Maria Emilia Telles Walter

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Reversal and transposition distance of linear chromosomes

In recent years we are seeing increasing interest in research on mutational events acting on large portions of chromosomes. Among these events, a reversal acts on a fragment of a chromosome, reversing the order and orientation of the genes, and a transposition moves fragments from one region to another within a chromosome. We analyze genomes evolving by reversals and transpositions. We present approximation algorithms to compute the reversal and transposition distance for linear permutations, and a lower bound on the reversal and transposition diameter of signed linear permutations.

Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome

Guarana (Paullinia cupana var. sorbilis) is a plant native to the central Amazon basin. Roasted seed extracts have been used as medicinal beverages since pre-Colombian times, due to their reputation as stimulants, aphrodisiacs, tonics, as well as protectors of the gastrointestinal tract. Guarana plants are commercially cultivated exclusively in Brazil to supply the national carbonated soft-drink industry and natural product stores around the world. In this report, we describe and discuss the annotation of 15,387 ESTs from guarana seeded-fruits, highlighting sequences from the flavonoid and purine alkaloid pathways, and those related to biotic stress avoidance. This is the largest set of sequences registered for the Sapindaceae family.

Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs)

Bothrops atrox is a highly dangerous pit viper in the Brazilian Amazon region. We produced a global catalogue of gene transcripts to identify the main toxin and other protein families present in the B. atrox venom gland. We prepared a directional cDNA library, from which a set of 610 high quality expressed sequence tags (ESTs) were generated by bioinformatics processing. Our data indicated a predominance of transcripts encoding mainly metalloproteinases (59% of the toxins). The expression pattern of the B. atrox venom was similar to Bothrops insularis, Bothrops jararaca and Bothrops jararacussu in terms of toxin type, although some differences were observed. B. atrox showed a higher amount of the PIII class of metalloproteinases which correlates well with the observed intense hemorrhagic action of its toxin. Also, the PLA2 content was the second highest in this sample compared to the other three Bothrops transcriptomes. To our knowledge, this work is the first transcriptome analysis of an Amazonian rain forest pit viper and it will contribute to the body of knowledge regarding the gene diversity of the venom gland of members of the Bothrops genus. Moreover, our results can be used for future studies with other snake species from the Amazon region to investigate differences in gene patterns or phylogenetic relationships.

A new approach for approximating the transposition distance

One of the proposed ways to compare genomes or other large DNA molecules is by computing a rearrangement distance, defined as the minimum number of rearrangement events necessary to transform one molecule into another taking into account only the relative order of similar genes. In this work, we study the problem of computing the transposition distance between two linear gene orders, represented by permutations. To help solve it, we present a very simple structure, the breakpoint diagram, and a 2.25-approximation algorithm for the problem based on this structure. While there are better approximation algorithms, they are based on more complex data structures. Our algorithm was implemented in the C programming language and we show experimental results obtained with it on all permutations of up to 11 genes, plus selected permutations of higher size.

Working on the problem of sorting by transpositions on genome rearrangements

In computational biology, genome rearrangements is a field in which we investigate the combinatorial problem of sorting by transpositions. This problem consists in finding the minimum number of transpositions (mutational event) that transform a chromosome into another. In this work, we implement the 1.5-approximation algorithm proposed by Christie [2] for solving this problem, introducing modifications to reduce its time complexity, and we also propose heuristics to further improve its performance. Comparing our experimental results with the best known results, we had better performance. This work targets to contribute for discovering the complexity of the problem of sorting by transpositions, which remains open.

Parallel strategies for the local biological sequence alignment in a cluster of workstations

Sequence comparison is a basic operation in DNA sequencing projects, and most of sequence comparison methods used are based on heuristics, which are faster but there are no guarantees that the best alignments are produced. On the other hand, the algorithm proposed by Smith-Waterman obtains the best local alignments at the expense of very high computing power and huge memory requirements. In this article, we present and evaluate our experiments with three strategies to run the Smith-Waterman algorithm in a cluster of workstations using a distributed shared memory system. Our results on an eight-machine cluster presented very good speedups and indicate that impressive improvements can be achieved, depending on the strategy used. Also, we present some theoretical remarks on how to reduce the amount of memory used.

Comparing Two Long Biological Sequences Using a DSM System

Distributed Shared Memory systems allow the use of the shared memory programming paradigm in distributed architectures where no physically shared memory exist. Scope consistent software DSMs provide a relaxed memory model that reduces the coherence overhead by ensuring consistency only at synchronisation operations, on a per-lock basis. Sequence comparison is a basic operation in DNA sequencing projects, and most of sequence comparison methods used are based on heuristics, that are faster but do not produce optimal alignments. Recently, many organisms had their DNA entirely sequenced, and this reality presents the need for comparing long DNA sequences, which is a challenging task due to its high demands for computational power and memory. In this article, we present and evaluate a parallelisation strategy for implementing a sequence alignment algorithm for long sequences in a DSM system. Our results on an eight-machine cluster presented good speedups, showing that our parallelisation strategy and programming support were appropriate.

Provenance in bioinformatics workflows.

In this work, we used the PROV-DM model to manage data provenance in workflows of genome projects. This provenance model allows the storage of details of one workflow execution, e.g., raw and produced data and computational tools, their versions and parameters. Using this model, biologists can access details of one particular execution of a workflow, compare results produced by different executions, and plan new experiments more efficiently. In addition to this, a provenance simulator was created, which facilitates the inclusion of provenance data of one genome project workflow execution. Finally, we discuss one case study, which aims to identify genes involved in specific metabolic pathways of Bacillus cereus, as well as to compare this isolate with other phylogenetic related bacteria from the Bacillus group. B. cereus is an extremophilic bacteria, collected in warm water in the Midwestern Region of Brazil, its DNA samples having been sequenced with an NGS machine.