Yersinia pestis, l'agent de la peste, est une bactérie à Gram-négatif classée comme agent pathogène ré-émergent et potentielle arme de bioterrorisme. De plus, l'apparition d'une souche multi-résistance de cette bactérie souligne la nécessité de mieux comprendre comment cette bactérie hyper-virulente interagit avec son hôte. Afin d'identifier des facteurs génétiques de vulnérabilité à la peste, notre laboratoire travaille sur la réponse de souris résistantes versus sensibles à Y. pestis. Notre stratégie pour identifier les facteurs génétiques impliqués dans la résistance/sensibilité à la peste combine une approche de cartographie de QTL (Quantitative Trait Locus) et d'analyse d'expression génique. Nous avons précédemment décrit la lignée SEG/Pas, issue de Mus spretus, comme la première résistante à une souche virulente de Y. pestis, alors que la plupart des lignées murines de laboratoire, telle que la lignée C57BL/6J, sont extrêmement sensible à la bactérie. Des croisements entre SEG/Pas et C57BL/6J nous ont permis d'identifier trois QTL impliqués dans la résistance à Y. pestis, localisés sur les chromosomes 3, 4 et 6. Deux des QTL (situés sur les chromosomes 4 et 6) ont pu être confirmés par l'analyse de lignées congéniques. Plus de 40 % des femelles bi-congéniques hétérozygotes pour ces deux QTL ont survécu à l'infection, alors que tous les témoins C57BL/6J ont succombé. La dissection de ces deux QTL par l'analyse de lignées sous-congéniques, nous a permis d'affiner l'architecture génétique de la résistance à la peste chez SEG/Pas. Nous avons conclu qu'un minimum de quatre facteurs génétiques, au sein de ces deux QTL, sont nécessaires pour augmenter la résistance à Y. pestis chez la Souris. Cependant, la production de plusieurs lignées congéniques portant le QTL situé sur le chromosome 3, dont une lignée triple congénique, ne nous a pas permis de confirmer l'existence de ce QTL. En parallèle de l'analyse génétique, nous avons déterminé que les macrophages de SEG/Pas et de C57BL/6J présentaient des caractéristiques différentes après exposition à Y. pestis. Une analyse différentielle du profil transcriptionnel des macrophages de ces deux lignées a été réalisée à l'aide de puces à ADN. Nos résultats montrent une forte activation de la production cytokinique dans les macrophages de SEG/Pas en réponse à la bactérie, activation qui n'est pas observée dans la lignée C57BL/6J. Ces résultats suggèrent que les souris SEG/Pas sont capables de mettre en place une réponse immune innée plus forte ou peut-être plus précoce que C57BL/6J. Nous avons ensuite étudié par qRT-PCR l'expression en cinétique de 44 gènes dans des macrophages de SEG/Pas, C57BL/6J et des bi-congéniques portant les QTL sur les chromosomes 4 et 6. Cette étude nous a permis de confirmer que les souris SEG/Pas sont capables se mettre en place une forte réponse inflammatoire lors de l'infection. Cependant, aucune différence significative n'a été observée entre la lignée bicongénique et la lignée parentale C57BL/6J. D'autres expériences seront nécessaires afin de mieux comprendre les mécanismes biologiques impliqués dans la résistance intermédiaire de cette lignée. La dissection génétique associée à l'analyse de l'expression génique de ces lignées résistante et sensible permet d'augmenter notre compréhension de la réponse de l'hôte à Y. pestis. / Yersinia pestis, the agent of plague, is a deadly gram-negative bacterium classified as a re-emerging pathogen and class A biological weapon. The appearance of a multi-resistant strain highlights the need to better understand how this pathogen kills its host. To identify genetic factors of host susceptibility to plague, our laboratory is investigating the response of resistant versus susceptible mice to Y. pestis. Our strategy to decipher genetic determinants involved in resistance to plague combines Quantitative Trait Loci (QTL) mapping with gene expression analysis. We previously described the Mus spretus-derived SEG/Pas strain as the first to resist fully virulent Y. pestis, while most inbred strains, such as C57BL/6, are highly susceptible. Crosses between these two strains identified three QTLs (located on chromosome 3, 4 and 6) contributing to resistance. Two of the QTLs (on chromosome 4 and 6) were confirmed through creation of congenic mice. Up to 40% of the congenic mice heterozygous at these two QTLs, on a C57BL/6J background, survived the infection while all C57BL/6J mice died. Further dissection of these two QTLs, through the use of subcongenic strains, enabled us to refine the genetic architecture of resistance to plague in SEG/Pas mice. We concluded that a minimum of four genetic factors, within these two QTLs, are required to increased resistance to Y. pestis in mice. Despite production of numerous congenic strains, including triple congenic mice, we were not able to confirm the existence of the third QTL identified on chromosome 3. In parallel to genetic studies, we determined that SEG/Pas and C57BL/6J macrophages exhibit distinct characteristics upon in vitro exposure to Y. pestis. The underlying molecular differences were investigated by using microarrays. Our results show strong activation of cytokines in SEG/Pas macrophages in response to Y. pestis, which is not found in C57BL/6J macrophages. These results suggest that SEG/Pas mice are able to better activate innate immune response to Y. pestis than C57BL/6J mice.We further studied the expression of 44 genes in a kinetic study on macrophages in vitro of SEG/Pas, C57BL/6J and bicongenic mice (carrying QTLs on chromosome 4 and 6). This study confirmed that SEG/Pas mice are able to build a stronger inflammatory response at early time of infection. Nevertheless no significant differences were observed in the bicongenic strain compared to C57BL/6J. Further studies will be required to understand the mechanisms involved in the intermediate resistance of this strain. This combination of genetic dissection and gene expression analysis of resistant and susceptible mouse strains will enhance our ability to better understand the host response to plague.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 144-167).

Objetivos: Investigar anomalias dentárias, presença de fissura de lábio e/ou palato e higiene bucal em indivíduos com síndrome de Kabuki (SK) e listar as alterações sistêmicas presentes. Metodologia: Grupo 1: 46 prontuários de indivíduos com SK matriculados no HRAC/USP analisados quanto a presença de fissura de lábio e/ou palato e listadas as alterações sistêmicas presentes (cardiopatias, doenças infecciosas e imunológicas, nefropatias, comprometimento neurológicos e repercussões, aspectos genéticos). Grupo 2: formado por 15 indivíduos com SK matriculados no HRAC/USP submetidos aos exames clínicos para a investigação das alterações bucais (anomalias dentárias, presença de fissura de lábio e/ou palato e avaliação da higiene bucal índice de placa) e doenças sistêmicas existentes. Resultados: Grupo 1 43 (93,47%) indivíduos apresentaram fissura de lábio e/ou palato, 36 apresentaram fissura de palato; 17 (36,95%) indivíduos apresentaram cardiopatia congênita, 36 (76,59%) indivíduos tiveram doenças infecciosas ou imunológicas, 8 (17,39%) indivíduos apresentaram nefropatias, 40 (86,95%) indivíduos tinham deficiência intelectual e 1 (2,77%) indivíduos apresentou cariótipo com alteração. Grupo 2 todos os indivíduos com SK apresentaram fissura de lábio e/ou palato, 11 (73,33%) indivíduos apresentaram anomalias dentárias, todos os indivíduos apresentaram comprometimento da higiene bucal, 5 (33,33%) indivíduos apresentaram cardiopatias congênitas, 12 (80%) tiveram doenças infecciosas ou imunológicas, em um indivíduo foi notada nefropatia, 14 (93,33%) indivíduos demonstraram deficiência intelectual e 1 (6,66%) apresentou cariótipo alterado. Conclusões: Os indivíduos com SK apresentaram anomalias dentárias, fissura de lábio e/ou palato, higiene bucal comprometida, cardiopatias congênitas, doenças infecciosas e deficiência intelectual. / Aim: To investigate dental anomalies, presence of lip and/or cleft palate and dental hygiene in individuals with Kabuki Syndrome (KS) as well as detail their systemic alterations. Methodology: GROUP 1: 46 prontuaries of individuals with KS (patients from HRAC/USP) were analyzed concerning to the presence of lip and cleft palate and their systemic alterations were described (cardiopathies, infectious and immunological diseases, nephropathies, neurological disorders and genetical aspects). GROUP 2: 15 individuals with KS (patients from HRAC/USP) submitted to the clinical examination to investigate buccal alterations (dental anomalies, presence of lip and/or cleft palate and dental hygiene - plaque index) and systemic alterations. Results: Group 1 - 43 (93.47%) individuals showed lip and/or cleft palate, 36 showed cleft palate; 17 (36.95%) individuals showed congenic cardiopathies, 36 (36.95%) individuals showed infectious or immunological diseases, 8 (17.39%) individuals showed nephropathies, 40 (86.95%) individuals had intellectual disability and 1 (2.77%) individuals showed cariotip with alteration. Group 2 - all the individuals with KS showed lip and/or cleft palate, 11 (73.33%) individuals showed dental anomalies, all the individuals showed compromised dental hygiene, 5 (33.33%) individuals showed congenic cardiopathies, 12 (80%) showed infectious or immunological diseases, in only 1 individual nephropathy was observed, 14 (93.33%) had intellectual disabilities and 1 (6.66%) showed a cariotip with alteration. Conclusions: Individuals with KS showed dental anomalies, presence of lip and/or cleft palate, compromised buccal hygiene, congenic cardiopathies, infectious diseases and intellectual disability.

Metabolic syndrome (MetS) is a complex condition with a number of interacting genes, epigenetic and environmental factors underlying its pathogenesis. The analysis of genetic component of MetS showed that number of defining parameters of the syndrome is linked to regions of rat chromosome 4. In order to verify these quantitative trait loci (QTL), a double congenic strain was derived with parts of chromosome 4 of spontaneously hypertensive rat (SHR, an inbred MetS model) origin introgressed onto genomic background of congenic Brown Norway strain (BN-Lx). The aim of the proposed thesis is comprise detail genetic mapping of differential segments of the above mentioned double congenic strain BN-Lx.SHR4 and comparison of its metabolic profile under different dietary conditions with varying carbohydrate and fat content. Utilizing DNA sequence and gene expression comparisons, candidate genes or polymorphisms for the MetS aspects and potential nutrigenetic interactions will be identified. Key words: nutrigenetics, experimental models, metabolic syndrome, congenic strain, genotyping, rat

A síndrome de Williams Beuren (SWB), doença congênita causada pela microdeleção do cromossomo 7, incluindo o gene da elastina, pode conferir às pessoas afetadas, facies típico, retardo mental, cardiopatia congênita, hipertensão, alterações gástricas, distúrbios de desenvolvimento de dentes, dentre outras alterações. O objetivo desse estudo foi conhecer as alterações faciais e bucais, a condição de saúde bucal, características oclusais e aspectos da ATM, bem como as alterações sistêmicas e condições médicas que afetam o manejo odontológico em uma amostra significativa de indivíduos brasileiros com a SWB. Para tanto, examinamos 25 indivíduos com SWB, com média de idade de 13 anos (4 a 26 anos). Os resultados mostraram que todos os participantes exibiam algum grau de retardo mental. A hiperacusia foi encontrada em 88% (22/25), cardiopatia congênita, em 76% (19/25); especialmente estenose aórtica supravalvar), hiperatividade em 68% (17/25) e hipertensão arterial em 40% (10/25) dos participantes. Distúrbios de desenvolvimento de dente foram encontrados em todos os participantes sendo que o mais frequente foi a retenção prolongada de dentes decíduos (64% - 16/25), seguido do diastemas generalizados (60% - 15/25), anodontia parcial (42% - 9/21), hipoplasia de esmalte (28% - 7/25), incisivos em forma de chave de fenda (24% - 6/25), microdontia (8% - 2/25). Em iguais porcentagens (4% - 1/25), foram encontrados casos de geminação, taurodontismo e dente conóide. Quanto à presença de alterações oclusais, todos os pacientes examinados apresentaram maloclusão. A maioria exibiu maloclusão classe III dentária de Angle (11/19,57 %) e mordida cruzada (11/25, 44%). Setenta e dois por cento dos pacientes exibiam algum hábito parafuncional. A experiência presente e passada de cárie, entre os 25 pacientes examinados, foi classificada como muito baixa, de acordo com o índice CPO-D e ceo-d, e 76% apresentaram gengivite. Os pacientes exibiram alteração da amplitude dos movimentos excursivos de mandíbula (abertura máxima - 12/22, 54%; lateralidade - 6/9, 66%; e protrusão - 7/9; 77%), bem como alterações nas ATMs, como dor à palpação (3/22, 13%) e ruídos articulares (4/25, 16%). Concluímos que distúrbios de desenvolvimento de dentes e maloclusão são frequentes em pessoas brasileiras afetadas pela SWB, e que a maioria delas apresenta alterações sistêmicas e comportamentais que podem interferir no manejo clínico odontológico. / The Williams Beuren syndrome (WBS), congenital disease caused by micro deletion of chromosome 7, including the elastin gene, is characterized by typical facies, mental retardation, congenital heart disease, hypertension, gastric alterations, teeth development disorders, among other changes. The aim of this study was to know facial and oral abnormalities, the oral health condition, occlusal features and aspects of temporomandibular joint, as well as systemic and medical conditions that affect the dental management on a significant sample of Brazilian individuals with WBS. For this, we examined 25 patients with SWB, with a mean age of 13 years (4-26 years). The results showed that all participants had some degree of mental retardation. The hyperacusis was found in 88% (22/25), congenital heart disease in 76% (19/25; especially supravalvular aortic stenosis), hyperactivity in 68% (17/25) and hypertension in 40% (10/25) of participants. Tooth development disorders were found in all participants with the most frequent was prolonged retention of the deciduous teeth (64% - 16/25), followed by generalized diastema (60% - 15/25), hypodontia (42% - 9/21), enamel hypoplasia (28% - 7/25), incisor-shaped screwdriver (24%- 6/25), microdontia (8% - 2/25). In equal percentages (4% - 1/25) were found cases of gemination, taurodontism and conoid tooth. For the presence of occlusal changes, all the patients examined presented malocclusion. Most showed malocclusion class III dental malocclusion (11/19, 57%) and crossbite (11/25, 44%). Seventy-two percent of patients had some parafunctional habit. Present and past caries experience among the 25 patients examined was classified as very low, according to the DMFT, and 76% had gingivitis. The patients exhibited change in amplitude of excursive movements of the jaw (maximun mouth opening - 12/22, 54%; laterality - 6/9, 66%; e protrusion - 7/9; 77%), as well as changes in ATMs, such as pain on palpation (3/22,13%) and joint sounds (4/25, 16%). We conclude that teeth and malocclusion development disorders are common in Brazilian people affected by WBS, and most of them have systemic and behavioral changes that can interfere with dental clinical management.

No description available.

A síndrome de Williams Beuren (SWB), doença congênita causada pela microdeleção do cromossomo 7, incluindo o gene da elastina, pode conferir às pessoas afetadas, facies típico, retardo mental, cardiopatia congênita, hipertensão, alterações gástricas, distúrbios de desenvolvimento de dentes, dentre outras alterações. O objetivo desse estudo foi conhecer as alterações faciais e bucais, a condição de saúde bucal, características oclusais e aspectos da ATM, bem como as alterações sistêmicas e condições médicas que afetam o manejo odontológico em uma amostra significativa de indivíduos brasileiros com a SWB. Para tanto, examinamos 25 indivíduos com SWB, com média de idade de 13 anos (4 a 26 anos). Os resultados mostraram que todos os participantes exibiam algum grau de retardo mental. A hiperacusia foi encontrada em 88% (22/25), cardiopatia congênita, em 76% (19/25); especialmente estenose aórtica supravalvar), hiperatividade em 68% (17/25) e hipertensão arterial em 40% (10/25) dos participantes. Distúrbios de desenvolvimento de dente foram encontrados em todos os participantes sendo que o mais frequente foi a retenção prolongada de dentes decíduos (64% - 16/25), seguido do diastemas generalizados (60% - 15/25), anodontia parcial (42% - 9/21), hipoplasia de esmalte (28% - 7/25), incisivos em forma de chave de fenda (24% - 6/25), microdontia (8% - 2/25). Em iguais porcentagens (4% - 1/25), foram encontrados casos de geminação, taurodontismo e dente conóide. Quanto à presença de alterações oclusais, todos os pacientes examinados apresentaram maloclusão. A maioria exibiu maloclusão classe III dentária de Angle (11/19,57 %) e mordida cruzada (11/25, 44%). Setenta e dois por cento dos pacientes exibiam algum hábito parafuncional. A experiência presente e passada de cárie, entre os 25 pacientes examinados, foi classificada como muito baixa, de acordo com o índice CPO-D e ceo-d, e 76% apresentaram gengivite. Os pacientes exibiram alteração da amplitude dos movimentos excursivos de mandíbula (abertura máxima - 12/22, 54%; lateralidade - 6/9, 66%; e protrusão - 7/9; 77%), bem como alterações nas ATMs, como dor à palpação (3/22, 13%) e ruídos articulares (4/25, 16%). Concluímos que distúrbios de desenvolvimento de dentes e maloclusão são frequentes em pessoas brasileiras afetadas pela SWB, e que a maioria delas apresenta alterações sistêmicas e comportamentais que podem interferir no manejo clínico odontológico. / The Williams Beuren syndrome (WBS), congenital disease caused by micro deletion of chromosome 7, including the elastin gene, is characterized by typical facies, mental retardation, congenital heart disease, hypertension, gastric alterations, teeth development disorders, among other changes. The aim of this study was to know facial and oral abnormalities, the oral health condition, occlusal features and aspects of temporomandibular joint, as well as systemic and medical conditions that affect the dental management on a significant sample of Brazilian individuals with WBS. For this, we examined 25 patients with SWB, with a mean age of 13 years (4-26 years). The results showed that all participants had some degree of mental retardation. The hyperacusis was found in 88% (22/25), congenital heart disease in 76% (19/25; especially supravalvular aortic stenosis), hyperactivity in 68% (17/25) and hypertension in 40% (10/25) of participants. Tooth development disorders were found in all participants with the most frequent was prolonged retention of the deciduous teeth (64% - 16/25), followed by generalized diastema (60% - 15/25), hypodontia (42% - 9/21), enamel hypoplasia (28% - 7/25), incisor-shaped screwdriver (24%- 6/25), microdontia (8% - 2/25). In equal percentages (4% - 1/25) were found cases of gemination, taurodontism and conoid tooth. For the presence of occlusal changes, all the patients examined presented malocclusion. Most showed malocclusion class III dental malocclusion (11/19, 57%) and crossbite (11/25, 44%). Seventy-two percent of patients had some parafunctional habit. Present and past caries experience among the 25 patients examined was classified as very low, according to the DMFT, and 76% had gingivitis. The patients exhibited change in amplitude of excursive movements of the jaw (maximun mouth opening - 12/22, 54%; laterality - 6/9, 66%; e protrusion - 7/9; 77%), as well as changes in ATMs, such as pain on palpation (3/22,13%) and joint sounds (4/25, 16%). We conclude that teeth and malocclusion development disorders are common in Brazilian people affected by WBS, and most of them have systemic and behavioral changes that can interfere with dental clinical management.

Recentemente desenvolvida, a biotecnologia denominada por Microarrays permite o monitoramento simultâneo dos valores de expressão gênica de centenas de milhares de genes, fator este que traz uma nova interpretação aos resultados obtidos em pesquisas desenvolvidas nas mais diversas áreas do conhecimento incluindo, por exemplo, a Farmacologia e Medicina, uma vez que os resultados obtidos são interpretados ao nível molecular. Contudo, apesar de muita tecnologia ser empregada à técnica de Microarrays, sua aplicação ainda ocasiona algumas complicações decorrentes, por exemplo, das inúmeras fontes de variação existentes, da escala das respostas ou da natural dificuldade de se analisar uma grande quantidade de fragmentos genéticos avaliados sob poucas unidades experimentais. Frente a estas complicações, atualmente, muitas são as propostas metodológicas de análises estatísticas para atenuar ou eliminar os problemas inerentes à técnica de Microarrays e propiciar a extração de resultados mais confiáveis a partir dos valores de expressão gênica, porém muitos desafios ainda persistem. Sob esta colocação, o presente trabalho procurou explorar duas metodologias de análise estatística alternativas no que diz respeito a seus conceitos, embora ambas tenham sido contextualizadas ao problema de Microarrays e aplicadas para se atingir o mesmo objetivo: possibilitar a identificação dos genes diferencialmente expressos sob distintas condições experimentais. A primeira metodologia consistiu da aplicação de Modelos de Análise de Variância de efeitos fixos com a adoção de modificações nas estatísticas de teste, metodologias de correções para múltiplos testes e a construção de gráficos vulcão. Já, a segunda metodologia consistiu da contextualização e aplicação da Teoria da Resposta ao Item TRI aos experimentos de Microarrays, abordagem esta pouco explorada na análise deste tipo de dado, mas a qual possibilita a seleção de genes diferencialmente expressos a partir de uma medida latente estimada para cada gene e a construção de uma escala para as categorias de resposta de expressão gênica. A motivação para este trabalho originou de um experimento de Microarrays com ratos congênicos disponibilizado pelo Laboratório de Cardiologia e Genética Molecular do Instituto do Coração (InCor-USP) cujo objetivo é identificar genes associados à hipertensão. / Recently developed, the biotechnology denominated Microarrays permits a simultaneous monitoring of the gene expression values of hundred thousands of genes; fact that introduces a new interpretation of the results obtained in researches developed in many distinct areas including, for example, Pharmacology and Medicine, once the obtained results are read according to the molecular level. However, despite the fact that much technology is used in the Microarrays technique, its application still causes some implications, for example, the countless sources of existing variance, the scale of answers or the natural difficulty in analyzing a large number of genetic fragments measured by few experimental units. Facing such complications, a lot of methodologies were suggested in order to reduce or eliminate the problems caused by the Microarrays technique and also foster the obtainment of more reliable results from the gene expression values, yet many challenges still persist. Under this perspective, the present work aimed at exploring two alternative methodologies regarding concepts, despite both were contextualized according to the Microarrays problem and applied with the same objective: enabling the identification of the genes differently expressed under different experimental conditions. The first methodology was composed by the application of Analysis of Variance Models of fixed effects with changes in the test statistics, correction methodologies for multiple tests and volcano plot. The second methodology consisted of the contextualization and application of the Item Response Theory IRT towards the Microarrays experiments, being this one not much explored in analysis that use this kind of data, but enabling the selection of genes differently expressed from an estimated latent trait for each gene and the construction of a scale for the categories of gene expression answers. The motivation for the present work came from an experiment of Microarrays with congenic mice made available by the Cardiology and Molecular Genetics Laboratory of the Heart Institute (InCor-USP) that aimed at identifying genes associated with hypertension.

Recentemente desenvolvida, a biotecnologia denominada por Microarrays permite o monitoramento simultâneo dos valores de expressão gênica de centenas de milhares de genes, fator este que traz uma nova interpretação aos resultados obtidos em pesquisas desenvolvidas nas mais diversas áreas do conhecimento incluindo, por exemplo, a Farmacologia e Medicina, uma vez que os resultados obtidos são interpretados ao nível molecular. Contudo, apesar de muita tecnologia ser empregada à técnica de Microarrays, sua aplicação ainda ocasiona algumas complicações decorrentes, por exemplo, das inúmeras fontes de variação existentes, da escala das respostas ou da natural dificuldade de se analisar uma grande quantidade de fragmentos genéticos avaliados sob poucas unidades experimentais. Frente a estas complicações, atualmente, muitas são as propostas metodológicas de análises estatísticas para atenuar ou eliminar os problemas inerentes à técnica de Microarrays e propiciar a extração de resultados mais confiáveis a partir dos valores de expressão gênica, porém muitos desafios ainda persistem. Sob esta colocação, o presente trabalho procurou explorar duas metodologias de análise estatística alternativas no que diz respeito a seus conceitos, embora ambas tenham sido contextualizadas ao problema de Microarrays e aplicadas para se atingir o mesmo objetivo: possibilitar a identificação dos genes diferencialmente expressos sob distintas condições experimentais. A primeira metodologia consistiu da aplicação de Modelos de Análise de Variância de efeitos fixos com a adoção de modificações nas estatísticas de teste, metodologias de correções para múltiplos testes e a construção de gráficos vulcão. Já, a segunda metodologia consistiu da contextualização e aplicação da Teoria da Resposta ao Item TRI aos experimentos de Microarrays, abordagem esta pouco explorada na análise deste tipo de dado, mas a qual possibilita a seleção de genes diferencialmente expressos a partir de uma medida latente estimada para cada gene e a construção de uma escala para as categorias de resposta de expressão gênica. A motivação para este trabalho originou de um experimento de Microarrays com ratos congênicos disponibilizado pelo Laboratório de Cardiologia e Genética Molecular do Instituto do Coração (InCor-USP) cujo objetivo é identificar genes associados à hipertensão. / Recently developed, the biotechnology denominated Microarrays permits a simultaneous monitoring of the gene expression values of hundred thousands of genes; fact that introduces a new interpretation of the results obtained in researches developed in many distinct areas including, for example, Pharmacology and Medicine, once the obtained results are read according to the molecular level. However, despite the fact that much technology is used in the Microarrays technique, its application still causes some implications, for example, the countless sources of existing variance, the scale of answers or the natural difficulty in analyzing a large number of genetic fragments measured by few experimental units. Facing such complications, a lot of methodologies were suggested in order to reduce or eliminate the problems caused by the Microarrays technique and also foster the obtainment of more reliable results from the gene expression values, yet many challenges still persist. Under this perspective, the present work aimed at exploring two alternative methodologies regarding concepts, despite both were contextualized according to the Microarrays problem and applied with the same objective: enabling the identification of the genes differently expressed under different experimental conditions. The first methodology was composed by the application of Analysis of Variance Models of fixed effects with changes in the test statistics, correction methodologies for multiple tests and volcano plot. The second methodology consisted of the contextualization and application of the Item Response Theory IRT towards the Microarrays experiments, being this one not much explored in analysis that use this kind of data, but enabling the selection of genes differently expressed from an estimated latent trait for each gene and the construction of a scale for the categories of gene expression answers. The motivation for the present work came from an experiment of Microarrays with congenic mice made available by the Cardiology and Molecular Genetics Laboratory of the Heart Institute (InCor-USP) that aimed at identifying genes associated with hypertension.

L’hypertension constitue un facteur majeur de risque de maladies cardiovasculaires et touche à un pourcentage important de la population humaine. Il s’agit d’une maladie complexe étant donné son caractère multifactoriel. La régulation de la pression artérielle (PA) est sous contrôle de plusieurs gènes, appelés loci pour traits quantitatifs ou QTL, et elle est le résultat de leur interaction. Étant donné que la PA est un trait quantitatif sous contrôle de plusieurs composantes comme les facteurs génétiques et environnementaux, l’étude de l’hypertension est limitée chez les populations humaines. Ainsi la stratégie principale pour l’étude de cette maladie est l’identification de QTL à PA chez des souches congéniques de rat construites à partir des lignées hyper- et normotendues; à savoir les souches Dahl salt-sensitive [1] et Lewis, respectivement. Des études précédentes dans notre laboratoire ont localisé trois QTL à PA au niveau du chromosome 18 chez le rat. Au cours de ce projet, de nouvelles sous-souches ont été construites afin de raffiner la cartographie de ces QTL. Ainsi, les C18QTL1, C18QTL3 et C18QTL4 ont été définis. Des analyses moléculaires ont été effectuées sur deux gènes candidats pour le C18QTL3; à savoir, Adrb2 et Nedd4l associés précédemment à l’hypertension. La comparaison des résultats de séquençage des régions régulatrices et codantes de ces deux gènes, ainsi que leur analyse d’expression par qRT-PCR chez les souches contrastantes DSS et Lewis, n’ont pas montré de différence significative pouvant expliquer la variation du phénotype observé. Des études plus poussées devront être effectuées sur ces deux gènes et, le cas échéant, l’analyse d’autres gènes contenus dans le C18QTL3 devra être entamée afin d’identifier le gène responsable de ce QTL. / Hypertension is a major risk factor in cardiovascular disease and affects a large percentage of human population. It is a complex disease because of its multifactorial nature. Blood pressure (BP) regulation is controlled by several genes, known as quantitative trait loci or QTL, and results from their interaction. Given that BP is a quantitative trait influenced by several components such as genetic and environmental factors, the study of hypertension is limited in humans. As a result, the main strategy in the study of this disease is the identification of BP QTL in congenic rat strains established from hyper- and normotensive breeds, namely, Dahl salt-sensitive [1] and Lewis. Previous studies showed the existence of three QTL in rat chromosome 18. For this project, new congenic sub-strains were developed in order to refine these QTL containing regions. Thus, C18QTL1, C18QTL3 and C18QTL4 were defined. Molecular analyses were carried out for two candidate genes contained in C18QTL3; namely Adrb2 and Nedd4l previously associated with hypertension. Comparison of the sequencing results from regulatory and coding regions of the two genes, as well as their expression analyses, showed no significant difference able to account for the variation of the observed phenotype distinguishing DSS from Lewis. Further investigation of these two genes must be conducted and, if needed, analyses of other genes contained in C18QTL3 should be undertaken in order to uncover the locus responsible for the QTL.