A Doença de Alzheimer (DA) é uma desordem neurodegenerativa influenciada por elementos genéticos e ambientais. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. A identificação de biomarcadores baseados em tecido periférico como o sangue permitiria um diagnóstico menos invasivo e mais preciso, como também poderia servir como marcador de predisposição, conversão, progressão e resposta à tratamento. Para atingir tal objetivo, métodos de prospecção em larga escala de perfis de expressão gênica e protéica têm sido extensamente aplicados. O presente estudo se propôs a selecionar e validar potenciais candidatos a biomarcadores na DA, e também, de identificar potenciais biomarcadores pelo desenvolvimento de perfis proteômicos de plaquetas de pacientes. Dados publicados de microarray para DA foram comparados e genes com perfil de expressão similar em pelo menos dois estudos independentes foram selecionados. Foram identificados 4 genes de expressão aumentada em pacientes (HLADRB1, HLAB, CIRBP, S100A4) e 4 genes de expressão diminuída (CALM1, ATP5J2, KIF1B, FLOT1), que posteriormente foram submetidos a validação por PCR em tempo-real em amostras de RNA extraído a partir de leucócitos de sangue periférico de 20 pacientes e 20 controles idosos. Ao mesmo tempo, padronizamos e traçamos o perfil proteômico de pools de proteínas de plaquetas de pacientes do sexo feminino e masculino e seus respectivos controles idosos por Eletroforese Bidimensional (2-D). Dentre os genes analisados por meio de PCR em tempo-real, três (ATP5J2, S100A4 e KIF1B) apresentaram expressão aumentada no grupo DA em relação ao grupo controle (p >0,05). Estes genes podem estar envolvidos na resposta inflamatória periférica e indução da apoptose. A padronização do perfil proteômico por 2-DE foi bem sucedida e um conjunto de 5 proteínas diferencialmente expressas para ambos os gêneros foram obtidas, mas não foi possível a sua identificação por Espectrometria de Massas devido a massa limitada dessas proteínas em cada spot. Por ambas as técnicas foi possível identificar perfis diferentes de expressão gênica e protéica entre pacientes e controles idosos, demonstrando que sangue periférico é uma boa matriz de prospecção de biomarcadores de doenças neurodegenerativas. / Alzheimer\'s disease (AD) is a neurodegenerative disorder influenced by genetic and environmental elements. The diagnosis is based on clinical parameters, but its confirmation needs post-mortem pathologic evaluation at autopsy. The identification of biomarkers based on peripheral tissue would allow a less invasive and more accurate diagnosis, but could also serve as a marker of predisposition, conversion, progression and response to treatment. To achieve this goal, methods of exploring large-scale gene expression profiles and protein have been extensively applied. The present study proposed to select and validate potential candidate genes for biomarkers in AD, and also identify potential biomarkers from proteomic profiles of platelets of these patients. Published microarray data for AD were compared and genes with similar expression profile in at least two independent studies were selected. We identified four up-regulated genes (HLADRB1, HLAB, CIRBP, S100A4) and four down-regulated genes in patients (CALM1, ATP5J2, KIF1B, FLOT1), which were then submitted to validation by real time PCR in RNA samples extracted from peripheral blood leukocytes of 20 patients and 20 elderly controls. At the same time, standardized, and traced the proteomic profile of platelet proteins pools of female patients and male elders and their respective controls by two-dimensional electrophoresis (2-D). Among the genes analyzed by real time PCR three of them (ATP5J2, S100A4 and KIF1B) showed increased expression in the AD group compared to the control group (p> 0.05). These genes may be involved in peripheral inflammatory response and induction of apoptosis. The standardization of proteomic profile by 2-DE has been successful and a set of five differentially expressed proteins in both genders were obtained, but could not be identified by mass spectrometry due to limited mass of these proteins in each spot. For both techniques were able to identify different patterns of gene and protein expression between patients and elderly controls, demonstrating that peripheral blood is a good prospect source of biomarkers for neurodegenerative diseases.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa influenciada por elementos genéticos e ambientais. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. A identificação de biomarcadores baseados em tecido periférico como o sangue permitiria um diagnóstico menos invasivo e mais preciso, como também poderia servir como marcador de predisposição, conversão, progressão e resposta à tratamento. Para atingir tal objetivo, métodos de prospecção em larga escala de perfis de expressão gênica e protéica têm sido extensamente aplicados. O presente estudo se propôs a selecionar e validar potenciais candidatos a biomarcadores na DA, e também, de identificar potenciais biomarcadores pelo desenvolvimento de perfis proteômicos de plaquetas de pacientes. Dados publicados de microarray para DA foram comparados e genes com perfil de expressão similar em pelo menos dois estudos independentes foram selecionados. Foram identificados 4 genes de expressão aumentada em pacientes (HLADRB1, HLAB, CIRBP, S100A4) e 4 genes de expressão diminuída (CALM1, ATP5J2, KIF1B, FLOT1), que posteriormente foram submetidos a validação por PCR em tempo-real em amostras de RNA extraído a partir de leucócitos de sangue periférico de 20 pacientes e 20 controles idosos. Ao mesmo tempo, padronizamos e traçamos o perfil proteômico de pools de proteínas de plaquetas de pacientes do sexo feminino e masculino e seus respectivos controles idosos por Eletroforese Bidimensional (2-D). Dentre os genes analisados por meio de PCR em tempo-real, três (ATP5J2, S100A4 e KIF1B) apresentaram expressão aumentada no grupo DA em relação ao grupo controle (p >0,05). Estes genes podem estar envolvidos na resposta inflamatória periférica e indução da apoptose. A padronização do perfil proteômico por 2-DE foi bem sucedida e um conjunto de 5 proteínas diferencialmente expressas para ambos os gêneros foram obtidas, mas não foi possível a sua identificação por Espectrometria de Massas devido a massa limitada dessas proteínas em cada spot. Por ambas as técnicas foi possível identificar perfis diferentes de expressão gênica e protéica entre pacientes e controles idosos, demonstrando que sangue periférico é uma boa matriz de prospecção de biomarcadores de doenças neurodegenerativas. / Alzheimer\'s disease (AD) is a neurodegenerative disorder influenced by genetic and environmental elements. The diagnosis is based on clinical parameters, but its confirmation needs post-mortem pathologic evaluation at autopsy. The identification of biomarkers based on peripheral tissue would allow a less invasive and more accurate diagnosis, but could also serve as a marker of predisposition, conversion, progression and response to treatment. To achieve this goal, methods of exploring large-scale gene expression profiles and protein have been extensively applied. The present study proposed to select and validate potential candidate genes for biomarkers in AD, and also identify potential biomarkers from proteomic profiles of platelets of these patients. Published microarray data for AD were compared and genes with similar expression profile in at least two independent studies were selected. We identified four up-regulated genes (HLADRB1, HLAB, CIRBP, S100A4) and four down-regulated genes in patients (CALM1, ATP5J2, KIF1B, FLOT1), which were then submitted to validation by real time PCR in RNA samples extracted from peripheral blood leukocytes of 20 patients and 20 elderly controls. At the same time, standardized, and traced the proteomic profile of platelet proteins pools of female patients and male elders and their respective controls by two-dimensional electrophoresis (2-D). Among the genes analyzed by real time PCR three of them (ATP5J2, S100A4 and KIF1B) showed increased expression in the AD group compared to the control group (p> 0.05). These genes may be involved in peripheral inflammatory response and induction of apoptosis. The standardization of proteomic profile by 2-DE has been successful and a set of five differentially expressed proteins in both genders were obtained, but could not be identified by mass spectrometry due to limited mass of these proteins in each spot. For both techniques were able to identify different patterns of gene and protein expression between patients and elderly controls, demonstrating that peripheral blood is a good prospect source of biomarkers for neurodegenerative diseases.

Les pollutions par les xénobiotiques, en particulier les pesticides, et les métaux lourds issus des activités agricoles présentent une grande complexité de composition chimique et de dynamique spatio-temporelle. La présence de bandes enherbées entre les parcelles cultivées et les cours d’eau permet une limitation de la diffusion de ces pollutions résiduelles vers les milieux naturels. Le compartiment végétal de ces bandes enherbées peut jouer de multiples rôles dans ce contexte de protection environnementale. L’étude comparative réalisée in situ et en conditions contrôlées de laboratoire a permis de mettre en évidence le rôle biologique du compartiment végétal avec son implication directe dans les processus in planta d’absorption, de stockage et/ou de dégradation au moins partielle. Un tel rôle phytoremédiateur est dépendant de la capacité des plantes à se maintenir sur ces milieux pollués, qui diffère selon l’espèce considérée et structure ainsi les communautés végétales des bandes enherbées. L’étude intégrative en conditions contrôlées des réponses des plantes aux interactions avec les xénobiotiques à faibles doses, à différentes échelles de complexité du fonctionnement végétal, a permis de montrer les effets de ces stress chimiques chez l’espèce modèle Arabidopsis thaliana et chez l’espèce prairiale Lolium perenne. Les xénobiotiques et les métaux lourds à des doses subtoxiques ont induit d’importants bouleversements métabolomiques et moléculaires chez ces espèces, avec des effets cryptiques de ces polluants et de leurs produits de dégradation. L’analyse en conditions de multi-pollution, qui reflètent de manière réaliste les pollutions péri-agricoles, a montré la complexité et la difficulté de prédiction des interactions entre les effets des contaminants en mélange. Ces mécanismes de réponses diffèrent selon l’espèce et le polluant et laissent supposer des divergences en termes de perception et/ou de transport des polluants, ou de coordination des réponses moléculaires et métaboliques. Arabidopsis a ainsi présenté une coordination de ses réponses orientée vers une augmentation des métabolites de stress, et une diminution des métabolites carbonés (sucres solubles), en parallèle de modifications de l’expression de gènes impliqués dans les défenses antioxydantes, les défenses contre les stress xénobiotiques, ou dans la dynamique des phytohormones. Le stress chimique a entraîné chez Lolium des modifications majeures du métabolisme azoté, ainsi qu’un remaniement des processus de photorespiration. L’analyse transcriptomique de cette espèce a de plus montré que la majorité des gènes identifiés sont impliqués dans des voies de transduction de signal, montrant ainsi la complexité des mécanismes de réponse et les couplages qui existent entre les signaux métaboliques, en particulier liés aux sucres, les voies de signalisation associées aux phytohormones, les signaux de stress et la photosynthèse. / Environmental pollutions by xenobiotics, especially by pesticides and heavy metals derived from agricultural activities, show an important complexity of chemical composition and of spatiotemporal dynamic. Vegetative filter strips between cultivated fields and streams limit the diffusion of these residual pollutions to natural environments. However, the exact biological role of plant in these buffer strips is poorly understood in this context of environmental and ecological protection. A comparative study carried out in situ and in controlled conditions highlighted the role of plant compartment in the processes of absorption, storage and/or partial degradation of pollutants in planta. Such capability of phytoremediation depends on the maintenance of a vegetal cover in area subjected to recurring flow of pesticides, it varies according to species and leads to the structuration of vegetative filter strip communities. An integrative study in controlled conditions of plant responses to low doses of pollutants allowed to analyze at different levels of complexity the impacts of chemical stresses on the model species Arabidopsis thaliana and the grassland species Lolium perenne. Low and sublethal doses of xenobiotics, associated degradation products and heavy metals induced cryptic perturbations at metabolic and molecular levels. Multi-pollution analyses, which reflect realistic conditions of environmental exposure, highlighted complex interactive effects between pollutants in mixture and the difficulty to predict them. The mechanisms of response to these chemical stresses differ according to the species and the pollutant, and suggest differences in term of perception and/or transport of pollutants, or of coordination of molecular and metabolic responses. Arabidopsis presented a coordination of its responses toward an increase of stress metabolites, a decrease of carbon metabolites (soluble carbohydrates), in parallel with modifications of gene expressions implicated on antioxidant defences, defence against xenobiotic stresses, or phytohormone dynamic. Chemical stress leads to major modifications of nitrogen metabolism in Lolium, and perturbations of processes of photorespiration. De novo transcriptomic analysis of Lolium therefore showed that a majority of identified genes are related to signal transduction pathways, highlighting the complexity of response mechanisms and the links between metabolic signals, especially linked to carbohydrate, hormonal signaling pathways, stress signals and photosynthesis. Subtoxic chemical stress induced cryptic re-engineering of plant processes that may explain the development of tolerance for some species and their persistence in area affected by residual pollution.

<p>The goal of this thesis is to algorithmically identify candidate modifiers for <i>retinitis pigmentosa</i> (RP) to help improve therapy and predictions for this genetic disorder that may lead to a complete loss of vision. A current research by (Chow et al., 2016) focused on the genetic contributors to RP by trying to recognize a correlation between genetic modifiers and phenotypic variation in female <i>Drosophila melanogaster</i>, or fruit flies. In comparison to the genome-wide association analysis carried out in Chow et al.’s research, this study proposes using a K-Means clustering algorithm on RNA expression data to better understand which genes best exhibit characteristics of the RP degenerative model. Validating this algorithm’s effectiveness in identifying suspected genes takes priority over their classification.</p><p>This study investigates the linear relationship between <i>Drosophila </i>eye size and genetic expression to gather statistically significant, strongly correlated genes from the clusters with abnormally high or low eye sizes. The clustering algorithm is implemented in the R scripting language, and supplemental information details the steps of this computational process. Running the mean eye size and genetic expression data of 18,140 female <i>Drosophila</i> genes and 171 strains through the proposed algorithm in its four variations helped identify 140 suspected candidate modifiers for retinal degeneration. Although none of the top candidate genes found in this study matched Chow’s candidates, they were all statistically significant and strongly correlated, with several showing links to RP. These results may continue to improve as more of the 140 suspected genes are annotated using identical or comparative approaches.</p>