Estratégias de Produção in vitro de Bioinseticida Viral: influências do Isolado, da Cinética e do Modo de operação

Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3±0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9±1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process.

ESTUDO da RETENÇÃO ENTRE RESINA ACRÍLICA PARA BASE DE DENTADURA E DENTES DE RESINA ACRÍLICA

Foi avaliada a resistência de união entre dentes e base de dentadura, ambos de resina acrílica. Os dentes foram incluídos em blocos cilíndricos de resina e, posteriormente, torneados, para se obterem cilindros de 5mm de diâmetro. Sobre a extremidade do dente, era adaptado um padrão de cera de mesmo diâmetro, que, após inclusão na mufla, era substituído por resina acrílica termicamente ativada pelo método de processamento convencional de base de dentadura. Como variáveis, usaram-se duas marcas de dentes, duas fases de inclusão (plástica e borrachóide) e aplicação ou não de detergente e monômero sobre os dentes. Os corpos-de-prova, antes dos ensaios de ruptura por tração, foram armazenados em água destilada, a 37ºC, por 2 semanas. Os resultados mostraram que: marca de dente e fase de condensação não influíram nos resultados; somente o uso de detergente ou de monômero aumenta a retentividade e o seu uso conjunto apresenta efeito acumulativo.

Influence of Different Base Thicknesses on Maxillary Complete Denture Processing: Linear and Angular Graphic Analysis on the Movement of Artificial Teeth

Objective: The purpose of this study was to compare the dental movement that occurs during the processing of maxillary complete dentures with 3 different base thicknesses, using 2 investment methods, and microwave polymerization.Methods: A sample of 42 denture models was randomly divided into 6 groups (n = 7), with base thicknesses of 1.25, 2.50, and 3.75 mm and gypsum or silicone flask investment. Points were demarcated on the distal surface of the second molars and on the back of the gypsum cast at the alveolar ridge level to allow linear and angular measurement using AutoCAD software. The data were subjected to analysis of variance with double factor, Tukey test and Fisher (post hoc).Results: Angular analysis of the varying methods and their interactions generated a statistical difference (P = 0.023) when the magnitudes of molar inclination were compared. Tooth movement was greater for thin-based prostheses, 1.25 mm (-0.234), versus thick 3.75 mm (0.2395), with antagonistic behavior. Prosthesis investment with silicone (0.053) showed greater vertical change compared with the gypsum investment (0.032). There was a difference between the point of analysis, demonstrating that the changes were not symmetric.Conclusions: All groups evaluated showed change in the position of artificial teeth after processing. The complete denture with a thin base (1.25 mm) and silicone investment showed the worst results, whereas intermediate thickness (2.50 mm) was demonstrated to be ideal for the denture base.

Optimization of cyclodextrin glucanotransferase production from Bacillus clausii E16 in submerged fermentation using response surface methodology

Cyclodextrin glucanotransferase production from Bacillus clausii E16, a new bacteria isolated from Brazilian soil samples was optimized in shake-flask cultures. A 2 4 full-factorial central composite design was performed to optimize the culture conditions, using a response surface methodology the combined effect among the soluble starch concentration, the peptone concentration, the yeast extract concentration, and the initial pH value of the culture medium was investigated. The optimum concentrations of the components, determined by a 2(4) full-factorial central composite design, were 13.4 g/L soluble starch, 4.9 g/L peptone, 5.9 g/L yeast extract, and initial pH 10.1. Under these optimized conditions, the maximum cyclodextrin glucanotransferase activity was 5.9 U/mL after a 48-h fermentation. This yield was 68% higher than that obtained when the microorganism was cultivated in basal culture medium.

Bacillus lehensis-an alkali-tolerant bacterium isolated from cassava starch wastewater: optimization of parameters for cyclodextrin glycosyltransferase production

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Use of Response Surface Methodology in Optimization of Lactic Acid Production: Focus on Medium Supplementation, Temperature and pH Control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

d(-)-Lactic Acid Production by Leuconostoc mesenteroides B512 Using Different Carbon and Nitrogen Sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3

Endo-polygalacturonase (endo-PG), exo-polygalacturonase (exo-PG) and pectin liase (PL) were produced by solid-state fermentation of a mixture of orange bagasse and wheat bran (1:1) with the filamentous fungus Penicillium viridicatum RFC3. This substrate was prepared with two moisture contents, 70% and 80%, and each was fermented in two types of container, Erlenmeyer flask and polypropylene pack. When Erlenmeyer flasks were used, the medium containing 80% of initial moisture afforded higher PL production while neither exo- nor endo-PG production was influenced by substrate moisture. The highest enzyme activities obtained were 0.70 U mL(-1) for endo-PG, 8.90 U mL(-1) for exo-PG, and 41.30 U mL(-1) for PL. However, when the fermentation was done in polypropylene packs, higher production of all three enzymes was obtained at 70% moisture (0.7 and 8.33 U mL(-1) for endo- and exo-PG and 100 U mL(-1) for PL). An increase in the pH and decrease in the reducing sugar content of the medium was observed. The fungus was able to produce pectin esterase and other depolymerizing enzymes such as xylanase, CMCase, protease and amylase. (c) 2005 Elsevier Ltd. All rights reserved.

Spot-test identification and rapid quantitative sequential analysis of dipyrone

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The use of Langmuir-Blodgett films of a perylene derivative and polypyrrole in the detection of trace levels of Cu2+ ions

Langmuir films of a tetracarboxylic perylene derivative and polypyrrole display condensed surface pressure isotherms that are shifted when Cu2+ ions are added to the ultrapure water subphase. These films were transferred onto interdigitated gold electrodes leading to Y-type Langmuir-Blodgett (LB) films. The electrodes modified with 5-layer LB films were immersed into a flask with ultrapure water and water containing Cu2+ ions at concentrations ranging from mM to muM. Impedance measurements indicated a distinct electrical response for the two types of films. Although the materials chosen have no specificity for ionic metals, they can be combined for detecting trace levels of Cu2+, which may be exploited in water quality monitoring. (C) 2004 Elsevier B.V. All rights reserved.

Change in the ascorbic-acid content of acerolas (Malpighia-glabra L) as function of harvest maturation degree and storage-temperature

The content of ascorbic acid was assayed in acerolas harvested in three phases of maturation: green-yellow fruits (I); light red (II) and wine-coloured (III). Phase I and Phase II fruit were packed in aluminium sheets and stoppered flasks and stored in freezer (-10o.C) and in refrigerator (8o.C). Samples of 8 fruits from each experimental condiction were analysed for ascorbic acid determination by 2-chlorophenol indophenol discolouration method. The averages of 1.393,5 mg./100g. for Phase I sample, 1024,9 for Phase II and 756,5 for Phase III fruits, showed a statistically significative linear decreasing of the ascorbic acid content related with the maturation extent Phase I samples stored in freezing showed statitically significative decreasing of that vitamin at 408 hours of storage in both: aluminium sheet and stoppered flask package; in chilling temperature there was significative reduction of ascorbic acid content after 240 and 312 hours, respectively, for fruits packed in aluminium sheet and stopped flasks. Phase Il samples showed significative lost at 72 hours of storage when maintained in freezing temperature either, in aluminium sheet or in stoppered flasks: When stored in chilling temperature showed progressive lost of ascorbic acid in all measuring periods in every package. After 144 hours suffered deterioration suggested by colour changes.

Effect of anaerobic digestion and initial pH on metal bioleaching from sewage sludge

The effects of anaerobic digestion and initial pH on the bioleaching of metals from sewage sludge were investigated in shake flask experiments. A strain of Acidithiobacillus thiooxidans was employed in the assays using secondary and anaerobic sludges, which resulted in similar solubilization yields of the metals chromium, copper, lead, nickel, and zinc for both the sludges investigated. The effect of initial pH (7.0 and 4.0) on metal bioleaching was assayed by using the anaerobic sludge inoculated with indigenous sulfur-oxidizing thiobacilli. Although the time required to reach the end of the experiment (final pH close to 1.0) was shortened at initial pH of 4.0, final metal solubilization was not significantly different for both initial pH values, resulting in higher solubilization yields for copper, nickel, and zinc (higher than 80%). Chromium and lead presented solubilization yields close to 50%. The results obtained in this work showed that the metal bioleaching process can be applied to sewage sludge regardless of the type of sludge and without the requirement of pH adjustment.

Effect of cooling procedure on final denture base adaptation

Well-fitted dentures prevent hyperplasic lesions, provide chewing efficiency and promote patient's comfort. Several factors may affect final adaptation of dentures, as the type of the acrylic resin, the flask cooling procedure and the water uptake. This investigation evaluated the effect of water storage and two different cooling procedures [bench cooling (BC) for 2 h; running water (RW) at 20 degreesC for 45 min] on the final adaptation of denture bases. A heat-cured acrylic resin (CL, Classico, Classico Artigos Odontologicos) and two microwave-cured acrylic resins [Acron MC, (AC) GC Dent. Ind. Corp.; Onda Cryl (OC), Classico Artigos Odontologicos] were used to make the bases. Adaptation was assessed by measuring the weight of an intervening layer of silicone impression material between the base and the master die. Data was submitted to ANOVA and Tukey's test (0.05). The following means were found: (BC) CL=0.72 +/- 0.03 a; AC=0.70 +/- 0.03 b; OC=0.76 +/- 0.04 c//(RW) CL= 1.00 +/- 0.11 a; AC=1.00 +/- 0.12 a; OC=0.95 +/- 0.10 a. Different labels join groups that are not statistically different (P > 0.05). Comparisons are made among groups submitted to the same cooling procedure (BC or RW). The conclusions are: interaction of type of material and cooling procedure had a statistically significant effect on the final adaptation of the denture bases (P < 0.05); water storage was not detected as a source of variance (P > 0.05) on the final adaptation.

Physicochemical parameters involved in the lethal toxicity of N,N-[(dimethylamino)ethyl]-4-substituted benzoate hydrochlorides: A QSAR study

A set of sixteen para-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides structurally related to procaine was synthesized. The apparent partition coefficients were determined by either shake-flask or HPLC methods and were taken as hydrophobic parameters. The IR stretching frequencies of the carbonyl group were determined in chloroform and taken as one of the electronic parameters. Additional physicochemical parameters were either taken from the literature: pi, sigma, T and R, MR4, or calculated: log P. The lethal potency was determined in the mouse via the LD50. In order to verify the nature and the relative contributions of the physicochemical parameters to lethal toxicity, QSAR equations were derived using regression analysis. A major contribution of hydrophobicity to ether with a smaller but still significant contribution of electronic or polar properties was found to a describe the toxicity within this set of compounds.

Determination of low-aliphatic aldehydes indoors by micellar electrokinetic chromatography using sample dissolution manipulation for signal enhancement

This work describes a novel approach for the analysis of selected aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and acrolein) and acetone in environmental samples using micellar electrokinetic chromatography (MEKC). The method is based on the reaction of carbonyl compounds with 3-methyl-2-benzothiazoline hydrazone (MBTH) that gives an azine intermediate with maximum absorbance at 216 nm. A systematic evaluation of sample dissolution medium was conducted as a means to enhancing sensitivity. In the best condition, samples were dissolved in 0.030 mol.L-1 tetraborate solution. This condition presented enhancement factors in the range of 35-54 for the aldehydes under investigation, computed as the improvement of the concentration limits of detection (LODs) with reference to the sample dissolved in pure water. The running buffer was 0.020 mol.L-1 tetraborate, pH 9.3, containing 0.050 mol-L-1 sodium dodecyly sulfate (SIDS). The overall methodology presented several advantages over established methods for aldehydes. Worthy mentioning that MBTH is available in high purity degree, dispensing laborious reagent purification procedures. A few method validation parameters were determined revealing good migration time repeatability (< 2.5% coefficient of variation, CV) and area repeatability (< 4% CV), excellent linearity (20-120 mug/L, r > 0.995) and adequate sensitivity for environmental applications. The LODs with respect to each single aldehyde were in the range of 0.54-4.0 mug.L-1 and 11 mug.L-1 for acetone. The methodology was applied to the determination of aldehydes indoors. Samples were collected in an impinger flask containing 0.05% MBTH solution, at a flow rate of 0.80 L.min(-1), during 2.5 h, at different times during the day. The most abundant carbonyls in the samples were acetone, followed by formaldehyde and acetaldehyde, with estimate peak concentrations of 452, 5.2 and 2.2 ppbv, respectively.