Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)

Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

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

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory

Estudo da cristalização de parafinas em sistemas solventes/tensoativos/água

The WAT is the temperature at the beginning of the appearance of wax crystals. At this temperature the first wax crystals are formed by the cooling systems paraffin / solvents. Paraffins are composed of a mixture of saturated hydrocarbons of high molecular weight. The removal of petroleum from wells and the production lines means a surcharge on produced oil, thus solubilize these deposits formed due to modifications of thermodynamics has been a constant challenge for companies of oil exploration. This study combines the paraffin solubilization by microemulsion systems, the determination of WAT systems paraffin / solvent and performance of surfactant in reducing the crystallization. We used the methods: rheological and the photoelectric signal, validating the latter which was developed to optimize the data obtained due to sensitivity of the equipment used. Methods developed for description of wax precipitation are often in poor agreement with the experimental data, they tend to underestimate the amount of wax at temperatures below the turbidity point. The Won method and the Ideal solution method were applied to the WAT data obtained in solvent systems, best represented by the second interaction of Won method using the solvents naphtha, hexane and LCO. It was observed that the results obtained by WAT photoelectric signal when compared with the viscosity occur in advance, demonstrating the greatest sensitivity of the method developed. The ionic surfactant reduced the viscosity of the solvent systems as it acted modifying the crystalline structure and, consequently, the pour point. The curves show that the WAT experimental data is, in general, closer to the modeling performed by the method of Won than to the one performed by the ideal solution method, because this method underestimates the curve predicting the onset of paraffin hydrocarbons crystallization temperature. This occurs because the actual temperature measured was the crystallization temperature and the method proposes the fusion temperature measurement.

Protein Hydrolysis by Immobilized and Stabilized Trypsin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Eficácia do bochecho de quitosana a 0,4% sobre o biofilme e bactérias orais

The purpose of this study was evaluate the effectiveness of the chitosan at 0.4 with high molecular weight and high deacetylation degree mouthrinse over the total decrease of the streptococci, Streptococcus mutans, lactobaci/li and over the perceptible bacterial film and gingival bleeding indices. For that, a total of 68 healthy students between 11 and 13 years old, not allergic to crustacean and not users of antibiotics or antimicrobial agent for the last three months or during the treatment, was selected. From those, thirty two individuaIs used the mouthrinse test, and thirty six, the control one. The participants rinsed 10 mL of the solutions twice a day, one during the moming (which was supervised), and another one during the aftemoon (which was not supervised), for fifteen days. The saliva collect for the microbiological analysis, as well as the perceptible bacterial film and gingival bleeding indices check, were made before the use ofthe mouthrinses (base line), immediately after the last mouthrinse on the day (zero time) and fifteen days after (fifteen time). These data were collected at school and the saliva was carried inside the ice to the laboratory. The samples were diluted, and 0.1 mL ofthe 10 -1 dilution was seeded in Rogosa SL agar, for further analysis of the total of lactobaci/lus~ 0.1 mL of the 10-4 dilution in Mitis Salivarius with bacitracin, for S. mutans analysis; and 0.1 mL of the 10-6 dilution in Mitis Salivarius for the analysis ofthe total of streptococcus. The Rogosa SL agar plates were incubated in aerobic at 37°C for 72 hours and the MSB and the MS were incubated in anaerobic in Gaspak@ jars at 37°C for 48 hours for further count ofColonies Former Units (CFUs). The assay was made in duplicate for each bacterial group analyzed. The number of CFUs transformed in LOGlO was analyzed according to the following tests: ANOV A, t of Paired and Not Paired Student, Friedman, Man-Whitney and square-qui test. On the base line, alI the variables analyzed were similar on both tested groups. On both groups, for the total of streptococcus there was no significant difference along the time and for S. mutans there was a statistic significant increase of the CFUs from the base line to the zero time. For the total of lactobaccilus there was no significant difference on the test group along the time, and on the control there was a significant increase ofthe CFUs ITom the base line to the zero time. For both groups, there was significant decrease ofthe perceptible bacterial film index along the time, and that can be explained by the mechanic effect of the mouthrinse over the bacterial film and by the participation of the students on the research which could have motivated him to a better toothbrushing (Hawthome effect). The gingival bleeding index also showed a decrease along the time, even though it was not significant. Therefore, the conclusion of this study was that the chitosan at 0.4 % mouthrinse was not effective on the CFUs reduction of the three bacterial groups analyzed, as well as on the reduction of the perceptible bacterial film and gingival bleeding indices

Characterization of excretory/secretory antigen from Toxocara vitulorum larvae

Toxocara vitulorum is a nematode parasite of the small intestine of cattle and water buffalo, particularly buffalo calves between one and three months of age, causing high morbidity and mortality. The purpose of this research was to characterize the excretory/secretory (ES) antigens of T vitulorum larvae by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot (WB), using immune sera and colostrum of buffalo naturally infected by T vitulorum. The parasitological status of the buffalo calves was also evaluated using sequential fecal examinations. The results showed that the ES antigen revealed eight (190, 150, 110, 90, 64, 56, 48, and 19 kDa) protein bands by SDS-PAGE. The majority of these bands were recognized in the sera and colostrum of infected buffalo with T vitulorum when analyzed by WB. However, particularly fractions of high molecular weight (190, 150, 110, and 90 kDa) were represented in more prominent bands and persisted in the groups of buffalo calves at the peak of egg output, as well as during the period of rejection of T vitulorum by the feces of the calves. During the period of post-rejection of the worms (between the day 118 and 210 of age) the serum antibodies did not react with any protein bands. on the other hand, sera from buffalo calves at one day of age (after suckling the colostrum and at the beginning of infection) reacted with the same bands detected in the serum and colostrum of the buffalo cows.

Degradação térmica e catalítica do resíduo atmosférico de petróleo (RAT), utilizando materiais nanoestruturados do tipo SBA-15

In this work were synthesized and characterized the materials mesoporous SBA-15 and Al- SBA-15, Si / Al = 25, 50 and 75, discovered by researchers at the University of California- Santa Barbara, USA, with pore diameters ranging from 2 to 30 nm and wall thickness from 3.1 to 6.4 nm, making these promising materials in the field of catalysis, particularly for petroleum refining (catalytic cracking), as their mesopores facilitate access of the molecules constituting the oil to active sites, thereby increasing the production of hydrocarbons in the range of light and medium. To verify that the materials used as catalysts were successfully synthesized, they were characterized using techniques of X-ray diffraction (XRD), absorption spectroscopy in the infrared Fourier transform (FT-IR) and adsorption nitrogen (BET). Aiming to check the catalytic activity thereof, a sample of atmospheric residue oil (ATR) from the pole Guamaré-RN was performed the process by means of thermogravimetry and thermal degradation of catalytic residue. Upon the curves, it was observed a reduction in the onset temperature of the decomposition process of catalytic ATR. For the kinetic model proposed by Flynn-Wall yielded some parameters to determine the apparent activation energy of decomposition, being shown the efficiency of mesoporous materials, since there was a decrease in the activation energy for the reactions using catalysts. The ATR was also subjected to pyrolysis process using a pyrolyzer with gas chromatography coupled to a mass spectrometer. Through the chromatograms obtained, there was an increase in the yield of the compounds in the range of gasoline and diesel from the catalytic pyrolysis, with emphasis on Al-SBA-15 (Si / Al = 25), which showed a percentage higher than the other catalysts. These results are due to the fact that the synthesized materials exhibit specific properties for application in the process of pyrolysis of complex molecules and high molecular weight as constituents of the ATR

Interações entre a carboximetilcelulose, carbonato de cálcio e bentonita: repercussões sobre as propriedades dos fluidos de perfuração aquosos

The role of carboxymethylcellulose (CMC) in association to calcium carbonate particles (CaCO3) in most water-based drilling fluids is to reduce the fluid loss to the surrounding formation. Another essential function is to provide rheological properties capable of maintaining in suspension the cuttings during drilling operation. Therefore, it is absolutely essential to correlate the polymer chemical structure (degree of substitution, molecular weight and distribution of substituent) with the physical-chemical properties of CaCO3, in order to obtain the better result at lower cost. Another important aspect refers to the clay hydration inhibitive properties of carboxymethylcellulose (CMC) in drilling fluids systems. The clay swelling promotes an undesirable damage that reduces the formation permeability and causes serious problems during the drilling operation. In this context, this thesis consists of two main parts. The first part refers to understanding of interactions CMC-CaCO3, as well as the corresponding effects on the fluid properties. The second part is related to understanding of mechanisms by which CMC adsorption occurs onto the clay surface, where, certainly, polymer chemical structure, ionic strength, molecular weight and its solvency in the medium are responsible to affect intrinsically the clay layers stabilization. Three samples of carboximetilcellulose with different molecular weight and degree of substitution (CMC A (9 x 104 gmol DS 0.7), CMC B (2.5 x 105 gmol DS 0.7) e CMC C (2.5 x 105 gmol DS 1.2)) and three samples of calcite with different average particle diameter and particle size distribution were used. The increase of CMC degree of substitution contributed to increase of polymer charge density and therefore, reduced its stability in brine, promoting the aggregation with the increase of filtrate volume. On the other hand, the increase of molecular weight promoted an increase of rheological properties with reduction of filtrate volume. Both effects are directly associated to hydrodynamic volume of polymer molecule in the medium. The granulometry of CaCO3 particles influenced not only the rheological properties, due to adsorption of polymers, but also the filtration properties. It was observed that the lower filtrate volume was obtained by using a CaCO3 sample of a low average size particle with wide dispersion in size. With regards to inhibition of clay swelling, the CMC performance was compared to other products often used (sodium chloride (NaCl), potassium chloride (KCl) and quaternary amine-based commercial inhibitor). The low molecular weight CMC (9 x 104 g/mol) showed slightly lower swelling degree compared to the high molecular weight (2.5 x 105 g/mol) along to 180 minutes. In parallel, it can be visualized by Scanning Electron Microscopy (SEM) that the high molecular weight CMC (2.5 x 105 g/mol e DS 0.7) promoted a reduction in pores formation and size of clay compared to low molecular weight CMC (9.0 x 104 g/mol e DS 0.7), after 1000 minutes in aqueous medium. This behavior was attributed to dynamic of interactions between clay and the hydrodynamic volume of CMC along the time, which is result of strong contribution of electrostatic interactions and hydrogen bounds between carboxylate groups and hydroxyls located along the polymer backbone and ionic and polar groups of clay surface. CMC adsorbs on clay surface promoting the skin formation , which is responsible to minimize the migration of water to porous medium. With the increase of degree of substitution, it was observed an increase of pores onto clay, suggesting that the higher charge density on polymer is responsible to decrease its flexibility and adsorption onto clay surface. The joint evaluation of these results indicate that high molecular weight is responsible to better results on control of rheological, filtration and clay swelling properties, however, the contrary effect is observed with the increase of degree of substitution. On its turn, the calcite presents better results of rheological and filtration properties with the decrease of average viii particle diameter and increase of particle size distribution. According to all properties evaluated, it has been obvious the interaction of CMC with the minerals (CaCO3 and clay) in the aqueous medium

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Histological evaluation of chitosan-based biomaterials used for the correction of critical size defects in rat's calvaria

Chitosan, a biopolymer obtained from chitin, and its derivates, such as chitosan hydrochloride, has been reported as wound healing accelerators and as possible bone substitutes for tissue engineering, and therefore these Substances could be relevant in dentistry and periodontology. The purpose of this investigation was to make a histological evaluation of chitosan and chitosan hydrochloride biomaterials (gels) used in the correction of critical size bone defects made in rat's calvaria. Bone defects of 8 mm in diameter were surgically created in the calviria of 50 Holtzman (Rattus norvegicus) rats and filled with blood clot (control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride, and high molecular weight chitosan hydrochloride, numbering 10 animals, divided into two experimental periods (15 and 60 days), for each biomaterial. The histological evaluation was made based on the morphology of the new-formed tissues in defect's region, and the results indicated that there was no statistical difference between the groups when the new bone formation in the entire defect's area were compared (p > 0.05) and, except in the control groups, assorted degrees of inflammation Could be Seen. In Conclusion, chitosan and chitosan hydrochloride biomaterials used in this study were not able to promote new bone formation in critical size defects made in rat's calvaria. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 107-114, 2016

Structural aspects, thermal behavior, and stability of a self-assembled supramolecular polymer derived from flunixin-meglumine supramolecular adducts

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Catálise estereoespecífica de estireno por titanocenos alquil-substituídos do tipo (RCp)2TiCl2

Uma série de sete complexos alquil substituídos do tipo (RCp)2TiCl2 (R= H, etila, n-propila, iso-propila, n-butila, iso-amila, ciclo-hexila) foi preparada juntamente com o ainda inédito contendo R= sec-butila, e o efeito do substituinte na polimerização do estireno foi investigada à temperaturas de polimerização de 0 e 50 ºC. Foi encontrado que R = n-butila maximiza a produção de poliestireno sindiotático. Ligantes alquilas com R = RCH2- são superiores aos R = RR CH- e ao precursor Cp2TiCl2 na produção de poliestireno sindiotático de alto peso molecular.

COMPORTAMENTO do ÓLEO DE GIRASSOL COM ALTO TEOR DE ÁCIDO OLÉICO em TERMOXIDAÇÃO E FRITURA

Óleos de sementes de girassol com diferentes níveis de ácido oléico foram utilizados sob condições de termoxidação e fritura para avaliar o comportamento do óleo de girassol com alto teor de ácido oléico quanto ao seu grau de insaturação quando comparado com o óleo de girassol convencional. Os métodos analíticos aplicados incluíram a determinação de compostos polares totais, quantificação de compostos glicerídicos menores, quantificação da composição triglicerídica, período de indução a 100oC e níveis de a -tocoferol. Os resultados para compostos polares totais apresentaram menores valores em óleo de girassol com alto teor de ácido oléico quando comparados com óleo de girassol convencional. Quanto à distribuição dos compostos glicerídicos menores, verificou-se, de modo geral, que o aumento dos compostos polares totais estava relacionado essencialmente aos compostos de maior peso molecular, isto é, polímeros, dímeros e triglicerídios oxidados, os quais são representantes das alterações térmica e oxidativa. Já os diglicerídios e ácidos graxos, indicativos da alteração hidrolítica, permaneceram praticamente nos níveis originais; uma vez que a umidade não se encontrava presente no meio. A perda quantitativa da composição triglicerídica dependeu do grau de insaturação do triglicerídio, sendo mais elevada em triglicerídios que contêm duas ou três moléculas de ácido linoléico. em todos os casos, o óleo de girassol convencional teve uma maior tendência à polimerização, um maior grau de alteração total e uma maior perda de triglicerídios majoritários que o óleo de girassol com alto teor de ácido oléico. Assim, dentre os óleos utilizados neste estudo, os resultados mostraram um excelente comportamento do óleo de girassol com alto teor de ácido oléico em relação à termoxidação, independente do tipo de aquecimento e à fritura descontínua. Tais resultados demonstraram que sua resistência à alteração termoxidativa estava relacionada com seu baixo teor em ácidos graxos polinsaturados e alto teor em ácido oléico e trioleína.