Les avancées en biotechnologie ont permis l’identification d’un grand nombre de mécanismes moléculaires, soulignant également la complexité de la régulation génique. Néanmoins, avoir une vision globale de l’homéostasie cellulaire, nous est pour l’instant inaccessible et nous ne sommes en mesure que d’en avoir qu’une vue fractionnée. Étant donné l’avancement des connaissances des dysfonctionnements moléculaires observés dans les maladies génétiques telles que la fibrose kystique, il est encore difficile de produire des thérapies efficaces. La fibrose kystique est causée par la mutation de gène CFTR (cystic fibrosis transmembrane conductance regulator), qui code pour un canal chlorique transmembranaire. La mutation la plus fréquente (ΔF508) induit un repliement incorrect de la protéine et sa rétention dans le réticulum endoplasmique. L’absence de CFTR fonctionnel à la membrane a un impact sur l’homéostasie ionique et sur l’hydratation de la muqueuse respiratoire. Ceci a pour conséquence un défaut dans la clairance mucocilliaire, induisant infection chronique et inflammation excessive, deux facteurs fondamentaux de la physiopathologie. L’inflammation joue un rôle très important dans l’évolution de la maladie et malgré le nombre important d’études sur le sujet, la régulation du processus inflammatoire est encore très mal comprise et la place qu’y occupe le CFTR n’est pas établie. Toutefois, plusieurs autres facteurs, tels que le stress oxydatif participent à la physiopathologie de la maladie, et considérer leurs impacts est important pour permettre une vision globale des acteurs impliqués. Dans notre étude, nous exploitons la technologie des puces à ADN, pour évaluer l’état transcriptionnel d’une cellule épithéliale pulmonaire humaine fibro-kystique. Dans un premier temps, l’analyse de notre expérience identifie 128 gènes inflammatoires sur-exprimés dans les cellules FK par rapport aux cellules non FK où apparaissent plusieurs familles de gènes inflammatoires comme les cytokines ou les calgranulines. L’analyse de la littérature et des annotations suggèrent que la modulation de ces transcripts dépend de la cascade de NF-κB et/ou des voies de signalisation associées aux interférons (IFN). En outre, leurs modulations pourraient être associées à des modifications épigénétiques de leurs loci chromosomiques. Dans un second temps, nous étudions l’activité transcriptionnelle d’une cellule épithéliale pulmonaire humaine FK en présence de DMNQ, une molécule cytotoxique. Notre but est d’identifier les processus biologiques perturbés par la mutation du gène CFTR en présence du stress oxydatif. Fondé sur une analyse canonique de redondance, nous identifions 60 gènes associés à la mort cellulaire et leur variance, observée dans notre expérience, s’explique par un effet conjoint de la mutation et du stress oxydatif. La mesure de l’activité des caspases 3/7, des effecteurs de l’apoptose (la mort cellulaire programmée), montre que les cellules porteuses de la mutation ΔF508, dans des conditions de stress oxydatif, seraient moins apoptotiques que les cellules saines. Nos données transcriptomiques suggèrent que la sous-activité de la cascade des MAPK et la sur-expression des gènes anti-apoptotiques pourraient être impliquées dans le déséquilibre de la balance apoptotique. / Biotechnical advances have allowed a large number of molecular mechanisms to be identified, and have also underlined the complexity of gene regulation. Nonetheless, we still only have a partial understanding of cellular homeostasis. Even with our current knowledge, molecular dysfunctionality observed in genetic illnesses such as cystic fibrosis remain largely misunderstood, prohibiting us from developing efficient treatments. Cystic fibrosis is caused by a mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene which codes for a chloric transmembrane channel. The most frequent mutation (ΔF508) causes the unfolding of the protein and its retention in the endoplasmic reticulum. This absence of a functional CFTR to the membrane has an impact on the ionic homeostasis and the hydration of the respiratory mucus. Consequently, the mucociliary clearance is disrupted, which leads to chronic infection and excessive inflammation - two fundamental factors of CF’s physiopathology. Inflammation plays a key role in the evolution of the illness and despite many studies on this subject, the regulation of the inflammatory process remains a mystery and CFTR’s role is not well established. Additionally, since the physiopathology cannot be explained by mutation alone, several authors suggest the importance of other factors (genetic and/or environmental) which are factors in the clinical picture of the illness. In our study, we use microarray technology to evaluate the transcriptional state of an epithelial lung cell issued from a human with cystic fibrosis. Initially, our analysis identifies 128 inflammatory genes which are over-expressed in the cystic fibrosis cells versus non-cystic fibrosis cells, showing several inflammatory gene families such as cytokines or calgranulins. An analysis of the publications and annotations of these transcripts suggests that their modulations depend upon the cascade of NF-κB and/or signalling pathways associated with interferons (IFN). In other words, their modulations could be associated with epigenetic modifications of their chromosomal loci. Secondly, we study the transcriptional activity of an epithelial lung cell issued from a human with cystic fibrosis in the presence of DMNQ, a cytotoxic molecule. Our goal is to identify the biological processus which are disturbed by the mutation of the CFTR gene in the presence of oxidative stress. Based on a canonical redundancy analysis, we identify 60 genes associated with cell death and their modulation in our study explained by the combined effect of mutation and oxidative stress. By measuring the activity of caspase 3/7, an effector of apoptosis (programmed cell death), we see that the cells containing the mutation ΔF508 could present a problem with apoptosis. Our transcriptomic data suggest that decreased activity of the MAPK cascade and over-expression of anti-apoptotic genes would be a factor in apoptotic imbalance.

Les avancées en biotechnologie ont permis l’identification d’un grand nombre de mécanismes moléculaires, soulignant également la complexité de la régulation génique. Néanmoins, avoir une vision globale de l’homéostasie cellulaire, nous est pour l’instant inaccessible et nous ne sommes en mesure que d’en avoir qu’une vue fractionnée. Étant donné l’avancement des connaissances des dysfonctionnements moléculaires observés dans les maladies génétiques telles que la fibrose kystique, il est encore difficile de produire des thérapies efficaces. La fibrose kystique est causée par la mutation de gène CFTR (cystic fibrosis transmembrane conductance regulator), qui code pour un canal chlorique transmembranaire. La mutation la plus fréquente (ΔF508) induit un repliement incorrect de la protéine et sa rétention dans le réticulum endoplasmique. L’absence de CFTR fonctionnel à la membrane a un impact sur l’homéostasie ionique et sur l’hydratation de la muqueuse respiratoire. Ceci a pour conséquence un défaut dans la clairance mucocilliaire, induisant infection chronique et inflammation excessive, deux facteurs fondamentaux de la physiopathologie. L’inflammation joue un rôle très important dans l’évolution de la maladie et malgré le nombre important d’études sur le sujet, la régulation du processus inflammatoire est encore très mal comprise et la place qu’y occupe le CFTR n’est pas établie. Toutefois, plusieurs autres facteurs, tels que le stress oxydatif participent à la physiopathologie de la maladie, et considérer leurs impacts est important pour permettre une vision globale des acteurs impliqués. Dans notre étude, nous exploitons la technologie des puces à ADN, pour évaluer l’état transcriptionnel d’une cellule épithéliale pulmonaire humaine fibro-kystique. Dans un premier temps, l’analyse de notre expérience identifie 128 gènes inflammatoires sur-exprimés dans les cellules FK par rapport aux cellules non FK où apparaissent plusieurs familles de gènes inflammatoires comme les cytokines ou les calgranulines. L’analyse de la littérature et des annotations suggèrent que la modulation de ces transcripts dépend de la cascade de NF-κB et/ou des voies de signalisation associées aux interférons (IFN). En outre, leurs modulations pourraient être associées à des modifications épigénétiques de leurs loci chromosomiques. Dans un second temps, nous étudions l’activité transcriptionnelle d’une cellule épithéliale pulmonaire humaine FK en présence de DMNQ, une molécule cytotoxique. Notre but est d’identifier les processus biologiques perturbés par la mutation du gène CFTR en présence du stress oxydatif. Fondé sur une analyse canonique de redondance, nous identifions 60 gènes associés à la mort cellulaire et leur variance, observée dans notre expérience, s’explique par un effet conjoint de la mutation et du stress oxydatif. La mesure de l’activité des caspases 3/7, des effecteurs de l’apoptose (la mort cellulaire programmée), montre que les cellules porteuses de la mutation ΔF508, dans des conditions de stress oxydatif, seraient moins apoptotiques que les cellules saines. Nos données transcriptomiques suggèrent que la sous-activité de la cascade des MAPK et la sur-expression des gènes anti-apoptotiques pourraient être impliquées dans le déséquilibre de la balance apoptotique. / Biotechnical advances have allowed a large number of molecular mechanisms to be identified, and have also underlined the complexity of gene regulation. Nonetheless, we still only have a partial understanding of cellular homeostasis. Even with our current knowledge, molecular dysfunctionality observed in genetic illnesses such as cystic fibrosis remain largely misunderstood, prohibiting us from developing efficient treatments. Cystic fibrosis is caused by a mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene which codes for a chloric transmembrane channel. The most frequent mutation (ΔF508) causes the unfolding of the protein and its retention in the endoplasmic reticulum. This absence of a functional CFTR to the membrane has an impact on the ionic homeostasis and the hydration of the respiratory mucus. Consequently, the mucociliary clearance is disrupted, which leads to chronic infection and excessive inflammation - two fundamental factors of CF’s physiopathology. Inflammation plays a key role in the evolution of the illness and despite many studies on this subject, the regulation of the inflammatory process remains a mystery and CFTR’s role is not well established. Additionally, since the physiopathology cannot be explained by mutation alone, several authors suggest the importance of other factors (genetic and/or environmental) which are factors in the clinical picture of the illness. In our study, we use microarray technology to evaluate the transcriptional state of an epithelial lung cell issued from a human with cystic fibrosis. Initially, our analysis identifies 128 inflammatory genes which are over-expressed in the cystic fibrosis cells versus non-cystic fibrosis cells, showing several inflammatory gene families such as cytokines or calgranulins. An analysis of the publications and annotations of these transcripts suggests that their modulations depend upon the cascade of NF-κB and/or signalling pathways associated with interferons (IFN). In other words, their modulations could be associated with epigenetic modifications of their chromosomal loci. Secondly, we study the transcriptional activity of an epithelial lung cell issued from a human with cystic fibrosis in the presence of DMNQ, a cytotoxic molecule. Our goal is to identify the biological processus which are disturbed by the mutation of the CFTR gene in the presence of oxidative stress. Based on a canonical redundancy analysis, we identify 60 genes associated with cell death and their modulation in our study explained by the combined effect of mutation and oxidative stress. By measuring the activity of caspase 3/7, an effector of apoptosis (programmed cell death), we see that the cells containing the mutation ΔF508 could present a problem with apoptosis. Our transcriptomic data suggest that decreased activity of the MAPK cascade and over-expression of anti-apoptotic genes would be a factor in apoptotic imbalance.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-05-11T20:59:15Z No. of bitstreams: 1 DISS_2015_Karen Janones da Rocha.pdf: 4327569 bytes, checksum: 1207959001ffdfc221d56edf601d488a (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-05-24T14:50:15Z (GMT) No. of bitstreams: 1 DISS_2015_Karen Janones da Rocha.pdf: 4327569 bytes, checksum: 1207959001ffdfc221d56edf601d488a (MD5) / Made available in DSpace on 2018-05-24T14:50:15Z (GMT). No. of bitstreams: 1 DISS_2015_Karen Janones da Rocha.pdf: 4327569 bytes, checksum: 1207959001ffdfc221d56edf601d488a (MD5) Previous issue date: 2015-02-06 / CAPES / O objetivo geral do presente estudo foi caracterizar um fragmento secundário de Floresta Estacional Semidecidual localizado em Tapurah-MT, quanto a sua estrutura e composição florestal, verificar a formação de grupos florísticos e, ainda, explorar possíveis relações com o ambiente. Aplicou-se o método de área fixa com conglomerados retangulares de dimensões de 10 x 250 m, foram alocados e medidos cinco conglomerados com cinco subunidades cada de 10 X 50 m. Em cada subunidade amostral foi considerada todas as espécies arbóreas e arbustivas com diâmetro à altura do peito (DAP) superior ou igual a 10 cm. A composição florística foi analisada quanto ao número de famílias, gêneros e espécies botânicas encontradas no levantamento do componente arbóreo e a suficiência amostral do levantamento florístico foi verificada pelo procedimento Bootstrap. A similaridade florística entre as subunidades amostrais foi obtida através do Índice de Jaccard e a diversidade de espécies nas subunidades amostrais foi medida pelo Índice de Shannon (H’) e pelo Índice de Equabilidade de Pielou (J’). A caracterização da estrutura horizontal da vegetação foi feita a partir dos parâmetros fitossociológicos e a estrutura diamétrica pelo procedimento de Spiegel. A presença de grupos florísticos foi verificada pelo método de associação das espécies e o número de grupos foi estabelecido pelo coeficiente de concordância de Kendall, onde para cada grupo florístico foi analisada a estrutura horizontal, o padrão de distribuição espacial das espécies pelo índice de Payandeh e a estrutura diamétrica dos indivíduos pelo procedimento de Spiegel. A construção da matriz das variáveis edáficas foi realizada através de uma análise preliminar para identificar variáveis semelhantes entre as subunidades amostrais, as quais não apresentaram influência foram retiradas. A correlação entre os dados de vegetação e dados ambientais foi realizada por meio da Análise de Correlação Canônica, que permitiu confirmar se os nutrientes do solo influenciam na presença das espécies e pela Análise de Redundância Canônica para avaliar quais as variáveis ambientais apresentaram maior influência sobre os indivíduos. No fragmento foi verificada uma alta variabilidade florística e estrutural, que pode ser explicada pelos históricos de perturbação local a que este fragmento foi submetido no passado. De uma forma geral, a vegetação corresponde a de florestas secundárias jovens e apresenta uma comunidade estável e autorregenerativa, além de preservar características da estrutura original. Através da análise de agrupamento foi verificado que as características autoecológicas das espécies assim como os DAP’s médios de cada espécie foram os principais responsáveis pela associação e similaridade entre os grupos. Também foi verificada que apesar das perturbações no ambiente que salientam a saturação do sítio florestal, o fragmento está se recuperando. A heterogeneidade das variáveis edáficas relacionadas influencia no comportamento florístico-estrutural do fragmento secundário de Floresta Estacional Semidecidual. Sendo, as espécies das famílias Fabaceae, Lauraceae, Moraceae e Vochysiaceae as mais influentes para o presente estudo. Destacando a Qualea paraensis Ducke quanto à importância ecológica e a sua adaptabilidade ao ambiente. / The general objective of this study was to characterize a secondary fragment of semideciduous forest located in Tapurah-MT, as its structure and forest composition, verify the formation of floristic groups and also explore possible relationships with the environment. Was applied the fixed area method with five rectangular clusters of 10 x 250 m, they were measured and allocated to five subunits of 10 x 50 m each. In each sample subunit was considered all tree and shrub species with diameter at breast height (DBH) greater than or equal to 10 cm. The floristic composition was analyzed for the number of families, genera and plant species found in the survey of the tree component and sampling sufficiency of floristic survey was tested by bootstrap procedure. The floristic similarity between plots was obtained through the Jaccard index and the diversity of species in the sample subunits was measured by the Shannon Index (H') and equability index of Pielou (J'). The characterization of the horizontal structure of vegetation was made from the phytosociological parameters and the structure diameter by Spiegel procedure. The presence of floristic groups was verified by the association method of species and the number of groups was established by Kendall concordance coefficient, where for each floristic group was analyzed horizontal structure, the pattern of spatial distribution of species by Payandeh index and the diameter distribution of individuals by Spiegel procedure. The construction of the matrix of the soil variables was performed in a preliminary analysis to identify variables similar to the sample subunits, which showed no influence were dropped. The correlation between the data of vegetation and environmental data was performed by Canonical Correlation Analysis, which allowed confirm that soil nutrients influence the presence of the species and the Canonical Redundancy Analysis to evaluate which environmental variables had the greatest influence on the individuals. In the studied fragment was observed high variability floristic and structural, which can be explained by historical local disturbance that this fragment was in the past. In general, the vegetation corresponds to young secondary forests and presents a stable and self-regenerative community, besides preserving the original structure characteristics. Through cluster analysis it was found that the ecological self characteristics of the species as well as the average DBH of each species were mainly responsible for the association and similarity between the groups. We also observed that despite the disturbances in the environment that emphasize the saturation of forest site, the fragment is recovering. The soil variables heterogeneity related influence the floristic-structural behavior of the secondary fragment of semideciduous forest. Being, the species of the families: Fabaceae, Lauraceae, Moraceae and Vochysiaceae the most influential for the present study. Highlighting the Qualea paraensis Ducke about its ecological significance and adaptability to the environment.