Browsing by Author "Meirinho, Sofia G."
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- An electronic tongue for gliadins semi-quantitative detection in foodstuffsPublication . Peres, António M.; Dias, L.G.; Veloso, Ana C.A.; Meirinho, Sofia G.; Morais, Jorge Sá; Machado, Adélio A.S.C.An all-solid-state potentiometric electronic tongue with 36 polymeric membranes has been used for the first time to detect gliadins, which are primarily responsible for gluten intolerance in people suffering from celiac disease. A linear discriminant model, based on the signals of 11 polymeric membranes, selected from the 36 above using a stepwise procedure, was used to semi-quantitatively classify samples of a “Gluten-free” foodstuff (baby milked flour), previously contaminated with known amounts of gliadins (<10, 20–50 or >50 mg/kg), as “Gluten-free”, “Low-Gluten content” or “Gluten-containing”. For this food matrix, the device had sensitivity towards gliadins of 1–2 mg/kg and overall sensitivity and specificity of 77% and 78%, respectively. Moreover, the device never identified an ethanolic extract containing gliadins as “Gluten-free”. Finally, the system also allowed distinguishing “Gluten-free” and “Gluten-containing” foodstuffs (15 foods, including breads, flours, baby milked flours, cookies and breakfast cereals) with an overall sensitivity and specificity greater than 83%, using the signals of only 4 selected polymeric membranes (selected using a stepwise procedure). Since only one “Gluten-containing” foodstuff was misclassified as “Gluten-free”, the device could be used as a preliminary tool for quality control of foods for celiac patients.
- Aplicação de um sistema de multi-sensores para a detecção de gliadinas: discriminação semi-quantitativa entre alimentos com glúten e sem glútenPublication . Meirinho, Sofia G.; Peres, António M.; Veloso, Ana C.A.A doença celíaca caracteriza-se pela intolerância ou hipersensibilidade à ingestão de prolaminas existentes no trigo, centeio, cevada e aveia. As proteínas do glúten do trigo contêm aproximadamente 50% de prolaminas, denominadas por gliadinas. O tratamento para a doença celíaca consiste em fazer uma dieta livre de glúten. Assim sendo, torna-se imperativo dispor de metodologias analíticas capazes de detectar e quantificar o teor de glúten em alimentos, especialmente em alimentos rotulados “sem glúten”. Neste trabalho, estudou-se a aplicabilidade de um sistema de multi-sensores (etongue), com 36 membranas poliméricas de sensibilidade cruzada como técnica analítica alternativa para análise qualitativa e semi-quantitativa de produtos alimentares. O objectivo foi estabelecer uma distinção, entre alimentos “com” e “sem glúten”. A discriminação entre os alimentos analisados baseou-se na capacidade do sistema multisensor fornecer diferentes perfis potenciométricos consoante diferentes conteúdos de gliadinas presentes nos alimentos, após extracção com uma solução aquosa de etanol a 70%. Os perfis de sinais do sistema de multi-sensores em conjunto com métodos estatísticos multivariados de reconhecimento de padrões, nomeadamente a análise discriminante, foram utilizados para diferenciar alimentos “com” e “sem glúten”. Foram analisados quinze alimentos comercializados em Portugal e, adquiridos em supermercados: 8 alimentos cujo rótulo indicava a presença de glúten e 7 alimentos, com indicação no rótulo de ausência de glúten. O teor de gliadinas nos alimentos estudados foi confirmado por cromatografia líquida de alta resolução, após a sua extracção. O sistema de multi-sensores utilizado, apresentou um desempenho satisfatório na diferenciação de extractos com diferentes teores de gliadinas, permitindo discriminar alimentos “com” e “sem glúten” com sensibilidade e especificidades globais superiores a 95% nos dados originais e de 75% no processo de validação cruzada. Além disso permitiu classificar de forma semi-quantitativa um alimento em 3 grupos: alimento sem glúten (<10 ppm), alimento com teor de glúten (20-40 ppm) e alimento com teor de glúten (100-400 ppm). Nesta discriminação obteve-se sensibilidades e especificidades globais de 100% e superiores a 79% para os dados originais e procedimento de validação cruzada (predição), respectivamente. Celiac disease is characterized by intolerance or hypersensitivity to ingested prolamins, which are composites of wheat, rye, barley and oats. Gluten proteins of wheat are constituted of approximately 50% of prolamins named gliadins. The treatment for celiac disease is gluten - free diet. A multisensor potentiometric (e-tongue) with 36 cross-sensibility polymeric membranes was applied for qualitative foodstuffs analysis. The objective was to distinguish gluten-containing from gluten-free foods. The discrimination was based on the capability of the e-tongue device to detect different gliadins contents. The e-tongue signal profiles were used together with supervised multivariate statistical methods for pattern recognition. A set of 15 Portuguese foods (7 foods samples with gluten-free indicating and 8, indicating gluten-containing), purchased in commercial supermarkets were analyzed. The multisensor systems used, showed satisfactory performance in the differentiation of extracts with different levels of gliadins, allowing discriminating food "with gluten" and "gluten free" with an overall sensitivity and specificity higher than 95% in the original data and 75% for the cross-validation procedure. Moreover it allowed to semi-quantitatively classify food samples into 3 groups: gluten-free food (<10 ppm), gluten-containing foods (20-40 ppm) and gluten-containing food (100-400 ppm). This discrimination was achieved with an overall sensitivity and specificity of 100% and greater than 79% for the original data and cross-validation (prediction) procedure, respectively. The satisfactory results obtained showed that the multisensor potentiometric device developed based on lipo-polymeric membranes can be used as an effective tool in the preliminary detection of gluten in foods. Os resultados satisfatórios obtidos mostraram que o sistema potenciomérico de multi-sensores desenvolvido com base em membranas lipo-poliméricas pode ser utilizado como uma ferramenta na detecção preliminar de glúten em alimentos.
- Application of an electronic tongue to detect gliadins in gluten-free foodsPublication . Peres, António M.; Dias, L.G.; Veloso, Ana C.A.; Meirinho, Sofia G.; Morais, Jorge Sá; Machado, Adélio A.S.C.Celiac disease is an autoimmune-mediated disorder triggered in genetically susceptible individuals by the ingestion of some gluten proteins, namely gliadins. To prevent inadvertent gluten consumption, the Commission Regulation (EC) N°41/2009 will implement labeling foods as ‘gluten-free” or “low-gluten content.
- Characterization of transglutaminase elicitor precursor from plants pathogen Phytophthora cinnamomiPublication . Carvalho, Marisa; Martins, Ivone; Meirinho, Sofia G.; Belo, Hélio; Choupina, AltinoThe oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that includes some of the most notorious pathogens of plants. Among these, members of the genus Phytophthora cause enormous economic losses on crop species as well as environmental damage in natural ecosystems. Phytophthora cinnamomi is the most widely distributed Phytophthora species, with nearly 1000 host species.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species. Associated with this oomycete is the ink disease of Castanea sativa Mill. Glucan endo-1,3-β-D-glucosidase catalyzes the hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defence by digesting wall components of the fungal pathogen. In oomycetes, glucanases have been studied at biochemical level for their possible role in hyphal tip growth and branching, where there is thought to be a delicate balance between the cell wall synthesis and hydrolysis. Fungal cell wall degrading enzyme production is influenced by a number of factors including the type of strain, the culture conditions and substrate type. The aim of this work was the analysis of homologous expression, in P. cinnamomi, and heterologous expression, in Pichia pastoris, of the endo-1,3-β-D-glucosidase encoding gene ENDO1 produced by P. cinnamomi. The expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production. Different plasmids were used to clone the gene on each organism and we used RT-PCR analysis to examine its expression. The major expression levels occurred at the medium with glucose as carbon source. These and other results will be presented.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is a soil-borne pseudofungus belonging to the Class Oomycetes or „water moulds' in the Kingdom Chromista (Figure 1). Is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species, as well as of some 900 other woody perennial plant species Associated with this oomycete is the ink disease of Castanea Sativa Mill. Glucan endo-1,3-β-D-glucosidase (EC 3.2.1.39) catalyzes de hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defense by digesting wall components of the fungal pathogen. In yeast, 1,3-- glucanases have been studied for their role in germination, sporulation, mating and cell growth since they are regulated in cell cycle dependent manner, and are differentially expressed during vegetative growth, mating and the late stages of sporulating diploid. In plant, 1,3--glucanases have been characterized for their major role in plant defence, as well as for their involvement in germination, microsporogenesis and embryogenesis. In oomycetes, glucanases have been studied on a biochemical level for their possible role in hyphal tip growth and branching where there is thought to be a delicate balance between cell wall synthesis and hydrolyses. In the present work, we obtained a fragment with 1231bp of the endo-1,3--glucanase gene by standard PCR, using conserved primers and the whole genomic sequence with 2586 bp was obtained by amplifying the previous sequence by asymmetric PCR. The gene expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production.
- Cloning, characterization, in vitro and in planta expression of a necrosis‑inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Costa, Rodrigo Arthur Fonseca; Cravador, Alfredo; Choupina, AltinoThe soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.
- Development of an Electrochemical Aptasensor for the Detection of Human OsteopontinPublication . Meirinho, Sofia G.; Dias, L.G.; Peres, António M.; Rodrigues, Lígia R.Electrochemical RNA aptasensor To obtain bio-sensor with high affinity for rhOPN aiming to be used for cancer prognosis Materials and methods RNA aptamer details Isolated by SELEX process [1] Sequence of the biotinylated RNA aptamer 5’-Biotin- CGGCCACAGAAUGAAAAACCUCAUCGAUGUUGCAUAGUUG-3’ Removal the interference of Rnase Working RNA aptamer solution 4 nM in PBS. RNA aptamer immobilization on a gold surfasse via streptavidin-biotin interaction
- Development of an electrochemical aptasensor for the detection of human osteopontinPublication . Meirinho, Sofia G.; Dias, L.G.; Peres, António M.; Rodrigues, Lígia R.Electrochemical aptasensors, an emerging technology, enables the detection of protein biomarkers, which may be indicative of tumour activity. Osteopontin is a protein present in body fluids, being a possible biomarker since its overexpression has been related with breast cancer progression. An RNA aptamer, described in the literature, with affinity for human osteopontin, was synthetized, immobilized in a microelectrode gold surface and used for development electrochemical aptasensor for human OPN detection in standard solutions. Cyclic voltammetry results showed that this aptasensor allowed detecting human osteopontin with a detection limit of 8 nM, showing a satisfactory selectivity towards the target in the presence of others proteins, except for thrombin.
- Development of an electrochemical RNA-aptasensor to detect human osteopontinPublication . Meirinho, Sofia G.; Dias, L.G.; Peres, António M.; Rodrigues, Lígia R.Electrochemical aptasensors may be used to detect protein biomarkers related to tumor activity. Osteopontin (OPN), a protein present in several body fluids, has been suggested as a potential biomarker since its overexpression seems to be associated with breast cancer progression and metastasis. In this work, a simple and label-free voltammetric aptasensor for the detection of OPN, using an RNA aptamer previously reported to have affinity for human OPN as the molecular recognition element, and the ferro/ferricyanide solution as a redox probe, was developed. The RNA aptamer was synthetized and immobilized in a working microelectrode gold surface (diameter of 0.8 mm) of a screen-printed strip with a silver pseudo-reference electrode and a gold counter electrode. The electrochemical behavior of the electrode surface after each preparation step of the aptasensor was studied using cyclic voltammetry and square wave voltammetry. The resulting voltammetric aptasensor was used to detect OPN in standard solutions. Cyclic voltammetry results showed that the aptasensor has reasonable detection and quantification limits(3.7+-0.6 nM and 11+-2 nM, respectively). Indeed, the detection limit falls within the osteopontin levels reported in the literature for patients with metastatic breast cancer. Moreover, the aptasensor is able to selectively detect the target protein in the presence of other interfering proteins, except for thrombin. Considering the overall results, a possible application of the aptasensor for cancer prognosis may be foreseen in a near future.
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