Syncytin-1 is an endogenous retroviral envelope glycoprotein specifically expressed in human placenta, where the protein was adopted for its physiological function. After interaction with specific receptors, transmembrane proteins ASCT1 and ASCT2, Syncytin-1 initiates cell-cell fusion leading to formation of multinucleated syncytiotrophoblast, which is essential for feto-maternal nutrients exchange. In this diploma thesis a new cell-cell fusion quantification assay was implemented for characterisation of Syncytin-1 fusion determinants. The assay uses Syncytin-1 and ASCT2 expressed separately with fragments of luciferase in heterologous cell-culture system. The assay enables to specifically quantify cell-cell fusions based on activity of reconstituted luciferase reporter. This study discovered new facts about the role of intracytoplasmic tail of Syncytin-1 in the process of the cell- cell fusion. This specific part of protein contains a tandem motif sensitive to changes in amino acid sequence that led to loss of fusogenic potential of Syncytin-1. It was further confirmed, that the protein Suppressyn works as an inhibitor of cell-cell fusions initiated by Syncytin-1. Suppressyn however does not bind to receptors of Syncytin-1 and the mechanism of its inhibition remains unsolved. Finally, it was demonstrated...

Oncolytic measles virus is a promising cancer therapeutic in clinical trials which possesses multiple characteristics that are advantageous over traditional therapies. Currently, clinical oncolytic measles virus vectors are unmodified or express reporter transgenes that benefit its therapeutic efficacy. The next phase in its development will see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.

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La fusion des myoblastes est une étape cruciale pour une bonne formation musculaire pendant l'embryogenèse et après une blessure à l'âge adulte. Le système génétique simpliste des mouches a été largement utilisé dans le passé pour identifier les acteurs essentiels impliqués dans la fusion des myoblastes. Chez la drosophile, la protéine d'échafaudage Antisocial (Ants)/Rols7 joue un rôle essentiel dans la fusion des myoblastes en connectant les protéines de surface d'adhésion cellulaire au cytosquelette. Même si la plupart des voies moléculaires régissant la fusion des myoblastes sont évolutives conservées entre les mammifères et les mouches, les contributions relatives de Tanc1 et Tanc2, les orthologues mammifères de Ants/Rols7, dans la fusion de myoblastes n'ont pas été établies. Le premier objectif de la thèse était d'évaluer les contributions potentielles de Tanc1 et Tanc2 dans la fusion de myoblastes en utilisant la lignée cellulaire de myoblastes murins C2C12 comme modèle de différenciation et de fusion de myoblastes. Nous avons constaté que l'expression de Tanc1 et Tanc2 n'est pas modulée lors de la différenciation C2C12, mais que les deux échafaudages sont enrichis au niveau du cortex lors de la prolifération des myoblastes. De plus, le knockdown de Tanc1 ou Tanc2 a altéré la fusion des myoblastes sans affecter la différenciation des myoblastes. Notamment, l'expression du défaut de fusion humain entièrement restauré Tanc1 ou Tanc2 observé dans les cellules C2C12 épuisées pour Tanc1 ou Tanc2 suggérant qu'un niveau seuil de leur expression est critique pour une fusion efficace des myoblastes. De plus, ni Tanc1 ni Tanc2 n'ont pu se substituer à Ants/Rols7 lors de la fusion des myoblastes chez la drosophile, ce qui suggère que différents acteurs pourraient être impliqués dans la régulation de la fusion des myoblastes chez les mammifères. Le deuxième objectif de la thèse était de caractériser davantage le rôle de Tanc1 et Tanc2 dans la fusion de myoblastes en utilisant des modèles murins de souris. À cette fin, des souris knock-out Tanc1 totales (Tanc1 KO) et des souris knock-out Tanc2 conditionnelles (Tanc2 cKO) ont été générées. Bien que les souris Tanc2 KO aient été précédemment signalées comme étant mortelles sur le plan embryonnaire, nous rapportons ici que ces souris sont viables contrairement à ce qui a été rapporté. L'expression de Tanc1 et Tanc2 a été détectée dans les somites ainsi que dans les fibres musculaires primaires. L'analyse du phénotype musculaire au stade embryonnaire a révélé une différenciation normale des somites et la formation de fibres musculaires chez les souris Tanc1 KO et Tanc2 cKO. De plus, lors de l'analyse au stade adulte, aucune différence dans la section transversale des fibres musculaires entre les souris de type sauvage et les souris mutantes n'a été détectée. Cela pourrait-il impliquer une redondance potentielle entre Tanc1 et Tanc2 dans la régulation de la myogenèse ? Pour répondre à cette question, des souris double knockout Tanc1 et Tanc2 sont actuellement en cours de génération. En conclusion, nous avons identifié dans cette étude un nouveau rôle pour les protéines d'échafaudage Tanc1 et Tanc2 dans la fusion de myoblastes chez les mammifères. L'identification de nouveaux acteurs essentiels dans la fusion des myoblastes nous rapproche de sa compréhension et de son ciblage thérapeutique à long terme. / Myoblast fusion is a crucial step for proper muscle formation during embryogenesis and after in injury during adulthood. The simplistic genetic system of flies has been extensively used in the past to identify essential players involved in myoblast fusion. In Drosophila, the scaffold protein Antisocial (Ants)/Rols7 plays an essential role in myoblast fusion by connecting the cell adhesion surface proteins to the cytoskeleton. Even though most molecular pathways governing myoblast fusion are evolutionary conserved between mammals and flies, the relative contributions of Tanc1 and Tanc2, the mammalian orthologs of Ants/Rols7, in myoblast fusion have not been established. The first aim of the thesis was to assess the potential contributions of Tanc1 and Tanc2 in myoblast fusion by using the murine myoblast C2C12 cell line as a model for myoblasts differentiation and fusion. We found that Tanc1 and Tanc2 expression is not modulated during C2C12 differentiation, but that both scaffolds are enriched at the cortex during myoblast proliferation. Furthermore, the knockdown of either Tanc1 or Tanc2 impaired myoblast fusion without affecting myoblast differentiation. Notably, the expression of human Tanc1 or Tanc2 fully restored fusion defect observed in C2C12 cells depleted for Tanc1 or Tanc2 suggesting that a threshold level of their expression is critical for efficient myoblast fusion. Furthermore, neither Tanc1 nor Tanc2 could substitute for Ants/Rols7 during Drosophila myoblast fusion suggesting that different players might be involved in regulating myoblast fusion in mammals. The second aim of the thesis was to further characterize the role of Tanc1 and Tanc2 in myoblast fusion by using murine mice models. For this purpose, total Tanc1 knockout mice (Tanc1 KO) and conditional Tanc2 knockout mice (Tanc2 cKO) were generated. Although Tanc2 KO mice were previously reported to be embryonically lethal, we report here that those mice are viable contrary to what has been reported. Tanc1 and Tanc2 expression was detected in the somites as well as in the primary muscle fibers. Analysis of the muscle phenotype at the embryonic stage revealed normal somites differentiation and muscle fiber formation in both Tanc1 KO and Tanc2 cKO mice. Furthermore, when analyzed at the adult stage, no difference in the cross-sectional area of the muscle fibers between wild-type mice and mutant mice was detected. Could this imply a potential redundancy between Tanc1 and Tanc2 in regulating myogenesis? To answer this question, Tanc1 and Tanc2 double knockout mice are currently being generated. In conclusion, we identified in this study a novel role for the scaffold proteins Tanc1 and Tanc2 in myoblast fusion in mammals. Identifying new and essential players in myoblast fusion brings us a step closer to understanding it and on the long run target it therapeutically.

Viruses cause human diseases by entering in to human cells. Many drugs have been developed that act at various stages of viral infection, but they fail due to their toxic side effects and high mutation rates of viruses. Recently, a new class of drugs called entry inhibitors has been developed which acts on the early stages of viral infection. These drugs have been developed by studying the entry process of viruses in to host cells. The success of these drugs, however, is still limited and research is being done to quantify the optimum dosage of these drugs and find new drugs targets. We developed a mathematical model based on chemical reaction kinetics to estimate the threshold number of complexes between viral and target cell surface proteins necessary for HIV-1 entry into target cells. Our model quantitatively describes data of HIV entry in the presence of several entry inhibitors and presents an avenue for identifying optimal drug levels for restricting HIV entry. Majority of viruses enter into host cells by either endocytosis of fusion. But when virus enters through endocytosis and when through fusion is still not clear. We developed a theory that predicts the virus entry pathway based on the underlying biophysical properties like membrane bending modulus, viral and cellular receptor concentration and the energy released by the formation of protein complexes. Through this theory of viruses we presented the entry of viruses through fusion or endocytosis on a phase diagram. We validated the phase diagram by comparing it with known pathways of existing viruses. This study may aid in unraveling the entry pathways of new viruses and may also help in identifying new drug targets.