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- Discriminative Protein Markers of Second-Hand Smoke Exposure Identified by Shotgun ProteomicsPublication . Neves, Sofia; Pacheco, Solange A.; Vaz, Fátima; Saraiva, Joana; James, Peter; Simões, Tânia; Penque, DeborahObjective: Chronic exposure to second-hand smoke (SHS) increases the risk of developing tobacco-related pathologies such as lung cancer and cardiovascular diseases. This study aimed to identify potential protein biomarkers for response and risk assessment of SHS exposure. Methods: A shotgun proteomics approach was employed to analyse protein expression profiles in nasal epithelium and plasma samples from healthy, non-smoking restaurant workers who were either exposed or not exposed to SHS in the workplace. A label-free quantification strategy was used to measure differential protein expression between the two groups. Logistic regression modelling was applied to identify the proteins that best discriminated exposed individuals from non-exposed controls, with the goal of establishing an expression profile indicative of SHS-related response. Results: In the nasal epithelium, SHS exposure was associated with modulation of proteins involved in HIF1α-regulated glycolytic pathways, xenobiotic metabolism, cell proliferation, and differentiation. In plasma, differentially expressed proteins were related to systemic inflammation and atherosclerosis. Among these, three plasma proteins—Histidine-rich glycoprotein (HRG), Vitamin D-binding protein (GC), and Leucine-rich alpha-2-glycoprotein (LRG1)—showed a significant discriminatory potential between SHS-exposed and non-exposed individuals. Conclusions: The identified proteins, particularly HRG, GC, and LRG1, are promising response biomarkers for SHS exposure. Their expression profiles may support the development of molecular tools for individual response assessment associated with environmental tobacco smoke.
- Gene editing as a tool for developing cell based models of a lysosomal storage disorder: preliminary resultsPublication . Duarte, Ana Joana; Moreira, Luciana; Gaspar, Paulo; Alves, Sandra; Bragança, José; Amaral, OlgaIn this work, we aimed to establish a Fabry Disease (FD, OMIM: #301500) disease model using the CRISPR/Cas 9 system by knocking out a HDFa iPSC line. We also aimed to correct a nonsense mutation (p. W 287 X) in the iPSCs derived from a patient with FD. The cell lines used were generated in our laboratory, and the FD iPSC line is registered in the Human Pluripotent Stem Cell Registry with identification "INSAi 002-A". To fully evaluate the molecular and cellular physiological changes, further studies are still required. The development of innovative cell models, particularly for rare diseases like Lysosomal Storage Disorders, is beneficial for studying the pathophysiology of the disease.
- Perceções e preocupações dos cidadãos portugueses sobre a exposição a substâncias químicas perigosasPublication . Namorado, Sónia; Silva, Maria João; Louro, Henriqueta; Isidro, Glória; Lobo Vicente, Joana; HBM4EU Citizen Survey Study GroupIntrodução e objetivo: É do conhecimento comum que a exposição a substâncias químicas pode causar efeitos adversos na saúde. Para minimizar a exposição é essencial informar a população e contribuir para a consciencialização da importância da tomada de decisões informadas no que respeita a comportamentos e estilos de vida promotores da saúde, através do desenvolvimento de intervenções dirigidas. Com o objetivo de obter informações sobre o conhecimento e perceções da população sobre a exposição a substâncias químicas foi realizado um inquérito aos cidadãos dos 30 países participantes na Iniciativa Europeia em Biomonitorização Humana (HBM4EU), incluindo Portugal.
- Economia circular e reciclagem de resíduos eletrónicos: o papel da biomonitorização humana na identificação de potenciais riscos ocupacionaisPublication . Silva, Maria João; Tavares, Ana; Rosário, Rita Lopes; Ventura, Célia; Namorado, Sónia; Martins, Carla; Louro, Henriqueta; Viegas, SusanaA reciclagem dos resíduos produzidos pela sociedade moderna, incluindo os resíduos elétricos e eletrónicos (REEE), é fundamental para a economia circular, um dos pilares do Pacto Ecológico Europeu. Porém, pode também representar um problema de saúde ocupacional, devido à exposição dos trabalhadores a metais (p.ex. chumbo e cádmio) e a compostos orgânicos (p.ex. ftalatos, PCBs) tóxicos. Como consequência, poderão surgir, a longo prazo, patologias respiratórias, renais, neurológicas ou oncológicas. Apresenta-se um estudo multicêntrico harmonizado de biomonitorização humana envolvendo indústrias de gestão de REEE localizadas em diversos países europeus, cujo objetivo consistiu em avaliar a exposição dos trabalhadores a substâncias químicas nocivas para a saúde e os seus efeitos precoces. Caracterizou-se a exposição através da medição dos compostos ou seus metabolitos em amostras de sangue e urina dos trabalhadores, comparativamente a um grupo de controlo, e os seus potenciais efeitos através da análise de biomarcadores de genotoxicidade em células bucais e sanguíneas. Os resultados evidenciaram a exposição de alguns grupos de trabalhadores a metais, com destaque para o chumbo, e a compostos orgânicos, incluindo PCBs e ftalatos. Relativamente aos biomarcadores de efeito, observou-se uma tendência para um acréscimo de instabilidade cromossómica local e sistémica nos trabalhadores expostos vs. controlos. Este estudo, iniciado no âmbito da Iniciativa Europeia em Biomonitorização Humana (HBM4EU) e continuado na Parceria para Avaliação do Risco de Químicos (PARC), permitiu identificar potenciais riscos para a saúde de trabalhadores envolvidos na gestão de REEE. Esses riscos deverão ser mitigados através da revisão/implementação de regulamentação, promoção do uso de novas tecnologias e/ou sistemas de proteção mais seguros e capacitação dos trabalhadores para a sua correta utilização.
- Assessing exposure and early biological effects in waste management workers using a harmonized occupational biomonitoring studyPublication . Tavares, Ana; Rosário, Rita; Aimonen, K.; Louro, Henriqueta; Martins, Carla; Viegas, Susana; Santonen, Tiina; Silva, Maria JoãoObjective: Human biomonitoring provides measurements of internal exposure to all chemicals by all routes of exposures and also addresses associated early biological effects. An occupational biomonitoring study requires a comprehensive and harmonised planning, including participants’ recruitment, sampling strategy, biomarkers definition and analysis, data collection, management, and reporting, in order to generate comparable and replicable data. In the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), a multi-centric biomonitoring campaign was planned to assess occupational exposure to hazardous chemicals from electronic and plastic waste management industries. We here present its design, comprising the assessment of external and internal exposure to several substances and characterization of early biological effects. Methods: Target participants are workers involved in e-waste and plastic recycling activities, and non-exposed individuals as controls. Besides industrial hygiene samples (e.g., air, settle dust) also biological samples (hair, blood, epithelial buccal cells and urine) are collected after informed consent, and a questionnaire is applied to all participants. Exposure biomarkers comprise measurements of heavy metals, flame retardants, per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), bisphenols, polychlorobiphenyls (PCBs), and other plasticizers in hair, blood, or urine samples by qualified laboratories. Effect biomarkers are assessed in blood (genotoxicity – micronucleus and comet assay - inflammation, epigenetic and oxidative stress markers), buccal cells (micronucleus) and urine samples (metabolic changes). Occupational hygiene measurements and contextual information will facilitate the interpretation of biomarkers data and the identification of potential confounding variables. Expected Results: The findings are expected to provide valuable information on exposure and associated early (preclinical) effects, and also to uncover groups at increased risk, supporting the promotion of good work practices and/or the implementation of efficient risk management strategies further improving worker’s health protection.
- Is exon skipping the key to correct N-acetylglucosamine-1-phosphotransferase deficiency? An antisense oligonucleotide therapeutic approachPublication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Gaspar, Paulo; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntroduction: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs the trafficking of lysosomal hydrolases. Of all ML II mutations, c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs). Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping in pre-mRNA, successfully generating an in-frame mRNA. Aims: Now, our aim is to determine whether this in-frame transcript leads to increased GlcNAc-PT levels improving ML II cellular phenotype. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts by a radioactive assay, but activity levels were similar in ML II and control fibroblasts (treated and non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without the exon 19) and a mutant (with the mutation c.3503_3504delTC) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Additionally, we have measured the activity of several lysosomal hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment of control and patient cells. To further help in the validation of this therapy we are also generating a novel GlcNAc-PT antibody in rabbits. Results: Our results demonstrated that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, the c.3503_3504delTC construct showed no expression, with no detectable α/β precursor band. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts treated with the ASO, particularly 24h and 48h post-treatment. However, only the values relatively to the α-Gal were statistically significant, but the WB analysis using an antibody against this enzyme did not detect any band in ASO-treated ML II fibroblasts. We also developed a novel antibody for GlcNAc-PT. Preliminary results revealed a band corresponding to the β-subunit in both control and ML II patient fibroblasts (unexpected), but in overexpression assays both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess their specificity. Conclusion: Our ASO-based approach effectively promotes the skipping of exon 19. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.
- A Multiplex Biomarker panel: A powerful tool for LSDs DiagnosisPublication . Neiva, Raquel; da Silva Gaspar, Paulo Jorge Miranda; Sousa e Silva, Lisbeth Elena; Gonçalves, Isabel; Ferreira, Sara; Diogo, Luisa; Vilarinho, LauraIntrodução / Descrição do Caso: Lysosomal Storage Disorders (DLSs) are a set of rare, chronic and multisystemic pathologies with a variable mode of presentation and severity. Acid sphingomyelinase deficiency (ASMD), historically known as Niemann–Pick disease (NPD) types A, A/B, and B, is a rare, progressive, potentially fatal lysosomal storage disease caused by pathogenic variants in SMPD1 gene. The disease manifestations frequently involve hepatosplenomegaly with progressive organ dysfunction, interstitial lung disease, and bleeding. The cellular damage caused by sphingomyelin accumulation can be irreversible and can lead to life-threatening complications with reduced life expectancy. ASMD can be underestimate and the diagnostic odyssey arise from an overlap in symptomology with other diseases, including primary hepatic disease, Gaucher disease, NPC, and lysosomal acid lipase deficiency.
- Early Diagnosis of Mucopolysaccharidoses in PediatricsPublication . da Silva Gaspar, Paulo Jorge Miranda; Neiva, Raquel; Sousa e Silva, Lisbeth Elena; Diogo, Luisa; Ferreira, A.; Miranda, A.; Ribeiro, S.; Antunes, D.; Garcia, P.; Rodrigues, E.; Campos, T.; Janeiro, P.; Lopes, Altina; Pereira, Cristina; Nogueira, Célia; Sousa, S.; Ferreira, S.; Alves, Sandra; Leão Teles, Elisa; Vilarinho, LauraIntroduction: Mucopolysaccharidoses (MPSs) are a group of Lysosomal Storage Disorders with multisystem involvement, presenting different degrees of severity and evolution. At early disease stages and late onset forms, diagnosis can be postponed for years or even missed. The FIND PROJECT was designed to claim awareness to the redflags of MPSs at pediatric age and to provide a useful tool for physicians to diagnose these pathologies, since most of them are amenable to enzyme replacement therapy.
- Differential Diagnosis of Alpha-Mannosidosis in MPSsPublication . da Silva Gaspar, Paulo Jorge Miranda; Gonçalves, Diana; Neiva, Raquel; Sousa e Silva, Lisbeth Elena; Vilarinho, LauraIntroduction: Mucopolysaccharidosis (MPSs) and oligosaccharidosis, two subgroups of lysosomal storage disorders (LSDs), face diagnostic challenges due to their wide spectrum of clinical presentations and overlapping symptoms. One of the oligosaccharidose is α-mannosidosis, an extremely rare and often undiagnosed disorder. It is characterized by a deficiency in the enzymatic activity of α-mannosidase, which is responsible for cleaving mannose from N-linked oligosaccharides. This study aimed to investigate the activity of α-mannosidase in dried blood spot (DBS) samples that had undergone screening for MPSs.
- Deleção intersticial 7q33q34 em fetos de gravidez gemelar monocoriónica diamnióticaPublication . Simão, Laurentino; Marques, Bárbara; Ferreira, Cristina; Serafim, Sílvia; Alves, Cristina; Silva, Marisa; Viegas, Mónica; Peliano, Ricardo; Brito, Filomena; Bernardeco, Joana; Cruz, Jader; Pedro, Sónia; Martins, Ana; Tarelho, Ana; Carvalho, Inês; Cohen, Álvaro; Correia, HildebertoIntrodução: O acompanhamento de gestações gemelares pode revelar-se desafiante se houver alterações ecográficas e discrepâncias entre os fetos. Deleções intersticiais 7q, abrangendo diferentes regiões e apresentando tamanho variável, são raras, e encontram-se quase exclusivamente descritas em pós-natal. Objectivos: Apresentamos o caso de uma gestante, de 32 anos, com gravidez gemelar monocoriónica e diamniótica de 16 semanas, referenciada por bolsas jugulares bilaterais, crescimento no P10-P20 e discrepância no pico sistólico de velocidade da artéria cerebral média (PSV-ACM). Foi efetuada colheita de liquido amniótico para estudo por microarray cromossómico (CMA). Metodologia: Foi efeituado diagnóstico rápido de aneuploidias (DRA) por QF-PCR (Devyser®), ao que se seguiu CMA com array CytoScan 750K (Thermo Fischer®) e cariótipo. Resultados: O DRA revelou um resultado normal. O CMA permitiu a identificação de uma deleção intersticial com 9,0 Mb em 7q33q34 - arr[GRCh37] 7q33q34(133411316_142427027)x1. A alteração engloba 12 genes mórbidos. O cariotipo confirmou o resultado: 46,XX,del(7)(q32.3q34)dn. Após aconselhamento genético, o casal optou por interrupção da gestação. Conclusões: Deleções intersticiais na região 7q32 a 7q35 apresentam grande variabilidade fenotípica. As características mais comuns são: atraso de desenvolvimento e da linguagem, défice intelectual, dismorfias faciais e atraso de crescimento. Nos raros casos descritos com alterações cromossómicas parcialmente sobreponíveis ao caso em estudo, a CNV tem sido classificada como patogénica. No único caso com referência ao período pré-natal e com alteração quase totalmente sobreponível, descreve-se decréscimo de movimentos fetais, baixo peso à nascença, atraso de desenvolvimento, défice intelectual, dismorfias faciais e infeções múltiplas. A alteração cromossómica encontrada poderá explicar a relativa restrição de crescimento fetal, não tendo as bolsas jugulares, presentes em ambos os fetos e a discrepância PSV-ACM sido até agora descritos. Gestações com alterações ecográficas e CNVs, mas com reduzida bibliografia, são desafiantes na interpretação dos resultados a nível laboratorial e clínico. Só a descrição de mais casos permitirá um ganho de conhecimento em saúde.