Detecção de cinco novas mutações em pacientes brasileiros com gangliosidose GM1

A Gangliosidose GM1 é um Erro Inato do Metabolismo (EIM) causado pela deficiência da enzima B-galactosidase ácida. Essa doença é caracterizada pelo acúmulo de metabólitos não degradados, principalmente gangliosídeo GM1, nos lisossomos de vários tipos celulares. Baseado na idade de início e na atividade residual da enzima, a Gangliosidose GM1 é classificada em três diferentes tipos: infantil, juvenil e adulto. O gene da B-galactosidase ácida (GLB1, GeneBank M27507) está situado no cromossomo 3 e possui mais de 60 kb, contendo 16 exons. Cerca de 50 mutações associadas à doença estão descritas na literatura. No sul do Brasil, há uma alta freqüência dessa doença (1:17.000 nascidos vivos). Neste trabalho, vinte pacientes diagnosticados no Hospital de Clínicas de Porto Alegre (Brasil) tiveram o gene GLB1 investigado por SSCP (Single Strand Conformational Polymorphism) usando DNA extraído de sangue periférico. Através desta triagem foram encontradas 52 alterações de mobilidade do DNA, indicando a presença de mutações. As amostras relativas aos exons 2 e 15 foram submetidas a sequenciamento direto com seqüenciador ABI31O(Applied Biosystens) utilizado kit BigDye 3.1. Cinco novas mutações no gene GLB1 (F63Y, R38G, Y36S, Y64F e R59C) e duas mutações já descritas (R59H e 1622-1627insG) foram encontradas. Este trabalho possibilitou a genotipagem completa de 6 pacientes e parcial de 5, e direcionou a investigação de mutações, contribuindo diretamente no diagnóstico da enfermidade e permitindo a realização de estudos de correlação genótipo/fenótipo destes pacientes.

Transferência de calor e controle de temperatura em tubos capilares utilizados em eletroforese capilar

Este trabalho tem por objetivo estudar a transferência de calor em tubos capilares cilíndricos utilizados na técnica de separação de moléculas denominada Eletroforese Capilar. Esta técnica é usada, por exemplo, na análise de biomoléculas e no sequenciamento de DNA, onde o controle da temperatura está diretamente ligado ao desempenho destes métodos e à qualidade dos resultados. Para empregar esta técnica, tensões elétricas da ordem de 20 kV são aplicadas entre as extremidades dos tubos capilares, que possuem normalmente 50 cm de comprimento, 350 µm de diâmetro externo e 50 µm de diâmetro interno, preenchidos por uma solução aquosa. Tais tensões geram uma corrente elétrica na solução, provocando aquecimento distribuído por Efeito Joule. Os tubos capilares são construídos em quartzo amorfo e protegidos por uma camada de material polimérico (poliimida). Para implementar o controle da temperatura, os tubos capilares são colocados em contato com um fluido de resfriamento. Num primeiro momento, os estudos são realizados por simulação numérica, empregando o Método dos Volumes Finitos em rotinas escritas em FORTRAN. São simulados casos onde os tubos são recobertos por camadas cilíndricas de materiais com uma condutividade térmica relativamente boa, com o objetivo de aumentar a superfície de troca de calor com o fluido de resfriamento. Como resultado, obtêm-se curvas da temperatura no centro dos tubos capilares em função do coeficiente de transferência de calor por convecção. Um caso de interesse é quando os tubos capilares são posicionados excentricamente ao recobrimento cilíndrico Num segundo momento, é utilizado o software de simulação numérica ANSYS CFX®, onde é simulado o resfriamento dos mesmos tubos capilares expostos a um escoamento transversal de ar a 15°C. Neste caso, também são aplicados os recobrimentos cilíndricos e, além disso, opta-se por simular o resfriamento de um arranjo de vários tubos (sistema multicapilar) dispostos entre placas de vidro, no formato de um sanduíche. Como resultados mais importantes salientam-se: a) o aumento do raio do recobrimento resulta no aumento da transferência de calor, fazendo com que a temperatura no núcleo do capilar fique estacionada em valores baixos que não comprometem as separações/análises; b) chegou-se a um valor de raio crítico da ordem de 10 mm para a condição de operação mais típicas na área da Eletroforese Capilar; c) as montagens com o tubo capilar concêntrico e excêntrico ao recobrimento não apresentam diferenças significativas no perfil de temperatura da solução tampão; e finalmente d) observa-se que o uso de duas placas de material dielétrico com os capilares posicionados em forma de sanduíche entre elas permite uma eficiente dissipação do calor gerado na solução tampão.

Metagenômica comparativa de solo de regiões de mata atlântica e caatinga do estado do Rio Grande do Norte - Brasil

The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored

Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Efeitos da microgravidade em plantas de cana-de-açúcar

Sugarcane (Saccharum spp.) is a plant from Poaceae family that has an impressive ability to accumulate sucrose in the stalk, making it a significant component of the economy of many countries. About 100 countries produce sugarcane in an area of 22 million hectares worldwide. For this reason, many studies have been done using sugarcane as a plant model in order to improve production. A change in gravity may be one kind of abiotic stress, since it generates rapid responses after stimulation. In this work we decided to investigate the possible morphophysiological, biochemical and molecular changes resulting from microgravity. Here, we present the contributions of an experiment where sugarcane plants were submitted to microgravity flight using a vehicle VSB-30, a sounding rocket developed by Aeronautics and Space Institute teams, in cooperation with the German Space Agency. Sugarcane plants with 10 days older were submitted to a period of six minutes of microgravity using the VSB-30 rocket. The morphophysiological analyses of roots and leaves showed that plants submitted to the flight showed changes in the conduction tissues, irregular pattern of arrangement of vascular bundles and thickening of the cell walls, among other anatomical changes that indicate that the morphology of the plants was substantially influenced by gravitational stimulation, besides the accumulation of hydrogen peroxide, an important signaling molecule in stress conditions. We carried out RNA extraction and sequencing using Illumina platform. Plants subjected to microgravity also showed changes in enzyme activity. It was observed an increased in superoxide dismutase activity in leaves and a decreased in its activity in roots as well as for ascorbate peroxidase activity. Thus, it was concluded that the changes in gravity were perceived by plants, and that microgravity environment triggered changes associated with a reactive oxygen specie signaling process. This work has helped the understanding of how the gravity affects the structural organization of the plants, by comparing the anatomy of plants subjected to microgravity and plants grown in 1g gravity

Co-infecção pelo vírus da hepatite G em pacientes infectados pelo Leihsmania chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Avaliação de parâmetros de estresse oxidativo em plantas de cana-de-açúcar tratadas com peróxico de hidrogênio

The genus Saccharum belongs to Poaceae family. Sugarcane has become important monocultures in Brazil due to their products: ethanol and sugar. The production may change between different regions from Brazil. This difference is related to soil, climatic conditions and temperature that promotes oxidative stress that may induce an early flowering. The aim of this work was to identify the effects of oxidative stress. In order to analyse this, sugarcane plants were submitted to oxidative stress using hydrogen peroxide. After this treatment, the oxidative stress were analyzed Then, the plant responses were analyzed under different approaches, using morphophysiological, biochemical and molecular tools. Thus, sugarcane plants were grown under controlled conditions and until two months they were subjected first to a hydroponics condition for 24 hours in order to acclimation. After this period, these plants were submitted to oxidative stresse using 0 mM, 10 mM, 20 mM and 30 mM hydrogen peroxide during 8 hours. The histomorphometric analysis allowed us to verify that both root and leaf tissues had a structural changes as it was observed by the increased in cell volume, lignin accumulation in cell walls. Besides, this observation suggested that there was a change in redox balance. Also, it was analyzed the activity of the SOD, CAT and APX enzymes. It was observed an increase in the SOD activity in roots and it was also observed a lipid peroxidation in leaves and roots. Then, in order to identify proteins that were differently expressed in this conditions it was used the proteomic tool either by bidimensional gel or by direct sequencing using the Q-TOF EZI. The results obtained with this approach identified more than 3.000 proteins with the score ranging from 100-5000 ions. Some of the proteins identified were: light Harvesting; oxygenevolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. All these proteins are involved in oxidative stress response, photsynthetic pathways, and some were classified hypothetical proteins and/or unknown (30% of total). Thus, our data allows us to propose that this treatment induced an oxidative stress and the plant in response changed its physiological process, it made changes in tissue, changed the redox response in order to survival to this new condition

Mutagênese induzida por flavonóides presentes do decocto das cascas da aroeira (Schinus terebinthifolius, Raddi)

The decoction of Brazilian pepper tree barks (Schinus terebinthifolius, Raddi), is used in medicine as wound healing and antiinflamatory. Once extracts from this plant are used for acceleration of scar s process, it is important to study their mutagenic and genotoxic potential. In previous works in our laboratory, it was observed mutagenicity caused by the decoction when in high concentrations. Among the chemical compounds of this plant that could be able to induce mutation, the flavonoids were the only group that was referred to have either an oxidant or antioxidant potential. The flavonoids were isolated, purified and quantified by adsorptive column chromatography under silica gel, bacterial and in vitro genotoxic tests were realized to determine if the flavonoids were the responsible agents for this mutagenicity found. The tests realized with plasmidial DNA were indicative that the flavonoids are probably genotoxic, due to the presence of correlation between increase of the flavonoid concentration and in plasmidial DNA double strand breakage visualized in agarose gel, as well as they were capable to generated abasic sites shown by the in vitro treatment with exonuclease III. The same tests with plasmidial DNA in the presence of copper [10 µM] and of a Tris-HCl pH 7.5 [10 µM] buffer were realized with the isolated flavonoids to determine if there would be or not participation of reactive oxygen species (ROS). The transformation of plasmidial DNA in different bacterial strains proficient and deficient in DNA repair enzymes in the presence or not of a Tris-HCl buffer, suggests that the enzymes that repair oxidative lesions are necessary to repair the lesions generated by the flavonoids and that ROS are generated and are necessary to promote the lesions. Bacterial tests with Escherichia coli strains of the CC collection (deficient or not for DNA repair enzymes), showed that the flavonoids are able to increase the frequency of mutations, mainly in strains mutated in repair enzymes (MutM, MutY-glicosylases and double mutant), suggesting that these agents are responsible for the enhancement in the mutation rate. In order to determine the mutation spectrum caused by the flavonoids of the Brazilian pepper tree stem bark, plasmidial DNA previously treated with the flavonoids were transformed in bacterial strains deficient and proficient in the DNA repair enzymes, followed by a blue-white selection with X-gal, DNA amplification by PCR and sequencing the positive mutant clones. Analysis of the mutants obtained from strains CC104, CC104mutM, CC104mutY, CC104mutMmutY, BW9101, BW9109 indicated a predominance of some mutations like G:C to C:G that can be correlated with the origin of 8-oxoG, due to oxidative lesions caused by the flavonoids. So it can concluded that the flavonoid isolated or in fractions enriched on them are genotoxic and mutagenic, and their mutations are predominantly oxidative, mediated by ROS, and the lesions are recognized by the BER system. In this way it is proposed that the flavonoids can act in two different ways to generate the DNA lesion: 1. in a Fenton-like reaction, when the flavonoid are in the presence of metal ions and that together with the water generate ROS that promotes the DNA lesions; 2. in another way the lesions can be generated by the formation of ROS due to the internal chemical structure of the flavonoid molecule due to the quantity and location of hydroxyl groups, and so producing the DNA lesions, those lesions can be directly (suggested by the in vitro experiments) or indirectly done (supported by the experiments using the CC bacterial strains)

Polimorfismos do gene MSX1 e a fenda labio e/ou palatina não-sindrômica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Imunorreatividade da p53 associada à ausência de mutações no gene TP53 em linfomas caninos

Sabendo-se da influência das mutações no gene TP53 no desenvolvimento das neoplasias e da discrepância entre os resultados obtidos pelas técnicas de sequenciamento e imunoistoquímica, esta pesquisa teve como objetivo relacionar a sequência do TP53 com a imunorreatividade da p53. Foram obtidas amostras de linfoma de 12 cães. O diagnóstico histopatológico foi determinado pela classificação de Kiel. O imunofenótipo e a imunomarcação da p53 foram determinados por imunoistoquímica. Para reação com a p53, utilizou-se anticorpo policlonal anti-p53 (CM1) na diluição de 1:500. A região do gene TP53 compreendida entre os exons quatro e nove foi amplificada por PCR e submetida ao sequenciamento. Apesar dos resultados obtidos pela imunoistoquímica, nenhuma mutação foi encontrada nas sequências analisadas. Conclui-se que a imunorreatividade da p53 pela imunoistoquímica não pode ser atribuída à presença de mutações no domínio central do gene TP53.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil

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

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

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

Detection of hemoplasma and Bartonella species and co-infection with retroviruses in cats subjected to a spaying/neutering program in Jaboticabal, SP, Brazil

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

Auxin-producing bacteria isolated from the roots of Cattleya walkeriana, an endangered Brazilian orchid, and their role in acclimatization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genetic characterization and nitrogen fixation capacity of Rhizobium strains on common bean

O objetivo deste trabalho foi a caracterização genética de quatro novas estirpes de Rhizobium e a avaliação de sua capacidade de fixação de N2 e nodulação, comparadas a estirpes comerciais e à população nativa de rizóbios de um Latossolo Vermelho. Dois experimentos foram conduzidos em blocos ao acaso, em casa de vegetação. No primeiro experimento, conduzido em tubetes com vermiculita, avaliaram-se a nodulação e a capacidade de fixação das novas estirpes, em comparação com as estirpes comerciais CIAT-899 e PRF-81 e com a população nativa do solo. Das colônias puras isoladas, extraiu-se o DNA genômico e realizou-se o seqüenciamento do espaço intergênico, para a caracterização genética das estirpes e da população nativa de rizóbios. O segundo experimento foi realizado em vasos com solo, para determinação da produtividade e da nodulação do feijoeiro, cultivar Pérola, com o uso das estirpes isoladamente ou em mistura com a PRF-81. A população nativa do solo foi identificada como Rhizobium sp. e se mostrou ineficiente na fixação de nitrogênio. Foram encontradas três espécies de Rhizobium entre as quatro novas estirpes. As estirpes LBMP-4BR e LBMP-12BR estão entre as que têm maior capacidade de nodulação e fixação de N2, e apresentam respostas diferenciadas quando misturadas à PRF-81.