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- Caracterização do potencial genotóxico de sedimentos estuarinos em sistemas experimentais in vitro e in vivoPublication . Sacadura, Joana Gomes; Silva, Maria João; Dias, Deodália Maria Antunes[PT] O estuário do Sado (Oeste de Portugal) está sujeito a várias fontes de poluição associadas a centros urbanos, industrias pesadas, actividades mineiras e agricultura. Este estuário permanece um local importante de actividade pesqueira para consumo próprio e para mercados locais e externos, assim com para actividades recreativas e turísticas. No entanto, estudos anteriores revelaram vários contaminantes presentes nos sedimentos aquáticos, maioritariamente metais, pesticidas e hidrocarbonetos aromáticos policíclicos. Estes compostos podem ser absorvidos e acumulados nas partes comestíveis das espécies estuarinas e nos produtos agrícolas locais entrando, deste modo, na cadeia alimentar humana, colocando em risco a saúde humana. Neste contexto, o presente estudo tem como objectivo analisar os efeitos genotóxicos, in vitro e in vivo, de extractos de sedimentos do estuário do Sado. Para esse fim, os sedimentos foram recolhidos de quatro locais de pesca (P, C, A e E) do estuário e os contaminantes orgânicos e inorgânicos totais foram extraídos com uma mistura de metanol:diclorometano. Células HepG2 foram expostas aos extractos e a um agente antimutagénico, para a análise da genotoxicidade utilizando o ensaio do cometa, a fim de analisar a possível reversão de danos oxidativos induzidos pelos contaminantes. Adicionalmente, recorreu-se a um modelo de ratinhos transgénicos baseados no plasmídeo lacZ expostos por via oral a duas doses de extracto P para testes de genotoxicidade in vivo. Neste modelo foi realizado o ensaio do micronúcleo, do cometa e de mutações em vários órgãos de ratinho. Nos resultados in vitro não se verificou a reversão dos danos oxidativos induzidos pelos extractos de sedimentos por parte da quercetina, nas condições testadas. In vivo, verificou-se a indução de micronúcleos, embora o ensaio do cometa não revelasse resultados positivos. Aparentemente, não se verificou mutagenecidade in vivo, embora sejam necessários mais estudos para concluir a análise de mutações. Globalmente, os resultados obtidos neste estudo reflectem o potencial efeito adverso para a saúde humana associado aos contaminantes encontrados nos sedimentos do estuário, contribuindo para a avaliação de risco.
- Tularaemia: a challenging zoonosisPublication . Carvalho, C.L.; Lopes de Carvalho, I.; Zé-Zé, Líbia; Núncio, M.S.; Duarte, E.L.In recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in Europe, including tularaemia, caused by Francisella tularensis. This remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. Also, a renewed concern has arisen with regard to F. tularensis: its potential use by bioterrorists. Based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. Moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals.
- Mucolipidosis II-Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc-1-Phosphotransferase Precursor Protein (GNPTAB)Publication . De Pace, Raffaella; Coutinho, Maria Francisca; Koch-Nolte, Friedrich; Haag, Friedrich; Prata, Maria João; Alves, Sandra; Braulke, Thomas; Pohl, SandraMucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/β-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients
- Rastreio Neonatal: novas tecnologias e novas patologiasPublication . Marcão, AnaO rastreio neonatal deve ser um programa sistemático e universal, destinado a todos os recém-nascidos. O seu principal objetivo é o diagnóstico pré-sintomático e a instituição precoce de terapia adequada, de forma a minimizar as consequências da patologia rastreada para o recém-nascido (RN). Neste sentido, deve ser um programa integrado, com vertentes política, social e ética, para além das vertentes clínica e técnico-científica. Os primeiros programas de rastreio surgiram no início da década de 60, com o rastreio da fenilcetonúria, e gradualmente outros rastreios foram incluídos nestes programas. Nos anos 90, com o desenvolvimento e aplicação da tecnologia de espetrometria da massa em tandem (MS/MS) ao rastreio, houve uma importante mudança no conceito de patologia rastreável, passando a ser possível o rastreio de doenças até aí não incluídas nos programas de rastreio, devido à sua raridade. Com esta tecnologia é possível o rastreio simultâneo de mais de 40 doenças hereditárias do metabolismo, utilizando a mesma amostra. O rastreio das doenças raras é atualmente considerado um problema de saúde pública, e nos últimos anos têm sido desenvolvidos esforços importantes ao nível dos decisores políticos europeus da área da saúde, no sentido da harmonização do rastreio neonatal em todos os países europeus e da sua disponibilização a todos os recém-nascidos. Apesar de instituídos na maioria dos países ditos desenvolvidos, existem enormes diferenças nos programas de rastreio neonatal efetuados em diferentes países ou mesmo em diferentes regiões do mesmo país. O Programa Nacional de Diagnóstico Precoce (PNDP) realiza-se em Portugal desde 1979 e atualmente inclui o rastreio neonatal de 24 Doenças Hereditárias do Metabolismo (DHM), por MS/MS, e do Hipotiroidismo Congénito (HC) utilizando um sistema automático de imunoensaio (autoDELFIA®). Em Outubro de 2013 iniciou-se, um estudo piloto para o rastreio neonatal da Fibrose Quística (FQ), que deverá ser efetuado em 80 000 recém-nascidos (RN) portugueses ao longo de aproximadamente um ano. Clinicamente a FQ é uma doença grave com atingimento multissistémico, frequentemente letal nos primeiros anos de vida. Diagnosticar precocemente a doença é uma fator decisivo no prognóstico, não só pela maior sobrevida, mas também para uma melhor qualidade de vida do doente. No final deste estudo deverá ser avaliada a inclusão da FQ no PNDP. Os programas de rastreio neonatal devem ser programas dinâmicos, com avaliação permanente das patologias a incluir e da tecnologia a utilizar. O desenvolvimento recente de novas terapias e de novas tecnologias, nomeadamente na área do metabolismo e da genética, abre infinitas possibilidades de rastreio, mas levanta também inúmeras questões. Os princípios básicos definidos por Wilson e Jungner para o rastreio neonatal, apesar de já terem quase meio século, não devem ser esquecidos e as recomendações internacionais devem ser seguidas.
- Mycotoxins and applicability of in vitro methods of digestion and absorption assessment – the patulin case studyPublication . Assunção, R.M.; Ferreira, M.; Martins, Carla; Dupond, D.; Alvito, PaulaMycotoxins are a wide group of fungal secondary metabolites that exert multiple toxic effects on humans and animals1. Patulin, a mycotoxin with significant public health risk, is a toxic secondary metabolite produced by a wide range of fungal species growing on rotten fruit2. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, although the total amount of an ingested contaminant does not always reflect the amount that is available to the body and exert its toxic effects3. In this study, two in vitro methods were applied to evaluate the bioaccessibility of patulin at different experimental conditions and the intestinal membrane integrity of Caco-2 cells exposed to patulin and cysteine (antioxidant) protective effects. Seven artificially contaminated fruit juices were assayed in the presence or absence of a standard meal showing a significant difference (p=0.001) for bioaccessibility values between contaminated samples alone and combinations with a standard meal4. Different concentrations of patulin and cysteine were assayed in a Caco-2 cells monolayers. At 95 µM, patulin produced a dramatic decrease of transepithelial electrical resistance. This effect was significantly (p=0.016) reduced when 400 µM and 4000 µM of cysteine was added to the cells4. The combination of in vitro digestion models with other techniques using intestinal cell lines offer a more complete picture of what is happening in the digestion and absorption process, as well as the study of beneficial effects of protective agents.
- Unverricht-Lundborg disease: development of splicing therapeutic approaches for a patient with an homozygous mutation in the cystatin B genePublication . Matos, L.; Duarte, A.J.; Ribeiro, D.; Jordan, P.; Prata, M.J.; Chaves, J.; Desviat, L.R.; Pérez, B.; Amaral, O.; Alves, S.Unverricht-Lundborg disease (ULD) is the most common form of progressive myoclonic epilepsy worldwide. It is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion, missense, nonsense, frameshift and mutations that may lead to alternative splicing have been described as causal of ULD. Recently, our group described an ULD patient who is homozygous for a new synonymous mutation (c.66G>A; p.Q22Q) located at the last nucleotide of exon 1. The transcriptional profile analysis allowed the identification of two CSTB splice variants, one of normal size with the G>A change and other with partial inclusion of intron 1 due to activation of a cryptic splice-site inside the intronic sequence. To correct the splice defect, here we developed antisense oligonucleotide and U1snRNA mediated therapeutic strategies. U1 is required for splice donor site (SDS) recognition of pre-mRNAs and initiates the splicing process. The mutation c.66G>A interferes with the recognition of the SDS by U1. In a first approach, to reduce missplicing we generated four U1 construct isoforms with increasing complementarity to the SDS. Transfection of patient-derived fibroblasts with different concentrations of the adapted U1 vectors did not allowed the correction of the aberrant transcript. In a second strategy, we have designed a specific lock nucleic-acid (LNA) oligonucleotide to block the activated cryptic splice-site in intron 1. Normal splicing pattern of a single transcript with the synonymous change G>A was successfully rescued after LNA transfection in patient cells. The therapeutic effect showed to be dose-dependent. These results suggest that antisense therapy might be a potential alternative or adjunct treatment strategy for patients holding splicing changes in CSTB gene. As far as we know this is the first report of a patient tailored therapy in cells of an ULD patient.
- Neuroprotective effect of steroidal alkaloids on glutamate-induced toxicity by preserving mitochondrial membrane potential and reducing oxidative stressPublication . Taveira, M.; Sousa, C.; Valentão, P.; Ferreres, F.; Teixeira, João Paulo; Andrade, P.B.Several evidences suggest that enhanced oxidative stress is involved in the pathogenesis and/or progression of several neurodegenerative diseases. The aim of this study was to investigate for the first time whether both extracts from tomato plant (Lycopersicon esculentum Mill.) leaves and their isolated steroidal alkaloids (tomatine and tomatidine) afford neuroprotective effect against glutamate-induced toxicity in SH-SY5Y neuroblastoma cells and to elucidate the mechanisms underlying this protection. Steroidal alkaloids from tomato are well known for their cholinesterases' inhibitory capacity and the results showed that both purified extracts and isolated compounds, at non-toxic concentrations for gastric (AGS), intestinal (Caco-2) and neuronal (SH-SY5Y) cells, have the capacity to preserve mitochondria membrane potential and to decrease reactive oxygen species levels of SH-SY5Y glutamate-insulted cells. Moreover, the use of specific antagonists of cholinergic receptors allowed observing that tomatine and tomatidine can interact with nicotinic receptors, specifically with the α7 type. No effect on muscarinic receptors was noticed. In addition to the selective cholinesterases' inhibition revealed by the compounds/extracts, these results provide novel and important insights into their neuroprotective mechanism. This work also demystifies the applicability of these compounds in therapeutics, by demonstrating that their toxicity was overestimated for long time.
- Cardiovascular risk assessment of dyslipidemic children: analysis of biomarkers to identify monogenic dyslipidemiaPublication . Medeiros, A.M.; Alves, A.C.; Aguiar, P; Bourbon, M.; on behalf of the Pediatric Investigators of the Portuguese Familial Hypercholesterolemia StudyThe distinction between a monogenic dyslipidemia and a polygenic/environmental dyslipidemia is important for the cardiovascular risk assessment, counseling, and treatment of these patients. The present work aims to perform the cardiovascular risk assessment of dyslipidemic children to identify useful biomarkers for clinical criteria improvement in clinical settings. Main cardiovascular risk factors were analyzed in a cohort of 237 unrelated children with clinical diagnosis of familial hypercholesterolemia (FH). About 40% carried at least two cardiovascular risk factors and 37.6% had FH, presenting mutations in LDLR and APOB. FH children showed significant elevated atherogenic markers and lower concentration of antiatherogenic particles. Children without a molecular diagnosis of FH had higher levels of TGs, apoC2, apoC3, and higher frequency of BMI and overweight/obesity, suggesting that environmental factors can be the underlying cause of their hypercholesterolem≥ia. An apoB/apoA1 ratio ≥0.68 was identified as the best biomarker (area under the curve = 0.835) to differentiate FH from other dyslipidemias. The inclusion in clinical criteria of a higher cut-off point for LDL cholesterol or an apoB/apoA1 ratio ≥0.68 optimized the criteria sensitivity and specificity. The correct identification, at an early age, of all children at-risk is of great importance so that specific interventions can be implemented. apoB/apoA1 can improve the identification of FH patients.
- Anomalias Cromossómicas: Síndromes e Estratégias de DiagnósticoPublication . Brito, Filomena; Marques, Bárbara; Correia, Hildeberto
- The effects of matrix proteins on the aflatoxin M1 bioaccessibility and the Caco-2 intestinal transportPublication . Tavares, A.M.; Egge, L.; Portmann, R.; Alvito, PaulaMycotoxins are fungal natural contaminants commonly found in food products that cause severe effects in human health, especially children. The mycotoxins occur in a great variety of foods, and can form complexes with the food matrix with a significant impact on their bioaccessibility. The bioaccessible fraction of the food contaminant contributes to the effective internal exposure depending on the contamination level, food matrix and the way the food is contaminated (spiked or naturally). To our knowledge, until now no studies were performed to disclose the possible role of milk proteins in the bioaccessibility of aflatoxin M1 (AFM1), a mycotoxin commonly found in milk products. On behalf of a Short Term Scientific Mission within the Infogest COST action and of the project Mycomix (FCT, Portugal), a collaboration study between the National Health Institute Doutor Ricardo Jorge (Portugal) and Agroscope Liebefeld- Posieux (Switzerland) was established. The recently submitted harmonized in vitro digestion protocol2 was for the first time applied to study the bioaccessibility of AFM1 in artificially contaminated infant formula and the protein profile of the samples analysed by LC-MS/MS. The results revealed a good performance of the harmonized method, showing a successful digestion of the proteins into smaller peptides. However, the presence of aflatoxin M1 contamination was not detected before and after digestion, suggesting an interaction with the food matrix. Moreover, in the transport assays, the presence of AFM1 did not impair the Caco-2 cells membrane integrity as shown by the Transepithelial Electrical Resistance. Further assays including an optimized AFM1 extraction method are in progress to evaluate toxin bioaccessibility and its presence in basolateral, apical cell media and cell cytoplasm.