Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A carcinicultura marinha vem assumindo um papel de grande relevÃncia na economia mundial, nÃo somente pela geraÃÃo de empregos e rendimentos, mas especialmente por representar uma alternativa viÃvel para o atendimento da crescente demanda mundial por alimentos. No entanto, a ocorrÃncia de enfermidades nos cultivos de camarÃes, principalmente as de etiologia viral, vem comprometendo significativamente a atividade, por provocarem elevados Ãndices de mortalidade e conseqÃente perda econÃmica. A identificaÃÃo e caracterizaÃÃo de genes relacionados a enfermidades de camarÃes tÃm auxiliado significativamente na compreensÃo das respostas imunes desencadeadas nos processos infecciosos, assim como dos mecanismos relacionados à interaÃÃo patÃgeno-hospedeiro, ainda de pouco conhecimento cientÃfico. O presente trabalho teve como objetivo principal a prospecÃÃo e caracterizaÃÃo de genes relacionados a enfermidades de camarÃes da espÃcie Litopenaeus vannamei sob condiÃÃes de cultivo. A investigaÃÃo sanitÃria de 40 camarÃes por mÃtodos histopatolÃgicos e moleculares de diagnÃstico resultou na detecÃÃo da IMN, IHHN, WSS, NHP, vibrioses e gregarinas, o que permitiu o agrupamento destes camarÃes em classes diferenciais destinadas ao estudo de prospecÃÃo gÃnica atravÃs da tÃcnica de Differential display RT-PCR (DDRT-PCR). A anÃlise da expressÃo diferencial de genes nos perfis de expressÃo dos camarÃes agrupados de acordo com o status sanitÃrio resultou na identificaÃÃo de trÃs sequÃncias genÃticas inÃditas e potencialmente relacionadas à ocorrÃncia de enfermidades no camarÃo L. vannamei, sendo duas sequÃncias (882-A1R2 e 730-A4R10) de homologias indeterminada e uma (842-A2R6) com 99% de similaridade nucleotÃdica com o gene mut-7 que codifica um tipo de RNAse envolvida no mecanismo de silenciamento pÃstranscricional de genes do nematÃide Caenorhabditis elegans. Este resultado sugere que o gene 842-A2R6 pode estar relacionado ao mecanismo de silenciamento de dsRNA associadas ao IMNV. / The marine shrimp culture has achieved great importance in the global economy as an agriculture business, not only for jobs and income generation, but especially because it represents a viable alternative source of animal protein for the growing global food demand. However, the occurrence of diseases in shrimp culture, especially those of viral aetiology, has significantly compromised the activity, causing high mortality rates and consequent economic loss. The identification and characterization of genes related to shrimp diseases has helped understanding the immune responses triggered in the infectious processes, and mechanisms related to host-pathogen interaction, about which very little is known at the moment. The main objective of the present work was the prospection and characterization of genes related to diseases of the shrimp species Litopenaeus vannamei under typical rearing conditions. The health status investigation of 40 shrimps by histopathological and molecular methods of diagnosis resulted in the detection of IMN, IHHN, WSS, NHP, vibriosis and gregarines in various degrees and combinations. Shrimps have been grouped in classes for gene prospecting through the technique of Differential Display RT-CR (DDRT-PCR). The analysis of differential gene expression in the profiles produced resulted in the identification of three previously undescribed gene sequences potentially related to the occurrence of diseases in shrimp L. Vannamei. Two of these sequences (882-A1R2 and 730-A4R10) have not had homologies to any annotated invertebrate sequences. Fragment 842-A2R6 however, showed 99% nucleotide similarity with the mut-7 gene, which encodes a type of RNAse involved in the mechanism of post-transcriptional gene silencing through RNAi in the nematode Caenorhabditis elegans. It is suggested hare that this kind of mechanism may be involved in the silencing of double stranded RNA from viruses like the IMNV.

Water stress affects vine productivity, disease tolerance, and enological characteristics of grape. Florida Hybrid Bunch grape are developed through hybridization of local grape spp with Vitis vinifera. These cultivars are mostly grown in southeast region of United States. Water deficit conditions resulted due to failure of rains in the region has developed concern among Florida grape growers to increase water use efficiency of grape. The goal of this research is to identify genes and proteins differentially expressed in response to water stress and to correlate these changes with enological characteristics. Investigating transcripts and proteins will allow us to correlate them and confirm the involvement of specific genes responding to stress. Florida hybrid bunch ‘Suwannee’ grape plants were maintained under green house conditions. Water stress was induced by withholding irrigation. The leaf samples were collected from both irrigated and stressed plants at 5, 10, 15 and 20 day interval. We generated over 200 Subtractive Hybridization PCR products from control and water stressed leaf tissues. Cloning, sequencing and transcript analysis revealed that, 54 genes related to drought and defense regulated pathways out of 125 characterized transcripts. Proteins were extracted from leaf tissue with trichloroacetic acid /acetone and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The proteins were sequenced in LC/Mass Spectrophotometer. The most important differentially expressed genes include sucrose synthase, actin, isoprene synthase, ABF3, SNF1 related protein kinase, WRKY type transcription factors, AP2, ASR2, glyoxalase I and, cytochrome b which play significant role in cell permeability, transportation, photosynthesis and, maintenance in osmotic stress. We have found that ribulose bisphosphate carboxylase and phosphoribulokinase, which play major role in photosynthesis, were suppressed in response to water stress in Florida hybrid bunch. The results suggested that water stress affects expression of cDNAs associated with defense and drought regulated functions. Such profiling studies will be used to explicate specific pathways disconcerted by water deficit treatments, and in the identification of varietal differences.