L'extinction post-transcriptionnelle de gènes ou PTGS (Posttranscriptional Gene Silencing) est un phénomène hautement conservé à travers l'évolution. Retrouvé chez des organismes allant des champignons aux vertébrés, ce système reconnaît spécifiquement les molécules de RNA double brin (db) et les dégrade en fragments de 21-23 nt. Chez les plantes, le PTGS pourrait défendre l'organisme contre les infections virales. En réponse à ce mécanisme de défense, un certain nombre de virus codent pour des protéines capables de supprimer le PTGS. Ces protéines dites "suppresseurs" de PTGS, comme les protéines HCPro des potyvirus et 2b des cucumovirus, ne partagent aucune homologie de séquence ni de structure et jouent des rôles différents dans le cycle de multiplication virale. Les résultats obtenus et décrits dans ce mémoire ont permis l'identification et la caractérisation de nouvelles protéines virales impliquées dans la suppression du PTGS, ceci afin de mieux cerner les mécanismes de suppression. Ainsi, nous avons montré que les protéines P15 et P14 de deux virus apparentés, le PCV (Peanut Clump Virus) et le BNYVV (Beet Necrotic Yellow Vein Virus) sont des suppresseurs de PTGS aux propriétés distinctes. En effet, la protéine P15 se localise dans les peroxysomes et semble être active sous une forme multimérique ; quant à la protéine P14, elle se localise dans le cytoplasme et le nucléole et supprime plus faiblement le PTGS. Par ailleurs, nous avons également découvert que la protéine P0 du BWYV (Beet Western Yellows Virus) est un suppresseur de PTGS très efficace hors de son contexte viral. De manière surprenante, son expression est fortement régulée par le virus au cours de l'infection. Enfin, nous avons confirmé que la protéine P0 jouait un rôle de protection du virus contre le PTGS grâce à l'utilisation de plantes transgéniques exprimant la protéine mineure de capside du BWYV. Ce virus déclenche sur ces plantes un PTGS dirigé contre le transgène, sans être affecté.

Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

En esta Tesis Doctoral se ha estudiado el efecto de una serie de ácidos grasos como el araquidónico, docosahexaenoico, eicosapentaenoico y oleico, así como un conjunto de derivados de DAG-lactonas en la activación de diferentes isoenzimas de PKC. El sistema modelo de estudio utilizado han sido diferentes líneas celulares de cáncer de mama (BT-474, MCF-7 y MDA-MB-231). Mediante técnicas de biología molecular y celular, microscopía confocal, ARN de interferencia y microarrays de expresión diferencial de genes se ha estudiado la función de la PKCalpha en la capacidad proliferativa, invasiva y de migración de las células modelo de cáncer de mama, revelando nuevos mecanismos moleculares por los que la PKCalpha se localiza en la membrana plasmática y se activa en dichas células. También se pone de manifiesto el efecto antiproliferativo e inductor de apoptosis de los diversos ácidos grasos estudiados, así como la implicación directa de la PKCalpha en la capacidad proliferativa, migr atoria e invasiva de dichas células / In this Doctoral Thesis, the role of several fatty acids like arachidonic, docosahexaenoic, eicosapentaenoic and oleic, as well as some DAG-lactones derivatives, on the activation of different PKC isoforms has been studied. Some breast cancer cell lines, specifically BT-474, MCF-7 and MDA-MB-231, have been used as a model. The role of PKC&#61537; in proliferation, invasion and migration has been studied by means of cellular and molecular techniques, confocal microscopy, siRNA and gene expression microarrays. The results obtained reveal new molecular mechanisms of PKCalpha; localization and activation in breast cancer cell lines. It is also interesting the anti-proliferative and pro-apoptosis role of several fatty acids tested, as well as the direct involvement of PKCalpha; in proliferation, migration and invasion of breast cancer cells

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

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Les virus exploitent la machinerie cellulaire de l’hôte de façon très variée et plusieurs types vont même jusqu’à incorporer certaines protéines cellulaires. Nous avons récemment effectué la première analyse protéomique du virion mature de l’Herpès simplex de type 1 (HSV-1), ce qui nous a permis de déterminer que jusqu’à 49 protéines cellulaires différentes se retrouvaient dans ce virus (Loret, S. et al. (2008). "Comprehensive characterization of extracellular herpes simplex virus type 1 virions." J Virol 82(17): 8605-18.). Afin de déterminer leur importance dans le cycle de réplication d’HSV-1, nous avons mis au point un système de criblage nous permettant de quantifier le virus produit et relâché dans le milieu extracellulaire en utilisant un virus marqué à la GFP ainsi que des petits ARN interférents (pARNi) ciblant spécifiquement ces protéines cellulaires. Cette approche nous a permis de démontrer que 17 des protéines identifiées précédemment jouaient un rôle critique dans la réplication d’HSV-1, suggérant ainsi que leur incorporation dans le virus n’est pas aléatoire. Nous avons ensuite examiné le rôle d’une de ces protéines, DDX3X (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked), une protéine multifonctionnelle connue pour son implication dans les cycles de réplication de plusieurs virus humains. À l’aide de pARNi ainsi que de différentes lignées cellulaires, dont une lignée DDX3X thermosensible, nous avons démontré que l’inhibition de DDX3X résultait en une diminution du nombre de capsides intracellulaires et induisait une importante diminution de l’expression des gènes viraux. Nous avons aussi démontré que la fraction de DDX3X incorporée dans le virion contribuait activement au cycle infectieux d’HSV-1. Ces résultats confirment l’intérêt de notre approche afin d’étudier les interactions hôte-pathogène en plus de démontrer la contribution des protéines cellulaires incorporées à HSV-1 dans l’infection virale. / Viruses exploit the cellular machineries in many ways and several viruses specifically incorporate host proteins. To understand their biological relevance, we recently performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify which of these cellular factors are critical for the HSV-1 life cycle. To this end, we performed a functional screen using small interfering RNA (siRNA) and a GFP-tagged virus, which indicated that at least 17 of the virion-incorporated host proteins alter HSV-1 proliferation in cell culture. Interestingly, these include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Among them, the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked protein (DDX3X) is a multifunctional molecule previously linked to several other viruses. Its relevance for HSV-1 was further confirmed with different siRNA reagents and cell lines, including a DDX3X thermosensitive cell line. We found that DDX3X inactivation reduced intracellular capsid abundance via a strong inhibition of viral gene expression. We also report evidence that the pool of DDX3X present in the mature virions actively contributes to HSV-1 life cycle. Altogether, this highlights a powerful and biologically relevant approach to characterize host-pathogen interactions and points to the important contribution host proteins within mature viral particles.

Deux différents types de vecteurs, les nanocapsules lipidiques (LNC) et les systèmes multimodulaires (MMS) ont été développés pour l'administration par voie systémique de deux types d'acides nucléiques, l'ADN et les petits ARN à interférence (siRNA). Ces vecteurs sont formulés à base de complexes d'acides nucléiques et de lipides cationiques (lipoplexes) qui ont été soit encapsulés au coeur des LNC, soit recouverts de stabilisateurs stériques afin de former des MMS. Une partie du travail a consisté à développer des vecteurs de siRNA et à les caractériser par des méthodes physico-chimiques. En fonction du lipide cationique utilisé, jusqu'à 65% de siRNA ont pu être encapsulés dans les LNC, en présentant des caractéristiques appropriées pour une administration par voie systémique. La seconde partie a consisté à approfondir la caractérisation des vecteurs d'ADN et à analyser leur profil de distribution en utilisant de l'imagerie par fluorescence in vivo. Chez la souris saine, les vecteurs d'ADN ont présenté des profils de biodistribution spécifiques à leur composition. Sur deux modèles tumoraux (gliome sous-cutané et mélanome), les vecteurs ayant un temps de circulation prolongé ont également montré une co-localisation intéressante avec les cellules tumorales. Afin de mettre en évidence l'efficacité de ces vecteurs, un plasmide codant pour la tymidine kinase du virus herpes simplex (HSV-tk) a été encapsulé et administré. Puis un traitement par le ganciclovir (GCV) basé sur l'approche par gène suicide a été effectué. Les premiers résultats sont concluants, montrant une baisse de croissance tumorale après quelques jours de traitement aussi bien dans le modèle de gliome que dans celui du mélanome. Ces résultats indiquent que ces outils sont prometteurs pour une variété d'applications en thérapie génique.