Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-08-30T11:54:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PerfilExpressaoMicroRNAs.pdf: 1891665 bytes, checksum: 8441547cef8f393c58ff8360d7f2a5c1 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-08-30T14:42:09Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PerfilExpressaoMicroRNAs.pdf: 1891665 bytes, checksum: 8441547cef8f393c58ff8360d7f2a5c1 (MD5) / Made available in DSpace on 2017-08-30T14:42:09Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PerfilExpressaoMicroRNAs.pdf: 1891665 bytes, checksum: 8441547cef8f393c58ff8360d7f2a5c1 (MD5) Previous issue date: 2016-03-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O carcinoma de células escamosas oral (CCEO) é uma doençaa mutifatorial que envolve fatores genéticos, epigenéticos e ambientais. Caracteriza-se por um padrão de crescimento agressivo, altamente metastático e elevadas taxas de mortalidade. Apesar dos avanços da tecnologia nos tratamentos cirúrgicos, rádio e quimioterápicos, a taxa de sobrevivência de cinco anos não houve melhora significativa nos últimos anos. Através da regulação da expressão de genes alvos, os microRNAs desempenham papel importante na iniciação e progressão em cânceres humano, consequentemente, são uma ferramenta promissora para investigação de biomarcadores de identificação de risco, prognóstico e alvos terapêuticos. Com objetivo de investigar o perfil de expressão dos miRNAs no cancêr gástrico, dez tecidos de CCEO foram caracterizados a partir de dados gerados de sequenciamento de alto desempenho. Além disso, por qRT-PCR avaliamos o tecido adjacente ao CCEO para quatro miRNAs. Os resultados mostraram 17 miRNAs diferecialmente expressos, os quais foram capazes de discriminar o tecido CCEO do sem câncer. Dentre esses, encontramos sete novos miRNAs (hsa-let-7c, hsa-miR-10a, hsa-miR- 199a, hsa-miR-381, hsa-miR-501, hsa-miR-654 e hsa-miR-941), os quais ainda não estão descritos na literatura suas participações no CCEO. Adicionalmente, verificamos que os quatro miRNAs hsa-miR-221, hsa-miR-21, hsa-miR-135b e hsa-miR-29c foram hiperexpressos no tecido adjacente, confirmando o efeito de campo de câncerização. Os resultados revelaram que esses miRNAs são potenciais biomarcadores de ocorrencia no CCEO com a capacidade de identificar indivíduos com maior risco de desenvolver este câncer, e indicam sua utilidade como possíveis alvos terapêuticos. / Oral squamous cell carcinoma (OSCC) is a multifactorial disease involving genetic, epigenetic and environmental factors. It is characterized by a pattern of aggressive growth, high metastatic potential and high mortality rates. Despite advances in technology in the surgical treatment, radiotherapy and chemotherapy, the five-year survival rate has no significant improvement in recent years. By regulating the expression of target genes, microRNAs play an important role in the initiation and progression of human cancers. Therefore, they are a promising tool for the identification of biomarkers of risk, as well as prognostic and therapeutic targets. In order to investigate the miRNAs expression profile in ten tissues of OSCC were characterized based on data generated from high-performance sequencing. Furthermore, by qPCR we assessed the adjacent tissue to the OSCC for four miRNAs. The results showed 17 differentially expressed miRNAs were able to discriminate the tissue without cancer. Among these, we found seven new miRNAs (hsalet- 7c, hsa-miR-10a, hsa-miR-199a, hsa-miR-381, hsa-miR-501, hsa-miR-654 and hsamiR- 941) which are not described in the literature their participation in OSCC. Additionally, we found that the four miRNAs hsa-miR-221, hsa-miR-21, hsa-miR-135b and hsa-miR-29c presented overexpression in the adjacent tissue, confirming the field cancerization effect. The results revealed that these miRNAs are potential biomarkers occurring in OSCC with the ability to identify individuals at high risk of developing this type of cancer, and indicate their utility as potential therapeutic targets.

Les Poaceae aussi appelées Graminées forment une importante famille botanique regroupant près de 12 000 espèces en plus de 700 genres dont les céréales. Cette famille présente un intérêt économique majeur car elle est importante dans la nutrition humaine et animale. De ce fait, cette famille a été très étudiée en génomique comparée depuis les années 1990 révélant une grande conservation de la structure de leur génome depuis leur divergence d’un ancêtre commun. Avec le séquençage de Brachypodium distachyon en 2009, nous avons réalisé l’analyse de son génome par l’identification de douze blocs de synténie avec les génomes séquencés du riz, du sorgho et du maïs ainsi que sept blocs de duplications partagées entre ces graminées. Ces données nous ont permis de suggérer que les cinq chromosomes modernes de Brachypodium sont issus de l’ancêtre commun des graminées constitué de douze chromosomes et ayant subi sept fusions au cours de l’évolution. Ces travaux nous ont permis de confirmer un possible génome ancêtre des graminées constitué de cinq chromosomes porteurs de près de 10 000 gènes et d’une taille minimale de près de 35Mb. Ensuite, sur la base des résultats de génomique comparée, nous nous sommes intéressés à l’évolution des différentes familles de micro-ARN (miARN). La comparaison de ces ARN non-codants réalisée pour le riz, le sorgho, le maïs et Brachypodium montre une conservation de cette famille chez les graminées avec 50% d’orthologues et 20% de paralogues. Sur la base des résultats de paléogénomique, nous avons proposé une modélisation de l’évolution des miARN qui corrobore l’hypothèse d’une origine très ancienne de ce mécanisme de « gene silencing ». Au-delà des nouvelles connaissances fondamentales générées au cours de ce travail de thèse sur l’évolution des génomes de graminées, les résultats que nous avons obtenus ont des applications potentielles dans le domaine de l’amélioration variétale, comme avec par exemple la possibilité de définir des marqueurs moléculaires de type COS (Conserved Orthologous Set). Ces marqueurs COS ont été mis en oeuvre pour l’étude de caractères agronomiques d’intérêt dans des espèces dont le génome n’est pas encore complètement séquencé comme le blé. / Poaceae also called Grasses are an important botanical family consisting in nearly 12,000 species in over 700 genres including cereals. This family is of major economic interest because it comprises cereals that are among the most important crops for human and animal nutrition. This family has been extensively studied in comparative genomics since the 1990s and showed a high degree of gene conservation among species since they diverged from a common ancestor. With the sequencing of Brachypodium distachyon in 2009, we performed an analysis of its genome by the identification of twelve synteny blocks with the sequenced genomes of rice, sorghum and maize and seven duplications blocks shared with these last grass species. These data allowed us to suggest the five chromosomes of Brachypodium are from the common ancestor composed of twelve chromosomes and having undergone seven fusions during the evolution. This work allowed us to confirm a possible grass ancestor with five chromosomes carrying almost 10,000 genes with a size of 35Mb. Then, based on these comparative genomics results, we studied more particularly the evolution of different families of microRNAs (miRNAs). The comparison of non-coding RNA from rice, sorghum, maize and Brachypodium showed conservation into this family for the grass species with 50% of orthologs and 20% of paralogs. Based on the paleogenomics results, we proposed an evolutionary scenario of miRNA genes, which supports the hypothesis of an ancient origin of this gene silencing mechanism in plants. Beyond the fundamental knowledge generated on the evolution of grass genomes during this PhD, these results have potential applications in breeding, for example with the possibility to identify COS (Conserved Orthologous Set) molecular markers. Such COS markers have been used for the study of agronomic traits in species not completely sequenced as wheat.

Unravelling biological processes is dependent on the adequate modelling of regulatory mechanisms that determine the timing and spatial patterns of gene expression. In the last decade, a novel regulatory mechanism has been discovered and its biological importance has been increasingly recognised. This mechanism is mediated by RNA molecules named miRNAs that are the product of the maturation of non-coding gene transcripts and act post- transcriptionally usually to dampen or abolish the expression of protein-coding genes. Despite having eluded detection for such a long time, it is now clear that the elucidation of the expression pattern of many genes cannot be achieved without incorporating the effects of miRNA-mediated regulation. The technical difficulties that the experimental detection of these regulators entailed prompted the development of increasingly sophisticated computational approaches. Gene finding strategies originally developed for coding genes cannot be applied since these non- coding molecules are subject to very different sequence restraints and are too short to exhibit statistical properties that can be easily distinguished from the background. As a result, com- putational tools came to rely heavily on the identification of conserved sequences, distant homologs and machine learning techniques. Recent developments in sequencing technology have overcome some of the limitations of earlier experimental approaches, but pose new computational challenges. At present, the identification of new miRNA genes is therefore the result of the use of several approaches, both computational and experimental. In spite of the advancement that this research field has known in the last several years, we are still not able to formally and rigourously characterise miRNA genes in order to identify whichever sequence, structure or contextual requirements are needed to turn a DNA sequence into a functional miRNA. Efforts using computational algorithms towards the enumeration of the full set of miRNAs of an organism have been limited by strong reliance on arguments of precursor conservation and feature similarity. However, miRNA precursors may arise anew or be lost across the evolutionary history of a species and a newly-sequenced genome may be evolutionarily too distant from other genomes for an adequate comparative analysis. In addition, the learning of intricate classification rules based purely on features shared by miRNA precursors that are currently known may reflect a perpetuating identification bias rather than a sound means to tell true miRNAs from other genomic stem-loops. In this thesis, we present a strategy to sieve through the vast amount of stem-loops found in metazoan genomes in search of pre-miRNAs, significantly reducing the set of candidates while retaining most known miRNA precursors. Our approach relies on precursor properties derived from the current knowledge of miRNA biogenesis, analysis of the precursor structure and incorporation of information about the transcription potential of each candidate. i Our approach has been applied to the genomes of Drosophila melanogaster and Anophe- les gambiae, which has allowed us to show that there is a strong bias amongst annotated pre-miRNAs towards robust stem-loops in these genomes and to propose a scoring scheme for precursor candidates which combines four robustness measures. Additionally, we have identified several known pre-miRNA homologs in the newly-sequenced Anopheles darlingi and shown that most are found amongst the top-scoring precursor candidates for that or- ganism, with respect to the combined score. The structural analysis of our candidates and the identification of the region of the structural space where known precursors are usually found allowed us to eliminate several candidates, but also showed that there is a staggering number of genomic stem-loops which seem to fulfil the stability, robustness and structural requirements indicating that additional evidence is needed to identify functional precursors. To this effect, we have introduced different strategies to evaluate the transcription potential of the remaining candidates which vary according to the information which is available for the dataset under study.

Atherosclerosis is a major contributor to cardiovascular disease and accounts for an estimated one third of deaths overall. In order to address the hemodynamic components of disease pathogenesis, researchers have focused on mechanotransduction of flow-dependent shear stress in the vascular endothelium as a source of novel pathological mechanisms. Understanding how unidirectional, laminar blood flow protects vessels from atherogenesis, while disturbed, oscillatory blood flow promotes it, stands to provide enormous insight into disease pathogenesis and may provide powerful, specific new therapies for cardiovascular disease intervention. The overall objective of this dissertation was to determine which microRNAs (miRNAs) and mRNAs are regulated by different flow conditions in vascular endothelial cells in vitro and in mouse carotid artery endothelium in vivo, and to identify which miRNAs mediate flow-dependent vascular inflammation. The overall hypothesis of this project was that oscillatory shear (OS) and laminar shear (LS) stress differentially alter the expression of mechanosensitive miRNAs each capable of regulating complex networks of gene expression, which in turn leads to inflammation in endothelial cells. This hypothesis was tested using both in vitro and in vivo approaches, high throughput microarray analyses, and functional validation of specific targets by PCR. The findings from the partial carotid ligation model show that acute exposure to disturbed flow results in accelerated endothelial dysfunction and atherosclerosis in vivo. High-throughput microarrays reveal distinct expression profiles of both miRNAs and mRNAs in mouse endothelium exposed to disturbed flow suggesting the regulatory mechanisms by which miRNAs regulate mRNAs resulting in EC inflammation, the earliest stage of atherosclerosis. This in vivo study provides new insight into the mechanisms of flow induced atherosclerosis. In particular, the upregulation of miR-663 due to OS in HUVEC causes monocyte adhesion, but not endothelial apoptosis, in an ICAM-1 dependent manner. miR-663 regulates a group of genes including transcriptional factors and inflammatory genes which may also mediate OS-induced EC inflammation. Collectively, revealing the profiles of miRNAs and mRNAs regulated by hemodynamic flow provides a better understanding in vascular diseases and provide potential target for developing effective preventative therapeutic approaches in cardiovascular diseases.

MicroARN (miARN) ont récemment émergé comme un acteur central du gène réseau de régulation impliqués dans la prise du destin cellulaire. L'apoptose, un actif processus, par lequel des cellules déclenchent leur auto-destruction en réponse à un signal, peut être contrôlé par les miARN. Il a également été impliqué dans une variété de maladies humaines, comme les maladies du cœur, et a été pensé comme une cible pour le traitement de la maladie. Tanshinone IIA (TIIA), un monomère de phenanthrenequinones utilisé pour traiter maladies cardiovasculaires, est connu pour exercer des effets cardioprotecteurs de l'infarctus du myocarde en ciblant l'apoptose par le renforcement de Bcl-2 expression. Pour explorer les liens potentiels entre le miARN et l'action anti-apoptotique de TIIA, nous étudié l'implication possible des miARN. Nous avons constaté que l'expression de tous les trois membres de la famille miR-34, miR-34a, miR-34b et miR-34c ont été fortement régulée à la hausse après l'exposition soit à la doxorubicine, un agent endommageant l'ADN ou de pro-oxydant H2O2 pendant 24 heures. Cette régulation à la hausse causé significativement la mort cellulaire par apoptose, comme déterminé par fragmentation de l'ADN, et les effets ont été renversés par les ARNs antisens de ces miARN. Le prétraitement des cellules avec TIIA avant l'incubation avec la doxorubicine ou H2O2 a empêché surexpression de miR-34 et a réduit des apoptose. Nous avons ensuite établi BCL2L2, API5 et TCL1, en plus de BCL2, comme les gènes nouveaux cibles pour miR-34. Nous avons également élucidé que la répression des ces gènes par MiR-34 explique l'effet proapoptotique dans les cardiomyocytes. Ce que la régulation positive de ces gènes par TIIA realisée par la répression de l'expression de miR-34 est probable le mécanisme moléculaire de son effet bénéfique contre ischémique lésions cardiaques. / MiRNAs (miRNAs) have recently emerged as a central player of gene regulatory network involved in decision of cell fate. Apoptosis, an active process that leads to cell death, has been shown to be controlled by miRNAs. It has also been implicated in a variety of human disease, such as heart disease, and established as a target process for disease therapy. Tanshinone IIA (TIIA), a monomer of phenanthrenequinones used to treat cardiovascular diseases, is known to exert cardioprotective effects in myocardial infarction by targeting apoptosis through enhancing Bcl-2 expression. To explore the potential link between miRNAs and the anti-apoptotic action of TIIA, we studied the possible involvement of miRNAs. We found that expression of all three members of the miR-34 family, miR-34a, miR-34b and miR-34c that have been known to mediate the apoptotic effect of p53 in cancer cells, were robustly upregulated after exposure to either the DNA-damaging agent doxorubicin or pro-oxidant H2O2 for 24 hr in cultured neonatal rat ventricular myocytes. This upregulation caused significant apoptotic cell death, as determined by DNA fragmentation, and the effects were reversed by the antisense to these miRNAs. Pretreatment of cells with TIIA prior to incubation with doxorubicin or H2O2 prevented upregulation of miR-34 and reduced apoptosis. We then established BCL2L2, API5 and TCL1, in addition to BCL2, as the novel target genes for miR-34. We further unraveled that repression of these genes by miR-34 accounts for its proapoptotic effect in cardiomyocytes whereas upregulation of these genes by TIIA through downregulating miR-34 is likely the molecular mechanism for its beneficial effect against ischemic myocardial injuries.

Background: Hereditary transthyretin amyloidosis is an autosomal dominant disease with a reduced penetrance. The most common mutation in Sweden is the V30M mutation in the transthyretin gene. Clustering areas of the disease can be found in Northern Sweden, Portugal, Brazil and Japan, although sporadic cases exist worldwide. Despite being caused by the same mutation, there are large differences in onset, penetrance and symptoms of the disease. Swedish V30M patients typically have a later onset with a lower penetrance compared to those from the clustering Portuguese V30M areas. The reasons for these differences have not been fully understood. The aim of this thesis is to study mechanisms that may influence onset and symptoms and investigate why patients carrying the same mutation have different phenotypes. Methods: Genealogy studies were performed on all known V30M carriers in Sweden using standard genealogy methods. DNA samples from patients, asymptomatic carriers and controls from different countries were collected and the transthyretin gene was sequenced. Liver biopsies from patients were used for allele specific expression analysis and a cell assay was used for miRNA analysis with the mutated allele. Gene expression analysis was performed on biopsies from liver and fat from patients and controls. Results and conclusions: Genealogic analysis of all known Swedish V30M carriers managed to link together 73% of the Swedish ATTR V30M population to six different ancestors from the 17th and 18th century, thus dating the Swedish V30M mutation to be more than 400 years old. A founder effect was also visible in descendants to one of the ancestors, producing a later age at onset. Sequencing of the transthyretin gene revealed a SNP in the 3’ UTR of all Swedish V30M carriers that was not found in any of the Japanese or French V30M carriers. The SNP was present on the Swedish transthyretin haplotype and defined the Swedish V30M population as separate from others. However, the SNP itself had no effect upon phenotype or onset of disease. Gene expression analysis of liver and fat tissue revealed a change in genetic profile of the patients’ livers, in contrast to the unchanged profile of the fat tissue. A changed genetic profile of the liver could explain why domino liver recipients develop the disease much earlier than expected.

The general public is at risk of ionising-radiation exposure. The development of high-throughput methods to triage exposures is warranted. Current biodosimetry techniques are low-throughput and encumbered by time and technical expertise. Although there has been an emergence of gene-profiling tools for the purpose of photon biodosimetry, similar capacities do not exist for alpha-particle radiation. Herein is the first genomic study useful for alpha-particle radiation biodosimetric triage. This work has identified robust alpha-particle induced gene-based biomarkers in isolated, ex-vivo irradiated leukocytes from multiple donors. It was found that alpha-particle and photon radiation elicited similar transcriptional responses, which could potentially be distinguished by aggregate-signature analysis. Although no distinct genes were sole indicators of exposure type, clustering algorithms and principal component analysis were able to demarcate radiation type with some success. By comparing the biological effects elicited by photon and alpha-particle radiation, significant contributions have been made to the field of radiation biodosimetry.

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.

[Truncated abstract] The phloem long distance translocation system is not only involved in the transport of nutrients and photo-assimilates to different organs of the plant, but it also appears to be important for the transport of information molecules including growth-regulators, proteins and RNA. Translocation of signals appears to be involved in the coordination of developmental processes and also in the response of the plant to environmental cues. Much of the information about macromolecules in phloem comes from analyses of exudates collected from the stylets of sap sucking insects or from incisions made to the vasculature. Among the legumes, members of the genus Lupinus exude phloem 'freely' from incisions made to the vasculature at most organs of the plant. This feature was exploited in this study to document some of the macromolecules present in exudate of L. albus and which might represent potential mobile signals. Phloem exudate was collected mainly from the sutures of developing pods and from inflorescence racemes. Two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry were used to identify 83 proteins in exudate. Analysis of a cDNA library constructed from exudate identified 609 unique transcripts. Both proteins and mRNA were classified into functional groups. The largest group was related to general and energy metabolism, suggesting some metabolic activity probably to support the sieve element (SE). Other significant functional groups were represented by proteins and transcripts involved in protein synthesis, turnover and sorting, and in redox homeostasis. Proteins in these categories could play a role in maintaining the functions and stability of proteins in SE. Macromolecules involved in signalling such as transcripts encoding proteins mediating calcium levels and the Flowering locus T (FT) protein were also identified in phloem exudate of L. albus. FT protein has been recently identified as a mobile signal that induces flowering. ... The hen1 mutant accumulates low, sometimes even undetectable levels of miRNA due to the lack of methylation. No translocation of the five miRNA assayed under nutrient replete (non stress) conditions was observed. Translocation of miR395 in response to sulphur (S) deficiency was also investigated, and while conclusive evidence of translocation was not obtained, the data suggested some movement from roots to shoots (possibly in xylem) of a signal in response to S-deficiency. Future work is required to provide greater insight into the translocation path and identity of this S-deficiency signal. This study suggests that not all miRNA identified in phloem exudates are mobile, which raises the question about their biological relevance in SE and how they reached this location (e.g. through the action of a non-selective transport mechanism). However, there is also the possibility that miRNA are translocated only in response to specific internal or external cues not tested in this study. This is the first study that provides information on macromolecules present in the phloem exudate of a member of the Fabaceae. The information obtained from this work, provides a basis for future studies in the identification of potential mobile signals that may play a role in a communication network that traffics information around the plant, regulating its various developmental processes and responding to environmental cues.

Les coxsackievirus du groupe B (CVB) sont des petits virus à ARN appartenant à au genre Enterovirus et à la famille des Picornaviridae. Chez, l’homme, les CVB sont responsables de nombreuses infections aiguës bénignes ou sévères. Ils sont également incriminés dans le développement de maladies chroniques telles que le diabète de type 1 (DT1). En effet, plusieurs données épidémio-cliniques sont en faveur d’un lien entre les entérovirus et notamment les CVB et le DT1. Deux mécanismes majeurs ont été proposés pour expliquer cette pathogenèse entérovirale du DT1. Il s’agit de l’activation « en passant » d’un environnement inflammatoire et la persistance virale qui concourent à l’initiation du processus auto-immun. Les études présentées dans cette thèse visent à comprendre les caractéristiques et conséquences de l’infection à CVB qui pourraient expliquer l’implication de ces mécanismes. Les résultats obtenus suggèrent que CVB4 (utilisé comme modèle des CVB) est un virus inflammatoire. In vitro, il induit la production de grandes quantités d’IFNα par les cellules mononuclées du sang (CMN). Néanmoins cette induction d’IFNα n’est possible qu’en cas de facilitation de l’infection par des anticorps non neutralisants, qui se traduit par une entrée importante du virus dans les cellules. Dans nos travaux, l’IFNα a été détecté dans le plasma de sujets diabétiques, et fréquemment associé à la présence d’ARN entéroviral. De même, parmi les CMN, les monocytes ont été identifiés comme les principales cellules cibles du virus. En dehors de l’IFNα, nous avons montré que CVB4 peut induire la synthèse de plusieurs autres cytokines pro-inflammatoires notamment l’IL-6 et le TNFα. De façon intéressante, l’infection des cellules n’est pas indispensable car cette induction est possible par des particules non infectieuses. Cette production de cytokines pro-inflammatoires par les CMN peut également être amplifiée par la facilitation de l’infection en présence de particules infectieuses de CVB4. Nous avons montré que les macrophages, cellules effectrices importantes de l’immunité innée au niveau tissulaire, peuvent produire en présence de CVB4 de l’IFNα et d’autres cytokines pro-inflammatoires. Les macrophages dérivés de CMN en présence de M-CSF (mais pas de GM-CSF) sont infectables par CVB4 et le virus peut persister dans ces cellules. CVB4 peut également établir une infection chronique productive de type « état porteur » dans des cellules canalaires pancréatiques. Les cellules chroniquement infectées peuvent être guéries grâce à un traitement par de la fluoxétine. Cette molécule utilisée dans le traitement de troubles psychiatriques, présente in vitro une activité antivirale vis-à-vis de certains entérovirus, et permet d’éliminer complètement en quelques semaines le virus des cellules chroniquement infectées par CVB4. Des modifications cellulaires ont été observées au niveau des cellules chroniquement infectées notamment une diminution de l’expression de PDX-1, une résistance à la lyse au cours d’une réinfection par CVB4, ainsi qu’une diminution très importante de l’expression du récepteur CAR. Ces modifications cellulaires acquises au cours de l’infection chronique pouvaient persister après l’élimination du virus. Les cellules chroniquement infectées présentent également un profil de microARNs très différent de celui des cellules non infectées. Une évolution du virus a été également observée avec des changements phénotypiques et génotypiques. L’ensemble de nos observations montre que les caractéristiques de l’infection à CVB4 sont compatibles avec les mécanismes évoqués dans la pathogenèse entérovirale du DT1 et renforcent l’hypothèse de l’implication des CVB dans cette maladie. / Group B coxsackieviruses (CVB) are small RNA viruses belonging to Enterovirus genus and to the Picornaviridae family. In humans, CVB can cause numerous mild and severe acute infections. They are also thought to be involved in the development of chronic diseases such as type 1 diabetes (T1D). Several epidemiological and clinical data support a link between enteroviruses, especially CVB and T1D. Two main mechanisms have been described to explain this enteroviral pathogenesis of T1D including a “bystander activation” of an inflammatory environment and viral persistence. These mechanisms contribute to initiation of the autoimmune process. Our studies aimed to understand the features and outcomes of CVB infection that could explain their involvement in these mechanisms. The results suggest that CVB4 (used as CVB model) is an inflammatory virus. CVB4 induces in vitro the production by peripheral blood mononuclear cells (PBMCs) of high amounts of IFNα. However this induction is only possible when CVB4 infection is enhanced by non-neutralizing antibodies, resulting in increased viral entry in cells. We also reported detection of IFNα in plasma of T1D patients, commonly associated with enteroviral RNA. In addition, monocytes have been identified as major targets of enteroviruses among PBMCs. Besides IFNα, CVB4 can induce the synthesis of other proinflammatory cytokines, mainly IL-6 and TNFα. Interestingly, infection is not needed, since inactivated viral particles can induce these proinflammatory cytokines. In addition, the enhancing of CVB4 infection in PBMCs results in increased production of these cytokines. We have shown that macrophages that are known as major innate immunity effectors can produce IFNα and other proinflammatory cytokines upon infection with CVB4. Macrophages derived from PBMCs in presence of M-CSF (but not GM-CSF) can be infected by CVB4, and the virus can persist in these cells. CVB4 can also establish a productive, carrier-sate persistent infection in pancreatic ductal-like cells. The virus can be completely cleared from chronically-infected cells using fluoxetine. This molecule already used in the treatment of depression and other mental disorders, has displayed antiviral activity against many enteroviruses, and can completely clear CVB4 from chronically-infected cells within few weeks. Cellular changes have been observed during chronic infection including a reduced expression of PDX-1, a resistant profile to lysis upon superinfection with CVB4, and an important decrease of CAR expression. These changes can linger even after the clearance of CVB4. In addition the miRNA profile in chronically-infected ductal-like cells was clearly different from that of mock-infected cells. Some phenotypic and genotypic changes were also observed in the virus derived from chronic infection. Altogether, these findings show the features of CVB4 infection are compatible with mechanisms reported in the enteroviral pathogenesis of T1D, and support the hypothesis of involvement of CVB in this disease.