Natural antioxidants extraction and their incorporation into model pharmaceutical systems

Dissertation to obtain a Master Degree in Biotechnology

Characterization of Nitric Oxide Reductase (NOR) from pseudomonas nautica, a study on biologic nitric oxide reduction

Dissertation submitted to obtain the phD degree in Biochemistry, specialty in Physical- Biochemistry, by the Faculdade de Ciências e Tecnologia from the Universidade Nova de Lisboa

Crystallographic studies on molybdopterin-dependent enzymes

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

A molecularly imprinted sensor for sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative stress biomarker

6th Graduate Student Symposium on Molecular Imprinting

Protein–polyphenol interaction on silica beads for astringency tests based on eye, photography or reflectance detection modes

Astringency is an organoleptic property of beverages and food products resulting mainly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers, but the only effective way of measuring it involves trained sensorial panellists, providing subjective and expensive responses. Concurrent chemical evaluations try to screen food astringency, by means of polyphenol and protein precipitation procedures, but these are far from the real human astringency sensation where not all polyphenol–protein interactions lead to the occurrence of precipitate. Here, a novel chemical approach that tries to mimic protein–polyphenol interactions in the mouth is presented to evaluate astringency. A protein, acting as a salivary protein, is attached to a solid support to which the polyphenol binds (just as happens when drinking wine), with subsequent colour alteration that is fully independent from the occurrence of precipitate. Employing this simple concept, Bovine Serum Albumin (BSA) was selected as the model salivary protein and used to cover the surface of silica beads. Tannic Acid (TA), employed as the model polyphenol, was allowed to interact with the BSA on the silica support and its adsorption to the protein was detected by reaction with Fe(III) and subsequent colour development. Quantitative data of TA in the samples were extracted by colorimetric or reflectance studies over the solid materials. The analysis was done by taking a regular picture with a digital camera, opening the image file in common software and extracting the colour coordinates from HSL (Hue, Saturation, Lightness) and RGB (Red, Green, Blue) colour model systems; linear ranges were observed from 10.6 to 106.0 μmol L−1. The latter was based on the Kubelka–Munk response, showing a linear gain with concentrations from 0.3 to 10.5 μmol L−1. In either of these two approaches, semi-quantitative estimation of TA was enabled by direct eye comparison. The correlation between the levels of adsorbed TA and the astringency of beverages was tested by using the assay to check the astringency of wines and comparing these to the response of sensorial panellists. Results of the two methods correlated well. The proposed sensor has significant potential as a robust tool for the quantitative/semi-quantitative evaluation of astringency in wine.

SPR based Studies for Pentagalloyl Glucose Binding to α-Amylase

Astringency is an organoleptic property resulting mostly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers but being typically measured by sensorial panels it turns out subjective and expensive. The main goal of the present work is to develop a sensory system to estimate astringency relying on protein/polyphenol interactions. For this purpose, a model protein was immobilized on a sensory gold surface and its subsequent interaction with polyphenols was measured by Surface Plasma Resonance (SPR). α-amylase and pentagalloyl glucose (PGG) were selected as model protein and polyphenol, respectively. To ensure specific binding between these, various surface chemistries were tested. Carboxylic terminated thiol decreased the binding ability of PGG and allowed covalent attachment of α-amylase to the surface. The pH 5 was the optimal condition for α-amylase immobilization on the surface. Further studies focus on Localized SPR sensor and application to wine samples, providing objectivity when compared to a trained panel.

New data on Echinococcus spp. in Southern Brazil

40 Echinococcus isolates from sheep and cattle in Southern Brazil were genetically analysed in order to obtain further data on the presence of different taxa of the Echinococcus granulosus complex. Differentiation was done using a PCR technique and sequencing of mitochondrial cytochrome c oxidase subunit 1 (CO1). Most samples (38) could be allocated to the sheep strain (G1) of E. granulosus, while two samples belonged to E. ortleppi, previously known as cattle strain (G5) of E. granulosus. Due to the shorter prepatent period in dogs of the latter taxon, this records have important implications for the design of control measures in this endemic region.

Depressão – Bases Biológicas e Princípios do Tratamento Farmacológico

Introdução: Na criança e no adolescente os episódios depressivos podem apresentar-se como tristeza generalizada, anedonia (característica bastante típica da 1ªInfância), tédio ou irritabilidade (que substitui muitas vezes o humor depressivo) e sentimento de indiferença perante actividades que anteriormente causavam prazer. É a presença de comprometimento funcional que distingue a depressão dos “altos e baixos” normais da infância e adolescência. Estima-se que a prevalência de depressão na 2ª Infância seja de 1-2%. Esta prevalência aumenta proporcionalmente com a idade, podendo atingir, no fim da adolescência, os 20%. O diagnóstico pode ser difícil: por vezes a irritabilidade e as alterações do comportamento dominam o quadro clínico. Objectivos: Este trabalho tem como principais objectivos fazer uma revisão das teorias biólogicas existentes e abordar, sucintamente, o tratamento farmacológico da depressão, dando ênfase aos fármacos mais utilizados em Psiquiatria da Infância e da Adolescência. Resumo: Trata-se de um trabalho de revisão bibliográfica onde serão abordados conceitos farmacológicos chave como as definições de Resposta, Remissão, Recuperação, Recaída, Recorrência, bem como o funcionamento dos neurónios e circuitos monoaminérgicos (noradrenérgicos, serotoninérgicos e dopaminérgicos). Nas bases biológicas da depressão serão enfatizadas as teorias da monoamina e dos receptores de neurotransmissores. Abordar-se-á de modo sucinto a influência dos receptores de monoaminas na transdução de sinal e regulação da expressão de genes. O vasto grupo farmacológico dos antidepressivos engloba, entre outros, os inibidores da monoamina oxidase, os antidepressivos tricíclicos e os inibidores selectivos da recaptação da serotonina. Não se pretendendo uma revisão exaustiva, tentar-se-á salientar os grupos mais importantes e os novos antidepressivos. Sendo a Infância e a Adolescência uma faixa etária para a qual existem poucos estudos de eficácia vs efeitos secundários dos antidepressivos, tentaremos salientar os fármacos melhor estudados e aqueles que estão recomendados.

Descrição de duas novas espécies do género Nevskia da classe Gammaproteobacteria

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Construção de um sensor eletroquímico molecularmente impresso para monitorização do cancro da mama

O Cancro da mama é uma doença cuja incidência tem vindo a aumentar de ano para ano e além disso é responsável por um grande número de mortes em todo mundo. De modo a combater esta doença têm sido propostos e utilizados biomarcadores tumorais que permitem o diagnóstico precoce, o acompanhamento do tratamento e/ou a orientação do tipo tratamento a adotar. Atualmente, os biomarcadores circulantes no sangue periférico recomendados pela Associação Americana de Oncologia Clinica (ASCO) para monitorizar os pacientes durante o tratamento são o cancer antigen 15-3 (CA 15-3), o cancer antigen 27.29 (CA 27.29) e o cancer embryobic antigen (CEA). Neste trabalho foi desenvolvido um sensor eletroquímico (voltamétrico) para monitorizar o cancro da mama através da análise do biomarcador CA 15-3. Inicialmente realizou-se o estudo da adsorção da proteína na superfície do elétrodo para compreender o comportamento do sensor para diferentes concentrações. De seguida, estudaram-se três polímeros (poliaminofenol, polifenol e polifenilenodiamina) e selecionou-se o poliaminofenol como o polímero a utilizar, pois possuía a melhor percentagem de alteração de sinal. Após a seleção do polímero, este foi depositado na superfície do elétrodo por eletropolimerização, formando um filme polimérico molecularmente impresso (MIP) à volta da proteína (molde). Posteriormente, foram analisados cinco solventes (água, mistura de dodecil sulfato de sódio e ácido acético, ácido oxálico, guanidina e proteinase K) e o ácido oxálico revelou ser mais eficaz na extração da proteína. Por último, procedeu-se à caraterização do sensor e analisou-se a resposta analítica para diferentes concentrações de CA 15-3 revelando diferenças claras entre o NIP (polímero não impresso) e o MIP.

Insight into the multicopper oxidases stability

Dissertation presented to obtain the PhD degree in Biochemistry

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448r

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Genetic characterization of Portuguese Fasciola hepatica isolates

Dissertation presented to obtain the Master Degree in Molecular, Genetics and Biomedicine