Browsing by Issue Date, starting with "2019-02"
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- Application of the Six Sigma methodology in the evaluation of the results in Cell Blood Count EQAS Program (PNAEQ)Publication . Ovelheiro, Ana; Miranda, Armandina; Correia, Helena; Cardoso, Ana; Faria, Ana Paula; Gomes Requeijo, JoséThe haemogram is one of the most frequently requested laboratory tests, in hospital and ambulatory It is important in the evaluation of anaemia, polycythaemia, leukaemia, infection, inflammation, among others Therefore, given the importance of the haemogram in the clinical context, an evaluation was performed on the results of the clinical laboratories participating in PNAEQ’s EQA Cell Blood program.
- Distribuição e dinâmica de ocorrências de cianobactérias e compostos associados na Albufeira do RoxoPublication . Dias, Elsa; Menezes, CarinaO presente relatório resultou dos trabalhos desenvolvidos no âmbito do Protocolo de Colaboração entre o Instituto Nacional de Saúde Doutor Ricardo Jorge e a AgdA- Águas Públicas do Alentejo, SA. Pretendeu avaliar-se a distribuição e dinâmica de ocorrência de cianobactérias potencialmente produtoras de COVs e de cianotoxinas na albufeira do Roxo. Especificamente, pretendia-se estudar três locais e três profundidades de colheita, de forma a identificar o ponto mais adequado à captação de água da albufeira. Foi também objetivo do trabalho, avaliar a persistência e viabilidade de fitoplâncton na água tratada à saída da ETA e nos reservatórios da Atalaia e Aljustrel, bem como a presença de COVs. O trabalho visava, ainda, a monitorização de parâmetros físico-químicos nas amostras de água bruta, de forma a identificar eventuais factores associados ao desenvolvimento de florescências cianobacterianas.
- Diferenças genotípicas entre o sudoeste da Europa e África: um estudo comparativo em genes relacionados com a hipertensãoPublication . Aguiar, Laura; Semente, Ildegário; Carvalho, Andreia; Ferreira, Joana; Caria, Helena; Damasceno, Albertino; Faustino, Paula; Inácio, Ângela; Bicho, ManuelIntrodução: Desde o surgimento do género Homo, os hominídeos ocuparam uma grande variedade de ambientes com relação ao clima. Assim, espera-se que as pressões seletivas variem amplamente entre diferentes regiões geográficas. Objetivo: O objetivo do presente trabalho foi comparar a frequência genotípica em genes que têm sido propostos como potenciais moduladores da hipertensão arterial (HTA), uma doença multifatorial cujo contributo genético é importante, no sudoeste da Europa e em África. Os genes analisados foram: sintase do óxido nítrico endotelial (eNOS), glicose-6-fosfato desidrogenase (G6PD), alfa-globina (HBA) e enzima conversora da angiotensina (ECA). Material e métodos: Foram analisadas 224 amostras de DNA de Portugal e 202 amostras de África (24 amostras de DNA de Moçambique e 178 amostras de DNA de São Tomé e Príncipe). Para o gene eNOS, a análise polimórfica do número variável de repetições em tandem (VNTR) no intrão 4 (repetição em tandem de 27 pb) foi realizada por reação em cadeia da polimerase (PCR). A caracterização do SNP rs1050829 no gene G6PD foi obtida por PCR seguida de análise do comprimento do fragmento de restrição. Para a pesquisa da deleção α-talassémica de -3,7kb no agrupamento génico da α-globina foi usada uma metodologia de Gap-PCR. A genotipagem do polimorfismo da ECA I/D (inserção/ deleção) foi realizada por PCR. Todas as análises estatísticas foram realizadas recorrendo ao software SPSS, versão 24.0, sendo o nível de significância estatística estabelecido para p <0,05. Resultados: Os resultados mostram que, em relação ao gene eNOS, os genótipos que apresentam o alelo 4a (quatro cópias de repetições de 27 pb) têm uma frequência menor em Portugal do que em África (p <0,001). Curiosamente, apenas em África encontramos os alelos raros 4c, 4d e 4y. Para o gene G6PD (cromossoma X) há uma menor frequência dos genótipos com o alelo G em Portugal, comparado com a população africana (p <0,001). A presença da deleção α-talassémica de -3,7kb é comum em África, mas rara em Portugal (p <0,001). Em relação ao gene ECA, não se encontram diferenças estatisticamente significativas entre as populações estudadas (p =0,151). Conclusão: Os nossos resultados mostram diferenças na distribuição geográfica de três polimorfismos que potencialmente influenciam o desenvolvimento da HTA. Estas diferenças podem estar relacionadas com diferentes pressões seletivas proporcionadas pelos diferentes climas no sudoeste da Europa e na África equatorial e subequatorial. De facto, condições como o calor e a humidade estão associadas a maior perda de sódio.
- Analysis of publicly available LDLR, APOB, and PCSK9 variants associated with familial hypercholesterolemia: application of ACMG guidelines and implications for familial hypercholesterolemia diagnosisPublication . Chora, J.R.; Medeiros, A.M.; Alves, A.C.; Bourbon, M.Familial Hypercholesterolemia (FH): Lipid metabolism autosomal dominant condition; Patients present elevated low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) values since birth - elevated cardiovascular risk.
- Polimorfismos genéticos e a sua interação na suscetibilidade para a hipertensão na população portuguesaPublication . Aguiar, Laura; Semente, Ildegário; Ferreira, Joana; Mascarenhas, Mário Rui; Barbosa, Ana Paula; Menezes Falcão, Luís; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntrodução: A Hipertensão Arterial (HTA) é um fator de risco cardiovascular muito prevalente em Portugal. Esta patologia é multifatorial, envolvendo fatores genéticos e ambientais. Objetivo: Este estudo teve como objetivo investigar a potencial implicação de polimorfismos genéticos nos genes da sintase do óxido nítrico endotelial (eNOS) e da enzima conversora da angiotensina (ECA) e da sua interação no desenvolvimento da HTA na população portuguesa. Material e métodos: Foi realizado um estudo de caso-controlo para uma amostra de 377 indivíduos portugueses, dos quais 243 hipertensos (90 hipertensos ligeiros e 153 hipertensos graves) e 134 normotensos. As amostras foram recolhidas no Centro Hospitalar Lisboa Norte, no Hospital de Santa Maria. As análises polimórficas do número variável de repetições em tandem (VNTR) no intrão 4 (repetição em tandem de 27 pb) do gene eNOS e do polimorfismo ECA I/D (inserção/ deleção) foram realizadas por reação em cadeia da polimerase (PCR). Todas as análises estatísticas foram realizadas recorrendo ao software SPSS, versão 24.0, sendo o nível de significância estatística estabelecido para p <0,05. Resultados: Quando os polimorfismos foram analisados isoladamente, encontrou-se uma associação entre alelo 4a (quatro cópias de repetições de 27 pb) do gene eNOS e a hipertensão, em especial a hipertensão grave (p =0,001). Em relação ao gene ECA, não se encontram diferenças estatisticamente significativas entre doentes e controlos. No entanto, para a interação entre os polimorfismos nos genes eNOS e ECA, encontrou-se uma associação do alelo 4a do gene eNOS e do alelo D do gene ECA e a hipertensão ligeira (p =0,049). Conclusão: Os nossos resultados mostram uma associação entre o gene eNOS e a HTA na população portuguesa. Os resultados ainda sugerem uma relação entre a interação epistática nos genes eNOS e ECA e a suscetibilidade para o desenvolvimento de hipertensão. A identificação de polimorfismos genéticos que possam influenciar o desenvolvimento e gravidade da HTA, bem como as suas interações epistáticas, pode permitir um diagnóstico mais precoce e específico, que pode proporcionar melhores estratégias terapêuticas e de prevenção, para esta doença tão prevalente em Portugal.
- CRISPR/Cas in iPSCs from sphingolipidoses patientsPublication . Moreira, Luciana; Duarte, Ana Joana; Ribeiro, Diogo; Amaral, OlgaBackground: Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Ultimately, the use of CRISPR/Cas-mediated gene editing can provide specific cellular models of disease, correct causal mutations in LSDs and create mutants for functional studies. Aim: In this work, experimental conditions were tested to validate a CRISPR/Cas9 approach to generate a KO. Conclusion: The cells were amenable to edition by CRISPR/Cas9 and the results obtained proved that editing was achieved. All results contribute to the improvement of our knowledge regarding this technology, broadening the validation of CRISPR application and making it an accessible option.
- Tracing the geographical origin of food products with multielement fingerprinting, isotope ratios and chemometrics: the case of Rocha pearPublication . Coelho, Inês; Matos, Ana Sofia; Nascimento, Ana; Bordado, João; Donard, Olivier F.X.; Epova, Ekaterina; Berail, Sylvain; Castanheira, IsabelRocha pear (Pyrus communis L. var. Rocha) is a traditional Portuguese variety of pear recognized by its organoleptic qualities. Adding to the known benefits of consuming fruit in general, pears are considered a good source of fiber and potassium with low sodium content, combined with low glycemic index (Barda, 2011; Foster-Powell et al., 2002) . The nutritional value of pears, in particular its mineral content, is of great relevance for human nutrition and public health. In this sense, Food Composition Databases (FCDBs) are a crucial tool. In this study, we analyze the mineral content of Rocha pears to promote the linkage between the nutritional profile and geographical origin, filling the gap of absent values in FCDBs. Rocha pears produced in the west part of Portugal can be certified as Protected Designation of Origin (PDO), under the label of “Pera Rocha do Oeste”. These represent the largest PDO production in the country (Globalagrimar, 2016). However, this variety is also cultivated in other areas at national level (nPDO) and is gaining interest at international level due to growing exportations. The mineral profile of plants is a reflection of the soil type and the environmental conditions under which plants were grown. Therefore, minerals and trace elements, as well as their isotopes, are expected to be suitable biomarkers in authenticity studies (Coelho et al., 2017; Gonzalvez et al., 2009). Nonetheless, to extract the most information from datasets these must to be combined with chemometric tools capable of performing multivariate data analysis. The present work aims to develop a chemical fingerprint of Rocha pears produced within the PDO region based on multielement analysis and isotope ratios (IR). For such purpose 150 pear samples were collected from 10 farms located in two regions (PDO and nPDO). Samples were analyzed in pools, each composed of 3 pears from the same tree, leading to 50 pools. Multielement analyses were carried out, in triplicate, in compliance with NP EN ISO/IEC 17025 and EN 1380. The content of twenty-four minerals and trace elements was measured by 2 inductively coupled plasma optical emission spectrometer (ICP-OES) (Fe, Mg, P, Ca, Na, K) and ICP mass spectrometer (ICP-MS) (Li, Be, B, Al, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Tl, Pb). A Multicollector-ICP-MS (MC-ICP-MS) was used to determine Sr IR. Results showed a low level of the analyzed contaminants indicating that these do not present a risk for consumers. K was the most abundant element, followed by P. Be, Se, Sn and Pb were systematically below the limit of quantification (LOQ) and were not included in the statistical treatment. The dataset for multielement content and Sr IR was analyzed resorting to chemometric techniques, namely Principal Component Analysis (PCA), Hierarchical Clustering Analysis (HCA) and Linear Discriminant Analysis (LDA). Differences were identified in the level of inorganic components between the regions under study. The most significant were in the levels of Al, Ni and Na, which were higher in samples originating from the PDO region, while the content of Sr and Tl were superior outside this region. The exploratory analysis, based on the unsupervised models PCA and HCA, identified relationships between samples and their geographical provenance. LDA confirmed the possibility of tracing pear samples to the correspondent origin based on mineral profile and Sr IR. Samples used for cross validation of the predictive model were matched to its origin with 100% accuracy. In conclusion, the analytical methods were suitable for the purpose and are recommended methods for the analysis of inorganic components even at low values. The chemometric techniques used allowed the differentiation of samples according to their geographical origin, contributing to its authenticity and traceability as well as to a deeper knowledge of the mineral content of Rocha pear. The combination of these two techniques is a proper tool for mineral fingerprinting of Rocha Pear’s geographical origin.
- First description of an outbreak of cattle intoxication by cyanobacteria (blue-green algae) in the South of PortugalPublication . Menezes, Carina; Nova, Rita; Vale, Micaela; Azevedo, Joana; Vasconcelos, Vítor; Pinto, CarlosA herd of 53 Angus x Charolais crossbred cows and 1 herd bull grazing a stubble field broke through an electric fence, and gained access to stagnant water from a small river (Ribeira de Oeiras). In less than 19 hours, 20 cows died and 5 were sick with clinical signs of ataxia, recumbence, abdominal distension, opisthotonus, paddling and masticatory movements, frothing at the mouth, and bloody diarrhea. The 5 sick cows later died, and the remaining 29 animals remained healthy. Clinical and pathological findings revealed the presence of hepatic and renal necrosis. In the water samples, several cyanobacterial species were identified, predominantly belonging to the toxic genera Microcystis. Altogether, these observations led to a tentative diagnosis of acute hepatoxicity caused by cyanotoxins. The diagnosis was confirmed by detection of microcystin-LR in the kidney from 1 animal. To our knowledge, this is the first report of microcystin-LR animal poisoning in Portugal.
- Occupational Exposure to Mycotoxins in Swine Production: Environmental and Biological Monitoring ApproachesPublication . Viegas, S.; Assunção, R.; Martins, Carla; Nunes, Carla; Osteresch, Bernd; Twaruzek, Magdalena; Kosicki, Robert; Grajewski, Jan; Ribeiro, Edna; Viegas, C.Swine production workers are exposed simultaneously to multiple contaminants. Occupational exposure to aflatoxin B1 (AFB1) in Portuguese swine production farms has already been reported. However, besides AFB1, data regarding fungal contamination showed that exposure to other mycotoxins could be expected in this setting. The present study aimed to characterize the occupational exposure to multiple mycotoxins of swine production workers. To provide a broad view on the burden of contamination by mycotoxins and the workers’ exposure, biological (urine) samples from workers (n = 25) and 38 environmental samples (air samples, n = 23; litter samples, n = 5; feed samples, n = 10) were collected. The mycotoxins biomarkers detected in the urine samples of the workers group were the deoxynivalenol-glucuronic acid conjugate (60%), aflatoxin M1 (16%), enniatin B (4%), citrinin (8%), dihydrocitrinone (12%) and ochratoxin A (80%). Results of the control group followed the same pattern, but in general with a lower number of quantifiable results (
- CRISPR/Cas in iPSCs from Sphingolipidoses patientsPublication . Amaral, Olga; Duarte, Ana; Ribeiro, Diogo; Moreira, LucianaClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by defects in lysosomal proteins leading to accumulation of undigested macromolecules within the cells. The lack of good in vitro models hinders research of the pathophysiologic mechanisms and the development of new therapies. Induced pluripotent stem cells (iPSCs) are patient-specific and can be differentiated in any cell type. The advantage of iPSCs is to enable targeted studies in cells with the patient’s own background leading to more straightforward results than other models. Combining CRISPR and iPSCs is, therefore, a promising strategy. We aim to use CRISPR/Cas-mediated gene editing to provide more specific cellular models of disease, to correct causal mutations in LSDs and to create mutants for functional studies. In this work, we generated and characterized iPSCs from human fibroblasts obtained from Gaucher and Fabry patients (through Gaslini Institute) and will edit them with a CRISP/Cas9 approach. Because both gene editing and iPSCs generation require manipulating the cell’s genome, we envisage multiple check points along the workflow. It will be useful to compare the “native” mutated cells with the corrected cells that modulate the “disease in a dish”. Gene editing is still recent and the methods require improvement, namely increasing transfection rates and mutagenesis efficiency with less off-targets. Nevertheless, CRISPR/Cas is a promising alternative to other therapies, and every result contributes to the enhancement of this technology, broadening the validation of CRISPR application and making it an accessible option.