Modernas tecnicas de biologia molecular sao utilizadas para quantificar os niveis de expressao genica de amostras celulares, frequentemente submetidas a diferentes condicoes experimentais. A genomica funcional e a area da bioinformatica responsavel pela caracterizacao do papel funcional dos genes e processos biologicos associados. A analise de expressao genica requer o uso (integrado) de diversas ferramentas de analise para a obtencao de respostas as questoes biologicas de interesse. Uma plataforma de analise pode ser utilizada para facilitar a integracao sintatica e semantica destas ferramentas. Tal plataforma deve oferecer acessibilidade, reprodutibilidade e transparencia, de modo a promover a colaboracao e a disseminacao da pesquisa cientifica. A plataforma SemanticSCo foi concebida para prover suporte a composicao semantica de servicos de analise de expressao genica. Contudo, esta plataforma, em sua versao standalone, apresenta baixa acessibilidade, alem de nao prover suporte para a reproducao e o compartilhamento dos workflows desenvolvidos por seus usuarios. O principal objetivo deste projeto foi ampliar a acessibilidade da plataforma SemanticSCo, bem como introduzir suporte para a reproducao e compartilhamento de workflows. Como resultado principal, desenvolvemos uma nova versao da plataforma SemanticSCo, chamada SemanticSCo Web. A plataforma SemanticSCo Web possui uma interface simples e amigavel, contribuindo para o desenvolvimento colaborativo de pesquisa cientifica reproduzivel no dominio da genomica funcional. Adicionalmente, desenvolvemos uma API chamada SIBRI para permitir a execucao automatica, via framework Activiti, de servicos RESTful modelados como processos BPMN. O uso desta API facilita a integracao de aplicacoes desenvolvidas na linguagem Java e que fazem uso de servicos RESTful, dado que esta API prove suporte a interacao com o usuario em um ambiente web para que o mesmo possa prover valores de parametros que serao utilizados na execucao de um dado servico. Desta forma, a API SIBRI simplifica a incorporacao de novos servicos na plataforma SemanticSCo Web. / Modern molecular biology techniques are used to quantify the levels of gene expression of cellular samples, often submitted to different experimental conditions. Functional genomics is the area of bioinformatics responsible for the characterization of the functional role of genes and associated biological processes. The analysis of gene expression requires the (integrated) use of several analysis tools to obtain answers to a given biological question. An analysis platform can be used to facilitate the syntactic and semantic integration of these tools. Such platform should offer accessibility, reproducibility and transparency in order to promote collaboration and dissemination of scientific research. The SemanticSCo platform was designed to provide support for the semantic composition of gene expression analysis services. However, this platform, in its standalone version, presents low accessibility, and does not provide support for the reproduction and sharing of developed workflows. The main objective of this project was to increase the accessibility of the SemanticSCo platform, as well as to introduce support for the reproduction and sharing of workflows (transparency). As a main result, we have developed a new version of the SemanticSCo platform, called SemanticSCo Web. The SemanticSCo Web platform has a simple and user-friendly interface, contributing to the collaborative development of reproducible scientific research in the functional genomics domain. Additionally, we have developed an API called SIBRI to allow the automatic execution, via Activity framework, of RESTful services modeled as BPMN processes. The use of this API facilitates the integration of applications developed in Java that uses RESTful services, since this API provides support for the interaction with the end user in a web environment so s/he can provide parameters to be used in the execution of a given service. In this way, the API SIBRI simplifies the incorporation of new services in the SemanticSCo Web platform.

Modernas tecnicas de biologia molecular sao utilizadas para quantificar os niveis de expressao genica de amostras celulares, frequentemente submetidas a diferentes condicoes experimentais. A genomica funcional e a area da bioinformatica responsavel pela caracterizacao do papel funcional dos genes e processos biologicos associados. A analise de expressao genica requer o uso (integrado) de diversas ferramentas de analise para a obtencao de respostas as questoes biologicas de interesse. Uma plataforma de analise pode ser utilizada para facilitar a integracao sintatica e semantica destas ferramentas. Tal plataforma deve oferecer acessibilidade, reprodutibilidade e transparencia, de modo a promover a colaboracao e a disseminacao da pesquisa cientifica. A plataforma SemanticSCo foi concebida para prover suporte a composicao semantica de servicos de analise de expressao genica. Contudo, esta plataforma, em sua versao standalone, apresenta baixa acessibilidade, alem de nao prover suporte para a reproducao e o compartilhamento dos workflows desenvolvidos por seus usuarios. O principal objetivo deste projeto foi ampliar a acessibilidade da plataforma SemanticSCo, bem como introduzir suporte para a reproducao e compartilhamento de workflows. Como resultado principal, desenvolvemos uma nova versao da plataforma SemanticSCo, chamada SemanticSCo Web. A plataforma SemanticSCo Web possui uma interface simples e amigavel, contribuindo para o desenvolvimento colaborativo de pesquisa cientifica reproduzivel no dominio da genomica funcional. Adicionalmente, desenvolvemos uma API chamada SIBRI para permitir a execucao automatica, via framework Activiti, de servicos RESTful modelados como processos BPMN. O uso desta API facilita a integracao de aplicacoes desenvolvidas na linguagem Java e que fazem uso de servicos RESTful, dado que esta API prove suporte a interacao com o usuario em um ambiente web para que o mesmo possa prover valores de parametros que serao utilizados na execucao de um dado servico. Desta forma, a API SIBRI simplifica a incorporacao de novos servicos na plataforma SemanticSCo Web. / Modern molecular biology techniques are used to quantify the levels of gene expression of cellular samples, often submitted to different experimental conditions. Functional genomics is the area of bioinformatics responsible for the characterization of the functional role of genes and associated biological processes. The analysis of gene expression requires the (integrated) use of several analysis tools to obtain answers to a given biological question. An analysis platform can be used to facilitate the syntactic and semantic integration of these tools. Such platform should offer accessibility, reproducibility and transparency in order to promote collaboration and dissemination of scientific research. The SemanticSCo platform was designed to provide support for the semantic composition of gene expression analysis services. However, this platform, in its standalone version, presents low accessibility, and does not provide support for the reproduction and sharing of developed workflows. The main objective of this project was to increase the accessibility of the SemanticSCo platform, as well as to introduce support for the reproduction and sharing of workflows (transparency). As a main result, we have developed a new version of the SemanticSCo platform, called SemanticSCo Web. The SemanticSCo Web platform has a simple and user-friendly interface, contributing to the collaborative development of reproducible scientific research in the functional genomics domain. Additionally, we have developed an API called SIBRI to allow the automatic execution, via Activity framework, of RESTful services modeled as BPMN processes. The use of this API facilitates the integration of applications developed in Java that uses RESTful services, since this API provides support for the interaction with the end user in a web environment so s/he can provide parameters to be used in the execution of a given service. In this way, the API SIBRI simplifies the incorporation of new services in the SemanticSCo Web platform.

<p>Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis.</p><p>The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization.</p><p>Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.</p>

<p>Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.</p><p>This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.</p><p>The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.</p><p>The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an <i>in vitro</i> model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, <i>Yersinia pseudotuberculosis</i>, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.</p>

Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis. The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization. Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.

Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine. The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system. The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.

The formation of blood, hematopoiesis, is a dynamic process originating from a small number of hematopoietic stem cells (HSCs). To sustain hematopoiesis throughout life HSCs have the unique capacity to differentiate into all mature hematopoietic lineages as well as generating more HSCs by a mechanism referred to as self-renewal. However, the regulation of these processes is largely unknown. During embryonic development HSCs expand in the fetal liver, indicating that this environment supports HSC self-renewal. The LIM-homeobox gene Lhx2 is expressed in the fetal liver during this period and Lhx2 null mutant mice die in utero due to severe anemia caused by an environmental defect in the fetal liver. Embryonic stem cells differentiate in vitro, forming embryoid bodies (EBs) containing various tissues including hematopoietic progenitor cells. Introduction of Lhx2 into this system by retroviral transfer led to the generation of cytokine dependent HSC-like cell lines that were multipotent and expressed surface markers similar to embryonic HSCs. However, the specificity and efficiency of this event could not be elucidated. To further evaluate the function of Lhx2 expression during hematopoietic development, Lhx2 was introduced into an ES cell system where expression could be efficiently turned on. This approach revealed that Lhx2 induce self-renewal of distinct multipotent hematopoietic progenitor/stem cells present in the EB, with the ability to form HSC-like cell lines. The Lhx2 induced self-renewal is growth factor specific since stem cell factor and interleukin-6 are necessary and sufficient for this process. However, Lhx2 expression blocked erythroid differentiation and interfered with early ES cell commitment, indicating that the effect of Lhx2 is cell type specific. Since HSCs of early embryonic origin are inefficient in engrafting adult recipients upon transplantation, we wanted to address whether we could generate cell lines retaining this capacity by expression of Lhx2 in hematopoietic cells from adult bone marrow. This led to the generation of clonal and cytokine dependent HSC-like cell lines capable of generating erythroid, myeloid and lymphoid cells upon transplantation into lethally irradiated recipients. When transplanted into stem cell-deficient mice, they contributed to circulating erythrocytes for at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. However, expression of Lhx2 was maintained in vivo and most engrafted mice developed a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia. Thus, elucidation of the mechanism for Lhx2 function in HSC-like cell lines would give insights into both normal and pathological regulation of HSCs. Down-regulation of Lhx2 expression in HSC-like cell lines with inducible Lhx2 expression led to rapid loss of stem cell characteristics and differentiation into various hematopoietic cell types. Thus, global gene expression analysis comparing Lhx2+ HSC-like cell lines to their Lhx2- progeny would give insights into the molecular basis for Lhx2 function in stem cells. A number of differentially expressed genes overlapped with previously reported HSC enriched genes, further emphasizing the resemblance between HSCs and the HSC-like cell lines also at the molecular level. Moreover, a number of genes were identified with functions or expression patterns related to Lhx2 in other organs. Collectively, these data suggest that these HSC-like cell lines represent a relevant model system for normal HSCs on the molecular and the functional level as well as for evaluating Lhx2 function in the development of various tissues in the embryo as well as in disease.

De plus en plus d’applications ont pour objectif d’automatiser l’analyse des comportements humains afin d’aider les experts qui réalisent actuellement ces analyses. Cette thèse traite de l’analyse des expressions faciales qui fournissent des informations clefs sur ces comportements.Les travaux réalisés portent sur une solution innovante, basée sur l’organisation des expressions, permettant de définir efficacement une expression d’un visage.Nous montrons que l’organisation des expressions, telle que définie, est universelle : une expression est alors caractérisée par son intensité et sa position relative par rapport aux autres expressions. La solution est comparée aux méthodes classiques et montre une augmentation significative des résultats de reconnaissance sur 14 expressions non basiques. La méthode a été étendue à des sujets inconnus. L’idée principale est de créer un espace d’apparence plausible spécifique à la personne inconnue en synthétisant ses expressions basiques à partir de déformations apprises sur d’autres sujets et appliquées sur le neutre du sujet inconnu. La solution est aussi mise à l’épreuve dans un environnement multimodal dont l’objectif est la reconnaissance d’émotions lors de conversations spontanées. Notre méthode a été mise en œuvre dans le cadre du challenge international AVEC 2012 (Audio/Visual Emotion Challenge) où nous avons fini 2nd, avec des taux de reconnaissance très proches de ceux obtenus par les vainqueurs. La comparaison des deux méthodes (la nôtre et celles des vainqueurs) semble montrer que l’extraction des caractéristiques pertinentes est la clef de tels systèmes. / More and more applications aim at automating the analysis of human behavior to assist or replace the experts who are conducting these analyzes. This thesis deals with the analysis of facial expressions, which provide key information on these behaviors.Our work proposes an innovative solution to effectively define a facial expression, regardless of the morphology of the subject. The approach is based on the organization of expressions.We show that the organization of expressions, such as defined, is universal and can be effectively used to uniquely define an expression. One expression is given by its intensity and its relative position to the other expressions. The solution is compared with the conventional methods based on appearance data and shows a significant increase in recognition results of 14 non-basic expressions. The method has been extended to unknown subjects. The main idea is to create a plausible appearance space dedicated to the unknown person by synthesizing its basic expressions from deformations learned on other subjects and applied to the neutral face of the unknown subject. The solution is tested in a more comprehensive multimodal environment, whose aim is the recognition of emotions in spontaneous conversations. Our method has been implemented in the international challenge AVEC 2012 (Audio / Visual Emotion Challenge) where we finished 2nd, with recognition rates very close to the winners’ ones. Comparison of both methods (ours and the winners’ one) seems to show that the extraction of relevant features is the key to such systems.

Made available in DSpace on 2014-06-12T15:50:15Z (GMT). No. of bitstreams: 2 arquivo1959_1.pdf: 2352925 bytes, checksum: 90760b286db4ed0dcc12ae48554413a9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos recentes têm demonstrado que as redes biológicas apresentam características nãoaleatórias, dentre as quais destacamos a arquitetura modular. Neste trabalho, estamos interessados na organização modular das redes de regulação transcricional (RRT), que modelizam as interações entre genes e proteínas que controlam a sua expressão no nível transcricional. Compreender os mecanismos de regulação transcricional é crucial para se explicar a diversidade morfológica e funcional das células. Nós nos propomos a abordar o problema da identificação de módulos regulatórios transcricionais, i.e. grupos de genes co-regulados e seus reguladores, com ênfase no aspecto computacional. Uma distinção importante deste trabalho é que estamos também interessados em estudar o aspecto evolutivo dos módulos transcricionais. Do ponto de vista biológico, a abordagem proposta está fundamentada em três premissas principais: (i) genes co-regulados são controlados por proteínas regulatórias comuns (fatores de transcrição FTs) e, portanto, eles devem apresentar padrões de sequência (motifs) comuns nas suas regiões regulatórias, que correspondem aos sítios de ligação desses FTs, (ii) genes co-regulados respondem coordenadamente a certas condições ambientais e de desenvolvimento e, logo, devem ser co-expressos sob essas condições, e (iii) uma vez que módulos transcricionais são presumivelmente responsáveis por funções biológicas importantes, eles estão sujeitos a uma maior pressão seletiva e, consequentemente, devem ser evolutivamente conservados. Nós definimos, portanto, o conceito de metamódulo regulatório transcricional (MMRT) como grupos de genes compartilhando motifs e exibindo um comportamento de expressão coerente em contextos específicos consistentemente em várias espécies e propomos modelos probabilísticos para descrever o comportamento modular em termos do compartilhamento de elementos regulatórios (motifs), da co-expressão e da conservação evolutiva das associações funcionais entre os genes com base em dados diversos tais como dados de sequência, de expressão e dados filogenéticos

Made available in DSpace on 2014-07-29T15:16:30Z (GMT). No. of bitstreams: 1 DISSERTACAO RACHEL SILVEIRA FREITAS.pdf: 844126 bytes, checksum: 0544e2973ae364fb2429cc263d62f87a (MD5) Previous issue date: 2011-06-30 / Species of the genus Trichoderma have been used as biocontrol agents against different pathogens. The mechanisms employed by Trichoderma species against these pathogens ranging from competition for nutrients, production of non-volatile and volatile antibiotics in the production of hydrolytic enzymes, in a mechanism denominated mycoparasitism. In addition to its characteristics, many strains of Trichoderma are competent rhizosphere are able to colonize and grow in association with plant roots. The root colonization by Trichoderma spp., often is associated with the induction of local and systemic resistance. Whereas fungi of the genus Trichoderma have been described as inducers of defense responses and systemic resistance in association with maize (Zea mays), cucumber, tomato and cotton, it was our interest to analyze the interaction between Trichoderma spp. and beans. Therefore, this study aimed to identify and evaluate gene expression of protein elicitors of defense (SM1) in different isolates of Trichoderma spp. obtained from Cerrado soils. Eight isolates were selected and all showed a band of approximately 250pb, corresponding to the expected size of the gene Sm1 from T. Virens. The complete sequence of SM1 gene was obtained using as template cDNA and genomic DNA of T. harzianum. The amplification products containing an ORF of 417 bp and the protein predicted from this sequence has 138 amino acids. The ORF showed identity with sequences of other protein elicitors isolated from Trichoderma spp., and also proteins belonging to the family of cerato-platanin. Studies of the expression of SM1 with the isolate Trichoderma 37 showed that this protein is expressed in different carbon sources. / Espécies do gênero Trichoderma têm sido utilizadas como agentes de controle biológico contra diferentes tipos de fitopatógenos. Os mecanismos utilizados pelas espécies de Trichoderma contra esses fitopatógenos vão desde a competição por nutrientes, produção de antibióticos voláteis e não voláteis à produção de enzimas hidrolíticas, em um mecanismo denominado micoparasitismo. Em adição a estas características, muitas linhagens de Trichoderma são rizosfera competentes e são capazes de colonizar e crescer em associação com as raízes das plantas. A colonização da raiz pelo Trichoderma spp., frequentemente, é associada com a indução de resistência local e sistêmica. Considerando que fungos do gênero Trichoderma já foram descritos como indutores de resposta de defesa e resistência sistêmica em associação com milho (Zea mays), pepineiro, tomateiro e algodoeiro, foi nosso interesse analisar a interação entre Trichoderma spp., e feijoeiro. Sendo assim, este trabalho teve por objetivo identificar e avaliar a expressão do gene de uma proteína elicitora de defesa (Sm1) em diferentes isolados de Trichoderma spp. obtidos de solos do Cerrado. Oito isolados foram selecionados e todos apresentaram uma banda de aproximadamente 250pb, correspondendo ao tamanho esperado do gene Sm1 de T. virens. A seqüência completa do gene da Sm1 foi obtida utilizando como molde cDNA e DNA genômico de T. harzianum. Os produtos da amplificação contém uma ORF de 417 pb e a proteína predita a partir dessa seqüência tem 138 aminoácidos. Esta ORF apresentou identidade com seqüência de proteínas elicitoras de outros isolados de Trichoderma spp. e também com proteínas pertencentes a família das ceratoplataninas. Estudos de expressão da Sm1 com isolado Trichoderma 37 mostrou que esta proteína é expressa em diferentes fontes de carbono.