Bioinformatics methodologies for detection and study of repetitive sequences in gene loci of chimeric transcripts

Orientador: Michel Eduardo Beleza YamagishiTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A grande quantidade de dados biológicos gerados recentemente permitiu verificar que os genomas são repletos de seqüências repetitivas (SR), como microsatélites e elementos genéticos móveis, altamente improváveis de ocorrer estatisticamente se os genomas fossem gerados a partir de uma distribuição aleatória de nucleotídeos. Tal comprovação motivou a classificação de tais seqüências e também a construção de diversas ferramentas de bioinformática, além de mecanismos de armazenamento baseados em sistemas de gerenciamento de bancos de dados (SGBD) para permitir localizá-las e armazená-las para posterior estudo. Entretanto, foi com a comprovação biológica da importância das SR, como no mecanismo de interferência por RNAi (SR reversa complementar), que as SR despertaram maior interesse por parte da comunidade científica. Atualmente, já há fortes evidências que associam as SR com fenômenos biológicos bastante interessantes, como o processamento de RNA por cis-splicing e a formação de transcritos quiméricos, freqüentes em organismos inferiores e muito raro em organismos superiores. Tais tipos de transcritos podem ser gerados a partir de trans-splicing ou, como conjecturamos nesse trabalho, pela transposição de elementos genéticos móveis (como por exemplo transposons ou retrotransposons). Em virtude disso, este projeto propõe a construção de metodologias de Bioinformática, disponibilizadas na WEB, para detectar transcritos quiméricos em genomas de organismos, tanto em versões draft ou em alta qualidade, e também estudar as SR que ocorrem no locus gênico dos transcritos envolvidos na formação de uma seqüência quimérica. As ferramentas propostas permitiram identificar, a partir de bibliotecas de transcritos de full-length cDNA, tanto de humanos quanto de bovinos, novos transcritos quiméricos provenientes de células de tecidos normais, e que não seguem splice-sites canônicos na região de fusão dos transcritos envolvidos. Além disso, as seqüências encontradas apresentam uma elevada taxa de concentração de pares de SR do tipo reverso complementar no locus gênico dos dois transcritos que formam a seqüência quimérica. As ferramentas propostas podem ser utilizadas para outros organismos e direcionar trabalhos experimentais para tentar comprovar em bancada novos transcritos quiméricos, tanto em organismos inferiores quanto em superioresAbstract: The recent availability of a huge amount of biological data allowed to know about the high concentration of repetitive sequences (SR) like microsatellites and genetic mobile elements in different genomes. Repetitive sequences are improbable to occur statistically if genome data were generated by a random distribution of nucleotides. Such observation motivated the classification of repetitive sequences, and the construction of several bioinformatics tools. Furthermore, several mechanisms to store repetitive sequences, which are based on data base management systems (DBMS) were proposed and created. They can be used to search for specific sequences to make a posteriori study. However, it was with the biological confirmation of the importance of repetitive sequences, like by the RNA interference (reverse complement, or inverted repeat) mechanism, that the scientific community gained more interest by such sequences. Actually, there is strong evidence that associates the repetitive sequences with some interesting biological phenomena, like in RNA processing by cis-splicing, and in chimeric transcript formation mechanism. This last one is very frequently in inferior organism, but rare in superior organisms. Such types of transcripts can be generated by trans-splicing, or like conjectured in this work, by the retrotransposition of mobile genetic elements (like transposons or retrotransposons). In this way, this work proposed the construction of several Bioinformatics methodologies, available in the WEB, to detect new evidences of chimeric transcripts in genomes of different organisms, both in draft genome and in high quality genome assemblage. We also studied repetitive sequences in gene loci of the involved transcripts in a chimeric sequence formation. The proposed tools allowed us to identify, using a full-length cDNA databank, new chimeric transcript candidates in human and in bovine genome. They are from cells of normal tissues, and do not follow canonical splice-sites in the fusion region of the involved transcripts. Moreover, it was possible to show that the detected sequences have high concentration pairs of reverse complement type of repetitive sequences in gene loci of the two involved transcripts, which originated a new chimeric transcript candidate. The created bioinformatics tools can be used in other organisms in addition to the one used in this work, leading to the proposition of new experimental work to try to prove in vivo new chimeric transcripts, both in superior organism and in inferior organismDoutoradoBioinformaticaDoutor em Genetica e Biologia Molecula

Saprochaete clavata invasive infection: characterization, antifungal susceptibility, and biofilm evaluation of a rare yeast isolated in Brazil

Rare emerging pathogens such as Saprochaete clavata are associated with invasive fungal diseases, high morbidity, mortality, rapidly fatal infections, and outbreaks. However, little is known about S. clavata infections, epidemiology, risk factors, treatment, biofilms, and disease outcomes. The objective of this study was to describe a new case of severe S. clavata infection in a patient diagnosed at a referral children’s hospital in Brazil, including antifungal minimal inhibitory concentration, S. clavata biofilm characterization, and molecular characterization. The S. clavata isolated from an immunocompromised 11-year-old male patient was characterized using MALDI-TOF, Gram staining, scanning electron microscopy (SEM), and next generation sequencing (NGS) of genomic DNA. Biofilm production was also evaluated in parallel with determining minimal inhibitory concentration (MIC) and biofilm sensitivity to antifungal treatment. We observed small to medium, whitish, farinose, dry, filamentous margin colonies, yeast-like cells with bacillary features, and biofilm formation. The MALDI-TOF system yielded a score of ≥ 2,000, while NGS confirmed S. clavata presence at the nucleotide level. The MIC values (in mg L-1) for tested drugs were as follows: fluconazole = 2, voriconazole ≤ 2, caspofungin ≥ 8, micafungin = 2, amphotericin B = 4, flucytosine ≤ 1, and anidulafungin = 1. Amphotericin B can be active against S. clavata biofilm and the fungus can be susceptible to new azoles. These findings were helpful for understanding the development of novel treatments for S. clavata-induced disease, including combined therapy for biofilm-associated infections

Proposal of a performance modeling and analytical prediction system for a parallel processing system

Orientador: Marco Aurelio Amaral HenriquesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de ComputaçãoResumo: A predição de desempenho é um importante mecanismo para avaliar a utilização de recursos e estimar o tempo de execução de aplicações em sistemas paralelos. Este trabalho apresenta uma ferramenta que permite criar modelos que representam características da aplicação e dos computadores utilizados para processá-la. Tais modelos são combinados para gerar um modelo de desempenho mais abrangente, cuja análise permite obter estimativas de tempo de execução que contemplem fatores de atraso tais como operações aritméticas sobre diferentes tipos de dados e efeitos de contenção, causados por concorrência. As estimativas podem ser geradas em poucos segundos e permitem analisar o impacto causado na aplicação pela utilização de diferentes configurações do sistema de processamento paralelo. A ferramenta foi implementada para um sistema de processamento paralelo baseado em Java, chamado JoiN, e ela mostrou ser possível obter estimativas satisfatórias de tempos de execução para diversos tipos de aplicações paralelasAbstract: Performance prediction is an important mechanism to evaluate the use of resources and predict the execution time of applications in parallel systems. This work presents a tool for the creation of models that represent applications and computers characteristics. Such models are combined to generate a performance model, whose analysis derives execution time estimates that include delay factors, such as arithmetic operations on different data types and contention effects caused by process concurrence. The estimates can be generated in a few seconds and allow the analysis of the impact caused in the application by changes in the parallel system configuration. The tool was implemented for a Java based parallel processing system called JoiN and it showed that satisfactory execution time estimates can be obtained for several types of parallel applicationsMestradoEngenharia de ComputaçãoMestre em Engenharia Elétric

Method And Use For Verification Of Mounting Errors In Genomes

Resumo não disponível.BR102012031096 (A2)G06F19/10BR20121031096G06F19/1

Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction

Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human’s health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (n = 3), CH (n = 4) and MD (n = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three TMD conditions showed different protein expression profiles, and novel proteins were identified in both synovial fluid and disc sample. TMD is a complex condition and the identification of the proteins expressed in the three different types of TMD may contribute to a better comprehension of how each pathology develops and evolutes, benefitting the patient with a focus–target treatment

Evaluation of Lipid Profile in Adolescents

<div><p>Abstract Background: Atherosclerosis is a chronic, multifactorial and insidious disease that can begin in childhood and adolescence, and whose major consequences appear during adulthood. Serum levels of lipoproteins, such as LDL-c, total cholesterol (TC), HDL-c, and non-HDL-c can be used as a screening method for disease diagnosis. In Brazil, few studies have correlated the serum levels of those lipoproteins with age. Objective: To evaluate the serum concentrations of TC, LDL-c, HDL-c, VLDL-c, non-HDL-c and triglycerides (TG) of adolescents aged 10 to 19 years in the municipality of Araucária, Paraná state. Methods: Cross-sectional retrospective study, collecting the following data from 600 adolescents: age, sex and serum levels of TC, LDL-c, HDL-c and TG from June to December 2016. Data were analyzed using the SPSS software 2.0, with Mann-Whitney U test and Spearman coefficient of correlation to identify statistical significance (p < 0.05). Results: The female sex showed higher serum levels of TC, TG and LDL-c than the male sex. The HDL-c levels were identical in both sexes, with 48% of desirable values and 52% of low values. This study identified a strong correlation between the lipids and association with the age group of 10 to 14 years. Conclusion: Non-HDL-c showed stronger correlation with the other lipids (TG, LDL-c and TC) as compared to LDL-c, suggesting that non-HDL-c can be used as an effective complementary diagnostic method to assess the risks for atherosclerosis in adolescents.</p></div

Transcriptional changes involved in kumquat (Fortunella spp) defense response to Xanthomonas citri subsp. citri in early stages of infection

Xanthomonas citri subsp. citri (Xcc), the causal agent of type A canker, differentially affects all commercial varieties of citrus plants. Among them, Fortunella spp (kumquat) have higher tolerance to disease development, although the molecular mechanisms involved in their defense are not fully understood. In this study, using RNA-seq approach, the transcriptional responses of kumquat leaves at 24 h after water (controls) and Xcc inoculation were analyzed. A total of 1439 Differentially expressed genes (DEG) with statistical significance (p-value<0.025) were identified, with 444 being upregulated. These genes were found to be involved in pathogen recognition, cell wall remodeling and reinforcement, lignin biosynthesis, reactive oxygen species (ROS) production, pathogenesis-related proteins (PR) and biosynthesis of secondary metabolites including phenylpropanoids, terpenoids and alkaloids. Genes related to disease resistance (R genes) and salicylic acid (SA)-dependent systemic acquired resistance (SAR) were also induced. In turn, the 995 downregulated genes were mainly associated with photosynthesis, photorespiration, chlorophyll synthesis and cell growth. This suggest that, once the pathogen is detected, the plant generates a strong oxidative burst and its cellular machinery is directed towards the synthesis of secondary metabolites and defense proteins while its own growth is inhibited. Overall, these transcriptional changes provide valuable information about the molecular basis of the defense in kumquat plants, which may be useful in the design of new control methods for citrus canker

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.121726072617Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FINEP in BrazilFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.FAPESPFAPESPFAPESP/CEPIDFAPESP (CEPID)CNPqCNPqCNPq (INCT)CNPq (INCT)FINEP in BrazilFINEP in BrazilFAPESP (INCT)FAPESP (INCT