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Vascular tissues provide both the mechanical support to the plant body and the conducting cells for the transport of water, mineral solutes, hormones and other signaling molecules, amino acids, and sugars. To identify genes that may regulate vascular tissue-specific functions, we isolated xylem, phloem-cambium, and nonvascular tissues from the Arabidopsis root-hypocotyl, performed a genome-wide comparative analysis of tissue-specific transcripts using the 24K Affymetrix Arabidopsis ATH1 Genome Array (24K GeneChip), and identified potential genes that are required for xylem and phloem differentiation or tissue-specific functions. Based on this comparative analysis, two phloem-specific G2-like transcription factors, MYR1 and MYR2, and a xylem-specific NAC domain family member, XND1, were selected for further characterization. Under continuous light, myr2 plants flowered early, while myr1 plants did not differ significantly from wild type controls. However, double mutant myr1myr2 plants exhibited a novel phenotype characterized by elongated petioles, semi-erect leaf orientation, and suppression of lateral shoot outgrowth. These characteristics are reminiscent of yucca, a dominant Arabidopsis mutant with elevated levels of free auxin. Preliminary results indicated that like yucca, myr1myr2 plants were more resistant than wt plants to 5-mT, a toxic tryptophan analog, suggesting that MYR1 and MYR2 may be involved in regulating tryptophan-dependent auxin biosynthesis. Overexpression of any one of MYR1 isoforms resulted in a phenotype that in some cases resembled that observed in the double mutant, indicating that the regulation mediated by MYR1 and MYR2 may depend on formation of specific heterodimers consisting of isoforms of MYR1 and/or MYR2, and that the dimerization was susceptible to disruption both by overexpression and loss-of-function of MYR1/MYR2. Overexpression of XND1 resulted in the absence of TEs as determined from the absence of both secondary cell wall deposition and TE death. Using 3 tissue-specific promoter-GUS lines as genetic backgrounds, we demonstrated that overexpression of XND1 suppressed only TE-specific GUS expression but not phloem-specific GUS expression. Three T-DNA/transposon insertion lines, xnd1-1, -2, and -3, were identified. Under normal conditions, xnd1 did not exhibit significantly different growth and development compared to wild type plants. However, preliminary data indicated that xnd1 plants were ABA and cold hypersensitive. Yeast-two hybrid screening using the N-terminal portion of XND1 as bait identified a novel RING finger protein, At3g62970 that may function as the ubiquitin ligase (E3). These results suggested that XND1 functions as a negative regulator of xylem cell differentiation, and that the regulation mediated by XND1 may be integrated with the ubiquitin/26S proteasome pathway. / Ph. D.

Microarray technology provides a quantitative assessment of the number of gene transcripts gene using a high-throughput hybridization assay. Reliable detection of gene expression therefore requires reliable design of probes used in the hybridization assay. It is noted that microarray gene expression measurements are often characterized by variability, even among a series of technical replicate arrays. Therefore, sequence verification, used as a low-level filter to exclude probes exhibiting sequence inaccuracies, has previously been shown to reduce gene expression variability.Building on this work, the effects of sequence- and annotation-based filtering methods were quantified, and shown to be effective in reducing microarray variability among a set of technical replicates. Further, appropriate thresholds for filtering are recommended. A significant interaction in an analysis of variation model was found when a combination of sequence- and annotation-based filtering methods were explanatory factors, suggesting the use of the combination of filtering methods might be most beneficial.

O genoma humano varia entre os indivíduos não somente na forma de sequência, mas também estruturalmente. Originalmente, organismos diploides possuem duas cópias de cada região autossômica, uma por cromossomo. Entretanto, com o avanço das técnicas moleculares de identificação do DNA, foram descritas sequências que se repetem em diferentes regiões do genoma em número maior ou menor do que as duas cópias esperadas. Essas variantes são denominadas copy number variants (CNVs) e definidas como segmentos genômicos, geralmente maiores do que 1 kilobase (kb), que variam em número de cópias em comparação com o genoma de referência. As CNVs podem contribuir para a variabilidade do risco entre os indivíduos na etiologia de doenças complexas. Nesse contexto, o Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune com forte componente genético, caracterizada por inflamação crônica e produção de autoanticorpos. Estudos de associação genômica em larga escala (GWAS) identificaram vários loci associados ao LES que contribuem para a susceptibilidade à patogênese. Entretanto, as pesquisas atuais com CNVs e LES focalizam apenas a análise individual de algumas variantes. O objetivo do presente trabalho foi conduzir o primeiro estudo de CNVs em larga escala em pacientes com LES. A detecção de CNVs foi feita por ensaio de Hibridação Genômica em arrays, utilizando a plataforma Affymetrix GeneChip® CytoScan HD em amostra de 23 pacientes com LES. Foram identificadas 406 CNVs distribuídas em todos os cromossomos, exceto no Y. A média foi de 18 CNVs por paciente. As deleções foram mais frequentes do que as duplicações, 311 e 95, respectivamente. O perfil de CNVs revelou 269 CNVs envolvendo genes, 152 CNVs únicas e 59 regiões de CNVs (CNVRs). Nove CNVs identificadas não haviam sido descritas em bancos de dados de variantes estruturais. Adicionalmente, encontramos CNVs em cinco genes previamente associados com LES: CFHR4, CFHR5, STAT4, MECP2 e HLA-DPB2. CNVs nestes genes foram reportadas em pacientes com LES pela primeira vez. O conhecimento das CNVs associadas com LES e autoimunidade podem contribuir para o entendimento da etiologia da doença. Em conclusão, o presente estudo foi o primeiro delineamento em larga escala de CNVs do genoma completo em pacientes com LES. / The human genome varies between individuals not only at the sequence level but also structurally. Originally, diploid organisms have two copies of each autosomal region, one per chromosome. Advances in molecular-based techniques for DNA identification enabled the description of many repeated sequences with higher or lower copy number than that two copies expected. Those sequences are termed copy number variants (CNVs) and are defined as genomic segments, usually greater than 1 kilobase (kb) in size, ranging in copy number when compared to reference genome. CNVs can contribute to risk variability among individuals in complex diseases etiology. In this context, Systemic Lupus Erythematosus (SLE) is an autoimmune disease with strong genetic component and is characterized by chronic inflammation and autoantibodies production. To date, genome-wide association studies (GWAS) have identified several loci associated with SLE that contribute to pathogenesis susceptibility. However, current CNVs studies associated with SLE focus only in few variants analysis. The aim of the present study was to conduct the first genome-wide CNVs study in SLE patients. CNVs detection was performed by high-resolution array Genomic Hybridization Assay, using the Affymetrix GeneChip® CytoScan HD platform, in 23 SLE patients samples. We identified 406 CNVs distributed in all chromosomes, except Y. The average was 18 CNVs per patient. Deletions were more frequent than duplications, 311 and 95, respectively. CNV profile showed 269 CNVs overlapped by genes, 152 unique CNVs and 59 CNV regions (CNVRs). Nine CNVs were never described in structural variants databases. We found CNVs in five genes previously associated with SLE: CFHR4, CFHR5, STAT4, MECP2 and HLA-DPB2. CNVs in these genes were reported in SLE patients for the first time. Knowledge of CNVs associated with SLE risk and autoimmunity could also improve our understanding of disease etiology. In conclusion, the present study was the first effort to search for CNVs in whole genome of SLE patients.

O genoma humano varia entre os indivíduos não somente na forma de sequência, mas também estruturalmente. Originalmente, organismos diploides possuem duas cópias de cada região autossômica, uma por cromossomo. Entretanto, com o avanço das técnicas moleculares de identificação do DNA, foram descritas sequências que se repetem em diferentes regiões do genoma em número maior ou menor do que as duas cópias esperadas. Essas variantes são denominadas copy number variants (CNVs) e definidas como segmentos genômicos, geralmente maiores do que 1 kilobase (kb), que variam em número de cópias em comparação com o genoma de referência. As CNVs podem contribuir para a variabilidade do risco entre os indivíduos na etiologia de doenças complexas. Nesse contexto, o Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune com forte componente genético, caracterizada por inflamação crônica e produção de autoanticorpos. Estudos de associação genômica em larga escala (GWAS) identificaram vários loci associados ao LES que contribuem para a susceptibilidade à patogênese. Entretanto, as pesquisas atuais com CNVs e LES focalizam apenas a análise individual de algumas variantes. O objetivo do presente trabalho foi conduzir o primeiro estudo de CNVs em larga escala em pacientes com LES. A detecção de CNVs foi feita por ensaio de Hibridação Genômica em arrays, utilizando a plataforma Affymetrix GeneChip® CytoScan HD em amostra de 23 pacientes com LES. Foram identificadas 406 CNVs distribuídas em todos os cromossomos, exceto no Y. A média foi de 18 CNVs por paciente. As deleções foram mais frequentes do que as duplicações, 311 e 95, respectivamente. O perfil de CNVs revelou 269 CNVs envolvendo genes, 152 CNVs únicas e 59 regiões de CNVs (CNVRs). Nove CNVs identificadas não haviam sido descritas em bancos de dados de variantes estruturais. Adicionalmente, encontramos CNVs em cinco genes previamente associados com LES: CFHR4, CFHR5, STAT4, MECP2 e HLA-DPB2. CNVs nestes genes foram reportadas em pacientes com LES pela primeira vez. O conhecimento das CNVs associadas com LES e autoimunidade podem contribuir para o entendimento da etiologia da doença. Em conclusão, o presente estudo foi o primeiro delineamento em larga escala de CNVs do genoma completo em pacientes com LES. / The human genome varies between individuals not only at the sequence level but also structurally. Originally, diploid organisms have two copies of each autosomal region, one per chromosome. Advances in molecular-based techniques for DNA identification enabled the description of many repeated sequences with higher or lower copy number than that two copies expected. Those sequences are termed copy number variants (CNVs) and are defined as genomic segments, usually greater than 1 kilobase (kb) in size, ranging in copy number when compared to reference genome. CNVs can contribute to risk variability among individuals in complex diseases etiology. In this context, Systemic Lupus Erythematosus (SLE) is an autoimmune disease with strong genetic component and is characterized by chronic inflammation and autoantibodies production. To date, genome-wide association studies (GWAS) have identified several loci associated with SLE that contribute to pathogenesis susceptibility. However, current CNVs studies associated with SLE focus only in few variants analysis. The aim of the present study was to conduct the first genome-wide CNVs study in SLE patients. CNVs detection was performed by high-resolution array Genomic Hybridization Assay, using the Affymetrix GeneChip® CytoScan HD platform, in 23 SLE patients samples. We identified 406 CNVs distributed in all chromosomes, except Y. The average was 18 CNVs per patient. Deletions were more frequent than duplications, 311 and 95, respectively. CNV profile showed 269 CNVs overlapped by genes, 152 unique CNVs and 59 CNV regions (CNVRs). Nine CNVs were never described in structural variants databases. We found CNVs in five genes previously associated with SLE: CFHR4, CFHR5, STAT4, MECP2 and HLA-DPB2. CNVs in these genes were reported in SLE patients for the first time. Knowledge of CNVs associated with SLE risk and autoimmunity could also improve our understanding of disease etiology. In conclusion, the present study was the first effort to search for CNVs in whole genome of SLE patients.

Traumatic brain injury (TBI) is the most common cause of mortality and disability in the younger (<50 years) Swedish population with an incidence rate of 20,000 cases per year. This thesis aims to increase the understanding of brain injury mechanisms, especially in a molecular and cellular context. Bone morphogenetic protein (BMP) signalling was examined in three genetically modified mice (two “loss-of-function”, one “gain-of-function”) exposed to TBI (controlled cortical impact, CCI) with CaMKII used as promoter for Cre-driven recombination in postnatal forebrain neurons. The mice survived, developed normally and did not show any obvious phenotypes except for an upregulation in Mtap2 mRNA in mice with impaired BMP signalling. Reactive Gfap and Timp1 mRNA expression measured using quantitative RT-PCR (qRT-PCR) was reduced in the mice overexpressing BMP signals. The BMP signalling pathway was further studied in cultured PC12 cells with BMP4 and NGF added. Egr3 expression was substantially increased by these growth factors. Blocking Egr or Junb functions reduced neurite outgrowth. TBI-induced mRNA expression changes in 100 selected genes in C57BL/6J mouse neocortex and hippocampus were measured using qRT-PCR at different time points post-injury. Several distinct gene clusters with similar expression patterns were identified. GeneChip analysis (Affymetrix) of the injured mouse neocortex at three days revealed 146 transcripts significantly upregulated, confirming and extending the qRT-PCR results. The findings demonstrate marked increases after injury among chemokine transcripts and activation of many genes involved in inflammation. In conclusion, the present study has revealed transcriptional changes in specific signalling pathways after brain injury. The results may help to identify novel targets for neuroprotective interventions after traumatic brain injury.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal