Les tumeurs de vessie suivent deux voies de progression tumorale. La voie des tumeurs papillaires qui progressent rarement vers des tumeurs invasives mais qui récidivent très fréquemment et la voie des carcinomes in situ (CIS) qui progressent pour envahir le chorion puis le muscle. Les tumeurs infiltrant le muscle sont de mauvais pronostic et les traitements par chimiothérapie restent d’efficacité limitée. Il est alors important d’en comprendre les bases moléculaires. Les microRNAs sont d’importants régulateurs de l’expression post-transcriptionnelle des gènes. Des perturbations de leur expression et/ou de leur activité contribuent au développement tumoral en dérégulant l’expression de gènes clés dans les cancers. Nos travaux ont porté sur l’étude de l’expression et des fonctions des microRNAs dans la carcinogenèse urothéliale. Dans la première partie, nous avons choisi d’étudier, par une approche de gènes candidats, miR-155, un oncomiR dont l’expression dérégulée a été rapportée dans plusieurs cancers mais pas encore dans le cancer de la vessie. J’ai identifié une surexpression significative de ce miARN dans un sous-groupe de cancers invasifs de vessie. Ensuite, j’ai montré par des analyses fonctionnelles, le rôle de miR-155 dans l’invasion et la migration tumorale mais pas dans la prolifération cellulaire. Dans la deuxième partie, nous avons utilisé une approche plus globale. J’ai d'abord effectué une revue extensive de la littérature pour rechercher les miR dont l'expression avait été montrée comme dérégulée dans les cancers de vessie et/ou les miR impliqués fonctionnellement dans ce cancer. J’ai ensuite réalisé une analyse multiparamétrique en intégrant les données d'expression de ces miR, les données anatomopathologiques et moléculaires (stade et grade, statut mutationel de TP53 et FGFR3, phénotype épigénétique MRES et signature CIS) et les données du transcriptome (puces Affymetrix U133 Plus 2.0), des altérations génomiques (puces Illumina 370.000 sondes) et de méthylation (puces Illumina 27.000 sondes). Ce travail m’a permis, d'identifier des miR associés à l'une des deux voies de progression identifiées dans les cancers de vessie et de proposer des cibles candidates de ces miR. La recherche des altérations épigénétiques pouvant affecter l’expression de ces miR a permis d’identifier une association significative entre l’expression d’un miARN (miR-17-5p) et la méthylation d’un promoteur. En revanche les altérations génétiques n’ont été associées à aucune expression de miR. Ce travail propose une liste de très bons candidats miARNs pour lesquels des études fonctionnelles pourront être envisagées au-delà de mon travail de thèse. / Bladder tumors are characterized by two progression pathways. The first pathway leads to the developpement of papillary tumors, which are at high risk of recurrence but that rarely progress to invasive tumors. Another pathway involves carcinoma in situ (CIS), which often progresses by first invading the lamina propria and then the muscle. Tumors infiltrating the muscle have a poor prognosis and chemotherapy regimens are of limited benefits. It is yet important to understand the molecular basis underlying these events. The microRNAs are important regulators of post-transcriptional gene expression. Alteration in their expression and /or activity is believed to contribute to tumor development by deregulating the expression of cancer-related genes. Our work has been focused on studying the expression and function of microRNAs in urothelial carcinogenesis. In the first part, we employed a candidate gene approach to study miR-155, a oncomiR whose dysregulated expression has been reported in many cancers, but not in bladder cancer. I identified a significant overexpression of this miRNA in a subgroup of invasive bladder cancers. Next, I demonstrated a role for miR-155 in tumor invasion and migration, without any apparent effect on cell proliferation. In the second part, we used a more comprehensive approach in which I first conducted an extensive review of the literature to search for miR whose expression was already found to be deregulated and/or miR functionally involved in bladder cancer. I then performed a multiparametric analysis by integrating expression data of miR, pathological and molecular data (stage and grade, mutational status of FGFR3 and TP53, MRES epigenetic phenotype and CIS signature) data of the transcriptome (Affymetrix U133 Plus 2.0), genomic alterations (370,000 chips Illumina probes) and methylation (Illumina chips 27,000 probes). This work allowed us to identify miRs associated with one or the other pathway linked to progression of bladder cancer and also, it revealed candidate targets for these miRs. The search for epigenetic alterations capable to affect the expression of those miRs showed significant association between expression of a particular miRNA (miR-17-5p) and the methylation of its promoter. Genetic alterations however, have failed to associate with expression of miR. Finally, this work suggests a list of good candidates miRNAs for which future functional studies should help to get insight into the role of these miRNAs

O timo é um órgão linfóide primário, responsável pela indução da tolerância imunológica central. Histologicamente esse órgão é formado por um estroma composto por células tímicas epiteliais (TECs) além de outros tipos celulares. Dentre as TECs encontramos as células tímicas epiteliais corticais (cTECs) e as células tímicas epiteliais medulares (mTECs), as quais são responsáveis pela seleção positiva e seleção negativa dos timócitos em desenvolvimento, respectivamente. Durante seu desenvolvimento intra-tímico, os timócitos modulam da expressão de genes de marcadores de diferenciação, os chamados clusters de diferenciação (CDs) além de outros genes. Neste projeto nós nos interessamos por este aspecto, ou seja, o controle da expressão gênica durante a diferenciação dos timócitos. Como os microRNAs (miRNAs) são elementos essenciais de controle fino da expressão gênica, atuando ao nível pós-transcricional de RNAs mensageiros (mRNAs) de células eucarióticas, nosso interesse foi o de estudar este tipo de controle em timócitos. Dentre as centenas de miRNAs já descritos no camundongo, o miRNA 155 (miR-155) é o mais expresso no timo e no baço sendo que seu papel foi já foi demonstrado em células T maduras periféricas, mas ainda não se estudou seu possível papel em timócitos em desenvolvimento. A partir das evidências do papel do miR-155 nas células T maduras, nós elaboramos a hipótese de que esse miRNA também atua no desenvolvimento de timócitos. Para testar essa hipótese, nós utilizamos a estratégia de silenciamento do miR-155 por meio de eletroporação (eletrotransfecção) do antagonista Anti-miR-155 diretamente no timo de camundongos BALB/c. Os timócitos de camundongos controle e silenciados foram então separados por citometria de fluxo e amostras de RNA dessas células foram então analisadas por meio de qRT-PCR para nos certificarmos da eficiência do silenciamento do miR-155 e por hibridizações com microarrays de mRNAs para a análise do transcriptoma. Observamos que o silenciamento do miR-155 provoca a modulação de um grande conjunto de mRNAs, os quais foram analisados com auxílio do banco de dados do \"Immunogical Genome Project\" (ImmGen) quanto aos seus aspectos funcionais focando no desenvolvimento de timócitos. Os mRNAs modulados e envolvidos neste processo, foram ainda reanalisados quanto sua capacidade de interagir por hibridização com o miR-155 (hibridização miRNA-mRNA) por meio da ferramenta computacional RNA-Hybrid. Por meio destas estratégias, conseguimos repertoriar um conjunto de mRNAs que codificam proteínas importantes para o desenvolvimento de timócitos e que são potencialmente controlados por miR-155. / The thymus is a primary lymphoid organ responsible for the induction of central immune tolerance. Histologically this organ is formed by a thymic stroma composed of thymic epithelial cells (TECs) and other cell types. The TEC cells are subdivided into cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs), which are responsible for positive selection and negative selection of developing thymocytes, respectively. During its intra-thymic development, thymocytes modulate the gene expression of differentiation markers, so-called clusters of differentiation (CD) and other genes. In this project we are interested in the control of gene expression during the differentiation of thymocytes. As microRNAs (miRNAs) are essential elements of fine control of gene expression, acting at the post-transcriptional level of messenger RNAs (mRNAs) of eukaryotic cells, our interest was to study this type of control in thymocytes. Among the hundreds of miRNAs been described in mice, miRNA 155 (miR-155) is strongly expressed in the thymus and spleen and its role was already demonstrated in peripheral mature T cells, but has not yet been studied during the thymocyte development. Taking into account the evidence for the role of miR-155 in mature T cells, we raise the hypothesis that this miRNA is also active in developing thymocytes. To test this, we use the miR-155 silencing strategy by using electroporation (electrotransfection) of anti-miR-155 antagonist directly into the thymus of BALB/c mice. The thymocytes of control or silenced mice were then separated by flow cytometry and RNA samples from these cells were initially analyzed by qRT-PCR to make sure of miR-155 silencing efficiency and then by hybridization with microarrays for mRNA transcriptomeanalysis. We note that miR- 155 silencing cause modulation of a large set of mRNAs, which were analyzed through \"Immunological Genome Project\" (ImmGen) database focusing on developing thymocytes. The modulated mRNAs that were involved in this process were also retested for their ability to interact with miR-155 (miRNA-mRNA hybridization) by means of RNA-Hybrid computational tool. Through these strategies we were able to found a set of mRNAs encoding proteins important for the development of thymocytes that are potentially controlled by miR-155.

O timo é um órgão linfóide primário, responsável pela indução da tolerância imunológica central. Histologicamente esse órgão é formado por um estroma composto por células tímicas epiteliais (TECs) além de outros tipos celulares. Dentre as TECs encontramos as células tímicas epiteliais corticais (cTECs) e as células tímicas epiteliais medulares (mTECs), as quais são responsáveis pela seleção positiva e seleção negativa dos timócitos em desenvolvimento, respectivamente. Durante seu desenvolvimento intra-tímico, os timócitos modulam da expressão de genes de marcadores de diferenciação, os chamados clusters de diferenciação (CDs) além de outros genes. Neste projeto nós nos interessamos por este aspecto, ou seja, o controle da expressão gênica durante a diferenciação dos timócitos. Como os microRNAs (miRNAs) são elementos essenciais de controle fino da expressão gênica, atuando ao nível pós-transcricional de RNAs mensageiros (mRNAs) de células eucarióticas, nosso interesse foi o de estudar este tipo de controle em timócitos. Dentre as centenas de miRNAs já descritos no camundongo, o miRNA 155 (miR-155) é o mais expresso no timo e no baço sendo que seu papel foi já foi demonstrado em células T maduras periféricas, mas ainda não se estudou seu possível papel em timócitos em desenvolvimento. A partir das evidências do papel do miR-155 nas células T maduras, nós elaboramos a hipótese de que esse miRNA também atua no desenvolvimento de timócitos. Para testar essa hipótese, nós utilizamos a estratégia de silenciamento do miR-155 por meio de eletroporação (eletrotransfecção) do antagonista Anti-miR-155 diretamente no timo de camundongos BALB/c. Os timócitos de camundongos controle e silenciados foram então separados por citometria de fluxo e amostras de RNA dessas células foram então analisadas por meio de qRT-PCR para nos certificarmos da eficiência do silenciamento do miR-155 e por hibridizações com microarrays de mRNAs para a análise do transcriptoma. Observamos que o silenciamento do miR-155 provoca a modulação de um grande conjunto de mRNAs, os quais foram analisados com auxílio do banco de dados do \"Immunogical Genome Project\" (ImmGen) quanto aos seus aspectos funcionais focando no desenvolvimento de timócitos. Os mRNAs modulados e envolvidos neste processo, foram ainda reanalisados quanto sua capacidade de interagir por hibridização com o miR-155 (hibridização miRNA-mRNA) por meio da ferramenta computacional RNA-Hybrid. Por meio destas estratégias, conseguimos repertoriar um conjunto de mRNAs que codificam proteínas importantes para o desenvolvimento de timócitos e que são potencialmente controlados por miR-155. / The thymus is a primary lymphoid organ responsible for the induction of central immune tolerance. Histologically this organ is formed by a thymic stroma composed of thymic epithelial cells (TECs) and other cell types. The TEC cells are subdivided into cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs), which are responsible for positive selection and negative selection of developing thymocytes, respectively. During its intra-thymic development, thymocytes modulate the gene expression of differentiation markers, so-called clusters of differentiation (CD) and other genes. In this project we are interested in the control of gene expression during the differentiation of thymocytes. As microRNAs (miRNAs) are essential elements of fine control of gene expression, acting at the post-transcriptional level of messenger RNAs (mRNAs) of eukaryotic cells, our interest was to study this type of control in thymocytes. Among the hundreds of miRNAs been described in mice, miRNA 155 (miR-155) is strongly expressed in the thymus and spleen and its role was already demonstrated in peripheral mature T cells, but has not yet been studied during the thymocyte development. Taking into account the evidence for the role of miR-155 in mature T cells, we raise the hypothesis that this miRNA is also active in developing thymocytes. To test this, we use the miR-155 silencing strategy by using electroporation (electrotransfection) of anti-miR-155 antagonist directly into the thymus of BALB/c mice. The thymocytes of control or silenced mice were then separated by flow cytometry and RNA samples from these cells were initially analyzed by qRT-PCR to make sure of miR-155 silencing efficiency and then by hybridization with microarrays for mRNA transcriptomeanalysis. We note that miR- 155 silencing cause modulation of a large set of mRNAs, which were analyzed through \"Immunological Genome Project\" (ImmGen) database focusing on developing thymocytes. The modulated mRNAs that were involved in this process were also retested for their ability to interact with miR-155 (miRNA-mRNA hybridization) by means of RNA-Hybrid computational tool. Through these strategies we were able to found a set of mRNAs encoding proteins important for the development of thymocytes that are potentially controlled by miR-155.

No description available.

Amyotrophic lateral sclerosis (ALS) is a progressive adult onset neurodegenerative disease characterized by selective death of the upper and lower motor neurons of the brain and spinal cord. Neuromuscular synapses are lost leading to paralysis and ultimately death. Non-neuronal cells, such as astrocytes, oligodendrocytes, and microglia, have been shown to contribute to ALS disease progression in mouse models. Microglia, the innate immune cells of the central nervous system, have been shown to be activated in ALS and contribute to disease progression. Hundreds of mRNAs have shown to be dysregulated in a variety of ALS cell types and tissues, including total spinal cord, acutely isolated microglia, and in vitro differentiated motor neurons. These mRNAs can be regulated post-transcriptionally by microRNAs (miRNAs), which are small endogenous non-coding RNAs with important regulatory roles in a wide range of cellular processes. This dissertation examines the contribution of miRNAs to ALS disease progression in microglia. I acutely isolated primary microglia from the spinal cords of transgenic mice overexpressing human wild type (WT) SOD1 and human G93A SOD1. I used small RNA sequencing to profile the miRNAs that are expressed during disease progression, and identified miRNAs that are differentially expressed. I confirmed these results by quantitative PCR and examined the expression changes of predicted targets in a microglia RNA-seq dataset. Here I show that miRNAs are dysregulated in acutely isolated microglia from SOD1 G93A transgenic mice, and that miR-155, a pro-inflammatory miRNA, and miR-210, a hypoxia-inducible miRNA, are significantly upregulated during disease progression. In addition, miR-1198-5p, miR-182, miR-503, and miR-668 are also dysregulated, and predicted mRNA targets of all six of these miRNAs are differentially expressed during disease progression. To my knowledge, this is the first analysis of miRNA expression in microglia during ALS disease progression. This work contributes to the understanding of the contribution of a non-neuronal cell type to ALS disease progression and serves as a paradigm for studies in other non-neuronal cell types, such as astrocytes and oligodendrocytes, and other ALS mouse models.

No description available.

Implanted biomaterials often induce inflammation that frequently leads to the foreign body response, fibrosis, and the failure of the implant. Thus, it is important to evaluate how cells interact with materials to promote a more regenerative response. It is critical to determine how to modulate the response of tissue resident innate immune cells, as they are among the first cells to interact with implanted materials. Among tissue resident innate immune cells are mast cells, which are inflammatory sentinels that degranulate and orchestrate the fate of other cell populations, such as monocytes/macrophages and lymphocytes. Mast cells have also been reported to play a vital role in the foreign body response of implanted biomaterials as well as angiogenesis. The goal of this study was to determine how to modulate mast cell responses to electrospun scaffolds by altering scaffold architecture and composition to promote anti-inflammatory and regenerative cell-scaffold interactions. Scaffold architecture was manipulated by changing either fiber diameter or pore diameter and mast cell responses were mediated by endogenous and exogenous DAMPs (i.e. IL-33 and LPS, respectively). Particularly in response to IL-33, scaffolds with increased fiber and pore diameter promoted less inflammatory cytokine and chemokine release while increasing angiogenic cytokine release. Additionally, taking scaffolds that promoted increased inflammatory cytokine expression and increasing the pore diameter alone dampened inflammatory cytokine expression. The next question we wanted to answer was how might the degradative byproducts of scaffolds alter mast cell inflammatory responses. Given the widespread use of polylactic acid, we decided to investigate this question using lactic acid as a degradative byproduct. In the presence of physiologically relevant levels of lactic acid, IL-33- and IgE-mediated inflammatory cytokines and chemokines are suppressed, while angiogenic cytokines are enhanced. This response was shown to be pH- and MCT1-dependent and was recapitulated in primary human skin mast cells as well as in vivo. In summary, scaffold architecture and the presence of select polymer degradative byproducts have the potential of selectively suppressing inflammatory cytokines and enhancing angiogenic cytokines.

Mast cells are resident immune cells abundantly found in the tissue at the host-environment interface, where they play a critical role in inflammatory allergic responses. Mast cell responses may be regulated by the cytokine milieu at the site of inflammation. Recent studies have revealed microRNAs to be important in altering cytokine signaling in immune cells. Here, we demonstrate for the first time that IL-10 and IL-33 induce miR-155 and miR-146a, respectively, to alter mast cell functions. We report that IL-10 enhanced IgE induced activation of mast cells. IL-10 effects are dependent on Stat3 activation, which elicits miR-155 expression, resulting in a loss of suppressor of cytokine signaling-1 (SOCS-1). The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155–deficient mice. Additionally, we show that IL-33 treatment greatly enhances miR-146a expression in mast cells and in mast cell derived exosomes. miR-146a induction is dependent on MyD88 and NFκB and seems to negatively regulate ST2 signaling, which is demonstrated by the hyperresponsiveness of miR-146a knockout BMMC in response to IL-33. Our preliminary data suggest that miR-146a serves as a feedback negative regulator of IL-33 signaling by targeting IRAK proteins. miR-155 and miR-146a are key microRNAs that regulate a range of immune functions. Taken together, our results reveal two novel microRNA pathways that regulate mast cell IgE and IL-33 induced responses.

Lymphomas are heterogenous class of diseases characterized by proliferation of a malignant lymphocyte clone. MicroRNA miR-155 was found to be a key molecule in immune response, namely in inflammation and germinal reaction of B cells. On the other hand, miR-155 can drive lymphoproliferation in mouse and its levels were found to be elevated in certain lymphoma types in human. MiR-155 down-regulates expression of its target gene PU.1, a hematopoietic transcription factor important for B cell differentiation. Expression of the gene encoding miR-155, known as MIR155HG, is controled by several transcription factors, among them MYB, a member of an oncogenic E-box protein family. Levels of MYB itself are controled by microRNA miR-150. In this study, we measured levels of miR-155, PU.1, MYB and miR-150 in lymph nodes of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, N=20), diffuse large B-cell lymphoma (DLBCL, N=24), follicular lymphoma (FL, N=29), Hodgkin lymphoma (HL, N=25), marginal zone lymphoma (MZL, N=13), and mantle cell lymphoma (MCL, N=10). We also measured levels of these molecules in lymph nodes with the finding of strong inflammation (N=4). We found that patients of all the diagnoses except of MCL display heterogeneously elevated levels of miR-155 and correspondingly...

Inflammation and aberrant microRNAs expressions promote colon cancer growth and progression. However, the molecular mechanisms that link these pathways remain to be determined. In this dissertation, the causal relationship between inflammation and aberrant microRNAs expressions were explored. Elevated expression of prostaglandin E₂ (PGE₂) receptor EP4 has been seen in human colon cancer. However, the mechanism by which EP4 receptor protein is deregulated is not known. Experiments in this dissertation demonstrate, for the first time, that the EP4 receptor is negatively regulated by miR-101.In previous work, we show that S100P is induced by stimulation of the PGE₂/EP4 receptor signaling pathway. S100P is a ligand for Receptor for Advance Glycation End-products (RAGE). However, little is known about the downstream targets of S100P/RAGE signaling. Here, we demonstrated that S100P/RAGE receptor signaling induces expression of miR-155 via the transcription factor AP-1. In addition, we investigated the genes that are downstream of S100P/RAGE/miR-155 pathway. Our microarrays and bioinformatics analyses identified two novel miR-155 targets, WNK1 and ZNF493 that are down-regulated upon activation of the S100P/RAGE/miR-155 pathway. Lastly, we investigated whether inhibition of S100P/RAGE signaling pathway would be beneficial as a cancer therapy using methyl-2-acetamidoacrylate (M2AA). M2AA treatments decreased colon cancer cells viability and also suppressed colon tumor growth and metastasis in vitro and also in the CAM assay in vivo. Taken together, our results suggest that modulation of S100P/RAGE signaling by M2AA offers therapeutic potential as anti-metastatic agents. In summary, this dissertation provides new insights on the molecular events that link inflammation pathways and microRNAs to colon cancer as well as show that therapeutic strategies targeting these pathways could be effective in treatment of neoplasia.