Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Avaliação do efeito da megadose de vitamina A no colostro humano

The mothers supplementation of vitamin A in the postpartum comes being a measure of intervention sufficiently used in the combat to the vitamin deficiency. The objective of this work was to evaluate the effect of the mother megadose of vitamin A under the levels of retinol in colostrum of postpartum mothers receiving care at the Januário Cicco Maternity School (MEJC), Natal, RN, as well as analyzing the influence of the maternal nutritional status in the reply to this supplementation. The study it was transversal type, with participation of 91 women in labor divided in group had participated of the study have controlled (44 women) and supplemented group (47 women). In the period of the morning blood and milk had been collected (milk 0h). After that a capsule of retinil palmitate of (200 000 UI or 60 mg) was supplied to the supplemented group. Another aliquot of colostro was after gotten 24h of the first collection (milk 24h). Retinol in milk and serum was quantified through the High Pressure Liquid Chromatography. The vitamin ingestion was evaluated by the questionnaire of frequency of alimentary consumption. The levels of serum retinol were 40.6 ± 10.6 and 35.9 ± 10.9 µg/dL in the groups controlled and supplemented, respectively. The women had presented a satisfactory average ingestion of vitamin (1492,4 µgRAE/dia), however with high prevalence of inadequate consumption (23%). Average values of retinol in milk 0h had been found and 24h of 93.5 ± 50.3 µg/dL and 99.1 ± 49.3 µg/dL has the group controlled group, respectively (p>0.05). After the supplementation had a significant increase in the levels of retinol of the supplemented group, being found values of 102.0 ± 56.0 µg/dL and 196.1 ± 74.0 µg/dL for milk 0h and 24h, respectively (p<0.0001). The women in labor presented different answers to the supplementation influenced for the basal levels of retinol in colostrum. It was possible to verify that women with deficient levels of retinol in milk had transferred more retinol to milk 24h than ones with adjusted levels, showing a percentage of reply equivalent to 326.1% and 86.5% of increase, respectively (p< 0.0001). Although the apparent normality found in the serum, the studied women are considered of risk to the development of the vitamin deficiency, and megadose was efficient in first 24h after the supplementation and wakes up with the mechanisms considered for transference of vitamin A to the milk

Influência da suplementação de retinol palmitato sobre os níveis de vitamina A no leite de puérperas saudáveis

The tendency towards reduction of serum retinol levels, an existing placental barrier and the increase of retinol demand, are factors that place puerperal and lactating women at risk for Vitamin A deficiency. This micronutrient is an essential component of vital processes such as differentiation, cellular proliferation, and apoptosis. The objective of this study is to evaluate the effect of palmitate retinol supplementation (100.000UI) upon the milk retinollevels in puerperal women at the Januário Cicco University Maternity Hospital. This intervention has been adopted by the Ministry of Health since 2002. The longitudinal experiment was conducted with 106 puerperal women (68 comprised the supplemented group and 38 the control group). The High Performance Liquid Chromatography (HPLC) method was used to dose the retinol of the milk and serum samples, and the creamtocrit method to determine the milk fat levels. The retinol means for the colostrums were 99.0 ± 64.4 ug/dL and 160.1 ± 94,4 ug/dl 6 hours afier supplementation; 68.9 ± 33.5 ug/dL for the transitional milk, and 30.6 ± 15.2 ug/dL for the mature milk of the supplemented group. Ali the difterences between means were statistically significant. The difterence between retinol means in the control group were also significant, with these being greater in the colostrum, 88.6 ± 62.1 ug/dL with 61.9 ± 30.1 ug/dl in the transition milk and 32.9 ±32.9 ± 17.6 ug/dL in the mature milk. No significant difference was observed in the retinol means of the three types ot milk in the supplemented group when compared to their respective means in the control group. The prevalence in serum (35.1 % and 81.1 % for the cutting point 20 ug/dL, respectively) and in milk (51.4%) revealed vitamin A deficiency as a public health problem. COlostrum, transition, and mature milk tats varied similarly in the supplemented group (1,92 ± 0,96; 3,25 ± 1,27 and 3,31 ± 1,36 grams) and in the control group (1,87 ± 1,14; 3,25 ± 1,31 and 3,36 ± 1,67 grams), with an observed difference between the colostrum/transition milk and the colostrum/mature milk fats. No difference was observed between the groups. The study showed that the 200.000UI supplementation was not sufficient to increase the milk retinol to the desired levels nor to meet the demands of the mothers with deprived hepatic reserves. It is suggested that another similar dose be offered within 30 days or less, and within 2 months post-partum, while continual/y monitoring for possible pregnancy

Avaliação da espécie vegetal Croton cajucara Benth como inibidor de biocorrosão em aço carbono Aisi 1020

Actually in the oil industry biotechnological approaches represent a challenge. In that, attention to metal structures affected by electrochemical corrosive processes, as well as by the interference of microorganisms (biocorrosion) which affect the kinetics of the environment / metal interface. Regarding to economical and environmental impacts reduction let to the use of natural products as an alternative to toxic synthetic inhibitors. This study aims the employment of green chemistry by evaluating the stem bark extracts (EHC, hydroalcoholic extract) and leaves (ECF, chloroform extract) of plant species Croton cajucara Benth as a corrosion inhibitor. In addition the effectiveness of corrosion inhibition of bioactive trans-clerodane dehydrocrotonin (DCTN) isolated from the stem bark of this Croton was also evaluated. For this purpose, carbon steel AISI 1020 was immersed in saline media (3,5 % NaCl) in the presence and absence of a microorganism recovered from a pipeline oil sample. Corrosion inhibition efficiency and its mechanisms were investigated by linear sweep voltammetry and electrochemical impedance. Culture-dependent and molecular biology techniques were used to characterize and identify bacterial species present in oil samples. The tested natural products EHC, ECF and DCTN (DMSO as solvent) in abiotic environment presented respectively, corrosion inhibition efficiencies of 57.6% (500 ppm), 86.1% (500 ppm) and 54.5% (62.5 ppm). Adsorption phenomena showed that EHC best fit Frumkin isotherm and ECF to Temkin isotherm. EHC extract (250 ppm) dissolved in a polar microemulsion system (MES-EHC) showed significant maximum inhibition efficiency (93.8%) fitting Langmuir isotherm. In the presence of the isolated Pseudomonas sp, EHC and ECF were able to form eco-compatible organic films with anti-corrosive properties

Estudo in silico da interação da albumina de soro humano com o ibuprofeno

Currently, computational methods have been increasingly used to aid in the characterization of molecular biological systems, especially when they relevant to human health. Ibuprofen is a nonsteroidal antiinflammatory or broadband use in the clinic. Once in the bloodstream, most of ibuprofen is linked to human serum albumin, the major protein of blood plasma, decreasing its bioavailability and requiring larger doses to produce its antiinflamatory action. This study aimes to characterize, through the interaction energy, how is the binding of ibuprofen to albumin and to establish what are the main amino acids and molecular interactions involved in the process. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant ε = 40 was used. The findings corroborate the literature in which the Sudlow site I (I-FA3) is the primary binding site and the site I-FA6 as secondary site. However, it differs in identifying the most important amino acids, which by interaction energy, in order of decreasing energy, are: Arg410, Lys414, Ser 489, Leu453 and Tyr411 to the I-Site FA3 and Leu481, Ser480, Lys351, Val482 and Arg209 to the site I-FA6. The quantification of interaction energy and description of the most important amino acids opens new avenues for studies aiming at manipulating the structure of ibuprofen, in order to decrease its interaction with albumin, and consequently increase its distribution

Caracterização fenotípica e genotípica de Staphylococcus aureus resistentes à meticilina isolados na cidade do Natal/RN

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais agentes de infecções associadas a serviços de saúde em todo o mundo. No Brasil, há a predominância de um clone de MRSA multirresistente denominando clone epidêmico brasileiro (CEB). Entretanto, novos clones nãomultirresistentes com alta virulência têm sido descritos em infecções comunitárias e hospitalares. O objetivo desse estudo foi realizar a caracterização fenotípica e genotípica de cepas de MRSA isoladas na cidade do Natal/RN. Inicialmente avaliamos 60 amostras de S. aureus quanto a resistência à meticilina através de diferentes técnicas fenotípicas, utilizando a detecção do gene mecA por PCR como padrão. O antibiograma de todas as cepas foi realizado utilizando 12 antimicrobianos conforme descrito pelo CLSI. As cepas de MRSA foram caracterizadas geneticamente através da tipagem do cassete cromossômico estafilocócico mec (SCCmec) e da eletroforese em campo elétrico alternado (PFGE). Dos 60 S. aureus estudados, 45 foram resistentes à meticilina. Observamos que para algumas cepas de MRSA os testes de triagem em ágar com 6μg/mL de oxacilina e difusão em meio sólido com oxacilina-1μg apresentaram dificuldades na sua interpretação. No entanto, todas as 45 amostras de MRSA, foram facilmente detectadas pelos testes com o disco de cefoxitina-30μg e pesquisa da PBP2a. A análise molecular das cepas de MRSA mostrou 8 padrões distintos de PFGE (A-H), com predominância do padrão A (73%), relacionado ao CEB. Estas carreavam o SCCmec tipo IIIA, e apresentaram uma considerável variedade de subtipos (A1-A16). Cinco cepas de MRSA portando SCCmec IV também foram xiv identificadas, três delas relacionadas geneticamente ao clone USA800 (Padrão B). Destas cinco, três (2 padrão F e 1 padrão B) foram altamente susceptíveis as drogas testadas, entretanto, dois outros isolados, padrão B, apresentaram multirresistência. As amostras restantes pertenciam a padrões de PFGE distintos dos clones internacionais predominantes em nosso continente. Para realização deste projeto de pesquisa, a metodologia exigiu a interação com pesquisadores de áreas como: infectologia, microbiologia e biologia molecular. Portanto, esta dissertação apresentou um caráter de multidisciplinaridade e transdiciplinaridade no seu desenvolvimento

Checklist das Cryptophyceae do estado de São Paulo, Brasil

Usando como base de informações a monografia sobre o grupo publicada pelo projeto Flora Ficológica do Estado de São Paulo (Programa BIOTA), o acervo depositado no Herbário Científico do Estado de São Paulo Maria Eneyda P. Kauffmann Fidalgo (SP), a Lista de Espécies da Flora do Brasil e artigos científicos, dissertações e teses (com descrições e ilustrações) temos: nove gêneros e 39 espécies de criptoficeas, sendo 16 espécies exclusivas para o Estado de São Paulo, todas registradas em ambientes de água doce. A carência de especialistas no Estado de São Paulo e no Brasil, além de problemas de estratégia amostral, necessidade de uso da microscopia eletrônica e ausência de estudos de biologia molecular, são fatores que devem ter subestimado o conhecimento taxonômico de Cryptophyceae no Estado.

Predição de promotores de Bacillus subtilis usando técnicas de aprendizado de máquina

One of the most important goals of bioinformatics is the ability to identify genes in uncharacterized DNA sequences on world wide database. Gene expression on prokaryotes initiates when the RNA-polymerase enzyme interacts with DNA regions called promoters. In these regions are located the main regulatory elements of the transcription process. Despite the improvement of in vitro techniques for molecular biology analysis, characterizing and identifying a great number of promoters on a genome is a complex task. Nevertheless, the main drawback is the absence of a large set of promoters to identify conserved patterns among the species. Hence, a in silico method to predict them on any species is a challenge. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. In this work, we present an empirical comparison of Machine Learning (ML) techniques such as Na¨ýve Bayes, Decision Trees, Support Vector Machines and Neural Networks, Voted Perceptron, PART, k-NN and and ensemble approaches (Bagging and Boosting) to the task of predicting Bacillus subtilis. In order to do so, we first built two data set of promoter and nonpromoter sequences for B. subtilis and a hybrid one. In order to evaluate of ML methods a cross-validation procedure is applied. Good results were obtained with methods of ML like SVM and Naïve Bayes using B. subtilis. However, we have not reached good results on hybrid database

Aplicação de técnicas de aprendizado de máquina no reconhecimento de classes estruturais de proteínas

Nowadays, classifying proteins in structural classes, which concerns the inference of patterns in their 3D conformation, is one of the most important open problems in Molecular Biology. The main reason for this is that the function of a protein is intrinsically related to its spatial conformation. However, such conformations are very difficult to be obtained experimentally in laboratory. Thus, this problem has drawn the attention of many researchers in Bioinformatics. Considering the great difference between the number of protein sequences already known and the number of three-dimensional structures determined experimentally, the demand of automated techniques for structural classification of proteins is very high. In this context, computational tools, especially Machine Learning (ML) techniques, have become essential to deal with this problem. In this work, ML techniques are used in the recognition of protein structural classes: Decision Trees, k-Nearest Neighbor, Naive Bayes, Support Vector Machine and Neural Networks. These methods have been chosen because they represent different paradigms of learning and have been widely used in the Bioinfornmatics literature. Aiming to obtain an improvment in the performance of these techniques (individual classifiers), homogeneous (Bagging and Boosting) and heterogeneous (Voting, Stacking and StackingC) multiclassification systems are used. Moreover, since the protein database used in this work presents the problem of imbalanced classes, artificial techniques for class balance (Undersampling Random, Tomek Links, CNN, NCL and OSS) are used to minimize such a problem. In order to evaluate the ML methods, a cross-validation procedure is applied, where the accuracy of the classifiers is measured using the mean of classification error rate, on independent test sets. These means are compared, two by two, by the hypothesis test aiming to evaluate if there is, statistically, a significant difference between them. With respect to the results obtained with the individual classifiers, Support Vector Machine presented the best accuracy. In terms of the multi-classification systems (homogeneous and heterogeneous), they showed, in general, a superior or similar performance when compared to the one achieved by the individual classifiers used - especially Boosting with Decision Tree and the StackingC with Linear Regression as meta classifier. The Voting method, despite of its simplicity, has shown to be adequate for solving the problem presented in this work. The techniques for class balance, on the other hand, have not produced a significant improvement in the global classification error. Nevertheless, the use of such techniques did improve the classification error for the minority class. In this context, the NCL technique has shown to be more appropriated

Estudo do Cultivo de dois clones de Escherichia coli recombinantes (eIF, LACK) para a Expressão de Antígenos da Leishmania chagasi

With advent of the technology of the recombinant DNA, the recombinant protein expression becomes an important tool in the studies of the structure, function and identification of new proteins, mainly with therapeutical purposes. The Escherichia coli has been procarioto predominant in the studies of genetic engineering due to wealth of information regarding its metabolism. Despite the expressivo advance of the studies of molecular biology and the immunology of the infections, it does not exist, currently, no prophylactic drug capable to prevent calazar. Of this form, it exists a great necessity of specific antigen identification for the vaccine development and kits for disgnostic against the visceral Leishmaniose. In this context, this work objectified to study the recombinant antigen expression of the Leishmania chagasi during the culture of Escherichia coli in shaker. A first set of assays was carried through with the objective of if knowing the kinetic behavior of the growth of two clones recombinant proteins (eIF, LACK) in two different compositions of culture medium (2xTY, TB) supplemented by antibiotics, without IPTG addition. In the second stage of the assays, the procedure of induction for IPTG was carried through, in order to verify the influence of the composition of the ways tested in the expression them recombinant proteins. On the basis of the gotten results, can be observed that the high complexity of culture medium favored the kinetic one of growth of clones recombinant (eIF, LACK), however, to if to deal with the assays submitted to the procedure of induction for IPTG, the raised complexity of culture medium did not favor the expression of recombinant proteins. On the other hand, they had been gotten resulted positive for all clones recombinant (eIF, LACK) tested, confirmed through the eletroforético profile

Influência das condições de cultivo na produção de antígenos recombinantes de Leishmania i. chagasi utilizando Escherichia coli M15 cultivada em incubador rotativo e biorreator

Escherichia coli has been one of the most widely used hosts in recombinant protein production, in both laboratory and industrial scale since the advent of recombinant DNA technology. Despite the substantial progress of studies on the molecular biology and immunology of infections, there is currently no medication-based prophylaxis capable of preventing leishmaniasis. As such, there is a great need to identify specific antigens for the development of vaccines and diagnostic kits against visceral leishmaniasis. Thus, the primary goal of the present study is to assess the influence of cultivation conditions on the production of Leishmania chagasi antigens, carried out in a rotating incubator and bioreactor. To that end, several assays were conducted to evaluate the kinetic behavior of antigens (648, 503) of Leishmania. i. chagasi in two different compositions of media (2xTY, TB), with and without an inducer. In order to improve expression, assays were performed in a benchtop bioreactor using the best conditions obtained in a rotating incubator, in addition to assessing the influence of stirring speed. Results show that high complexity of the cultivation medium favored kinetic growth of clones (648, 503). However, in assays submitted to induction by IPTG, this elevated complexity did not promote the expression of recombinant proteins. Expression of antigens 648 and 503 exhibited behavior associated with growth and, in terms of location, proteins 648 and 503 are intracellularly stored. Lactose may be the most adequate inducer in protein expression, when considering factors, cost, toxicity and stability. Elevated stirring may increase cell growth in clone 53, although it may not result in high concentrations for the protein of interest. On the other hand, positive results were obtained for all recombinant clones (648, 503) tested, confirmed by the electrophoretic profile

Biossíntese e recentes avanços na produção de celulose bacteriana

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

Análise do potencial genotóxico da superfície de titânio modificada por plasma

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Prospecção de genes associados ao processo de floração em tomateiro.

Flowering is a process marked by switch of shoot apical meristem to floral meristem, and it involves a complex regulation by endogenous and environmental factors. Analyses of key flowering genes have been carried out primarily in Arabidopsis thaliana and have provided a foundation for understanding the underlying molecular genetic mechanisms controlling different aspects of floral development. Several homologous have been found in other species, but for crops species such as tomatoes this process is not well known. The aim of this work was to use the genetic natural variation associated to the flowering process and use molecular tools such as subtractive libraries and real time PCR in order to identify and analyze the expression from genes that may be associated to flowering in these two species: L. esculentum cv Micro-Tom and L. pimpinellifolium. Our results showed there were identified many genes related to vegetative and possibly to the flowering process. There were also identified many sequences that were unknown. We ve chosen three genes to analyze the expression by real time PCR. The histone H2A gene gave an expression higher in L. pimpinellifolium, due to this the expression of this gene may be associated to flowering in this specie. It was also analyzed the expression of an unknown gene that might be a key factor of the transition to flowering, also in L. pimpinellifolium. For the elongation factor 1-α expression, the expression results were not informative, so this gene may have a constitutive expression in vegetative and flowering state. The results observed allowed us to identify possible genes that may be related to the flowering process. For further results it will be necessary a better characterization of them.

Análise de polimorfismos dos genes da rota quinureniana em pacientes com meningite bacteriana.

Bacterial meningitis (BM) is still an important infectious disease causing death and disability. Invasive bacterial infections of the central nervous systems (CNS) generate some of the most powerful inflammatory responses known, which contributes to neuronal damage. The DNA microarray technology showed alterations in the kynurenine (KYN) pathway that is induced in BM and other diseases associated with inflammation, leading to brain injury. Our main aim was to search SNPs previously described in the KYN path enzymes to investigate a putative association of this SNPs with imbalanced in this pathway in patients with BM. The patients included in this study were 33 males and 24 females, with ages varying from 02 months to 68 years. SNPs were located inside of the domain conserved in KYNU, IDO, KATI and KATII. Primers were designed for analysis of SNPs already described by PIRA-PCR followed by RFLP. The analysis of KYNU+715G/A SNP found a heterozygous frequency of 0.033. We did not found the variant allele of SNP KYNU+693G/A, KATI+164T/C, KATII+650C/T and IDO+434T/G. Despite of previews studies showing the importance of KYN pathway we did not found one association of these SNPs analyzed with susceptibility or severity of MB in study population.