expressed LiNOS folds as a homotetramer. It was also possible to conclude that the tetramer is stabilized when in presence of heme and tetrahydrobiopterine. A protein sequence analysis revealed the existence of a disordered region between residues 210 and 270 and a transmembrane region between residues 424 and 446 was predicted. When analysing disordered regions in LiNOS a potential binding site upsteam and/or downstream the predicted region was identified and the bond might me established with caveoline. The tridimentional structure of LiNOS monomer was predicted by homology, and it was possible to predict it for the majority of the sequence (residues 95 to 584). The acquired results led to the generation of crutial information to lead the next experimental steps for the structure’s complete elucidation, as wel as other function related aspects

Conflito trabalho-família, horário de trabalho e actividades familiares

Dissertação de Mestrado apresentada ao ISPA - Instituto UniversitárioO presente estudo pretendeu, em primeiro analisar se o tempo que os indivíduos dedicam à sua actividade profissional contribui para a percepção de conflito trabalho família. O segundo propósito desta investigação, pretendeu averiguar se existe uma relação entre a percepção de conflito trabalho família e a participação em actividades familiares. Foram analisadas dimensões específicas referentes às horas que os participantes passam a trabalhar, definidas actividades familiares concretas e analisados quatro factores referentes à percepção de conflito trabalho família. Os participantes considerados para este estudo tinham de cumprir quatro critérios de inclusão/exclusão, tendo sido a amostra recolhida em Jardins de Infância. Os resultados obtidos no primeiro estudo, sugerem que o tempo de deslocações e o tempo total de trabalho contribuem de forma significativa para a percepção de conflito trabalho-família, excepto nas dimensões emoções internas e pressão do indivíduo. Na segunda hipótese, concluise que a percepção de conflito trabalho-família em indivíduos não tem uma relação significativa com a participação em actividades escolares e lúdicas relacionadas com a interacção com os filhos, nem com a participação em tarefas domésticas dentro de casa.ABSTRACT: In the present study it´s pretended, firstly to analyze if the time dedicated to professional activities by an individual contribute to a perception of work-family conflict. Secondly was pretended to investigate if there is a relation between the perception of work family conflict and the participation in family activities. Specific dimensions was analyze concerning the hours that subjects spend at work, concrete family activities were defined and four factors belonging to the work family perception were also analyzed. The participants considered for this study had to fulfill four criteria of inclusion/exclusion, the sample used were collected in Kindergartens. The results gotten in the first study indicates that time spent going to work and the total working hours highly contribute to the perception of work family conflict, except in the dimensions internal emotions and pressure in individuals. In the second hypothesis, it is concluded that the perception of work family conflict wasn`t related to the participation of those in leisure and school activities related to the intersection with their children, or the collaboration in housekeeping tasks

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics andmore specialized ‘OMICS’ such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system.Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and awider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltrationcentrifugationmethod that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv ‘Trincadeira’ and ‘Regent’, was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteinswere identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classesinfo:eu-repo/semantics/publishedVersio

Uma proposta de formação em serviço de professores em interculturalidade para acompanhar estudantes imigrantes

O quadro teórico desta contribuição baseia-se em estudos de migrações, no paradigma das novas mobilidades, nos contributos da educação intercultural crítica e do multiculturalismo, e na aprendizagem colaborativa. Este texto discute o aumento da migração e da diversidade cultural e consequente o desenvolvimento da educação intercultural no contexto espanhol. Esta situação representa o grande desafio da formação de professores para a promoção de uma educação intercultural inclusiva e crítica. O texto está estruturado em torno das contribuições do projeto europeu Quammelot, que desenvolveu pesquisa, intervenção, formação e boas práticas sobre inclusão educacional e social de estudantes imigrantes e Menores Estrangeiros Desacompanhados, com um foco específico no sistema educacional, na faixa etária de 12 a 18 anos, e nas relações entre escolas, população imigrante e território. O artigo oferece uma revisão da literatura sobre a inclusão de estudantes imigrantes e menores imigrantes nas escolas secundárias. Posteriormente, é apresentada a metodologia inovadora do projeto, descrevendo o curso online destinado aos professores do ensino médio. Finalmente, são comentadas as contribuições do projeto sobre cooperação transnacional, inovação na educação formal, melhoria da formação de professores e visibilidade das necessidades dos atores educacionais sobre inclusão e diversidade

Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistance.

Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat