Desenvolvimento de pastilha potencialmente probiótica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi- walled carbon nanotubes(MWCNTs)paste electrode modified by dispersion of laccase(3%,w/w) within the optimum composite matrix(60:40%,w/w,MWCNTs and paraffin binder)showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate4- aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 ×10- 7 to 1.15 ×10- 5 molL 1 using 4- aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%).The limit of detection obtained was 1.8 × 10-7 molL 1 (0.04 mgkg 1 on a fresh weight vegetable basis).The high activity and catalytic properties of the laccase- based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electro- analysis were observed by the presence of pro-vitamin A, vitamins B1 and C,and glucose in the vegetable extracts. The proposed biosensor- based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Laccase–Prussian blue film–graphene doped carbon paste modified electrode for carbamate pesticides quantification

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussianblue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectro- scopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device.The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 23 full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0,LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra-and inter-day repeatability (1.8–3.8% RSD), reproducibility (4.1 and 6.3%RSD),selectivity(13.2% bias at the higher interference: substrate ratios tested),accuracy and stability(ca. twenty days)for quantification of five carbamates widely applied on tomato and potato crops.The attained detection limits ranged between 5.2×10−9 mol L−1(0.002 mg kg−1 w/w for ziram)and 1.0×10−7 mol L−1 (0.022 mg kg−1 w/w for carbofuran).Recovery values for the two tested spiking levels ranged from 90.2±0.1%(carbofuran)to 101.1±0.3% (ziram) for tomato and from 91.0±0.1%(formetanate)to 100.8±0.1%(ziram)for potato samples.The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

The computational role of short-term plasticity and the balance of excitation and inhibition in neural microcircuits: experimental and theoretical analysis

The computations performed by the brain ultimately rely on the functional connectivity between neurons embedded in complex networks. It is well known that the neuronal connections, the synapses, are plastic, i.e. the contribution of each presynaptic neuron to the firing of a postsynaptic neuron can be independently adjusted. The modulation of effective synaptic strength can occur on time scales that range from tens or hundreds of milliseconds, to tens of minutes or hours, to days, and may involve pre- and/or post-synaptic modifications. The collection of these mechanisms is generally believed to underlie learning and memory and, hence, it is fundamental to understand their consequences in the behavior of neurons.(...)

Comparison of indirect immunofluorescence test for measles antibodies with haemagglutination inhibition and plaque neutralization tests

Indirect Immunofluorescence (IFA), Plaque Reduction Neutralization (PRN) and Haemagglutination Inhibition (HI) tests for measles antibodies were carried out in 197 sera obtained from umbilical cord and vaccinated children. The IFA was also applied to blood samples collected with filter paper. IFA results demonstrated that the test is relatively simple to perform, with good reproducibility for different antigen lots. Good correlation was obtained between IFA, PRN and HI antibody titers. Better correlation was demonstrated with IFA and PRN than with HI and PRN tests. Sensitivity of IFA in detecting antibody was less effective than PRN, however more effective than HI using rhesus monkey red blood cells. PRN antibody titers over 100 were detected by IFA but not by HI (9.7% with negative results). IFA may be of considerable practical use and able to substitute HI in Seroepidemiological surveys and to evaluate vaccine efficacy. It also can be simplified by employing filter paper collected samples.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection

A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68×10− 9 ± 1.18×10− 10 – 2.15×10− 7 ± 3.41×10− 9 M), high accuracy, sensitivity (1.13×106 ± 8.11×104 – 2.19×108 ± 2.51×107 %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproductibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.

Surveillance of arbovirus infections in the atlantic forest region, State of São Paulo, Brazil: I. detection of hemagglutination-inhibition antibodies in wild birds between 1978 and 1990

We report data related to arbovirus antibodies detected in wild birds periodically captured from January 1978 to December 1990 in the counties of Salesópolis (Casa Grande Station), Itapetininga and Ribeira Valley, considering the different capture environments. Plasmas were examined using hemagglutination-inhibition (HI) tests. Only monotypic reactions were considered, except for two heterotypic reactions in which a significant difference in titer was observed for a determined virus of the same antigenic group. Among a total of 39,911 birds, 269 birds (0.7%) belonging to 66 species and 22 families were found to have a monotypic reaction for Eastern equine encephalitis (EEE), Venezuelan equine encephalitis (VEE), Western equine encephalitis (WEE), Ilheus (ILH), Rocio (ROC), St. Louis encephalitis (SLE), SP An 71686, or Caraparu (CAR) viruses. Analysis of the data provided information of epidemiologic interest with respect to these agents. Birds with positive serology were distributed among different habitats, with a predominance of unforested habitats. The greatest diversity of positive reactions was observed among species which concentrate in culture fields.

Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

Chlorella sp. coagulation‐flocculation by inducing a modification on the pH broth medium

Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Bioenergia

Alterações de pH após adição de pulsos de glucose a Saccharomyces cerevisiae: estimativa de parâmetros a partir da modelação matemática do comportamento cinéticometabólico

Dissertação para a obtenção do grau de mestre em Bioquímica Estrutural e Funcional