Browsing by Issue Date, starting with "2011-04"
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- Antagonistic Regulation of CFTR Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine KinasePublication . Mendes, A.I.; Matos, P.; Amaral, M.D.; Jordan, P.Members of the WNK (with no lysine (K)) subfamily of protein kinases regulate various ion channels involved in sodium, potassium and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe that WNK4 promotes the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with spleen tyrosine kinase (Syk), which we find to phosphorylate Tyr512 in the first nucleotide-binding domain (NBD) 1 of CFTR. The presence of WNK4 prevents the phosphorylation of NBD1 by Syk in vitro in a kinase-independent manner. In baby hamster kidney cells stably expressing CFTR, catalytically active Syk reduces while WNK4 promotes the cell surface expression of CFTR. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. Together, our results identify that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and describe an antagonistic role of WNK4 and Syk in this process.
- Parental socioeconomic and educational level and chil overweight - lessons from a municipality based studyPublication . Silva, Ana Lúcia; Lopes, Sara; Breda, João; Rito, AnaINTRODUCTION: The prevalence of childhood obesity is rising rapidly in Portugal, reaching one of the worst positions in the European Union. Inadequate food habits with high intake of high energy dense foods, seems to be one of the main factor behind this epidemic. Family socioeconomic status also appears to be an obesogenic factor, particularly parents’ educational level and their income. To better understand the association of unbalanced dietary habits and socioeconomic environment on children nutritional status and to develop effective prevention strategies, are the main aims of this municipality study METHODS: A cross-sectional study was conducted on five municipalities (Viana do Castelo, Fundão, Oeiras, Seixal and Montijo), which included 3275 children (6-9 years old), attending the universe of public elementary schools. Childhood nutritional status was assessed by anthropometric parameters (weight and height) considering the CDC growthcharts criteria percentiles of BMI. Children’s food habits, socioeconomic condition and parent’s level education were collected by a family questionnaire. RESULTS: Prevalence of childhood overweight (BMI≥P85) was 32,1%, and over 14% of children were obese (BMI≥P95). Pre-obesity (P85≤ BMI< P95) prevalence was higher among girls (18,1%) than boys (17,8%). The consumption of fast food group and candies occurred mainly in every 15 days (48,4% and 35,5% respectively), pastry products showed a higher frequency of 1-3 times a week (41,5%). The percentage of children consuming vegetables, at least daily, varied with nutritional status: 23,0%; 25,7%; 26,2%; 21,0% in underweight, normal, pre-obese and obese children. Overweight was associated with parent´s low socioeconomic status, and middle parents educational level. CONCLUSION: Further careful analysis will help to better understand the role of obesogenic factors. This knowledge may identify additional potential effective interventions at local level in order to reverse the obesity trends in Portuguese children.
- EuroFIR Nexus WP1Publication . Calhau, Maria Antónia; Castanheira, Isabel; Becker, Wulf; Finglas, Paul; Roe, Mark; Columbani, Paolo; Moller, Anders; Ireland, Jayne; Bell, Simone; Unwin, IanPresentation of quality workpackage objectives, partners, timeschedule and tasks
- Co-relação entre perfil lipídico/risco cardiovascular e tipo de mutação em doentes com Hipercolesterolemia FamiliarPublication . Alves, A.C.; Medeiros, A.M.; Bourbon, M.A Hipercolesterolemia Familiar (FH) é uma doença autossómica dominante que se caracteriza a nível clínico por níveis elevados de LDLc, levando ao aparecimento prematuro de doenças cardiovasculares. A nível genético esta doença caracteriza-se por mutações em três genes: LDLR, APOB e PCSK9. O fenótipo de doentes com mutações nestes 3 genes é variável, estando descrito que doentes (dts) com mutações no gene PCSK9 apresentam um fenótipo mais grave e dts com mutação no gene APOB apresentam o fenótipo menos grave. Diferentes mutações no gene LDLR estão também associadas a diferentes fenótipos. Consequentemente o risco cardiovascular destes dts varia de acordo com a severidade do fenótipo apresentado por cada um. O objectivo deste estudo foi analisar o perfil bioquímico de dts com FH diagnosticada geneticamente de acordo com as mutações encontradas nos diferentes genes e com os diferentes tipos de mutações identificadas no gene LDLR para identificar se existe uma co-relação entre estas variáveis nos dts portugueses. Os parâmetros bioquímicos, colesterol total (CT), LDLc, HDLc, trigliceridos, ApoB e ApoAI) de 325 dts (225 adultos e 100 crianças) com FH foram analisados por SPSS, utilizando os testes ANOVA e de Tukey. Doentes com mutação no gene PCSK9 têm valores de CT e LDLc significativamente mais elevados do que dts com mutações nos restantes 2 genes. Não existe diferença significativa nestes parâmetros entre dts com mutações nos genes APOB e LDLR, embora todos os valores sejam mais elevados nos dts com mutações no gene LDLR. Doentes com mutações nonsense no gene LDLR apresentam valores de CT, LDLc e ApoB estatisticamente mais elevados do que dts com mutações missense. A comparação entre as outras categorias de mutações não revelou diferenças significativas nos parâmetros bioquímicos. Doentes com mutações no gene PCSK9 têm um fenótipo mais grave do que dts com mutações nos genes LDLR e APOB, tendo por esta razão um risco cardiovascular mais elevado. Nos dts portugueses com FH não foram observadas diferenças estatisticas significativas entre o fenótipo de dts com mutação nos genes LDLR e APOB. Doentes com mutações nonsense no LDLR que levam à não produção da proteína apresentam um fenótipo mais grave do que dts com mutações no gene LDLR que dão origem uma proteína que retêm alguma funcionalidade (mutações missense e splicing). O tipo de mutação (em diferentes genes ou diferentes mutações no LDLR) é importante para determinar o risco cardiovascular de cada doente.
- Projecto Obesidade Zero [comunicação oral]Publication . Carvalho, Maria Ana; Ramos, Carlos; Breda, João; Rito, Ana IsabelINTRODUÇÃO: A obesidade infantil constitui um dos mais sérios desafios de saúde pública, tendo atingido níveis epidémicos em vários países do Mundo. A sua prevenção e tratamento são prioritários. As intervenções em ambiente familiar de base comportamental que incorporam modificações ao nível da alimentação e da actividade física parecem ser as mais efectivas no controlo do peso corporal. A participação da comunidade constitui um eixo estratégico indispensável no combate a esta doença, apesar de serem escassos os projectos que assentam no desenvolvimento de respostas inovadoras às famílias com crianças com excesso de peso. Neste sentido, foi implementado um projecto inovador de base municipal, o Projecto Obesidade Zero (POZ), cujo principal objectivo foi desenvolver um programa de intervenção e abordagem do excesso peso e obesidade em crianças dos 6 aos 10 anos de idade em ambiente familiar. MÉTODOS: Trata-se de um estudo quasi-experimental, multicêntrico, desenvolvido em 2009/2010 em cinco Concelhos do país (Melgaço, Mealhada, Cascais, Beja e Silves). O programa foi desenvolvido com articulação entre as cinco Câmaras Municipais e os respectivos Centros de Saúde e compreendeu as seguintes fases de desenvolvimento: 1) Consultas de Obesidade Infantil; 2) Workshops de Cozinha Saudável; 3) Sessões de Aconselhamento Alimentar em grupo; 4) Sessão Aconselhamento Alimentar dirigida às famílias. RESULTADOS: Foram inscritas 294 crianças com excesso de peso no programa com uma média de idades de 8,62 anos. 155 (52,9%) eram do sexo feminino. 80,5% das crianças reduziram o seu percentil relativo ao IMC/idade (CDC,2000) durante o período do projecto. Em média registou-se uma redução do percentil 93,6 para o percentil 91,3, sendo as diferenças estatisticamente significativas (p<0,05). CONCLUSÃO: O POZ instituiu e fortaleceu parcerias multissectoriais destacando o papel dos Municípios. Os resultados obtidos sugerem que as intervenções de base comunitária e familiar parecem ter efeitos significativos ao nível da prevalência da obesidade infantil.
- Protein kinase WNK2 regulates cell migration in malignant gliomasPublication . Moniz, S.; Martinho, O.; Reis, R.M.; Jordan, P.Malignant glioblastomas are the most common and lethal adult brain tumours, with patients dying within two years from diagnosis. Little is known about the molecular mechanisms underlying the formation and/or development of these tumours, which present a very invasive phenotype within the brain and are genetically heterogeneous and highly resistant to both chemo- and radio-therapies. Recently, the promoter region of the protein kinase WNK2 gene was found to be hypermethylated in 29 of 31 infiltrative gliomas and about 80% of meningiomas. We have previously described that the experimental depletion of WNK2 expression decreases RhoA activity whilst leading to increased Rac1 activity. Because RhoA/Rac1 activities are important for cell migration and glioblastomas are very invasive tumours, we tested the effects of WNK2 on wound-healing assays in glioma cell lines SW1088 and A172. SW1088 cells express endogenous WNK2 and we observed that wound closure was increased upon experimental depletion of endogenous WNK2. In contrast, A172 cells display complete promoter region methylation and WNK2 re-expression was found to decrease migration. Consistently, we observed an increase in Rac1 activity in SW1088 cells upon WNK2 down-regulation, but lower levels of active Rac1 in A172 cells stably expressing WNK2 cDNA when compared with an equivalent cell line stably transfected with the same empty vector. Our studies indicate that loss of WNK2 expression promotes Rac1 activation and may contribute to the highly invasive phenotype that glioblastomas present. We also observed that, in a panel of glioblastoma cell lines, WNK2 promoter methylation correlates with a marked deregulation in Akt, MEK1/2 and ERK1/2 activities, suggesting WNK2 may also be important for tumour cell survival and cell cycle progression.
- Arsenic content in fish and associated measurement uncertainty as a metrological parameter for Total Diet StudiesPublication . Coelho, Inês; Gueifão, Sandra; Matos, Ana Sofia; Santos, Rui; Castanheira, IsabelTotal Diet Studies (TDS) are the most important tools to assure that chemical contaminants presence in foodstuffs remains within safety levels. Reliability of data provided by laboratories to assess the effectiveness of measurements aiming at reducing the risk of exposure to chemical hazards is of paramount importance. Evaluation of measurement uncertainty associated with a result is an important parameter in assessing the sources of analytical data variability. Two methods to estimate uncertainty in analytical measurement of arsenic in fish are discussed. Measurement uncertainty was estimated for the determination of arsenic content in fish samples by Inductively Coupled Mass Spectrometry (ICP-MS). The work addressed both approaches accepted by Eurolab and NIST: modeling (bottom up) and empirical (top down). The Ishikawa diagram was used to identify the most significant sources of uncertainty. The mathematical modeling techniques to assess uncertainty components based on a classical model that accounts for all recognized significant sources of errors was similar to the top down based on validation data from interlaboratory comparison studies. Both methods showed that analytical variability in arsenic content was less than 10%.
- A WNK4 gene variant relates to osteoporosis and not to hypertension in the Portuguese populationPublication . Mendes, Ana Isabel; Mascarenhas, Mário Rui; Matos, Sónia; Sousa, Inês; Ferreira, Joana; Barbosa, Ana Paula; Bicho, Manuel; Jordan, PeterGermline mutations in the WNK4 gene originate Gordon syndrome or pseudohypoaldosteronism type II, a familial form of hypertension with hyperkalemia and hypercalciuria. In order to elucidate the contribution of WNK4 genetic variants to hypertension and/or osteoporosis, we analyzed 271 control individuals and a cohort of 448 hypertensive and 372 osteoporosis patients from the Portuguese population. Ten genetic variants were detected in 4.3% of the population under study, none of which revealed any significant association to the hypertension phenotype. In contrast, a rare missense alteration within exon 17 in a highly conserved arginine residue showed a possible tendency for association to the osteoporosis group. Our data suggest that WNK4 polymorphism rs56116165 is a rare allelic variant in a candidate gene with a biological function in renal calcium homeostasis that may contribute to a genetic predisposition to osteoporosis.
- A proposal to guarantee metrological traceability in determination of heavy metals contents in cork stoppers by ICP-MSPublication . Gueifão, Sandra; André, Catarina; Coelho, Inês; Castanheira, IsabelThe compliance of heavy metals contents in cork with European Legislation for materials in contact with food and beverages are an important requirement of safety and quality of cork stoppers. However there are few specific regulations for cork. Therefore metrological strategies to assure reliability of analytical measurement results are crucial to ensure that the quantification of chemical contaminants are effectively in line with limits established in law. In this work a general approach is described for implementation of metrological traceability covering all analytical procedure for determination of Arsenic, Lead, Nickel, Chromium and Cadmium contents in cork stoppers with the potential to migrate to beverages. A flowchart diagram to identify the critical points of experimental procedure that need to be under metrological conditions is described. The experimental procedure encompasses the test conditions to simulate migrations for beverages, digestion procedure and determination by ICP-MS. The most important metrological control points to assure that measurements will be traceable to SI were: validation of analytical method to assure correct identification and quantification of metal under study; measurement of recovery of the spikes to estimate analyte lost during the digestion of samples; use of appropriate calibrants to calibrate the ICP-MS; regular participations in PT schemes for contaminants under study; estimation of associated measurement uncertainties. This metrological approach seems realistic to guarantee the traceability of assigned values of inorganic contaminants in cork stoppers and to demonstrate concordance of test conditions with ISO/IEC 17025 requirements.
- Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approachPublication . Giardine, Belinda; Borg, Joseph; Higgs, Douglas R; Peterson, Kenneth; Philipsen, Sjaak; Maglott, Donna; Singleton, Belinda; Anstee, David J; Basak, Nazli; Clark, Barnaby; Costa, Flavia C; Faustino, Paula; Fedosyuk, Halyna; Felice, Alex E; Francina, Alain; Galanello, Renzo; Gallivan, Monica VE; Georgitsi, Marianthi; Gibbons, Richard J; Giordano, Piero C; Harteveld, Cornelis L; Hoyer, James D; Jarvis, Martin; Joly, Philippe; Kanavakis, Emmanuel; Kollia, Panagoula; Menzel, Stephan; Miller, Webb; Moradkhani, Kamran; Old, John; Papachatzopoulou, Adamantia; Papadakis, Manoussous N; Papadopoulos, Petros; Pavlovic, Sonja; Perseu, Lucia; Radmilovic, Milena; Riemer, Cathy; Satta, Stefania; Schrijver, Iris; Stojiljkovic, Maja; Thein, Swee Lay; Traeger-Synodinos, Jan; Tully, Ray; Wada, Takahito; Wayne, John S; Wiemann, Claudia; Zukic, Branda; Chui, David HK; Wajcman, Henri; Hardison, Ross C; Patrinos, George PWe developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic disease.
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