In silico analysis of the coffee transcriptome : identification of SNPs and inference of mechanisms of gene expression regulation

Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O café é uma das culturas mais importantes do mundo, sendo consumido mundialmente e com significativa participação na economia em países em desenvolvimento. Coffea arabica e Coffea canephora são responsáveis por 70% e 30% da produção comercial, respectivamente. Análise citogenética indicou que C. arabica é uma planta alotetraploide autógama formada por uma hibridação (1 milhão de anos atrás) entre os diplóides C. canephora e Coffea eugenioides. C. eugenioides é uma espécie silvestre que cresce em maiores altitudes próximo das bordas de florestas e produz poucas e pequenas sementes com baixo teor de cafeína. Por outro lado, C. canephora é alógama e cresce melhor em terras baixas, é também caracterizada por maior produtividade, maior tolerância a pragas e maior teor de cafeína, mas tem uma bebida considerada de qualidade inferior em comparação com a produzida por C. arabica. Durante a última década, algumas iniciativas de pesquisa têm sido lançadas para produzir dados genômicos e transcritômicos de algumas espécies de café. Esta coleção de ESTs representa uma boa visão do transcriptoma de C. arabica e C. canephora, sendo um importante recurso para análise molecular dessas espécies. Este trabalho teve como objetivo obter mais informações sobre algumas espécies do gênero Coffea, incluindo a estrutura dos genes, análise de expressão e identificação de genes e famílias gênicas que são específicos ou expandidos em café. Além disso, também foi proposto estudar a regulação da expressão gênica nos genes homeólogos da alotetraploide C. arabica. A fim de investigar estes conjuntos de dados de EST foram realizadas duas montagens: (i) a primeira montagem com cada espécie individualmente, com o objetivo de fazer uma análise comparativa entre as C. arabica, C. canephora e outras culturas, e (ii) com as duas espécies de café juntas, permitindo a identificação de SNPs entre C. arabica e C. canephora, e avaliar questões evolutivas em C. arabica. A identificação dos transcritos diferencialmente expressos e novas famílias gênicas foram utilizados como ponto de partida para a correlação de características de desenvolvimento e de perfis de expressão gênica em Coffea sp.. Domínios de proteínas e análises de Gene Ontology sugerem diferenças significativas entre os dados das espécies de café analisadas, principalmente em relação a síntese de açúcares, ligação de proteínas a nucleotídeos, retrotransposons e proteínas de resposta a estresse. A ferramenta OrthoMCL identificou as famílias de proteínas específicas ou predominante de café quando comparado com outras cinco espécies de plantas. Usando as discrepâncias de alta qualidade encontradas em ESTs sobrepostos de C. arabica e C. canephora, os perfis de diversidade de seqüência foram avaliados em ambas as espécies e utilizados para deduzir a contribuição de C. canephora e C. eugenioides na transcrição de C. arabica. A identificação de genes homeologous de C. arabica aos genomas ancestrais permitiu analisar as contribuições de expressão gênica de cada subgenoma. Nós sugerimos que este fenômeno tem uma questão importante na expressão dos genes e fisiologia de Coffea.Abstract: Coffee is one of the most important crops in the world, being worldwide consumed and having significant participation in under development economies. Coffea arabica and Coffea canephora are responsible for 70% and 30% of commercial production, respectively. Cytogenetic analysis established that C. arabica is an autogamous alotetraploid formed by a recent (1 mya) hybridization between the diploids C. canephora and Coffea eugenioides. C. eugenioides is a wild species which grows in higher altitudes near forest edges, and produces few berries with small beans of low caffeine content. On the other hand, C. canephora is alogamous and grows better in lowlands. It is also characterized by higher productivity, more tolerance to pests, and higher caffeine content, but it has an inferior beverage compared with C. arabica. During the last decade, research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. This EST collection represents a good overview of C. arabica and C. canephora transcriptome, being appropriate as a resource for Coffea molecular analysis. This work aimed to obtain further information about Coffea spp. gene structure and expression and to identify genes that are specific or expanded in coffee plants. Moreover, it also intended to study the homeologous gene expression regulation in the alotetraploid C. arabica. In order to investigate these data two different EST assemblies were performed: (i) with each species individually, aiming the comparative analysis between the C. arabica, C. canephora and other crops; and (ii) with both coffee species together, allowing the identification of SNPs between C. arabica and one of its direct ancestors C. canephora and the examination of evolutive issues in C. arabica. The identification of differentially expressed transcrip ts and new gene families offered a starting point for the correlation of gene expression profil es and Coffea sp. development traits. Protein domain and Gene Ontology analyzes suggested significant differences between the data of coffee species analyzed, mainly in relation to complex sugar synthases, nucleotide binding proteins, retrotransposons and stress response. OrthoMCL tool identified specific or prevalent coffee protein families when compared with other five plant species. Using the high quality discrepancies, found in overlapped ESTs from C. arabica and C. canephora, sequence diversity profiles were evaluated within both species and used to deduce the transcript contribution of the C. canephora and C. eugenioides ancestors in the C. arabica. The assignment of the C. arabica homeologous genes to the ancestral genomes allowed us to analyze gene expression contributions of each subgenome. We suggest that this phenomenon has an important issue in Coffea gene expression and physiology.DoutoradoBioinformaticaDoutor em Genetica e Biologia Molecula

Software development for gasometric data interpretation applicable in intensive care units

The objective of this study was to develop a software for data interpretation of gasometric applicable in Intensive Care Units that can provide a diagnosis for disorders of acid-base balance. This is an experimental study being selected on a theoretical basis with the Java IDE NetBeans 6.8 through a partnership with professionals trained in Information Systems. The program development was based on creating an algorithm, a sequence of well-defined and unambiguous to be performed mechanically in order to provide a desired diagnosis. We created a software applicable in intensive care units called InterGas, which is a program easy to install, has an interface easy to understand and use, and to process the data quickly and accurately providing as the final diagnosis for the disorder the acid-base balance. The lack of another tool that brings together all the components of InterGas makes it a pioneering software that facilitates decision-making, as it characterizes the occurrence of mixed disturbances using compensation formulas.O objetivo deste estudo foi desenvolver um software para interpretação de dados gasométricos aplicável em UTIs. Trata-se de estudo de caráter experimental, sendo selecionada uma base teórica em Java com a IDE NetBeans 6.8 por meio de parceria com profissionais capacitados em Sistemas de Informação. O desenvolvimento do programa foi baseado na criação de um algoritmo, uma sequência de instruções bem definidas e não ambíguas a serem executadas mecanicamente com a finalidade de fornecer um diagnóstico desejado. Foi criado um software aplicável em UTIs denominado InterGas, que é um programa de fácil instalação, possui interface de fácil compreensão e utilização, além de processar os dados rapidamente e de forma precisa, oferecendo como resultado final o diagnóstico para o distúrbio do equilíbrio ácido-básico. O desconhecimento de outra ferramenta que reúna todos os componentes do InterGas o torna um software pioneiro que facilita a tomada de decisão à medida que caracteriza a ocorrência de distúrbios mistos utilizando fórmulas de compensação. Com isso, futuros estudos deverão ser feitos com o objetivo de avaliar aspectos relacionados à implementação e eficácia do software desenvolvido

First dinosaur record from the Marília Formation (Maastrichtian) in the Gurinhatã municipality, Minas Gerais state, Brazil

Titanosaurs are one of the most common dinosaurs found in Cretaceous outcrops, especially in Brazil. In this article we describe a proximal portion of an ulna (Paleo-UFG/V-0039) which was found isolated Paleo-UFG/V-0039 comes from a sandstone outcrop, with fine to medium granulation, of the Marília Formation (Bauru Group) that appears irregularly in the municipality of Gurinhatã, state of Minas Gerais, Brazil. The occurrence described here is the first dinosaur osteological remains documented in this municipality. Although incomplete, Paleo-UFG/V-0039 could be identified as an indeterminate lithostrotian titanosaur whose morphology is similar to some appendicular elements of European species than South American ones. However, the incompleteness of the specimen has difficult complex interpretations. Finally, Paleo-UFG/V-0039 highlights the importance of the Gurinhatã outcrops and other sites in this region for future discoveries

Identification of SNPs in RNA-seq data of two cultivars of Glycine max (soybean) differing in drought resistance

The legume Glycine max (soybean) plays an important economic role in the international commodities market, with a world production of almost 260 million tons for the 2009/2010 harvest. The increase in drought events in the last decade has caused production losses in recent harvests. This fact compels us to understand the drought tolerance mechanisms in soybean, taking into account its variability among commercial and developing cultivars. In order to identify single nucleotide polymorphisms (SNPs) in genes up-regulated during drought stress, we evaluated suppression subtractive libraries (SSH) from two contrasting cultivars upon water deprivation: sensitive (BR 16) and tolerant (Embrapa 48). A total of 2,222 soybean genes were up-regulated in both cultivars. Our method identified more than 6,000 SNPs in tolerant and sensitive Brazilian cultivars in those drought stress related genes. Among these SNPs, 165 (in 127 genes) are positioned at soybean chromosome ends, including transcription factors (MYB, WRKY) related to tolerance to abiotic stress.331334Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system

In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation

PREVALÊNCIA E FATORES ASSOCIADOS À ADESÃO AO TRATAMENTO MEDICAMENTOSO POR PACIENTES COM HIPERTENSÃO ARTERIAL

Objetivou-se avaliar a prevalência e os fatores associados à adesão ao tratamento entre pessoas acometidas por hipertensão arterial sistêmica. Estudo transversal, descritivo, realizado com 92 usuários atendidos em uma Unidade Básica de Saúde. Utilizou-se questionário contendo características sociodemográficas, econômicas, de saúde e o Teste de Morisky-Green. Aferiu-se a pressão arterial pelo método indireto. A prevalência de adesão ao tratamento foi 72,8%. Evidenciou-se diferenças significativas na adesão ao tratamento segundo situação marital de união estável (p= 0,043), forma de controle da pressão arterial por meio de tratamento medicamentoso combinado com mudanças no estilo de vida (p=0,003) e tempo de diagnóstico da hipertensão inferior a 10 anos (

Global Analyses Of Ceratocystis Cacaofunesta Mitochondria: From Genome To Proteome.

The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated omics approaches. The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization. The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity.149

A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

<p>Abstract</p> <p>Background</p> <p>The basidiomycete fungus <it>Moniliophthora perniciosa </it>is the causal agent of Witches' Broom Disease (WBD) in cacao (<it>Theobroma cacao</it>). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. <it>M. perniciosa</it>, together with the related species <it>M. roreri</it>, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of <it>M. perniciosa </it>was analyzed to evaluate the overall gene content of this phytopathogen.</p> <p>Results</p> <p>Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that <it>M. perniciosa </it>has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that <it>M. perniciosa </it>have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in <it>M. perniciosa </it>genome survey.</p> <p>Conclusion</p> <p>This genome survey gives an overview of the <it>M. perniciosa </it>genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the <it>M. perniciosa</it>/cacao pathosystem.</p