Repositório RCAAP

O trabalho realizado é baseado em estudos imagiológicos de ressonância magnética nuclear das bolhas resultantes de cirurgias de glaucoma por dispositivos de filtração onde existem diversos critérios morfológicos para a sua avaliação, contudo são de valor limitado uma vez que essa bolha não é visível por técnicas rotineiras como a lâmpada de fenda por terem uma localização muito posterior. Uma da forma de as estudar é através do uso de técnicas de imagem, como a ressonância magnética nuclear. Existem diversos estudos que correlacionam estes parâmetros morfológicos das bolhas à redução da pressão intraocular e portanto com a eficácia clinica da cirurgia sendo o objetivo desta tese de final de mestrado explorar as diferenças entre os diversos estudos tentando estabelecer um padrão entre a morfologia e os valores da pressão intraocular pós operatórios bem como explorar outros fatores que possam ter impacto clínico como o uso de diferentes dispositivos de filtração (por exemplo valvulados- Ahmed vs não valvulados – Baerveldt), o tamanho e posição da placa terminal ou o volume da orbita.

Phosphorylation regulates the conformation, stability, homo- and heterotypic protein interactions, localization, and activity of the tumor suppressor PTEN. From a simple picture, at the beginning of this millennium, recognizing that CK2 phosphorylated PTEN at the C-terminus and thereby impacted on PTEN stability and activity, research has led to a significantly more complex scenario today, where for instance GSK3, Plk3, ATM, ROCK or Src-family kinases are also gaining the spotlight in this evolving play. Here, we review the current knowledge on the kinases that phosphorylate PTEN, and on the impact that specific phosphorylation events have on PTEN function.

The post-translational addition of C-16 long chain fatty acids to protein cysteine residues is catalysed by palmitoyl-S-acyl-transferases (PAT) and affects the affinity of a modified protein for membranes and therefore its subcellular localisation. In apicomplexan parasites this reversible protein modification regulates numerous biological processes and specifically affects cell motility, and invasion of host cells by Plasmodium falciparum merozoites and Toxoplasma gondii tachyzoites. Using inhibitor studies we show here that palmitoylation is key to transformation of zygotes into ookinetes during initial mosquito infection with P. berghei. We identify DHHC2 as a unique PAT mediating ookinete formation and morphogenesis. Essential for life cycle progression in asexual blood stage parasites and thus refractory to gene deletion analyses, we used promoter swap (ps) methodology to maintain dhhc2 expression in asexual blood stages but down regulate expression in sexual stage parasites and during post-fertilization development of the zygote. The ps mutant showed normal gamete formation, fertilisation and DNA replication to tetraploid cells, but was characterised by a complete block in post-fertilisation development and ookinete formation. Our report highlights the crucial nature of the DHHC2 palmitoyl-S-acyltransferase for transmission of the malaria parasite to the mosquito vector through its essential role for ookinete morphogenesis.

This article documents the public availability of (i) microbiomes in diet and gut of larvae from the dipteran Dilophus febrilis using massive parallel sequencing, (ii) SNP and SSR discovery and characterization in the transcriptome of the Atlantic mackerel (Scomber scombrus, L) and (iii) assembled transcriptome for an endangered, endemic Iberian cyprinid fish (Squalius pyrenaicus).

A quantidade e qualidade da água disponível no mundo tem vindo a ser um tema em constante destaque a nível global. Com as alterações climáticas e o constante aumento da população mundial, existe uma forte probabilidade de as reservas de água potável atingirem os seus limites nas próximas décadas. Consequentemente, existe uma maior consciencialização da sociedade para os perigos que daí possam advir, o que proporciona a criação de programas de sustentabilidade com o objetivo de tornar o uso da água o mais eficiente e o menos poluente possível, de forma a não comprometer as gerações vindouras. É nesse âmbito que em Portugal, na região do Alentejo, a Comissão Vitivinícola Regional Alentejana desenvolveu o Plano de Sustentabilidade dos Vinhos do Alentejo, um plano para ser seguido pelas entidades associadas. Esse plano inclui várias áreas de foco, sendo a componente hídrica uma das mais importantes. Neste trabalho são analisadas as pegadas da água de 4 vinhos, dois colhidos em 2017 (vinhos tintos: Reserva e Touriga Nacional) e outros dois colhidos em 2018 (vinhos brancos: Seleção e Verdelho). O cálculo da pegada da água para cada um dos vinhos, engloba todo o processo, desde o cultivo até ao engarrafamento, numa perspetiva cradle-to-gate, excluindo os materiais inerentes aos edifícios, maquinaria e do fabrico da garrafa, rotulo, rolha e embalagem. A unidade funcional deste trabalho é uma garrafa de vinho habitual de 0,75 litros. Existem várias metodologias para o cálculo da pegada da água, daí a necessidade de criar a Norma Internacional ISO 14046 de forma a definir diretrizes a seguir sempre que se procede ao cálculo de uma pegada da água de um processo, produto ou instituição. Utilizou-se duas metodologias propostas por duas comunidades distintas. Aplicou-se a metodologia proposta no Water Footprint Assessment Manual (comunidade Water Footprint Network), um projeto liderado por Arjen Y. Hoekstra e publicado em 2011. O outro método aplicado é o utilizado pelo software V.I.V.A., um programa desenvolvido pelo Ministério da Transição Ecológica de Itália com o objetivo de tornar as práticas vitivinícolas do país mais sustentáveis. De notar que ambas as metodologias aplicadas seguem uma avaliação de ciclo de vida (LCA) e na prática os métodos são bastante semelhantes, diferenciando-se principalmente na apresentação de resultados. Os resultados do trabalho mostram uma pegada da água média em 2018, #% maior do que em 2017. Em ambos os anos de produção, a componente da água verde (referente à evapotranspiração) é a mais significativa, representando em média, #% para os vinhos Seleção e Verdelho e #% para o vinho Reserva e Touriga. Todos os vinhos apresentam uma componente da água cinzenta (água necessária para diluir os poluentes) nula. A componente azul (água de consumo) para a colheita de 2017 é em média 140 L H2O/garrafa, por sua vez, para a colheita de 2018 toma o valor de 59 L H2O/garrafa. Em termos dos processos onde existe consumo de água azul, a rega é em todos os vinhos onde existe um maior consumo de água. O volume de água para rega dos vinhos Reserva e Touriga é em média #% maior que o volume de água para rega dos vinhos Seleção e Verdelho.

The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 μs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 μs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ ∼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).

The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established.

Amyloid fibers, implicated in a wide range of diseases, are formed when proteins misfold and stick together in long rope-like structures. As a natural mechanism, osmolytes can be used to modulate protein aggregation pathways with no interference with other cellular functions. The osmolyte sucrose delays fibrillation of the ribosomal protein S6 leading to softer and less shaped-defined fibrils. The molecular mechanism used by sucrose to delay S6 fibrillation was studied based on the two-state unfolding kinetics of the secondary and tertiary structures. It was concluded that the delay in S6 fibrillation results from stabilization and compaction of the slightly expanded tertiary native structure formed under fibrillation conditions. Interestingly, this compaction extends to almost all S6 tertiary structure but hardly affects its secondary structure. The part of the S6 tertiary structure that suffered more compaction by sucrose is known to be the first part to unfold, indicating that the native S6 has entered the unfolding pathway under fibrillation conditions.

DNA phenotyping research is one of the most emergent areas of forensic genetics. Predictions of externally visible characteristics are possible through analysis of single nucleotide polymorphisms. These tools can provide police with "intelligence" in cases where there are no obvious suspects and unknown biological samples found at the crime scene do not result in any criminal DNA database hits. IrisPlex, an eye color prediction assay, revealed high prediction rates for blue and brown eye color in European populations. However, this is less predictive in some non-European populations, probably due to admixing. When compared to other European countries, Portugal has a relatively admixed population, resulting from a genetic influx derived from its proximity to and historical relations with numerous African territories. The aim of this work was to evaluate the utility of IrisPlex in the Portuguese population. Furthermore, the possibility of supplementing this multiplex with additional markers to also achieve skin color prediction within this population was evaluated. For that, IrisPlex was augmented with additional SNP loci. Eye and skin color prediction was estimated using the multinomial logistic regression and binomial logistic regression models, respectively. The results demonstrated eye color prediction accuracies of the IrisPlex system of 90 and 60% for brown and blue eye color, respectively, and 77% for intermediate eye color, after allele frequency adjustment. With regard to skin color, it was possible to achieve a prediction accuracy of 93%. In the future, phenotypic determination multiplexes must include additional loci to permit skin color prediction as presented in this study as this can be an advantageous tool for forensic investigation.

O ensino da História activo, criativo e crítico deve ter por base a utilização do documento histórico em contexto de sala de aula. Os documentos escritos e iconográficos surgem como recursos didácticos que mais utilizados são pelo professor para promover um ensino activo. Por isso, a sua utilização em sala de aula deve obedecer a vários critérios, entre os quais, a sua adaptação ao nível cognitivo e etário dos alunos, a sua adequação aos objectivos e competências definidos quer na planificação de aula quer nas orientações curriculares, e a sua adaptação de modo a torná-lo claro, inteligível e o menos complexo possível. A sua utilização deve ainda procurar responder a várias situações didácticopedagógicas, como diminuir o carácter verbalista e expositivo do professor, e deve obedecer a um conjunto vasto de metodologias e estratégias no seu manuseamento e tratamento, de modo a fortalecer o espírito crítico dos alunos e iniciá-lo no método de investigação histórica. Deste modo, a análise, interpretação, comentário e sínteses históricas são competências que devem estar intimamente relacionadas com a origem e a natureza epistemológica do documento. Noções que se obtêm através de informação explícita e implícita ao nível da estrutura formal e temática do documento.

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Introduction. Codonoblepharon forsteri (Dicks.) Goffinet is a rare epiphytic moss characteristically associated with water-filled holes in trees. We reviewed its range and population and assessed effects of climate change. Methods. An inventory of sites from where Codonoblepharon forsteri has been recorded was compiled. Extent of occurrence (EOO) and area of occupancy (AOO) were calculated. Population size was estimated, treating an occupied tree as an ‘individual-equivalent’ of the moss. Climatic conditions of the species’ current distribution were characterised, and an ensemble model of its distribution was generated. The latter was projected onto present and future climatic layers. Key results. Codonoblepharon forsteri has been recorded from 19 countries and 205 sites in Europe, North Africa and Southwest Asia. It has been undergoing an overall decline. Most sites have few occupied trees, and a world population of 1000–10,000 individual-equivalents is estimated. Model projections suggest that the species will experience a range increase of +0.36–0.65 by 2050 and +0.35–0.68 by 2070, especially in its northwest range, particularly across France and the UK. Range loss is predicted to be between –0.20 and –0.39 in 2050 and –0.21 and –0.65 in 2070, affecting the driest areas of the current range around the Mediterranean, especially in North Africa. Conclusions. Codonoblepharon forsteri has a relatively large EOO but relatively small AOO, probably due to habitat specialism. A major reason for recent declines appears to be widespread abandonment of traditional ‘pollarding’ of trees. The potential climatic range of the species will shift significantly northwards over the next few decades.

No final do ano de 2019, foi descoberto um novo coronavírus, Sars-Cov-2, responsável por milhões de casos de pneumonias atípicas. A transmissão rápida e sem fronteiras do vírus causou 79 milhões de casos e 1.7 milhões de mortes, até ao dia 29 de dezembro de 2020. A comunidade científica já conhecia outros coronavírus, o que permitiu inferir muitos mecanismos e características deste novo, de entre as quais saliento a relação com patologia neurológica. Apesar de ser um vírus maioritariamente respiratório, surgiram muitos casos de sintomas à apresentação, complicações secundárias e sequelas do foro neurológico. Mais que uma revisão da literatura neste âmbito, este trabalho pretende contextualizar a aquisição de conhecimento acerca do vírus, da doença e do seu impacto no sistema nervoso, em tempo de pandemia. Neste sentido, é necessário realizar uma cronologia de eventos científicos, políticos e sociais, pela enorme influência entre si, neste período crítico. Seguidamente, são descritos casos clínicos de patologia neurológica, no contexto de uma infeção por Sars-Cov-2, publicados na literatura internacional. Desses, será dado particular ênfase a neuropatias periféricas autoimunes, eventos cerebrovasculares e encefalopatias. Durante este período foram estudados os mecanismos subjacentes aos quadros clínicos descritos. Tal permitiu averiguar a relação de causalidade entre a infeção a Sars-Cov-2 e o desenvolvimento destas patologias, que foi mais clara em alguns casos do que noutros. No geral, o impacto desta infeção é real, quer seja por neuroinvasão do sistema nervoso ou pelo desencadear de reações imunológicas nocivas para o organismo humano.

A gestação é um período muito importante. Todas as ações da gestante vão influenciar o desenvolvimento embrionário e fetal que é particularmente vulnerável à ação de agentes extrínsecos. Nestes inclui-se a toma de fármacos, que é comum especialmente no primeiro trimestre, período em que ocorre a organogénese. A exposição pré-natal a determinados medicamentos pode ter efeitos nefastos como aborto, prematuridade, alterações do crescimento intrauterino, malformações e até distúrbios do comportamento e/ou da aprendizagem. Contudo, existem patologias que requerem um tratamento continuado, e nem sempre há dados suficientemente robustos para a decisão de manter ou suspender uma terapêutica em curso previamente à gravidez. A hipertensão arterial, uma condição frequente, está associada a complicações maternas e fetais major, se não tratada. Dentro dos fármacos usados existem vários com consequências negativas, ao induzirem fetopatia, como é o caso dos antagonistas dos recetores da angiotensina II (ARA II). Pretende-se, com base num caso clínico de exposição in útero a ARA II com consequente insuficiência renal neonatal, fazer uma revisão da literatura sobre os efeitos teratogénicos da utilização de alguns fármacos durante a gravidez. Será dado maior enfoque à utilização de anti-hipertensores como ARA II e consequências do seu uso para o feto. Pretende-se ainda abordar a comunicação de más notícias e a adoção de medidas de cuidados paliativos na fase final de vida.

The epidemiology of human infection with swine-origin influenza A(H1) viruses suggests that the virus must adapt to replicate and transmit within the human host. PB1 is essential to the replication process. The objective of this study was to identify whether PB1 retains genetic traces of interspecies transmission and adaptation. We have found that the evolutionary history of PB1 is traceable. Lineage appears to be distinguished by amino acid changes between the conserved motifs of the viral polymerase, which can have major impact in PB1 protein folding, and by changes in the expression of the Mitochondrial Targeting Sequence and in the predicted helical region, that putatively affect induction of cellular apoptosis by PB1-F2. Furthermore, we found genomic markers that possibly relate to viral adaptation to new hosts and to new cellular environment and, additionally, to an enhanced compatibility with HA. We found no specific trend in the amino acid substitutions. Viral fitness appears to be favored by less reactive amino acids in some positions, while in others more reactive ones are fixed. Also, more flexible conformations appear associated with higher protein stability in general, although often more restrictive conformations appear to have favored protein folding and binding. Several aspects of PB1 mapping domains and the specific roles and interaction of PB1, PB1-F2 and N40 with each other and with other viral proteins and host cellular molecules remain unclear. Tracing the genetic evolution is critical to further understand the mechanisms by which PB1 affects vital fitness and adaptation. This analysis now permits putative adaptive related polymorphisms to be experimentally evaluated for phenotypic impact.

Background: Excess 5-aminolevulinic acid (ALA) and α-aminoacetone (AA) are implicated in ketosis, porphyrinpathies and diabetes. Pathologic manifestations involve O₂⁻, H₂O₂, OH, enoyl radicals (ALA and AA) and their oxidation end products. Methods: To characterize enoyl radicals resulting from reaction of OH radicals with ALA and AA, micromolar OH concentrations were produced by pulse radiolysis of ALA and AA in aqueous solutions. Results: ALA and AA react with OH at k=1.5 × 10⁹ M⁻¹s⁻¹. At pH7.4, the ALA absorbance spectrum has a maximum at 330 nm (ε=750 M⁻¹cm⁻¹). This band appears as a shoulder at pH8.3 where two ALA species are present: (NH3)⁺-CH₂-CO-CH₂-CH₂-COO⁻ and NH₂-CH₂-CO-CH₂-CH₂-COO⁻ (pKa=8.3). At pH8.3, ALA reacts with oxygen (k=1.4 × 10⁸ M⁻¹s⁻¹) but not with O₂⁻. At pH8.3, AA oxidation produces two AA species characterized by an absorbance spectrum with maxima at 330 and 450 nm. ALA and AA are repaired by antioxidants (quercetin (QH), catechin, trolox, ascorbate) which are semi-oxidized (k>10(8)M⁻¹s⁻¹). QH bound to HSA or to apoferritin and ferritin repairs ALA and AA. In O₂-saturated apoferritin solutions, Q, O₂⁻, AA and reaction product(s) react with QH. Conclusions: The optical absorption properties and the time evolution of ALA and AA were established for the first time. These radicals and their reaction products may be neutralized by antioxidants free in solution or bound to proteins. General significance: Adjuvant antioxidant administration may be of interest in pathologies related to excess ALA or AA production.

The burden of hepatitis C virus infection remains very high despite huge progress in the cure of the infection. The high prevalence of hepatitis C, especially in vulnerable groups and particularly drug users, may compromise the achievement of the 2030 WHO targets with a 90% reduction in new infections and a 65% reduction in mortality. Therapy with the latest pangenotypic direct-acting antivirals provides cure rates in the order of 97% with short-term oral treatment (8-12 weeks) and with an excellent safety and tolerability profile. Curing the infection causes significant health gains derived from preventing complications from cirrhosis, especially hepatocellular carcinoma, and from liver transplantation. Elimination of hepatitis seems feasible with the implementation of a massive therapy program, focusing particularly on vulnerable populations, through micro-elimination strategies, and in the general population with age-based screening. The reduction of the virus reservoir (humans are the only reservoir) is a determining factor in eradicating the virus.

The authors report the clinical and genetic investigation of a family with hypertrophic cardiomyopathy (HCM). The individuals described are three affected first-degree relatives (father, daughter and son), one affected niece and unaffected nephew and niece. Those affected all share a very similar phenotype consisting of asymmetric HCM, with hypertrophy particularly affecting the septum and the anterior wall, and similar electrocardiographic features, including a short PR interval. Case 1 (proband) presented with obstructive HCM and had undergone myectomy and mitral valve replacement. Case 2 (oldest offspring of Case 1) had non-obstructive HCM with exertional angina and NYHA II heart failure (HF) symptoms; she developed non-sustained ventricular tachycardia during follow-up and received a single-chamber ICD for primary prevention of sudden cardiac death. Case 3 (son of case 1) presented with asymptomatic non-obstructive HCM and developed NYHA II HF symptoms during follow-up. Case 4 had non-obstructive HCM, mainly with NYHA II HF symptoms. Testing of the proband for sarcomeric mutations and phenocopies was initially negative. After eight years of clinical follow-up, the suspicion of an undiscovered pathogenic gene mutation shared among the members of this family led us to enroll the proband in a whole-genome sequencing research project, which revealed a heterozygous pathogenic intronic MYBPC3 variant (c.1227-13G>A [rs397515893]), cosegregating with the phenotype.

Insect Bite Hypersensitivity (IBH) is a common cutaneous disease, affecting a large number of horses worldwide. Several studies have identified Culicoides spp. saliva as a clinically relevant allergen source. The prevalence of IBH in Portugal, particularly in Lusitano horses, is still not known. However, the environmental characteristics of the national territory are favorable to the activity of Culicoides, and several species of this genus can be found, namely C. imicola and C. obsoletus/C. scoticus. In this study we characterized the Culicoides population present in Lusitano stud farms with a history of IBH. Thirteen stud farms with Lusitano horses were selected in several regions of mainland Portugal for having a previous history of IBH-affected horses, with a minimum of 5 affected horses. Culicoides were collected in May and June 2016 using OVI traps, placed in these stud farms, and we were able to identify several Culicoides species. We could also verify that C. obsoletus/C. scoticus, and C. imicola were the ones most frequently found, but other species like C. pulicaris were also found.

Brugada syndrome (BrS) is one of the most common inherited arrhythmogenic disorders, with a worldwide prevalence of approximately 0.05%. It is characterized by a typical electrocardiographic pattern (ST-segment elevation with coved morphology in the right precordial leads and right bundle branch block) combined with malignant ventricular arrhythmias (MVA), that can lead to syncope or sudden cardiac death (SCD). This electrocardiographic pattern is intermittent and may be unmasked by fever or pharmacological challenge with intravenous sodium channel blockers, of which flecainide or ajmaline are most often used.