Influenza A virus (IAV) is a global health threat, causing seasonal epidemics and potential pandemics leading to morbidity, death and economic losses. Currently, there are two main classes of licensed antivirals against IAV available in the US and Europe; adamantanes and neuraminidase inhibitors, both of which are hindered by the generation of resistant virus variants. The viral polymerase has a high error rate leading to mutations that allow the virus to overcome selection pressures directed at its own genome from conventional antivirals. The prospect of inhibiting host proteins that the virus exploits to facilitate its replication is of increasing interest as an antiviral strategy as the emergence of resistance has been predicted to be slower when targeting a host cellular factor. IAV utilizes the host nuclear export protein CRM1 to transport viral ribonucleoproteins (vRNPs) from the nucleus to the cytoplasm of an infected cell, a critical late stage of the influenza lifecycle. Leptomycin B (LMB), a Streptomyces metabolite, has been previously shown to target this pathway, resulting in reduced viral propagation; however, LMB's potent cytotoxicity has limited its use as a therapeutic agent. This thesis examined two novel selective inhibitors of nuclear export (SINE), KPT-335 and KPT-185, with less cytotoxicity. In vitro, KPT-335 inhibited replication of human and animal IAV strains in a dose-dependent manner with minimal cytotoxicity. To assess the resistance potential of KPT-335, IAV viruses were serially passaged in the presence of a sub-optimal concentration of the compound and assayed for the development of resistance. Resistance to KPT-335 became evident at 8-10 rounds of passage. Sequencing analysis of independently derived resistant virus clones identified 4 single amino acid changes on a surface exposed patch of the viral nucleoprotein (NP). Introduction of these amino acid changes, into otherwise wild type viruses by reverse genetics, confirmed that changes Q311R and N309T conferred a drug-resistant phenotype. However, these substitutions came at a fitness cost to virus replication. The molecular basis for resistance was unclear but Q311R and N309T NP-mutant viruses produced increased levels of M1 during infection as well as producing virus particles with increased M1:NP ratios. Furthermore, the KPT-335-resistance mutations were surprisingly similar to NP sequence polymorphisms previously associated with susceptibility to the innate defence protein MxA. Consistent with this, viruses harbouring the Q311R mutation displayed increased susceptibility to MxA inhibition compared to wild-type virus. Altogether this study confirms that SINEs have the potential to be successful therapeutic agents against IAV replication and that although resistance could be generated, it may be difficult for the virus to overcome both drug selection pressures and the human innate immune response restrictions by escape mutations.

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La leucémie lymphoïde chronique (LLC) est la plus fréquente des leucémies de l'adulte, caractérisée par l'accumulation de lymphocytes B CD5+ anormaux dans le sang, la moelle osseuse, et les organes lymphoïdes secondaires. Les événements oncogéniques à l'origine du développement de la LLC sont peu connus. L'identification de nouveaux gènes dérégulés est importante afin d'améliorer notre compréhension du développement et de l'évolution de la LLC, et de proposer de nouvelles stratégies ciblées. Tout d'abord, nous avons montré que la LLC se développe à partir de progéniteurs hématopoïétiques multipotentes pré-leucémiques portant des mutations somatiques. J'ai également analysé la délétion 14q dans une large série de patients, et nous avons conclu que la del(14q) est associé à la trisomie 12 et à des facteurs pronostiques péjoratifs : IGHV non mutée, mutations NOTCH1, et une survie sans traitement plus courte. Enfin, la partie principale de ma thèse portrait sur la caractérisation du gain du bras court du chromosome 2 (2p). J'ai identifié une région minimale de gain chez les patients LLC/2p+ et démontré que CRM1/XPO1 (Exportin-1) se trouve dans cette région, et muté de façon récurrente dans la LLC. J'ai démontré que le gain 2p provoque la résistance aux traitements RFC, Ibrutinib, R-Idélalisib, et au Selinexor. Nos résultats révèlent également que le Selinexor est inefficace pour induire l'apoptose dans les cellules LLC-B muté pour XPO1. Au total, mon travail préconise l'évaluation du gain 2p et des mutations de XPO1 avant de décider d'un traitement de la LLC. / Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by an accumulation of abnormal CD5+ B-lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs. The origin and pathogenic mechanisms of CLL are not fully understood. Identifying the deregulation of new genes is important to improve our understanding about CLL development and evolution, and to propose new targeted strategies. First, we have shown that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. I have also analyzed the deletion 14q in a large series of patients, and we have concluded that del(14q) was associated with trisomy 12 and with pejorative prognostic factors: unmutated IGHV, NOTCH1 mutations, and a short treatment-free survival. Finally, the main part of my thesis was about the characterization of the short arm of chromosome 2 (2p). I identified a minimal region gained in 2p+/CLL patients and demonstrated that CRM1/XPO1 (Exportin-1) is located in this region, and recurrently mutated in CLL. I demonstrated that 2p gain provokes FCR, Ibrutinib, R-Idelalisib, and Selinexor drug resistance. Our results also reveal that Selinexor is inefficient in inducing apoptosis in CLL B-cells with mutations in XPO1. Altogether, my work advocates for the assessment of the 2p+ and XPO1 mutations before deciding a CLL therapy.

The content of this diploma thesis is description of semi-automatic assembly line, description of design and realization of electroprojection including electrical switchboard and control panel, together with description of design and implementation of control system including communications and user interface. Part of the thesis is also the analysis and solution of safety risks using a safety controller and overall verification and evaluation of the functionality of the whole system on described line.

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has functional activity similar to human F9 and Fixl is functionally similar to Fx. Thus, the zebrafish could be used to identify factors controlling f9 gene expression with age and for modeling Hemophilia B in the quest to develop gene therapy protocols. In zebrafish, dilute plasma with exogenously added human fibrinogen was used for kinetic coagulation assays. Here, we developed a microkinetic assay using 25% zebrafish or 30% human plasma followed by the addition of coagulation activators and CaCl2. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established human kinetic curves. Moreover, clotting times derived from these kinetic curves were identical to human PT, PTT, and Russel's viper venom time. Thus, the microkinetic assay developed here could measure blood coagulation activity in small animal models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.