L’intérêt des composés organiques fluorés est de nos jours de plus en plus important en raison de leur large domaine d’application (agrochimie, nucléaire, matériaux, chimie médicinale…). Par exemple, en chimie médicinale, la présence d’un ou plusieurs atomes de fluor au sein de biomolécules conduit très souvent à une amélioration de leur profil thérapeutique. Par ailleurs, le cyclopropane, le plus petit et le plus tendu des cycloalcanes, permet également de modifier les caractéristiques pharmacologiques de composés biologiques de par sa géométrie inhabituelle. En effet, la rigidification structurale apportée par ce motif influe sur la biodisponibilité d’une biomolécule en améliorant sa sélectivité et son affinité pour un récepteur biologique. Dans ce contexte, nous avons choisi d’associer les propriétés remarquables de l’atome de fluor à la contrainte structurale du cyclopropane dans le but d’élaborer deux nouvelles classes de fluoropeptidomimétiques.Tout d’abord, nous nous sommes intéressés à la modification de la chaîne latérale d’acides aminés naturels en développant la synthèse des analogues cyclopropaniques fluorés de la méthionine, de la leucine, de la lysine et de l’arginine. Nous avons ensuite appliqué l’un de nos acides aminés cyclopropaniques fluorés à la synthèse totale de l’analogue fluoré d’un inhibiteur de la sérineprotéase NS3/4A, le TMC 435.Enfin, dans le but de proposer une voie de synthèse générale permettant l’accès aux pseudopeptides fluorés comportant un monofluorocyclopropane à la place du lien peptidique, nous avons développé une nouvelle stratégie basée sur une étape d‘addition nucléophile de réactifs organométalliques sur des N-(tert-butanesulfinyl)-α-fluoroimines chirales. / Fluoroorganic compounds are increasingly popular owing to their wide range of applications. For instance, in the field of medicinal chemistry, fluorinated molecules often lead to an improvement of the therapeutic profile compared to non-fluorinated derivatives. Besides, with its unique bonding properties, the cyclopropane ring provides unusual physical and pharmacological properties to structures that incorporate it. Indeed, the structural constraint provided by the cyclopropane ring clearly alters the selectivity and the affinity for a binding site. In this context, we decided to combine the cyclopropane and the fluorine atom to develop two new classes of peptidomimetics. First, we focused on the modification of the side chain of natural aminoacids (methionine, leucine,lysine and arginine) and the synthesis of fluorinated cyclopropyl analogues was achieved. Then, we applied our strategy to the synthesis of the fluorinated analogue of the TMC 435, a NS3/4A serine protease inhibitor involved in the replication cycle of Hepatitis C virus. Finally, in our project aiming at proposing a general method to access pseudopeptides featuring a fluorinated cyclopropane moiety as the peptide bond isostere, we develop a new strategy based on the nucleophilic addition of organometallic reagents to N-(tert-butanesulfinyl)-α-fluoroimines. This methodology allows us to control the asymmetric center on the N-terminal side of the peptide.

The hepatitis A crisis in Kentucky is unprecedented compared to other states. With thousands of hospitalizations and over 60 deaths in the state of Kentucky alone, there is a need to evaluate the government’s response to the hepatitis A virus (HAV) epidemic. Therefore, the instructional risk communication messages that are being conveyed by the Kentucky Cabinet for Health and Family Services (KCHFS) are instrumental in the education and action plans of Kentuckians who are at risk of contracting HAV. This study utilizes the IDEA Model Thematic Analysis Codebook as a guide to analyze the KCHFS’ risk communication regarding the hepatitis A crisis, identifying the strengths and weaknesses of its campaign. Through this research, I identify the weaknesses in the KCHFS’ risk communication campaign including: the need to focus on a more general audience, the need to foreground all facets of the IDEA model on its website, the need to promote the vaccination of the HAV more strongly, and the need to make the website more user-friendly for all populations.

Hepatitis C Virus (HCV) is a small RNA virus that progresses to chronicity in 50-80% of infected individuals. Direct-acting antivirals (DAAs) are revolutionary treatments for HCV with 90-98% cure rates. However, over time, chronic HCV infections can result in advanced liver disease, including cirrhosis. Patients with advanced fibrosis experience a poor response to vaccination, recurrent infections and increased risk for hepatocellular carcinoma (HCC). These outcomes are, in part, a consequence of immune dysfunction. Increased inhibitory receptor and Galectin-9 (GAL-9) expression is a possible mechanism promoting lymphocyte dysfunction. In this study, blood samples were collected from chronic HCV patients with different degrees of liver fibrosis. I conducted a 13-parameter flow stain on the peripheral blood mononuclear cells (PBMC) of these patients. Next, I measured the expression of inhibitory receptors (PD-1, CTLA-4, LAG-3, TIGIT and TIM-3) and GAL-9 on bulk T cell and NK cells of 15 chronic HCV patients with no to moderate fibrosis (F0-F2) and 15 with advanced fibrosis (F3-F4). To analyze receptor co-expression, I employed t-distributed stochastic neighbor embedding (t-SNE) analysis to dimensionally reduce the multi-parametric data. Notably, I found that F3-F4 patients had higher frequencies of >3 inhibitory receptor co-expression on NK cells. Moreover, t-SNE analysis of bulk T cells revealed that F3-F4 patients manifest a higher frequency of cells in the clusters with CD25+TIGITmed-hi CD4+ T cells and PD-1medLAG-3med-hiGAL-9med-hi CD4+ T cells. t-SNE analysis of NK cells also showed that F3-F4 patients manifest a higher frequency of cells in the cluster with CD25+TIGITmed-hiTIM-3med-hi CD56Dim NK cells and CCR7+ PD-1medLAG-3med-hiGAL-9med-hi CD56Dim NK cells. Lastly, the frequency of cells in these clusters was found to positively correlate with patient’s extent of liver damage. In conclusion, I identified phenotypes of immune dysregulation that could explain the increased susceptibility to infection and HCC in chronic HCV patients with advanced fibrosis. These phenotypes could identify targets for combinatorial checkpoint blockade therapy to potentially improve immune function in these patients.

INTRODUCTION: Despite rising hepatitis C virus (HCV) incidence in the United States in recent years among young adults, little data describe HCV testing in youth. My objective was to characterize the HCV care cascade in adolescents and emerging adults in a large US sample and to describe the association between diagnosed substance use disorders (SUDs) and HCV testing. METHODS: In this retrospective cohort study, I describe HCV care cascade outcomes for youth 13–21 years old seen at least once from 1/2012–9/2017 at an OCHIN-participating federally qualified health center. Using electronic health record data, I analyzed odds of HCV testing by number of concurrent diagnosed SUDs associated with HCV risk (those associated with injection or intranasal use: opioids, amphetamines, and cocaine). RESULTS: Among 269,124 youth who met inclusion criteria, (54.7% female, 62.5% non-white, mean age [SD] at testing 18.5 [2.2] years), 6812 (2.5%) were tested for HCV antibody, 122/6812 (1.8%) of those tested were anti-HCV positive, and of anti-HCV positive youth, 75.4% had additional diagnostic testing. Only 1 had documented HCV treatment. Each additional HCV risk-associated SUD was associated with higher odds of HCV testing, particularly in younger (OR 9.12, 95% CI 6.78, 12.4 in 13–15 year-olds, and OR 8.37, 95% CI 7.48, 9.36 in 16–18 year-olds) compared with older youth (OR 3.9, 95% CI 3.59, 4.24 in 19–21 year-olds). CONCLUSION: This study highlights important gaps in recommended HCV testing during the current opioid crisis. As the first step in the care cascade, addressing missed testing opportunities is critical for reducing hepatitis C burden. / 2020-09-28T00:00:00Z

Myeloid‐derived suppressor cells (MDSC s) and microRNA s (miRNA s) contribute to attenuating immune responses during chronic viral infection; however, the precise mechanisms underlying their suppressive activities remain incompletely understood. We have recently shown marked expansion of MDSC s that promote regulatory T (Treg) cell development in patients with chronic hepatitis C virus (HCV ) infection. Here we further investigated whether the HCV ‐induced expansion of MDSC s and Treg cells is regulated by an miRNA ‐mediated mechanism. The RNA array analysis revealed that six miRNA s were up‐regulated and six miRNA s were down‐regulated significantly in myeloid cells during HCV infection. Real‐time RT ‐PCR confirmed the down‐regulation of miR‐124 in MDSC s from HCV patients. Bioinformatic analysis suggested that miR‐124 may be involved in the regulation of signal transducer and activator of transcription 3 (STAT ‐3), which was overexpressed in MDSC s from HCV patients. Notably, silencing of STAT ‐3 significantly increased the miR‐124 expression, whereas reconstituting miR‐124 decreased the levels of STAT ‐3, as well as interleukin‐10 and transforming growth factor‐β , which were overexpressed in MDCS s, and reduced the frequencies of Foxp3+ Treg cells that were developed during chronic HCV infection. These results suggest that reciprocal regulation of miR‐124 and STAT ‐3 in MDSC s promotes Treg cell development, thus uncovering a novel mechanism for the expansion of MDSC and Treg cells during HCV infection.

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14+ M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14+ M/Mφ incubated with HCV+ Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 – an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.

The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57. To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.

L’infection par le virus de l’hépatite E (VHE) peut entraîner une hépatite aiguë chez l’homme évoluant en hépatite fulminante dans 1-4% des cas, voire 20% chez les femmes enceintes dans les régions endémiques. Le VHE, transmis par voie entérique, est responsable de grandes épidémies d’origine hydrique dans les pays en voie de développement et de nombreux cas sporadiques d’origine zoonotique dans des pays industrialisés. La description récente de cas d’hépatites E chroniques ou d’atteintes neurologiques graves souligne l’importance de caractériser les interactions du VHE avec son hôte. L’objectif de ce projet de thèse était de caractériser les interactions entre le VHE et la réponse immune innée de l’hôte et en particulier avec le système interféron de type I (IFN-I).La première partie de ce projet a consisté en l’étude de la modulation des voies de signalisation de l’IFN-I par la polyprotéine non-structurale ORF1 du VHE. Celle-ci est constituée de plusieurs domaines fonctionnels putatifs tels qu’une methyltransférase (Met) ou une protéase à cystéine de type papaïne (PCP) dont les fonctions sur la signalisation IFN-I restent encore peu connues. Les résultats obtenus ont montré que le domaine MetPCP de l’ORF1 est capable d’inhiber l’activation du promoteur de l’IFN-β et de celui des gènes sous le contrôle de l’IFN contenant des éléments de réponse à l’IFN (ISRE), ainsi que l’expression de certains des gènes induits par l’IFN (ISGs). En recherchant le mécanisme impliqué dans l’inhibition du promoteur ISRE, nous avons montré que le domaine MetPCP inhibe la phosphorylation de STAT1 et sa relocalisation nucléaire. Nous avons également montré que le domaine MetPCP n’inhibe pas la phosphorylation de STAT2. Le mécanisme d’action du domaine MetPCP reste encore à préciser. La deuxième partie de ce projet a été de déterminer si l’infection par le VHE entraîne la production d’IFN-I par les cellules dendritiques plasmacytoïdes (pDCs). En effet, les pDCs sont la principale source d’IFN-I et jouent un rôle crucial dans la réponse immunitaire innée et adaptative. Les résultats obtenus suggèrent que les pDCs ne produisent que modérément de l’IFN-I lorsqu’elles sont co-cultivées avec des cellules infectées par le VHE. L’ensemble des résultats obtenus pendant ce travail de thèse suggère que le VHE utilise plusieurs mécanismes pour moduler la signalisation IFN-I de l’hôte. / Hepatitis E virus (HEV) is the causative agent of acute hepatitis in humans that can lead to fulminant hepatitis in 1-4% of cases, and in 20% of pregnant women in endemic regions. HEV is an enterically-transmitted virus responsible for large waterborne epidemics in developing countries and numerous cases of zoonotic hepatitis E in industrialized countries. The recent description of cases of chronic hepatitis E and severe neurological disorders highlight the importance to characterize the interactions between HEV and the host. The objective of this PhD project was to characterize the interactions between HEV and the host innate immune response and particularly with the type I interferon system (IFN-I).The first part of this project aimed to study the ability of the ORF1 non-structural polyprotein of HEV to modulate the IFN-I signalling pathways. HEV ORF1 contains several putative functional domains including a methyltransferase (Met) and a papain-like cysteine protease (PCP) whose functions on the IFN-I signalling remain poorly understood. The results obtained showed that the MetPCP domain of ORF1 inhibits IFN-β and IFN-stimulated response element (ISRE) promoter activation and the expression of some IFN-stimulated genes (ISGs). We then investigated the mechanism involved in this inhibition of ISRE promoter activation. We showed that the MetPCP domain inhibits STAT1 phosphorylation and nuclear translocation. In contrast, we found that the MetPCP domain does not inhibit STAT2 phosphorylation. However, the mode of action of MetPCP remains to be fully characterised. The second part of this project aimed to determine the ability of plasmacytoid dendritic cells (pDCs) to produce IFN-I in response to HEV infection. Indeed, pDCs are the main IFN-I source and play a crucial role in innate and adaptive responses. The results obtained suggest that pDCs produce IFN-I moderately when co-cultured with HEV-infected cells. Taken together, the results obtained during this PhD project suggest that HEV has evolved different mechanisms to modulate the IFN-I host response.

Madagascar fait partie de la zone de haute endémicité pour l‟hépatite B dont le profil de circulation varie selon la ruralité de la zone d‟habitation. De par son insularité et les origines de ses peuplements, nous avons supposé que ce profil de circulation du VHB était dû à la variabilité du VHB et à l‟hétérogénéité du profil de transmission. Ce projet de thèse a pour objectif principal de déterminer les facteurs épidémiologiques et moléculaires influençant la dynamique de transmission et l‟évolution vers les complications de l‟infection par le virus de l‟hépatite B à Madagascar. Résultats : la séroprévalence globale pondérée en Ag HBs est de 6,9% avec des variations allant de 0% à 26% selon les zones géographiques considérées. La prévalence augmente en s‟éloignant des grandes villes et des principales routes nationales les reliant et chez les individus à faible statutsocio-économique. L‟étude du flux génétique des souches virales de l‟hépatite B montre que les zones les plus reculées représentent un réservoir pour la dissémination du virus. L‟infection par le virus de l‟hépatite B est responsable de 31% des maladies hépatiques chroniques rencontrées dans les services hospitaliers investigués à Antananarivo. L‟introduction du VHB s‟est probablement faite au cours du XIXème siècle. Sa propagation à l‟intérieur du pays a pris une allure exponentielle durant les années 80s probablement durant les épidémies de paludisme et suite à des réutilisations des matériels d‟injections. Conclusion : Les résultats de ces différents travaux nous ont permis de plaider pour une politique de lutte visant en particulier les zones très reculées de l‟île où la prévalence en AgHBs est la plus importante. / Madagascar is part of endemic region of HBV. Distribution is different in rural and urban area. The historic of human settlement and its insularity might impact distribution and molecular characteristic of HBV in Madagascar, we then supposed that difference observed in distribution and prevalence of HBV were due to viral variability and different pattern of viral transmission. Therefore, the main objective of this thesis was to determine molecular and epidemiological pattern that may influence dynamic transmission and complications of infection. Results: weighted prevalence of HBsAg was 6.9%. It varied from 3% to 26% according to area of sampling. Populations with a low socio-economic status and those living in rural areashad a significantly higher seroprevalence of HBsAg. Gene flow study showed rural area remain important in virus diffusion.HBV infection was found to be responsible of 31% of chronic liver disease encountered in the main public hospital in the capital of the country. Because of its recent emergence, its introduction dated from XIX century during colonization period. Its expansion during 1980s might be due to use of unsafe injection material mainly during malaria epidemic. Conclusion: The result of these work allowed us to advocate for a policy of struggle, in particular in the very remote areas of the island where the HBsAg prevalence is the most important and where care and preventive measures such as vaccinations are scarce.

Au sein du laboratoire, par une stratégie de conception de médicaments par la méthode des fragments, nous avons généré une nouvelle famille d'inhibiteurs de cyclophilines, les SMCypI (« Small-Molecule Cyclophilin Inhibitors »), non liée aux autres inhibiteurs de cyclophilines existants. Les cyclophilines sont des protéines cellulaires impliquées dans un grand nombre de processus biologiques. Toutefois, les inhibiteurs de cyclophilines disponibles possèdent de nombreux inconvénients qui rendent leur utilisation clinique difficile. Au cours de ma thèse nous nous sommes intéressés au développement des SMCypI dans deux domaines en particulier, la Virologie et l’Hépatologie.Dans le domaine de la Virologie, les cyclophilines sont impliquées dans la réplication de plusieurs virus et constituent donc une cible de choix dans le développement d'antiviraux à large spectre. Dans un premier temps, nous nous sommes intéressés à la caractérisation de l’activité antivirale de ces molécules sur le virus de l’Hépatite C, avec comme objectif de démontrer leur activité pangénotypique, leur haute barrière à la résistance, leur mécanisme d’action et leur activité antivirale à large spectre pour d’autres virus de la famille des Flaviviridae.Dans le domaine de l’Hépatologie, les lésions d’ischémie-reperfusion hépatique sont rencontrés pendant la chirurgie hépatique et la transplantation hépatique. La mitochondrie est un acteur majeur via l’ouverture du pore de transition de perméabilité mitochondrial. L’ouverture du pore est modulée par la cyclophiline D. Dans un deuxième temps, nous avons étudié les effets des SMCypI sur cette deuxième cible. Cela nous a permis de démontrer leur effet hépatoprotecteur dans un modèle murin d’ischémie-reperfusion hépatique.L’ensemble de ces résultats ouvre la porte pour le concept des antiviraux à large spectre, et l’utilité dans le domaine de l’hépatologie comme molécules hépatoprotectrices.... / In our laboratory we previously reported a rational design of a new family of smallmolecule cyclophilins inhibitors, SMCypI, unrelated to other cyclophilins inhibitors by means of a complex fragment-based drug discovery approach. Cyclophilins are cellular proteins involved in multiple biological processes. Unfortunately, different disadvantages have limited their clinical development. The aim my thesis was to study the SMCypI in two particulars fields, Virology and Hepatology.In the field of Virology, cyclophilins inhibitors are involved in viral replication of multiple viruses, which make them a convenient target for the development of “broad-spectrum antivirals”. Here, we first characterized the pangenotypic anti-HCV activity of this new family of SMCypI, with high resistance barrier. We studied its mechanism of action andits broad antiviral activity on other members of the Flaviviridae family.In the field of Hepatology, ischemia-reperfusion injuries occur during liver surgery and liver transplantation. Mitochondria play a central role in the opening of mitochondrial permeability transition pore. The opening of this pore is mainly regulated by cyclophilin D. The aim of the second part of our work was to demonstrated a hepatoprotective effect of the SMCypl in a murine model of hepatic ischemia reperfusion injury.Overall, these results are leading the way to the development of broad-spectrumantiviral drugs and their use in hepatology as hepatoprotective drugs....