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- Preliminary characterization of Vibrio cholerae strains isolated from seafood samples marketed in PortugalPublication . Lopes, Teresa; Lopes, David; Moura, Isabel Bastos; Sousa, Isabel; Rodrigues, João; Fernandes, Camila; Barreira, Maria João; Maia, Carla; Correia, Cristina Belo; Pintado, Cristina; Saraiva, Margarida; Batista, Rita; ElsevierVibrio cholerae, a natural inhabitant of aquatic ecosystems, has been related with gastrointestinal infections, particularly those associated with seafood consumption. This preliminary study aimed to evaluate the presence and characteristics of Vibrio cholerae, in seafood marketed in Portugal, given its potential role as a foodborne pathogen. Thus, the occurrence of Vibrio cholerae in 129 seafood raw samples (105 of shrimp and 24 of oysters), marketed in Portugal, was assessed. Isolates’ characterization regarding the presence of antimicrobial resistance (AMR) genes and of pathogenicity-specific genetic traits was attained by whole-genome sequencing (WGS). Core-genome Multi Locus Sequence Typing (cgMLST) analyses to evaluate the genetic relatedness among the isolates, and with other V. cholerae strains isolated in the world, as well as phenotypic AMR (performed by disc diffusion), were also attained. Overall, 43/129 (33.3 %) of the samples tested positive for V. cholerae (41/105 (39.1 %) of the shrimp and 2/24 (8.3 %) of the oysters’ samples). WGS analyses classified the studied strains as non-O1/non-O139 Vibrio cholerae (NOVC), lacking the main cholerae virulence factors encoded by the CTX phage. However, they carry diverse virulence factors similar to those found in O1 and O139 strains and/or in NOVC clinical strains. Furthermore, eight strains were classified as multidrug-resistant (MDR). The cg-MLST analyses revealed six genetic clusters among the 43 isolates (three identified sequence types - ST829, ST833, ST1085). Although it was not possible to find a close genetic relatedness between the studied V. cholerae strains and other deposited in PuBMLST database, a high genetic proximity among some strains isolated in different countries and from different sources (environmental and human) was observed, reinforcing the importance of a One Health approach. Assessing occurrence, pathogenic potential and genetic relatedness of Vibrio cholerae strains in the Portuguese food supply chain, this study contributes to understand their public health significance and supports a One Health approach to prevent foodborne outbreaks, contributing to food safety.
- Re-classification of LDLR Variants through High-Throughput Functional Characterisation: Advancing Diagnosis in Familial HypercholesterolaemiaPublication . Chora, Joana Rita; Islam, Mohammad Majharul; Alves, Ana Catarina; Pfisterer, Simon; Bourbon, MafaldaBackground: Familial hypercholesterolaemia (FH) is the most common autosomal disorder of lipid metabolism, affecting approximately 1 in 300 individuals. FH is characterised by markedly elevated plasma cholesterol levels from birth, predisposing to premature atherosclerotic cardiovascular disease. The identification of a pathogenic variant in a causative gene provides a definitive diagnosis and enables cascade screening of family members. However, a substantial proportion of FH variants remain classified as variants of uncertain significance (VUS), creating a critical gap in genetic diagnosis and patient management. Purpose: This study aims to implement a high-throughput pipeline for the functional classification of LDLR variants, thereby enabling their clinical re-interpretation and integration into diagnostic practice. Methods: Selected LDLR variants were divided into two groups: a validation set and a test set. The validation group included 7 variants previously classified as benign/likely benign and 50 variants previously classified as pathogenic/likely pathogenic. The test group comprised 131 VUS with no prior functional assessment. Functional activity was evaluated using an automated analysis platform based on multiplexed high-content imaging to quantify LDL uptake, as described previously (Islam, Tamlander, Hlushchenko, Ripatti, & Pfisterer, 2024). Variant classification followed FH VCEP LDLR-specific guidelines (Chora et al., 2022). Results: In total, 187 LDLR variants were analysed. In the validation group, 44 variants demonstrated completely concordant results with previous classifications, 8 fell within an intermediate "grey zone" (70–90% LDL uptake), and only 5 pathogenic variants were misclassified, yielding a sensitivity of 88.4%, specificity and precision of 100%, and overall accuracy of 78.9%. Among the test group, 51 variants exhibited <70% of wild-type LDL uptake, 41 showed >90% of wild-type uptake, and 39 fell into the intermediate range, requiring additional functional studies. Integrating these findings with other ACMG/AMP evidence codes, 47 variants were considered “hot VUS”, amenable to reclassification. Of these, 9 were reclassified as likely benign and 11 as likely pathogenic. Conclusions: This large-scale functional study of LDLR variants demonstrates the feasibility and clinical utility of integrating high-throughput functional evidence with high accuracy into variant interpretation. The ability to reclassify previously unresolved VUS represents a major step forward in reducing diagnostic uncertainty in FH. The PerMedFH project paves the way for a personalised medicine approach in FH, improving diagnostic precision, and ultimately enhancing patient care and cardiovascular outcomes.
- Development of Cellular and Animal Models for Mucopolysaccharidosis Type IIIPublication . Gonçalves, Francisca; Alves, Sandra; Coutinho, Maria FranciscaA mucopolissacaridose tipo III (MPS IIIA, B, C e D), ou Síndrome de Sanfilippo, é um subgrupo de doenças lisossomais de sobrecarga (DLS). São doenças raras, monogénicas e neurodegenerativas causadas por mutações nos genes que codificam as enzimas responsáveis pela degradação intralisossomal do sulfato de heparano (HS). Os doentes apresentam um fenótipo clínico grave, caracterizado por demência infantil de início precoce e por morte prematura. Embora tenham sido alcançados progressos significativos no seu conhecimento e algumas novas terapias tenham avançado até à fase de ensaios clínicos, ainda existem muitos aspetos dos mecanismos patofisiológicos inerentes a esta síndrome por desvendar, não estando atualmente disponível um tratamento eficaz. Modelos celulares e animais que reproduzam o fenótipo da doença, constituem recursos de grande valor, possibilitando estudos mais aprofundados e incisivos e, a identificação de novos alvos terapêuticos, além de uma triagem mais eficiente de novos fármacos. Neste trabalho procurámos responder a essa necessidade através de: (i) uma caracterização aprofundada de duas linhas de células estaminais pluripotentes induzidas (iPSCs, do inglês: induced pluripotent stem cells), obtidas por reprogramação de fibroblastos de doentes MPS IIIC e IIID, previamente desenvolvidas no nosso grupo; e (ii) do estabelecimento de duas novas linhas de células estaminais de dentes decíduos esfoliados (SHEDs, do inglês: stem cells from human exfoliated deciduous teeth) derivadas de doentes com MPS IIIB e IIIC. Paralelamente, desenvolvemos um novo modelo animal para MPS IIIB, através da geração de um mutante nulo funcional na geração F0 de peixe-zebra (Danio rerio) através da tecnologia de CRISPR-Cas9.
- Micronucleus assay in buccal and urothelial epithelial cells of wildland firefighters exposed to wildfire smokePublication . Esteves, Filipa; Madureira, Joana; Barros, Bela; Alves, Sara; Vaz, Josiana; Oliveira, Marta; Slezakova, Klara; Fernandes, Adília; do Carmo Pereira, Maria; Morais, Simone; Bonassi, Stefano; Teixeira, João Paulo; Costa, SolangeOccupational exposure as a firefighter is classified as carcinogenic to humans. However, information on the biological effects of wildland firefighting remains limited. This study aimed to assess genotoxicity in a group of wildland firefighters and evaluate the contribution of total concentration of urinary hydroxylated polycyclic aromatic hydrocarbons (ΣOHPAHs) to selected endpoints. A group of 59 northern Portuguese wildland firefighters (mean age: 35.5 ± 9.0 years) was evaluated before and during the wildfire season. Sociodemographic, lifestyle, occupational-related information was collected via questionnaire. The micronucleus (MN) assay in buccal and urothelial cells was applied to paired samples to assess the association with exposure, as indicated by urinary ΣOHPAHs levels. The risk of MN formation in urothelial cells was over twofold higher during the wildfire season [Frequency Ratio (FR): 2.13, 95 % CI: 1.99-2.27, p = 0.01]. A 35 % increase in MN frequency in buccal cells was observed during the wildfire season (FR: 1.35, 95 % CI: 0.76-2.40, p > 0.05). Urinary ΣOHPAHs exhibited a significant positive association with MN‰ in urothelial cells (FR:1.04, 95 % CI: 1.01-1.08, p < 0.05). This study provides the first assessment of MN frequency in urothelial cells of wildland firefighters, offering novel evidence of genotoxic risks and potential long-term health impacts associated with wildland firefighting.
- Acrilamida em Alimentos: Validação do Método Analítico e MonitorizaçãoPublication . Jesus, Susana; Copeto, Sandra; Delgado, Inês; Coelho, InêsApresentação realizada no âmbito dos Encontros com Ciência do Departamento de Alimentação e Nutrição do INSA, sobre a atualização em validação de métodos analíticos e monitorização de acrilamida em alimentos.