MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA molecules that regulate cellular messenger RNA (mRNA) and protein levels by binding to specific mRNAs. Aberrant miRNA expression has been shown to be implicated in several diseases, including cancer. Extracellular miRNAs have been found to circulate in the bloodstream and some of their levels have been associated with different diseases. Furthermore, they hold promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility. New miRNA analysis techniques will add toward the understanding of their biological functions. In this thesis, I investigate the utility of capillary electrophoresis (CE) and mass spectrometry (MS) for analysis of miRNAs through proof-of-concept experiments. In the fi rst part of this work, we developed a Protein-Facilitated Affinity Capillary Electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum (see List of publications (6)). We also implemented a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) method with online sample pre-concentration for detection of endogenous microRNAs in human serum and cancer cells. 3' modification of miRNA is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species. Recent studies have shown that post-transcriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for miRNA activity regulation. For example, such modifications in plants and C. elegans influence miRNA stability. In humans, effects on miRNA stability and on mRNA target repression have both been observed. Thus, there is a need for miRNA detection techniques which are direct and multiplexed, require minimal sample preparation and provide qualitative information regarding these modifications. We developed a multiplexed miRNA detection technique based on capillary electrophoresis coupled on line with electrospray ionization mass spectrometry (CE-ESI-MS). This method allowed a label-free, direct detection of multiple miRNAs extracted from cancer serum as well as their post-transcriptional modifications with a high mass accuracy.

Le rétrovirus HTLV-1 (Human T-cell Leukemia virus type 1) est l'agent étiologique de la leucémie T de l'adulte (ATLL) et de maladies inflammatoires. Il infecte principalement les lymphocytes T-CD4+ et T-CD8+, et se réplique essentiellement via l'expansion clonale de sa cellule hôte selon deux mécanismes distincts : HTLV-1 induit une résistance à l'apoptose des cellules T-CD8+ alors qu'il favorise la prolifération des cellules T-CD4+. Au stade chronique de l'infection, en comparaison à leur contreparties T-CD8+, les cellules T-CD4+ infectées non transformées présentent des caractéristiques pré-leucémiques telles que des anormalités génomiques et des défauts d'activation de la télomérase, ce qui explique vraisemblablement pourquoi les cellules d'ATLL sont régulièrement de phénotype T-CD4+. L'infection par HTLV-1 s'accompagne de reprogrammations drastiques du transcriptome cellulaire. Parmi elles, les modifications d'expression de micro-ARN (miARN). Les miARN sont de petits ARN non-codants qui contrôlent négativement la traduction des ARNm, et qui ont été récemment mis en évidence dans les cellules transformées par le virus, et semblent participer au maintien du phénotype tumoral. Ces données posent la question de l'origine de ces perturbations et de leurs implications dans les processus d'expansion clonale et d'initiation de la transformation des cellules infectées. Pour adresser cette question, nous avons réalisé une étude intégrée de l'expression des miARN et des ARNm de cellules T, issues de patients infectés sans malignité / Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia / lymphoma (ATLL) hat regularly occurs after a prolonged period of viral latency. In vivo, HTLV-1 replication relies on the clonal expansion of its host CD4+ and CD8+ T-cells, yet the virus causes adult T-cell leukemia / lymphoma (ATLL) that is regularly of the CD4+ phenotype. Infected cells express Tax and HBZ viral oncoproteins. Tax is mainly expressed in untransformed cells, where it promotes cell proliferation, genetic instability, and miRNA dysregulation, whereas HBZ is expressed in both untransformed and malignant T-cells where it contributes to promote cell proliferation and to silence virus expression. Here, we show that an HBZ / miRNA axis promotes cell proliferation and genetic instability. MicroRNAs (miRNAs) are evolutionarily conserved, small (~21 nucleotides), noncoding RNAs that are encoded within the genomes of almost all eukaryotes from plants to mammals. In general, miRNAs, especially in animals, post-transcriptionally regulate protein synthesis by base pairing to partially complementary sequences in the 3’ untranslated regions (UTRs) of target mRNAs. Furthermore, whereas human lymphocyte subsets are known to possess specific miRNA signatures involved in T-cell differentiation and activation, little is known about the role of miRNA dysregulation in the clonal expansion of untransformed, infected CD4+ and CD8+ T-cells in vivo, including its role, if any, in viral persistence, inflammation, genetic instability, and early leukemogenesis. To our knowledge, no study to date has assessed the effects of HBZ on the biogenesis and activity of miRNAs. In order to assess the effect of HTLV-1 infection on the miRNA expression profiles of host cells in vivo, we performed an integrated analysis of miRNA- and mRNA-expression profiles of cloned CD4+ and CD8+ T-cells derived from infected individuals without malignancy