Ação de indutores bióticos e abióticos no controle da ferrugem do eucalipto, atividade enzimática e expressão gênica durante o processo de infecção

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

Glucose and Fructose Production by Saccharomyces cerevisiaeInvertase Immobilized on MANAE-Agarose Support

Produção de glicose e frutose por invertase de Saccharomyces cerevisiae imobilizada em suporte MANAE-Agarose Invertase de Saccharomyces cerevisiaefoi imobilizada em agarose ativada com diferentes grupos (glioxil, MANAE ou glutaraldeído) e suportes epóxidos comerciais (Eupergit e Sepabeads). Derivados de invertase ativos e estabilizados foram produzidos pela adsorção da enzima em suportes MANAE-agarose, MANAE-agarose tratado com glutaraldeído e glutaraldeído-agarose. Em pH 5,0 estes derivados retiveram total atividade até 24h a 40 ºC e 50 ºC. Quando os ensaios foram a 40 °C e 50 °C com o pH alterado para 7,0, o derivado invertase-MANAE-agarose tratado com glutaraldeído apresentou 80% da atividade inicial. As atividades recuperadas dos derivados foram 73,5%, 44,4% e 36,8%, respectivamente para MANAE, MANAE tratado com glutaraldeído e glutaraldeído. Essas três preparações foram empregadas com sucesso em 3 ciclos de hidrólise da sacarose para produzir glicose e frutose.

Clonagem e expressão do gene engXCA de Xanthomonas campestri pv. campestris em Saccharmyces cerevisiae e estudos das endoglucanases nativa e recombinante

Pós-graduação em Biotecnologia - IQ

Uso de invertase imobilizada em pó de sabugo de milho para produção de açúcar invertido

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Determinação de comunidades em redes biológicas integradas

To understand how biological phenomena emerge, the nonlinear interactions among the components envolved in these and the correspondent connected elements, like genes, proteins, etc., can be represented by a mathematical object called graph or network, where interacting elements are represented by edges connecting pairs of nodes. The analysis of various graph-related properties of biological networks has revealed many clues about biological processes. Among these properties, the community structure, i.e. groups of nodes densely connected among themselves, but sparsely connected to other groups, are important for identifying separable functional modules within biological systems for the comprehension of the high-level organization of the cell. Communities' detection can be performed by many algorithms, but most of them are based on the density of interactions among nodes of the same community. So far, the detection and analysis of network communities in biological networks have only been pursued for networks composed by one type of interaction (e.g. protein-protein interactions or metabolic interactions). Since a real biological network is simultaneously composed by protein-protein, metabolic and transcriptional regulatory interactions, it would be interesting to investigate how communities are organized in this type of network. For this purpose, we detected the communities in an integrated biological network of the Escherichia coli and Saccharomyces cerevisiae by using the Clique Percolation Method and we veri ed, by calculating the frequency of each type of interaction and its related entropy, if components of communities... (Complete abstract click electronic access below)

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator

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

Energy values of traditional ingredients and sugarcane yeast for laying hens

An experiment was conducted to determine the chemical composition and apparent metabolizable energy (AME) and apparent metabolizable energy corrected for nitrogen balance (AMEn) values of corn, soybean meal (SBM), soybean oil (SO) and sugarcane yeast (SY) (Saccharomyces cerevisiae). A metabolism trial was performed with 120 Dekalb White laying hens at 65 weeks of age, using the method of total excreta collection. Birds were housed in metabolism cages and distributed according to a completely randomized design into five treatments with, six replicates of four birds each. The experimental period consisted of four days of adaptation and four days of excreta collection. The experimental diets included: a reference diet based on corn and SBM and four test diets containing 40% corn, 30% SBM, 10% SO or 30 % SY. The chemical compositions of the tested ingredients, expressed on "as-is" basis were: 86.9, 87.29, 87.32 and 99.5% dry matter; and 3.51, 2.08, 99.31 and 0.03 ether extract for corn, SBM, SO and SY, respectively. Corn, SBM, and SO presented 7.33, 43.61 and 24.64% crude protein, and 0.58, 5.07 and 6.77% ash, respectively; and crude fiber contents of corn and SBM were, respectively, 2.24% and 3.56%. The following AME and AMEn (kcal/kg dry matter) values were obtained: 3,801 and 3,760 kcal/kg for corn, 2,640 and 2,557 kcal/kg for SBM, 8,952 and 8,866 kcal/kg for SO, and 1,023 and 925 kcal/kg for sugarcane yeast, respectively.

Caracterização funcional do fator da tradução eIF5A por meio de interações genéticas

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

Using Pichia pastoris to produce recombinant glycerol kinase

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

Immunomodulation with β-glucan in matrinxã (Brycon amazonicus)

Pós-graduação em Zootecnia - FCAV

Physiological Studies on Candida albicans

Candida albicans is a common opportunistic, dimorphic human fungal pathogen. One of its virulence factors is the morphological switch between yeasts and hyphal or pseudohyphal forms, which can invade tissues and cause damage. Our studies focus on factors regulating pseudohyphae and epigenetic modifications of C. albicans. Regulating factors of pseudohyphae are aromatic alcohols and high phosphate. At low concentrations, exogenous aromatic alcohols induced pseudohyphae, as did high phosphate. For addressing the pathways involved in inducing pseudohyphae by aromatic alcohols or high phosphate, we used mutants defective in cAMP dependent PKA pathway (efg1/efg1), MAP kinase pathway (cph1/cph1), or both (cph1/cph1/efg1/efg1). These mutants failed to produce either hyphae or pseudohyphae in the presence of aromatic alcohols; but high phosphate still stimulated pseudohyphae. Gcn4, a transcription activator of more than 500 amino acid related genes, is turned-on in response to amino acid starvation. The accumulation of aromatic alcohols sends nitrogen starvation signals, which inhibit eIF2B, which in turn derepresses Gcn4p. High phosphate also induces pseudohyphae by derepressing Gcn4p, although the pathways involved are still unknown. In sum, aromatic alcohols and high phosphate induce pseudohyphae by derepressing Gcn4. In this study we found a novel posttranslational histone modification in C. albicans, which is biotinylation. Western blot and Mass spectrometry techniques were used to find that Histones H2B and H4 were biotinylated at every condition tested such as yeast vs. hyphae, aerobic growth vs. anaerobic growth, rich medium vs. defined medium. In C. albicans lysines K8, K11 in histone H4 and lysines K17, K18, K31 in histone H2B are biotin attachment sites as found using mass spectrometry. Biotin was also found to enhance the germ tube formation of C. albicans. Germ tube formation assays with biotin-starved cells as inoculum showed low percent of germ tubes (1-5%). Addition of biotin to the media showed 100% germ tubes. Biotinylation of histones were not detected from biotin-starved cells. Appendix-A details work related to Farnesol quantification assays in several strains of C.albicans and Ceratocystis ulmi, and growth studies of class E VPS strains of Saccharomyces Cerevisiae. Adviser: Kenneth W. Nickerson

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the alpha-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8 angstrom resolution of Tsa1(C47S) in the decameric form [(alpha(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that G1u50 and Arg146 participate in the stabilization of the Cys(P) alpha-helix. As a consequence, we raised the hypothesis that G1u50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6) M-1 S-1. Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that G1u50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx. (C) 2012 Elsevier Ltd. All rights reserved.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes.

Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The Delta sebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA:GFP (SebA:green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Delta sebA mutant. The A. fumigatus Delta sebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Delta sebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.