<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

High-throughput time series data characterizing magnitudes of gene expression, levels of protein activity, and the accumulation of select metabolites in vivo are being generated with increased frequency. These time profiles contain valuable information about the structure, dynamics and underlying regulatory mechanisms that govern the behavior of cellular systems. However, extraction and integration of this information into fully functional, computational and explanatory models has been a daunting task. Three types of issues have prevented successful outcomes in this inverse task of system identification. The first type pertains to the algorithmic and computational difficulties encountered in parameter estimation, be it using a genetic algorithm, nonlinear regression, or any other technique. The second type of issues stems from implicit assumptions that are made about the system topology and/or the functional model representing the biological system. These include the choice of intermediate pathway steps to be accounted for in the model, decisions on the irreversibility of a step, and the inclusion of ill-characterized regulatory signals. The third type of issue arises from the fact that there is often no unique set of parameter values, which when fitted to a model, reproduces the observed dynamics under one or several different sets of experimental conditions. This latter issue raises intriguing questions about the validity of the parameter values and the model itself. The central focus of my research has been to design a workflow for parameter estimation and system identification from biological time series data that resolves the issues outlined above. In this thesis I present the theory and application of a novel framework, called Dynamic Flux Estimation (DFE), for system identification from biological time-series data.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

Orientador: Eduardo Galembeck / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T17:08:58Z (GMT). No. of bitstreams: 1 Oliveira_ElaineAparecidade_M.pdf: 4028860 bytes, checksum: 12a75ba90b00d8eded4dc1107cc6168a (MD5) Previous issue date: 2010 / Resumo: Representações externas são muito utilizadas no ensino de bioquímica. As vias metabólicas constituem um tipo de representação externa e envolvem habilidades visuais; estão, portanto, intimamente relacionados à literacia visual (capacidade de interpretar imagens). Há uma preocupação crescente em diagnosticar a dificuldade dos estudantes com a bioquímica. Este tipo de diagnóstico é importante para poder propor soluções pedagógicas que possam facilitar o aprendizado. O presente trabalho tem como objetivo descrever o desenvolvimento e a aplicação de um teste informatizado para diagnosticar a literacia visual, relacionada ao estudo de vias metabólicas, de estudantes de cursos das áreas biológica e da saúde. Inicialmente foram desenvolvidos testes piloto com itens (modelos e questões) que contemplavam habilidades visuais para a compreensão de vias metabólicas mais frequentemente representadas em livros didáticos de bioquímica. Os testes piloto foram aplicados em 2007 e 2008 com turmas de graduação de cursos da área biológica e da saúde. Os resultados obtidos serviram de subsídio para que alguns itens fossem reformulados e outros adicionados. Os resultados obtidos foram tratados com uso do modelo de Rasch, tanto no processo de equalização dos testes piloto (para definir os itens do teste final), como para o tratamento dos resultados obtidos com a calibração de itens-respondentes após as aplicações do teste final. Três grupos participaram da aplicação do teste final e compreenderam 79 estudantes distribuídos entre os cursos de educação física (UNICAMP), enfermagem (UNICAMP) e fisioterapia/nutrição (FAJ). A partir dos dados obtidos utilizou-se o processo de calibração de itens-respondentes, proposta pelo modelo de Rasch, obtendo-se a dificuldade dos itens e as habilidades visuais apresentadas pelos grupos. Os resultados observados sugerem que entre as dificuldades apresentadas pelos estudantes no aprendizado de metabolismo pode ser incluída a dificuldade em se compreender as representações externas de vias metabólicas. Essa dificuldade pode ser minimizada com explicações dos modelos antes de se entrar nos conteúdos específicos. O teste produzido permite o diagnóstico de estudantes ou de grupos. A necessidade de desenvolver práticas de visualização para melhorar a compreensão de conceitos bioquímicos pelos estudantes mostrou-se um aspecto importante no estudo de vias metabólicas / Abstract: External representations are used in teaching of biochemistry. The metabolic pathways are a type of external representation and involve visual skills, are therefore closely related to visual literacy (ability to interpret images). There is an increasing concern regarding the diagnosis of students with difficulty to the biochemistry, this type of diagnosis is important to be able to propose solutions that would facilitate learning. This paper aims to describe the development and implementation of a computerized test to diagnose the visual literacy, related to the study of metabolic pathways for undergraduate students enrolled in courses in the biological and health. Initially, pilot tests have been developed with (models and items) that looked visual skills for the understanding of metabolic pathways most often represented in textbooks of biochemistry. The pilot tests were applied in 2007 and 2008 on undergraduate classes of various courses in the biological and health. The results served as a subsidy for some items were reformulated and others added. The results are processed using the Rasch model, both in the process of equalizing the pilot tests (for set of the final test) and for the treatment of the results obtained with calibration of items-respondents the applications for the final test. Three groups participated in the implementation of the final test and included 79 students distributed among the courses of physical education (UNICAMP), nursing (UNICAMP) and physiotherapy/nutrition (FAJ). From the data we used the calibration of items-respondents proposed by Rasch model resulting in the difficulty of items and visual skills presented by the groups. The results suggest that among the difficulties presented by the students in the learning of metabolism may be included the difficulty in understanding the external representations of metabolic maps. This difficulty can be minimized with the explanations of the models before they enter the specific content. The test produced allows the diagnosis of students or groups. The need of developing imagery to improve the understanding of biochemical concepts by students were an important aspect in the study of metabolic pathways / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

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Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

RNA-Seq is an increasingly popular approach to transcriptome profiling that uses the capabilities of next generation sequencing technologies and provides better measurement of levels of transcripts and their isoforms. In this thesis, we apply RNA-Seq protocol and transcriptome quantification to estimate gene expression and pathway activity levels. We present a novel method, called IsoDE, for differential gene expression analysis based on bootstrapping. In the first version of IsoDE, we compared the tool against four existing methods: Fisher's exact test, GFOLD, edgeR and Cuffdiff on RNA-Seq datasets generated using three different sequencing technologies, both with and without replicates. We also introduce the second version of IsoDE which runs 10 times faster than the first implementation due to some in-memory processing applied to the underlying gene expression frequencies estimation tool and we also perform more optimization on the analysis. The second part of this thesis presents a set of tools to differentially analyze metabolic pathways from RNA-Seq data. Metabolic pathways are series of chemical reactions occurring within a cell. We focus on two main problems in metabolic pathways differential analysis, namely, differential analysis of their inferred activity level and of their estimated abundance. We validate our approaches through differential expression analysis at the transcripts and genes levels and also through real-time quantitative PCR experiments. In part Four, we present the different packages created or updated in the course of this study. We conclude with our future work plans for further improving IsoDE 2.0.