Avaliação da α-glicosidase Cqm1 de Culex quinquefasciatus e o impacto de suas mutações na fisiologia de larvas

Lysinibacillus sphaericus (Lsp) é uma bactéria entomopatógena que produz a toxina Bináriav(Bin) com atividade larvicida para culicídeos. A sua ação em Culex quinquefasciatus depende da ligação da toxina Bin à α-glicosidase (Aglu) Cqm1, que atua como receptor no epitélio intestinal de larvas. Na colônia R2362, foram caracterizados dois alelos de resistência ao Lsp: cqm1REC e cqm1REC-2, cujas mutações impedem a expressão da Aglu Cqm1. O objetivo deste trabalho foi avaliar a atividade catalítica da Cqm1 e comparar a atividade α-glicosidase e o desenvolvimento pré-imaginal de larvas de indivíduos susceptíveis (S) e resistentes (R) para cada alelo. Para isto, foram avaliados os seguintes parâmetros: atividade catalítica da Cqm1 recombinante; padrão de transcrição de outras Aglus parálogas à Cqm1; atividade de Aglus nativas em larvas; sobrevivência de indivíduos frente a diferentes dietas. A Aglu Cqm1 mostrou atividade enzimática ótima à 37o C, pH 7,5-8,0 e utilizando o substrato sintético pNαG. A atividade α-glicosidase total em larvas S e R foi similar, apesar da ausência de expressão da Cqm1 nas larvas R. A investigação in silico revelou 18 proteínas parálogas à Cqm1 e, dentre 11 investigadas, nove são expressas em larvas S e R. A análise quantitativa de três parálogas demonstrou que duas tem um padrão de transcrição mais elevado em larvas resistentes, sugerindo a existência de um mecanismo de compensação de expressão de α-glicosidases. O desenvolvimento pré-imaginal de larvas S foi decrescente nas seguintes dietas: ração de gatos, ração de peixes, leite desnatado, extrato de levedura e sacarose. De uma forma global, a taxa de sobrevivência de larvas R foi inferior à S em todas as dietas testadas. Os dados obtidos mostram que as mutações ligadas aos alelos cqm1REC e cqm1REC-2 não parecem impactar a atividade Aglu nas larvas e que o custo biológico observado poderia estar relacionado a outros genes e vias metabólicas.

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response

Metagenômica: busca de novos genes envolvidos com a biodegradação de hidrocarbonetos

Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ± 118 bp) and 127.563 sequences (352 ± 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well

Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20

Metabolic flux analysis (MFA) is a powerful tool for analyzing cellular metabolism. In order to control the growth conditions of a specific organism, it is important to have a complete understanding of its MFA. This would allowed us to improve the processes for obtaining products of interest to human and also to understand how to manipulate the genome of a cell, allowing optimization process for genetic engineering. Streptomyces olindensis ICB20 is a promising producer of the antibiotic cosmomycin, a powerful antitumor drug. Several Brazilian researchers groups have been developing studies in order to optimize cosmomycin production in bioreactors. However, to the best of our knowledge, nothing has been done on metabolic fluxes analysis field. Therefore, the aim of this work is to identify several factors that can affect the metabolism of Streptomyces olindensis ICB20, through the metabolic flux analysis. As a result, the production of the secondary metabolite, cosmomycin, can be increased. To achieve this goal, a metabolic model was developed which simulates a distribution of internal cellular fluxes based on the knowledge of metabolic pathways, its interconnections, as well as the constraints of microorganism under study. The validity of the proposed model was verified by comparing the computational data obtained by the model with the experimental data obtained from the literature. Based on the analysis of intracellular fluxes, obtained by the model, an optimal culture medium was proposed. In addition, some key points of the metabolism of Streptomyces olindensis were identified, aiming to direct its metabolism to a greater cosmomycin production. In this sense it was found that by increasing the concentration of yeast extract, the culture medium could be optimized. Furthermore, the inhibition of the biosynthesis of fatty acids was found to be a interesting strategy for genetic manipulation. Based on the metabolic model, one of the optimized medium conditions was experimentally tested in order to demonstrate in vitro what was obtained in silico. It was found that by increasing the concentration of yeast extract in the culture medium would induce to an increase of the cosmomycin production

Estudos in silico da proteína AlkB e prospecção de bactérias alcanotróficas em solos da bacia petrolífera potiguar

Some microorganisms from virgin ecosystems are able to use petroleum it as source of carbon and energy. The knowledge of microbial biodiversity can help to reveal new metabolic systems for utilization alkanes with biotechnological importance. The aim of this study is: i) Accomplish an in silico study of the AlkB protein aimed to understand the probable mechanism involved on selectivity of alkanes in Gram positive and Gram negative bactéria. ii) prospect and analyze the response of the microbial alkanotrophics communities in soil and mangrove sediments of BPP RN and soil of Atlantic forest in the Horto Dois Irmãos Reserve area/PE using the molecular biomarker, gene alkB; with the PCR and PCR-DGGE approach

Polímeros biorreabsorvíveis como substrato para cultura de células e engenharia tecidual

Biomateriais poliméricos são desenvolvidos para uso como substitutos de tecidos danificados e/ou estimular sua regeneração. Uma classe de biomateriais poliméricos são os biorreabsorvíveis, compostos que se decompõem tanto in vitro quanto in vivo. São empregados em tecidos que necessitam de um suporte temporário para sua recomposição tecidual. Dentre os vários polímeros biorreabsorvíveis, destacam-se os alfa-hidróxi ácidos, entre eles, diferentes composições do poli(ácido lático) (PLA), como o poli(L-ácido lático) (PLLA), poli(D-ácido lático) (PDLA), poli(DL-ácido lático) (PDLLA), além do poli(ácido glicólico) (PGA) e da policaprolactona (PCL). Estes polímeros são considerados biorreabsorvíveis por apresentarem boa biocompatibilidade e os produtos de sua decomposição serem eliminados do corpo por vias metabólicas. Diversas linhas de pesquisa mostram que os diferentes substratos à base de PLA estudados não apresentam toxicidade, uma vez que as células são capazes de crescer e proliferar sobre eles. Além disso, diversos tipos de células cultivadas sobre diferentes formas de PLA são capazes de se diferenciarem sobre os diferentes polímeros e passar a produzir componentes de matriz extracelular. Neste trabalho, é revisada a utilização de substratos à base de alfa-hidróxi ácidos, com destaque para diferentes formas de PLA, utilizados como substratos para cultura de células, bem como suas aplicações.

Prospecting sugarcane genes involved in aluminum tolerance

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

Caracterização bioquímica de uma serino-protease produzida pelo fungo termofílico Myceliophthora sp

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos do hormônio de crescimento sobre o metabolismo energético e estresse oxidativo no miocárdio de ratos portadores de insuficiência cardíaca secundária à estenose aórtica

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

Indicadores nutricionais e níveis de homocisteína em pacientes com doença arterial periférica

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

Produção de prostaglandinas e leucotrienos por Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aspectos reprodutivos de ratos machos submetidos ao bloqueio androgênico perinatal por flutamida

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da orquidectomia e da reposição com testosterona sobre as respostas vasomotoras de veias isoladas de rato a agonistas simpatomiméticos

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Identificação e caracterização de possiveis marcadores moleculares em carcinoma renal de células claras

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Perfil de expressão e organização genômica de micrornas músculo-específicos na tilápia-do-nilo (Oreochromis nicoticus)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)