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.

Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric Transduction

JORNADAS DE ELECTROQUÍMICA E INOVAÇÃO 2013

Espresso beverages of pure origin coffee: Mineral characterization, contribution for mineral intake and geographical discrimination

Espresso coffee beverages prepared from pure origin roasted ground coffees from the major world growing regions (Brazil, Ethiopia, Colombia, India, Mexico, Honduras, Guatemala, Papua New Guinea, Kenya, Cuba, Timor, Mussulo and China) were characterized and compared in terms of their mineral content. Regular consumption of one cup of espresso contributes to a daily mineral intake varying from 0.002% (sodium; Central America) to 8.73% (potassium; Asia). The mineral profiles of the espresso beverages revealed significant inter- and intra-continental differences. South American pure origin coffees are on average richer in the analyzed elements except for calcium, while samples from Central America have generally lower mineral amounts (except for manganese). Manganese displayed significant differences (p < 0.05) among the countries of each characterized continent. Intercontinental and inter-country discrimination between the major world coffee producers were achieved by applying canonical discriminant analysis. Manganese and calcium were found to be the best chemical descriptors for origin.

Mineral and Bone Disease (MBD) on a Kidney Transplant Patient

A 50-year-old post-menopausal recipient of a kidney allograft with bone pain, osteoporosis, persistent hypercalcaemia and elevated parathormone (PTH) levels, despite a satisfactory graft function, was treated with bisphosphonates and cinacalcet starting, respectively, 5 and 6 months after renal transplantation (RT). Sixteen months after treatment, there was improvement of bone mineral density (BMD) measured by dualenergy X-ray absorptiometry (DEXA). A bone biopsy was taken, unveiling a surprising and worrisome result. Post-RT bone disease is different from classic CKD-MBD and should be managed distinctly, including, in some difficult cases, an invasive evaluation through the performance of a bone biopsy, as suggested in the KDIGO guidelines.

Integrated study by NMR and X-ray Crystallography on the analysis of the molecular interactions in heme-binding proteins

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

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica Estrutural

Peptidoglycan assembly machines: The Staphylococcus aureus Penicillin-Binding Proteins

Dissertation presented to obtain the Ph.D degree in Biology

Modelação 3D de teores de depósitos minerais condicionados por tipos de mineralização. O caso de estudo do depósito mineral do Zambujal, Minas de Neves-Corvo

Dissertação para obtenção do Grau de Mestre em Engenharia Geológica (Georrecursos)

Functional and structural studies of two enzymes: membrane bound kinase and Z-DNA/Z-RNA-binding protein

Dissertação para obtenção do Grau de Doutor em Sistemas de Bioengenharia

Inibição da nodulação por N-mineral em soja.

Apesar da indicação da pesquisa de que a inoculação adequada com bactérias do gênero Bradyrhizobium é suficiente para suprir a demanda de N pela cultura da soja pela fixação biológica de nitrogênio (FBN), alguns agricultores ainda têm feito o uso de N mineral. Entretanto, a adubação nitrogenada aumenta os custos de produção, apresenta riscos de danos ambientais, e pode reduzir a nodulação e a eficiência da FBN. O objetivo desse trabalho foi avaliar o efeito de doses crescentes de N mineral na nodulação da soja. Utilizaram-se duas cultivares de soja: BMX Potência e BRS 360, inoculadas com Bradyrhizobium no momento da semeadura. Os tratamentos compreenderam 0, 5, 10, 15, 25, 50, 75, 100 e 125 mg de N vaso-1, em seis repetições. Para cada cultivar, um experimento foi conduzido simultaneamente, em delineamento inteiramente casualizado, em vasos de Leonard contendo solução nutritiva de Hoagland desprovida de N, fazendo-se a sua complementação, sem troca, sempre que necessário. As doses de N foram aplicadas a cada seis dias na solução nutritiva, totalizando 5 aplicações até 30 dias. Aos 35 dias após a emergência (DAE), no florescimento das plantas, foi realizada a desinstalação do experimento, com a contagem dos nódulos (NN) e obtenção da massa de nódulos secos (MNS) da região da coroa e das raízes secundárias, separadamente. Para a cultivar BMX Potência, o NN se manteve estável, tanto na região da coroa quanto nas raízes secundárias, até a dose 50 mg de N vaso-1, mas houve decréscimo acentuado com o aumento das doses, chegando à inibição quase total em 125 mg de N vaso-1. No entanto, a MNS apresentou redução linear com o aumento das doses de N. Para a cultivar BRS 360, o aumento da dose de N reduziu o NN e a MNS, tanto na região da coroa quanto nas raízes secundárias. A adubação nitrogenada reduz a contagem de nódulos, mas o efeito mais evidente se dá na massa de nódulos da região da coroa e da nodulação secundária. Sugere-se a massa de nódulos seja empregada como característica mais sensível à avaliação de efeitos negativos à nodulação, como o emprego de fertilizante nitrogenado, do que o número de nódulos.

Electron Transfer and Ligand Binding Properties of Cytochromes

Dissertation presented to obtain the Ph.D degree in Biochemistry

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9%) mantiveram-se viáveis e 24 (92,3%) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50%) apresentaram atividade proteásica, duas (16,7%) em caseína do leite, uma (8,3%) em gelatina e nove (75%) em ambos os substratos; 16 (66,7%) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Aptamers binding to Bradykinin

Bradykinin is a peptide of the kinin group, involved in a number of receptor-mediated physiological actions, including inflammation and vasodilation, as well as neuromodulation, neuroprotection and promotion of neurogenesis. Bradykinin is the main ligand of the B2 receptor- the main kinin receptor- which is involved in the cardiac and renal protective effects of kinins in diseases. Antibodies have been considered for a long time as promising therapeutic agents in various fields, especially cancer-related ones. Aptamers, on the other hand, have proven to be an excellent alterative, since they have similar properties to those of monoclonal antibodies, such a high-specificity of recognition and high-affinity binding. Plus, they are developed using in vitro selection procedures and can be reproduced by enzymatic reactions. SELEX is a powerful tool for the development of both DNA and RNA aptamers. The main goal of this project was to design a method to select aptamers against bradykinin using capillary electrophoresis alongside the SELEX technique. The selection was done by comparing the aptamers’ (ssDNA-target complex) electrophoretic mobility with that of the ssDNA and the target, which allowed us to define an appropriate collection window that took into consideration the analytes’ detection time, thus enabling the collection of the desired oligonucleotides. After two selection rounds, the collected pool was sequenced, the affinity was measured and the aptamers’ secondary structure was predicted. We concluded that with only two selection cycles, the original DNA library’s bulk affinity grew around 0.4%. The structural characterization of the aptamers, performed with the aid of the Mfold software, revealed that there are many repetitive motifs amongst them, indicating that the selection process was successful. We have obtained 16 sequences of candidate aptamers as bradykinin ligands of similar sequences and secondary structures whose biological activity should be analyzed after synthesis; mainly in regard to their role as bradykinin inhibitors.