No description available.

Orientador: Vera Fernanda Martins Hossepian de Lima / Resumo: O processo transcricional em embriões extremamente complexo, nosso trabalho estimou o impacto de perturbações nos processos de transcricionais, durante as fases de ativação do genoma embrionário sobre o desenvolvimento embrionário in vitro de embriões; analisamos dados de sequenciamento de rna (RNA-seq) depositados nos bancos públicos (GEO) desde o estágio de oóocito até o dia 19 do desenvolvimento embrionário; Isolamos e caracterizamos a massa celular interna (ICM) e a trofectoderma (TE) do sexo masculino e feminino, oriundos de um mesmo blastocisto produzido in vitro com espermatozoides sexados (X e Y) e com sêmen convencional e caracterizamos e exploramos o transcriptoma desses isolados celulares. Concluímos então que a EGA menor é essencial para o desenvolvimento embrionário bovino, blastocistos possuem a maior atividade transcricional de um total de 6457 genes diferentemente expressos entre os contrastes avaliado encontramos; 2065 genes diferencialmente expressos entre a ICM e a TE, enquanto a ICM está voltada para a manutenção da pluripotência, a TE está voltada ao metabolismo energético. Os nossos dados sugerem que os embriões fêmeas são mais sensíveis ao cultivo in vitro. / Abstract: Transcription process in embryos is a complex process, our work estimated the impact of perturbations in the transcriptional processes during genome activation of vitro produced bovine embryos on their development; we analyzed public data (GEO) from rna sequencing data (RNA-seq) of oocyte up to the 19th day of embryonic development; We’d performed isolation and characterization of male and female inner cell mass (ICM) and trofectoderma (TE) from the same blastocyst produced in vitro with sorted semen (X and Y) and with conventional semen. We did the characterization and exploratory analysis of the transcriptome of these cells. We conclude that minor EGA is essential for bovine embryonic development. Blastocysts possess the highest transcriptional activity of 6457 differentially expressed genes among analyzed contrasts. We found 2065 genes differentially expressed between ICM and TE, while ICM is maintaining pluripotency, TE is focused on energy metabolism. Our data suggest that female embryos are more sensitive to in vitro culture. / Doutor

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Canola (Brassica napus) is a $19.3 billion industry for the Canadian economy annually, largely because of the demand for the oil derived from the seeds of this crop plant. Seed development and accumulation of important nutrients requires coordinated interactions between all seed regions, including the funiculus. The funiculus is a structure of the seed that serves as the only connection between the filial seed and the parent plant, yet its development and underlying transcriptional programs have not been explored. Using light and transmission electron microscopy, I completed an anatomical study of the funiculus over the course of development, from the mature ovule to the post-mature green seed stage. My results show that all three plant tissue systems of the funiculus undergo profound changes at the histological and ultrastructural level. To understand the programs that orchestrate these changes in the globular stage funiculus, I used laser microdissection coupled with RNA-sequencing. This produced a high-resolution dataset of the mRNAs present in each of the three tissue systems of the funiculus. Various clustering analyses and gene ontology term enrichment analysis identified several important biological processes associated with each tissue system. My data show that cell wall growth occurs in the epidermis, photosynthesis occurs in the cortex, and tissue proliferation and differentiation occurs in the vasculature. The importance of these processes in supporting overall seed growth and development is discussed, which can have profound implications in the genetic modification of the canola seed through manipulating the transcriptional activity of the funiculus.

Transcription and translation are the two most important central mechanisms to control gene regulation in living organism. Although these two mechanisms have been studied intensively for last several decades, it is still not clear how all the information encoded on genomic DNA is converted to mRNA and proteins, the molecular functional components that change the characteristics of cells, depending on their needs. Here, I investigated the gene regulation of opportunistic bacterium Pseudomonas aeruginosa, using recently developed high-throughput techniques, microarray for transcriptomics and LC-MS/MS for proteomics. By analyzing transcriptome of 17 strains isolated over time from three individuals with cystic fibrosis, I identified 24 genes showing significant expression changes consistently across all strains, as evidence of parallel evolution of common traits that were beneficial in establishing chronic infection. Also, by analyzing proteome and transcriptome of two reference Pseudomonas aeruginosa strains, PAO1 and PA14, under growth condition mimicking in vivo nutrition environment in cystic fibrosis patients, I revealed that protein abundances are less correlated than mRNA abundance between them, and many proteins known as virulence factors showed different abundances only in protein level, demonstrating that post-transcriptional regulation is important to understand pathogenesis of Pseudomonas aeruginosa. To boost sensitivity both in identification and quantification in shotgun proteomics, I also created a novel integrative database search algorithm, and released freely available software package termed in MSblender. These results would be valuable information for the research community to understand the regulatory mechanism of gene expression both in transcription and translation, especially related to infectious diseases caused by pathogenic bacteria. Also, I present an integrative analysis method would be generally beneficial to extract more information from conventionally used shotgun proteomics experiments. / text

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

This thesis is a follow-up of my Bachelor thesis about the mitochondrial genome of kinetoplastid protist Perkinsela sp. This work introduces a novel approach in high-throughput analysis method of uridine insertion and deletion RNA editing, describes its background and proposes its further development. Its effect on the interpretation of U-indel editing, both in Perkinsela and in general, is demonstrated via attached manuscript which also introduces other biologically relevant aspects of Perkinsela mitochondrion.

O splicing alternativo é um processo pelo qual os exons de um transcrito primário são ligados de diferentes maneiras durante o processamento do RNA, levando à síntese de proteínas distintas. Compreende um importante mecanismo na expressão gênica de eucariotos, responsável pelo aumento da diversidade proteômica e, portanto da capacidade codificante do genoma. Diferentes mecanismos parecem afetar a regulação do splicing alternativo, incluindo estresse metabólico. No presente estudo, foi realizada uma análise detalhada de sequências ORESTES de tecidos de cabeça e pescoço. Essa análise revelou que o ganho de sequências exônicas é mais freqüente que sua perda, e que a regra GT/AG é predominante em sítios de splicing. Nós observamos que o splicing alternativo geralmente não altera a matriz de leitura, mas pode afetar um domínio protéico e remover ou adicionar novos sítios de fosforilação e glicosilação. Elementos reguladores potenciais e elementos repetitivos foram freqüentes nas sequências alternativas e nas suas vizinhas. A expressão de isoformas de splicing potenciais foi investigada em diferentes tecidos, incluindo sob condições de estresse. Foram validados cerca de 50 eventos de splicing novos em células normais e tumorais. Diversas variantes, tais como aquelas dos genes HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 e TRIP6 mostraram padrões de expressão distintos em diferentes tipos celulares, em amostras normais e tumorais de pacientes com carcinoma de cabeça e pescoço e, em alguns casos, em diferentes estágios do tumor. Também foi validado um transcrito novo do gene RIPK2, responsável por codificar uma quinase de serine/treonine que ativa a via de sinalização NF-kB, e foi observada uma mudança na expressão dessa variante em resposta ao estresse térmico in vitro. Ainda não está claramente definido se o splicing alternativo é causa ou conseqüência do processo neoplásico. Nossos dados adicionam informações novas a esse tópico e fornecem alguns exemplos que evidenciam a importância do processamento do RNA na regulação da expressão gênica, tanto em condições normais como de doença. / Alternative splicing is a process by which the exons of the primary gene transcript are linked in different ways during RNA processing resulting in distinctive proteins. It is an important mechanism in eukaryotic gene expression that enhances proteome diversity and, therefore, the coding capacity of the genome. Different mechanisms seem affect alternative splicing regulation, including metabolic stresses. In the present study, a detailed informatics analysis of ORESTES sequences from head and neck tissues was performed. This in silico analysis revealed that gain of exon sequences is more frequent than exon skipping and GT/AG rule is predominant in splice sites. We observed that alternative splicing usually does not alter the reading frame but may disrupt a protein domain and remove or add new phosphorylation and glycosylation sites. Repetitive and potential regulator elements were frequent in the alternative sequences or in their neighbors. The expression of putative splicing isoforms was investigated in different tissues, including upon stress conditions. We validated approximately 50 new splicing events in normal and tumor cells. Several variants, such as those from HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 and TRIP6 genes showed distinctive expression pattern in different cell types, in normal and cancer samples from head and neck carcinoma patients and, in some cases, in different tumor stages. We also validated a new transcript of RIPK2 gene, which codes a serine/threonine kinase that activates the NF-kB pathway, and observed a shift in the expression of this variant as a response to temperature stress in vitro. It is currently not clear whether alternative splicing is the cause or the consequence of the neoplastic process. Our data add new information to this topic and provide some examples on the importance of RNA processing in gene expression regulation, both in normal and disease conditions.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.