Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...

Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...

La peau est organisée en niche de cellules souches/progénitrices qui contribuent au maintien des lignées épidermiques pendant l’homéostasie permettant ainsi de conserver l’intégrité du tissue. Les différentes cascades de signalisation qui régulent cet équilibre sont essentielles et la perturbation de ces voix peuvent amener à une différentiation anormale des kératinocytes, pouvant engendrer des cancers de la peau. Le but de cette thèse était d’identifier et de caractériser la population de progéniteurs responsables de la maintenance d’une niche épidermique spécialisée dans la mechanotransduction du toucher léger appelée les cellules de Merkel. Mon étude a porté sur le rôle des progéniteurs épithéliaux localisés dans le dôme du toucher (DT) de l’épiderme dans des conditions d’homéostasie ainsi que sur le développement du carcinome des cellules de Merkel. Basé sur l’analyse de données de microarray, j’ai identifié une nouvelle population de progéniteurs qui expriment de manière spécifique Kératine 17 (K17). Des expériences de traçage de lignées cellulaires démontrent que ces cellules donnent naissance aux cellules de Merkel (CM) ainsi qu’aux cellules squameuses. De plus, l’ablation génétique sélective des progéniteurs des CMs dans le DT a montré que cette niche est isolée et indépendante du reste de l’épiderme. Ces résultats établissent les CMs comme la quatrième lignée cellulaire de la peau. Cette découverte a permis l’établissement de nouveaux outils pour l’étude de conditions pathologiques associées à la lignée des cellules de Merkel, telles que les carcinomes des cellules de Merkel et le déclin du toucher léger avec l’âge. / The skin is organized in highly regionalized stem or progenitor cell niches that are in charge of maintaining all epidermal lineages during homeostasis. Disruption of molecular pathways that tightly regulate this balance leads to abnormal specification and differentiation of keratinocytes, eventually causing skin cancer. The goal of this thesis was to identify and characterize the progenitor population responsible for the maintenance of an epidermal niche specialized for mechanosensory signaling: the Merkel cell lineage. This work focused on the role of the epithelial progenitors located in the touch dome (TD) of hairy skin under homeostatic conditions and in a Merkel cell carcinoma (MCC) context. Based on previous microarray data, I first identified a distinct population of the interfollicular epidermis uniquely expressing Keratin 17 (K17). By lineage tracing analysis, I demonstrated that these cells give rise to the Merkel cell (MC) and squamous lineage. More importantly, selective genetic ablation of K17+ TD keratinocytes (TDKC) showed that the TD is a self-autonomous niche defining it as the 4th lineage of the skin. Interestingly, TDKCs may be involved in maintaining innervation of the Merkel cell-neurite complex. These critical results have established a new plateform for the field to allow studies of pathological skin conditions such as Merkel cell carcinoma and the loss of tactile acuity with age.

Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...

Le polyomavirus à cellules de Merkel est aujourd’hui reconnu comme l’agent étiologique du carcinome à cellules de Merkel (CCM). Le cycle viral et les mécanismes de l’oncogenèse viro-induite sont peu connus et les connaissances se basent essentiellement sur les études menées notamment sur le polyomavirus SV40. L’objectif des travaux de thèse était d’identifier les interactions entre les protéines virales et les protéines cellulaires lors de l’infection ou dans le contexte du carcinome à cellules de Merkel (CCM). Pour identifier ces interactions, nous avons réalisé des cribles double hybride en levures sur les oncogènes du MCPyV et du BKPyV. Afin valider les interactions obtenues en levures, nous avons utilisé une méthode orthogonale de validation par complémentation en cellules de mammifères reposant sur la restauration de la luciférase de Gaussia princeps. La combinaison de ses deux techniques nous a permis de valider des interactions avec des partenaires cellulaires impliqués dans la régulation du cycle cellulaire ou encore de la voie Akt-mTOR. Les précédents travaux du laboratoire, qui portaient sur l’interactome des protéines mineures de capsides VP2/VP3, avaient également permis d’identifier des interactions avec des protéines de la voie NF-kB. Nous avons alors testé les interactions entre les oncogènes et la protéine mineure de capside VP2 avec des protéines cellulaires impliquées dans cette voie. Ces travaux nous ont conduits à tester l’activation de la voie, l’expression des gènes sous le contrôle de NF-kB et la régulation de l’apoptose. Les résultats obtenus montrent une action de la protéine VP2 sur l’activation de la voie NF-kB et une induction de l’apoptose. / The Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis.