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

As moléculas co-estimuladoras CD80 e CD86, expressas na superfície de células dendríticas (DCs), as principais células apresentadoras de antígenos profissionais (APCs), possuem participação fundamental na indução de resposta e manutenção de tolerância, motivo pelo qual são consideradas alvos terapêuticos promissores. Essas moléculas promovem o segundo sinal necessário à ativação e proliferação dos linfócitos T por meio da ligação ao receptor CD28, ou inibem a resposta por essas células por meio da ligação ao receptor CTLA-4, ambos expressos na superfície dos linfócitos. Muitos são os relatos da literatura indicando diferenças tanto quantitativas quanto qualitativas entre CD80 e CD86 na capacidade de ativação de linfócitos T, os mais relevantes apontando diferenças na capacidade de indução de diferenciação de linfócitos para os padrões Th1 e Th2 de secreção de citocinas. Porém, tais relatos são muitas vezes contraditórios, e o verdadeiro papel funcional dessas moléculas ainda está por ser estabelecido. Assim, propusemo-nos a estabelecer as metodologias necessárias para silenciar as moléculas CD80 e CD86 em células dendríticas (DCs) humanas, derivadas de monócitos do sangue periférico, por meio da tecnologia de RNA de interferência. Isso possibilitaria esclarecer o papel desempenhado por cada uma dessas moléculas na capacidade de ativação de linfócitos T. Para tanto, padronizou-se a transfecção reversa de DCs do quarto dia da cultura com siRNA fluorescente e os agentes de transfecção lipídicos siPORT e iMAX, tendo sido obtidas eficiências de transfecção de 64,7% ± 5,2 e 69,7% ± 14,5%, respectivamente. DCs do quarto dia de cultura foram transfectadas com siRNAs específicos para CD80, e o fenótipo avaliado após 48 horas da transfecção. Foi possível identificar, além do eficiente silenciamento de CD80 por dois dos três siRNAs testados, também uma diminuição, inesperada, de células CD86+. Para o silenciamento de CD86, células CD14+ selecionadas positivamente por beads magnéticas foram transfectadas com siRNAs específicos para CD86, ativadas após 24 horas da transfecção e o silenciamento avaliado após 24 horas da ativação. Embora o silenciamento conseguido por um dos dois siRNAs testados tenha sido muito pequeno, observou-se fenômeno equivalente, com diminuição de células CD80+. Embora inconclusivos, esses dados sugerem a possibilidade de modulação recíproca dessas moléculas. Assim, pudemos obter a transfecção eficiente de DCs com siRNAs de interesse e, através deles, modular a expressão de CD80 e CD86. Com estes instrumentos, portanto, podemos agora desenvolver estudos quanto ao papel de cada uma destas moléculas na fisiologia da apresentação antigênica pelas DCs. / The costimulatory molecules CD80 and CD86, expressed on surface of dendritic cells (DCs), are essential to trigger T cell activation and to maintain self tolerance, indicating that these molecules are promising therapeutic targets. They can either bind to CD28 on T cells, promoting T cell activation and leading to their proliferation and cytokine production, or to CTLA-4, which is expressed following T cell activation, and can inhibit T cell response. Though CD80 and CD86 are thought to provide equivalent T cell costimulation, a growing body of evidence suggests that there are different functional consequences of CD28 engagement by these two molecules. Many reports point to variations in their ability to stimulate different lymphocyte subsets. However, there is still controversy in the literature and the actual role of CD80 and CD86 remains to be elucidated. Therefore, the aim of this study was to establish the methodology necessary to silence, by small interfering RNAs (siRNAs), both CD80 and CD86 expression on monocyte-derived dendritic cells. These findings would be the base of studys that could better elucidate the function of these two coestimulatory molecules in T cell activation. Therefore, transfection of 4th day DCs with fluorescent siRNA and lipidic transfection agents siPORT and iMAX was established, an d transfection efficiency observed was 64,7% ± 5,2 e 69,7% ± 14,5%, respectively. 4th day DCs were transfected with specific CD80 siRNAs and phenotype was observed after 48 hours of transfection. Besides CD80 efficient silencing by two from three siRNAs tested, there was an unexpected decrease in CD86+ cells. To establish CD86 silencing, CD14+ cells were positively selected with magnetic beads and immediately transfected with CD86 specific siRNAs, activated after 24 hours of transfection, and phenotype was observed after 24 hours of activation. Despite the fact that silencing conferred by one of two siRNAs was very low, equivalent phenomenon was observed, with a decrease in CD80+ cells. Although the observed effects were inconclusive, these data suggests a possible reciprocal modulation by these two molecules. Therefore, we were able to obtain efficient DC transfection with siRNAs of interest, as well as modulate CD80 and CD86 expression. With these instruments we can now develop studies regarding the real physiological role of these two costimulatory molecules in DCs antigen presentation.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

As moléculas co-estimuladoras CD80 e CD86, expressas na superfície de células dendríticas (DCs), as principais células apresentadoras de antígenos profissionais (APCs), possuem participação fundamental na indução de resposta e manutenção de tolerância, motivo pelo qual são consideradas alvos terapêuticos promissores. Essas moléculas promovem o segundo sinal necessário à ativação e proliferação dos linfócitos T por meio da ligação ao receptor CD28, ou inibem a resposta por essas células por meio da ligação ao receptor CTLA-4, ambos expressos na superfície dos linfócitos. Muitos são os relatos da literatura indicando diferenças tanto quantitativas quanto qualitativas entre CD80 e CD86 na capacidade de ativação de linfócitos T, os mais relevantes apontando diferenças na capacidade de indução de diferenciação de linfócitos para os padrões Th1 e Th2 de secreção de citocinas. Porém, tais relatos são muitas vezes contraditórios, e o verdadeiro papel funcional dessas moléculas ainda está por ser estabelecido. Assim, propusemo-nos a estabelecer as metodologias necessárias para silenciar as moléculas CD80 e CD86 em células dendríticas (DCs) humanas, derivadas de monócitos do sangue periférico, por meio da tecnologia de RNA de interferência. Isso possibilitaria esclarecer o papel desempenhado por cada uma dessas moléculas na capacidade de ativação de linfócitos T. Para tanto, padronizou-se a transfecção reversa de DCs do quarto dia da cultura com siRNA fluorescente e os agentes de transfecção lipídicos siPORT e iMAX, tendo sido obtidas eficiências de transfecção de 64,7% ± 5,2 e 69,7% ± 14,5%, respectivamente. DCs do quarto dia de cultura foram transfectadas com siRNAs específicos para CD80, e o fenótipo avaliado após 48 horas da transfecção. Foi possível identificar, além do eficiente silenciamento de CD80 por dois dos três siRNAs testados, também uma diminuição, inesperada, de células CD86+. Para o silenciamento de CD86, células CD14+ selecionadas positivamente por beads magnéticas foram transfectadas com siRNAs específicos para CD86, ativadas após 24 horas da transfecção e o silenciamento avaliado após 24 horas da ativação. Embora o silenciamento conseguido por um dos dois siRNAs testados tenha sido muito pequeno, observou-se fenômeno equivalente, com diminuição de células CD80+. Embora inconclusivos, esses dados sugerem a possibilidade de modulação recíproca dessas moléculas. Assim, pudemos obter a transfecção eficiente de DCs com siRNAs de interesse e, através deles, modular a expressão de CD80 e CD86. Com estes instrumentos, portanto, podemos agora desenvolver estudos quanto ao papel de cada uma destas moléculas na fisiologia da apresentação antigênica pelas DCs. / The costimulatory molecules CD80 and CD86, expressed on surface of dendritic cells (DCs), are essential to trigger T cell activation and to maintain self tolerance, indicating that these molecules are promising therapeutic targets. They can either bind to CD28 on T cells, promoting T cell activation and leading to their proliferation and cytokine production, or to CTLA-4, which is expressed following T cell activation, and can inhibit T cell response. Though CD80 and CD86 are thought to provide equivalent T cell costimulation, a growing body of evidence suggests that there are different functional consequences of CD28 engagement by these two molecules. Many reports point to variations in their ability to stimulate different lymphocyte subsets. However, there is still controversy in the literature and the actual role of CD80 and CD86 remains to be elucidated. Therefore, the aim of this study was to establish the methodology necessary to silence, by small interfering RNAs (siRNAs), both CD80 and CD86 expression on monocyte-derived dendritic cells. These findings would be the base of studys that could better elucidate the function of these two coestimulatory molecules in T cell activation. Therefore, transfection of 4th day DCs with fluorescent siRNA and lipidic transfection agents siPORT and iMAX was established, an d transfection efficiency observed was 64,7% ± 5,2 e 69,7% ± 14,5%, respectively. 4th day DCs were transfected with specific CD80 siRNAs and phenotype was observed after 48 hours of transfection. Besides CD80 efficient silencing by two from three siRNAs tested, there was an unexpected decrease in CD86+ cells. To establish CD86 silencing, CD14+ cells were positively selected with magnetic beads and immediately transfected with CD86 specific siRNAs, activated after 24 hours of transfection, and phenotype was observed after 24 hours of activation. Despite the fact that silencing conferred by one of two siRNAs was very low, equivalent phenomenon was observed, with a decrease in CD80+ cells. Although the observed effects were inconclusive, these data suggests a possible reciprocal modulation by these two molecules. Therefore, we were able to obtain efficient DC transfection with siRNAs of interest, as well as modulate CD80 and CD86 expression. With these instruments we can now develop studies regarding the real physiological role of these two costimulatory molecules in DCs antigen presentation.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.