Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

The major hurdle in the development of transdermal route as a versatile drug delivery method is the formidable transport barrier provided by the stratum corneum. Despite decades of research to overcome the stratum corneum barrier, limited success has been achieved. The objectives of this research were to develop and characterize two different strategies to overcome the stratum corneum barrier for transdermal delivery of biopharmaceuticals and vaccines. In the first strategy, coated microneedles (sharp-tipped, micron-sized structures) were developed to enable delivery of drugs directly into the skin by bypassing the stratum corneum barrier. In the second strategy, instead of bypassing the barrier, microdermabrasion was used to selectively abrade stratum corneum with sharp microparticles for topical drug application. Coated microneedles For developing painless microneedles, the first detailed study was performed to characterize the effect of microneedle geometry on pain caused by microneedle insertions in human volunteers. This study demonstrated that microneedles are significantly less painful than a 26-gage hypodermic needle and that decreasing microneedle length and numbers reduces pain. Next, the first in-depth study of microneedle coating methods and formulations was performed to (i) develop a novel micron-scale dip-coating process, (ii) test the breadth of compounds that can be coated onto microneedles, and (iii) develop a rational basis to design novel coating formulations based on the physics of dip-coating. Finally, a plasmid DNA-vaccine was coated onto microneedles to immunize mice, to provide the first evidence that microneedle-based skin immunization can generate a robust in vivo antigen-specific cytotoxic-T-lymphocyte response using similar, or lower, DNA doses on microneedles as when using the gene gun or intramuscular injection. Microdermabrasion We demonstrated for the first time that microdermabrasion in monkeys and humans can selectively, yet completely remove the stratum corneum layer. Using a mobile mode of microdermabrasion, an increase in the number of treatment passes led to greater tissue removal. Furthermore, topical application of Modified Vaccinia Ankara virus after microdermabrasion induced virus-specific antibodies in monkeys. In conclusion, both coated microneedles and microdermabrasion were developed to enable delivery of biomolecules into the skin, indicating their potential for transdermal delivery of a wide range of biopharmaceuticals and vaccines.

El virus de l'hepatitis C (VHC) provoca una hepatitis crònica que afecta a més de 170 milions de persones d'arreu del món. És un virus petit que es classifica dins de la família Flaviviridae i és un virus d'RNA de cadena positiva amb un genoma d'aproximadament 9.600 nucleòtids. A l'extrem 5' del genoma viral s'hi troba una regió no codificant (5'NCR) que comprèn els primers 341 nucleòtids i la seva funció està relaciona amb la traducció. Immediatament després hi ha una pauta de lectura oberta ORF que acaba en un únic codó d'aturada i codifica una poliproteïna de 3.010 aminoàcids. A continuació l'extrem 3' no codificant (3'NCR), que malgrat es desconeixen les seves funcions exactes, s'ha demostrat que és essencial per a la replicació vírica. La única poliproteïna generada és processada co- i postraduccionalment mitjançant proteases de l'hoste i víriques, donant lloc a les proteïnes estructurals (Core, E1 i E2-p7) i no estructurals (NS2-NS5B). Igual que la majoria de virus RNA, el VHC es caracteritza per tenir una taxa de mutació elevada. De fet, el genoma del virus no es pot definir com una única seqüència sinó per una població de variants molt relacionades entre sí. A aquesta manera d'organitzar la informació genètica se l'anomena quasiespècie viral i una de les seves implicacions principals és la facilitat amb què sorgeixen resistents al tractament. Els tractaments disponibles són llargs, cars, provoquen efectes secundaris considerables i només es resolen completament el 40% dels casos. Per aquesta raó es busquen altres solucions terapèutiques per combatre el virus entre les quals s'hi inclouen diferents estratègies. Una de les més innovadores i prometedores és la utilització de ribozims dirigits directament contra el genoma del virus. Aquest treball es centra en l'estudi de les noves estratègies terapèutiques basades en ribozims, concretament la ribonucleasa P. La ribonucleasa P és un ribozim que està present en tots els organismes ja que és l'enzim responsable de la maduració dels precursors d'RNA de transferència. El més interessant a nivell terapèutic és que s'ha demostrat que es pot dirigir la seva activitat cap a qualsevol RNA utilitzant una seqüència guia d'RNA que quan hibrida amb l'RNA diana, l'híbrid imita l'estructura secundària del substrat natural. En el cas del VHC, s'han estudiat ribozims dependents de seqüència (ribozims derivats d'RNAs satèl·lits i de viroides de plantes), sempre dirigits contra la regió més conservada del virus per evitar una disminució de l'eficiència del ribozim deguda a la variació de la diana. La ribonucleasa P és una endonucleasa d'activitat molt específica i es diferencia dels altres ribozims naturals en el sistema de reconeixement del substrat, reconeix elements estructurals i no de seqüència. L'objectiu final del treball és tallar in vitro l'RNA del VHC aprofitant la propietat que presenta aquest ribozim de reconèixer elements estructurals i no de seqüència ja que per a un mateix nombre de seqüències, el nombre d'estructures viables que pot adoptar l'RNA genòmic és molt més petit i per tant la variabilitat de la diana disminueix. S'han estudiat dos models d'RNasa P, la RNasa P humana guiada per seqüència guia externa (EGS) i l'RNA M1 de l'RNasa P d'E.coli unit a la seqüència guia per l'extrem 3' (ribozim M1GS). Abans però de dirigir el ribozim, s'han estudiat l'estructura i la variabilitat d'una regió del genoma del virus ja que s'ha descrit que són factors que poden limitar l'eficiència de qualsevol ribozim. Derivat d'aquests estudis s'aporten dades sobre accessibilitat i variabilitat d'una regió interna del genoma del virus de l'hepatitis C, la zona d'unió de la regió E2/NS2 (regió 2658-2869). L'estudi d'accessibilitat revela que la regió 2658-2869 del genoma del virus conté dominis oberts i tancats i que la transició entre uns i altres no és brusca si es compara amb altres regions d'estructura coneguda (regió 5' no codificant). Els resultats dels assajos in vitro amb els dos models de RNasa P mostren que s'ha aconseguit dirigir tant la ribonucleasa P humana com el ribozim M1GS cap a una zona, predeterminada segons l'estudi d'accessibilitat, com a poc estructurada i tallar l'RNA del virus. De l'anàlisi de mutacions, però, es dedueix que la regió estudiada és variable. Tot i dirigir el ribozim cap a la zona més accessible, la variació de la diana podria afectar la interacció amb la seqüència guia i per tant disminuir l'eficiència de tall. Si es proposés una estratègia terapèutica consistiria en un atac simultani de vàries dianes.D'altra banda i derivat d'un resultat inesperat on s'ha observat en els experiments control que l'extracte de RNasa P humana tallava l'RNA viral en absència de seqüències guia externes, s'ha caracteritzat una nova interacció entre l'RNA del VHC i la RNasa P humana. Per a la identificació de l'enzim responsable dels talls s'han aplicat diferents tècniques que es poden dividir en mètodes directes (RNA fingerprinting) i indirectes (immunoprecipitació i inhibicions competitives). Els resultats demostren que la ribonucleasa P humana, i no un altre enzim contaminant de l'extracte purificat, és la responsable dels dos talls específics observats i que es localitzen, un a l'entrada interna al ribosoma (IRES) i molt a prop del codó AUG d'inici de la traducció i l'altre entre la regió codificant estructural i no estructural. La ribonucleasa P és un dels enzims del metabolisme del tRNA que s'utilitza per identificar estructures similars al tRNA en substrats diferents del substrat natural. Així doncs, el fet que la ribonucleasa P reconegui i talli el genoma del VHC en dues posicions determinades suggereix que, a les zones de tall, el virus conté estructures semblants al substrat natural, és a dir estructures tipus tRNA. A més, tot i que el VHC és molt variable, els resultats indiquen que aquestes estructures poden ser importants per el virus, ja que es mantenen en totes les variants naturals analitzades. Creiem que la seva presència podria permetre al genoma interaccionar amb factors cel·lulars que intervenen en la biologia del tRNA,particularment en el cas de l'estructura tipus tRNA que es localitza a l'element IRES. Independentment però de la seva funció, es converteixen en unes noves dianes terapèutiques per a la RNasa P. S'ha de replantejar però l'estratègia inicial ja que la similitud amb el tRNA les fa susceptibles a l'atac de la ribonucleasa P, directament, en absència de seqüències guia externes. / Hepatitis C virus is a human pathogen causing chronic liver disease in 170 million people worldwide. The virus is classified within the family Flaviviridae. The RNA genome is single-stranded and functions as the sole mRNA species for translation. It comprises a 5'-untranslated region, which functions as an internal ribosome entry site, and a long open reading frame, which encodes a polyprotein precursor of about 3010 amino acids, that is cleaved into structural (core, envelope 1, envelope 2 and p7) and non-structural (NS-2, NS-3, NS-4 and NS-5) proteins; followed by a 3' non-coding region. Analyzing significant numbers of cDNA clones of hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. In the infected patient, a master (the most frequently represented sequence) and a spectrum of mutant sequences may be isolated at any given time during chronic infection. This manner of organizing genetic information, which characterizes most RNA viruses, is referred to as quasispecies. HCV resistance to treatment (either alone or in combination with ribavirin) is one of the most important clinical implications predicted by the quasispecies model suggesting the necessity to seek new therapies. HCV therapeutic strategies based on ribozyme cleavage are leading candidates. The ribozyme activity of Ribonuclease P (RNase P) is among proposed antiviral agents. RNase P is a ubiquitous cellular endonuclease and one of the most abundant and efficient enzymes in the cell. This enzyme is a ribonucleoprotein complex that catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA to generate the mature tRNA. Substrate recognition by the RNase P ribozyme does not rely on sequence requirements but on structural features of the RNA substrate. Custom-designed ribo-oligonucleotides, which hybridize with the target, called external guide sequences (EGSs), may provide the RNA structure which RNase P recognizes and cleaves in the hybridized complex. Recognition of structures instead of sequences may represent a great advantage in the fight against variable viruses because single or even double mutations in the target may be tolerated for RNase P recognition. One of the major aims of this work is to cleave HCV RNA using the RNase P ribozyme guided by EGS. To expand investigation of targeting in the HCV genome we assessed accessibility and low potential of variation of the target RNA since it is described that are crucial requirements for ribozyme therapy against viral infections. In the hepatitis C virus, the sequence of the 5' non coding region is conserved but the highly folded RNA structure severely limits the number of accessible sites. We have considered an internal genomic region whose sequence variation has been widely investigated. We have first mapped the accessibility of the genomic RNA to complementary DNAs within an internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P ribozymes targeted to the selected sites. Considerations on RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack. While performing targeting experiments on HCV RNA transcripts with RNase P we have found that, surprisingly, purified RNase P (peak activity) from HeLa cells cleaved HCV genomic RNA efficiently at two sites in the absence of EGSs. We report the techniques used to prove that the cleavage is specific to human RNase P (indirect methods: immunoprecipitation and competitive inhibition), and to show where cleavage occurs (direct method: RNA fingerprinting). We have confirmed that human RNase P is responsible for HCV RNA processing and that the two cleavages sites are in the IRES HCV domain, close to AUG initiator triplet, and in the E2/NS2 junction fragment (between structural and non structural coding region). To define cleavage by RNase P as a general property of HCV, viral sequences obtained from different patients were compared for RNase P cleavage accessibility. Cleavage was consistently observed in all sequences tested although with different efficiencies. Since RNase P recognizes and cleaves tRNA-like structures, we believe that such recognition by RNase P is an indication for the presence of two possible tRNA-like structures in the HCV genome. Comparison of such results at the two HCV RNase P cleavage sites should help us to understand in greater detail HCV substrate structure, tRNA mimicry, rules underlying recognition by human RNase P, and, in the particular case of the IRES motif, possible participation in translation. Whatever the role of such tRNA-like structures, such a strong tendency to maintain them might be important in the development of therapeutic strategies against the virus because they can represent highly susceptible targets for RNase P.

Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

L'hépatite chronique virale C est associée à une défaillance du système immunitaire. Nous nous sommes intéressés aux cellules NK et aux lymphocytes Treg, partenaires de la réponse immunitaire innée. Le nombre des NK, particulièrement les CD56dim, est significativement diminué chez les patients infectés, dans le foie plus que dans le sang, et s'accentue avec la fibrogenèse. Le nombre de CD3-CD56+brightNKG2A+ circulantes est corrélé à la sévérité de l'inflammation et de la fibrose et celui des CD3-CD56+dimNKG2A+ inversement corrélé à la charge virale. Les NK sont fonctionnelles, en capacité de produire de l'IFN-γ et d'engager un processus de cytolyse. L'expression de CD158 est significativement diminuée à la surface des NK hépatiques mais conservée dans les NK circulantes. L'expression de NKG2A,C,D dans les NK circulantes et hépatiques est identique à celles de patients non infectés. Les Treg intrahépatiques FoxP3+ sont quasi-exclusivement de phénotype CD4+. En analyse multivariée, le nombre de FoxP3+ est indépendamment associé à celui de CD8+, surtout dans les lésions nécrotico-inflammatoires et une corrélation forte est observée entre les transcrits CD8, FoxP3, IL-10 et TGF-β, suggérant que les Treg bloquent l'expansion et la cytotoxicité des TCD8 par contact cellulaire ou par le biais de cytokines immunosuppressives. L'équilibre entre FoxP3 et CD8 est rompu dans les grades et stades Métavir A>2 et F>3, avec un effondrement du rapport FoxP3/CD8. L'inflammation hépatique chronique s'accompagne de fibrose, aboutissant à la cirrhose, principale cause de CHC. Dans les cirrhoses virales C avec ou sans CHC, les lymphocytes CD3+, CD4+, CD8+, CD20+, CD56+, TCRγδ +, FoxP3+ sont plus nombreux dans la fibrose que dans le parenchyme. Le nombre de CD20+, CD3+, CD4+, CD8+ et l'expression d'IFN-γ et RANTES sont plus élevés dans les cirrhoses qui développent un CHC. En analyse multivariée, CD8 est le seul facteur indépendament associé à la récidive tumorale et à une diminution de la survie sans récidive à 5 ans. Les CD20+, CD3+, CD4+, CD8+, CD56+, TCRγδ+, FoxP3+ sont significativement moins nombreux dans le CHC que dans la cirrhose. Mais les FoxP3+ sont significativement plus nombreux et les CD56+ moins nombreux dans le CHC que dans le nodule parenchymateux, sans modification des LT, conduisant à une augmentation du rapport FoxP3/CD8 dans la tumeur. Les CD56+ diminuent de la cirrhose au CHC. Aucune corrélation n'est observée entre la densité intra-tumorale des lymphocytes étudiés et la récidive carcinomateuse. Conclusion. Un infiltrat inflammatoire dense au sein de la cirrhose C, particulièrement riche en CD8, favorise le développement et/ou la récidive du CHC. / Chronic hepatitis C is associated with the failure of the immune system. We were interested to NK cells and Treg cells, partners in the innate immune response. The number of NK, particularly the CD56+dim, is significantly reduced in infected patients, in the liver more than in the blood, and increases during the process of fibrogenesis. The number of circulating CD3- CD56+brightNKG2A+ correlates with the severity of inflammation and fibrosis and that of CD3- CD56+dimNKG2A+ inversely correlates with viral load. The NK functional capacity to produce IFN-γ and initiate a process of cytolysis is maintened. The CD158 expression is significantly reduced on the surface of intrahepatic NK, whereas NKG2A,C,D expression in circulating and hepatic NK is identical to that of non-infected patients. The intrahepatic Treg FoxP3+ are almost exclusively CD4+ phenotype. In multivariate analysis, the number of FoxP3+ is independently associated with that of CD8+, especially in necroinflammatory lesions and a strong correlation is observed between CD8, FoxP3, IL-10 and TGF-β, suggesting that Treg could inhibit CD8 expansion and cytotoxicity by cell contact or through immunosuppressive cytokines. The balance between FoxP3 and CD8 is broken in the most severe stages of the disease (METAVIR A>2 and F>3), which results in a drop in the FoxP3/CD8 ratio. Chronic inflammation is accompanied by liver fibrosis, leading to cirrhosis, the main cause of HCC. In viral C cirrhosis with or without HCC, CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ lymphocytes are more numerous in fibrosis than in parenchyma. The number of CD3+, CD4+, CD8+, CD20+ and the expression of IFN-γ and RANTES were higher in cirrhosis developing HCC. In multivariate analysis, CD8 is the only independent predictor of tumor recurrence and is associated with a significant decrease in the 5 years disease free survival. The CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ tumor infiltrating lymphocytes were significantly lower than in distant cirrhosis. However, FoxP3+ are significantly higher and CD56+ significantly lower in HCC than in parenchymatous nodules, without LT changes, leading to an increase in the FoxP3/CD8 ratio into the tumor. The number of CD56+ decreases from cirrhosis to HCC. No correlation was found between the density of studied tumor infiltrating lymphocytes and HCC recurrence. Conclusion. A dense inflammatory infiltrate in viral C cirrhosis, particularly rich in CD8, promotes HCC development and/or recurrence.

Afin de se répliquer et de se propager efficacement, les virus ont développé de multiples stratégies leur permettant d’échapper au système de défense innée : le système IFN de type I. Ce travail de thèse a alors consisté à étudier les interactions entre protéines virales et protéines de ce système de défense afin de mieux comprendre les mécanismes de subversion virale et d’identifier d’éventuelles cibles cellulaires thérapeutiques. La reconstruction d’un réseau d’interactions entre ces protéines nous a permis d’identifier des stratégies différentielles de subversion pour 4 familles virales et de montrer un ciblage massif et significatif des protéines du système IFN de type I par les virus. Les protéines en interaction directe avec ces protéines sont également fortement touchées par les virus et sont de potentiels modulateurs du système IFN de type I. Parmi ces modulateurs, le processus biologique sur-représenté est le transport nucléocytoplasmique et la protéine KPNA1 impliquée dans ce processus a retenu notre attention. L’étude fonctionnelle de l’interaction entre la protéine KPNA1 et la protéine NS3 du VHC a montré que la protéine NS3 associée à son cofacteur NS4A inhibe partiellement la réponse IFN de type I en empêchant l’import nucléaire de STAT1. Ce phénotype pourrait résulter de la dégradation de KPNA1 par NS3/4A. Par ailleurs, l’identification de nouveaux inter-acteurs de la protéine NS1 du virus influenza par criblage double-hybride levure a révélé la protéine induite par les IFN de type I, ADAR1, comme partenaire de la protéine NS1 de multiples souches virales et nous avons montré qu'ADAR1 est un facteur pro-viral dont la fonction editing est activée par NS1 / To replicate and propagate efficiently, viruses have developed multiple strategies allowing them to escape the innatedefense system: the type I IFN system, This work of thesis then consisted in studying the interactions between viralproteins and proteins of this defence system in order to understand better the mechanisms of viral subversion andidentifY possible therapeutic cellular tatgets. The reconstruction of a network of interacting proteins involved in the typeI IFN system allowed us to identifY differentiai subversion strategies for 4 viral families and to show a massive andsignificant targeting of proteins of the type I IFN system by viruses. Proteins directly interacting with the type Iinterferon system network are also strongly targeted by viruses and are potential modulators of the type I IFN system.Among these modulators, the most tatgeted function conesponds to the transport of NLS-bearing substrates to thenucleus and the KPNAI protein involved in this process held our attention. The functional study of the interactionbetween KPNA1 and NS3 protein of the HCV showed that NS3 protein associated with its cofactor NS4A inhibitsprutially the type I IFN response by preventing the nuclear translocation of ST A Tl. This phenotype could result fromthe degradation of KPNAI by NS3/4A. Besides, the identification of new cellular prutners ofNS 1 prote in of influenzavirus by yeast two-hybrid screens revealed ADARI, an interferon-stimulated prote in, as partner of NS 1 of ali testedvirus strains and we showed that ADARI is an essential host factor for viral replication and its editing function isactivated by NS 1 protein

Le traitement du virus de l'hépatite C (VHC) a connu une révolution récente, rapide et exemplaire grâce à l'arrivée des agents antiviraux directs (AAD) en plusieurs vagues depuis 2011, détrônant ainsi la bithérapie interféron-pégylé/ribavirine. Ces nouveaux traitements ont été rapidement confrontés à des limites concernant leur efficacité et leur tolérance notamment à leurs débuts avec les inhibiteurs de la protéase NS3/4A de première génération. L'arrivée de nouveaux AAD sur le marché lors d'une 2ème vague en 2014 a permis toutefois de surpasser celles-ci et de devenir le traitement de référence du VHC.Leur efficacité remarquable a laissé naître l'idée d'une potentielle élimination du VHC grâce à l'utilisation universelle de ces traitements. Cependant, leur coût élevé et les comportements à risque observés dans des sous-groupes de population (utilisateurs de drogues intraveineuses et homosexuels) restent encore des problématiques cruciales à surmonter pour espérer atteindre les objectifs fixés par l'Organisation Mondiale de la Santé en 2030 concernant l'élimination du VHC. De plus ces traitements, permettant l'élimination virale quasi-systématique et donc consécutivement une diminution du risque de complications hépatiques, ont été récemment confrontés à une polémique concernant un potentiel risque de récidive précoce de carcinome hépatocellulaire (CHC) suite à une exposition à ces derniers.Le travail présenté dans cette thèse s'articule autour de trois problématiques ayant toutes pour objectif principal d'optimiser l'utilisation de ces traitements dans l'optique de contrôler l'histoire naturelle de la maladie à l'échelle individuelle et à l'échelle populationnelle par l'intermédiaire de diverses méthodes statistiques.Nos résultats ont permis de montrer au sein d’une première problématique ayant exploré l'efficacité et la tolérance de ces traitements antiviraux à l’échelle individuelle: (i) une efficacité antivirale moindre que celle annoncée dans les essais de phase III des inhibiteurs de protéase de première génération(télaprévir et bocéprévir), (ii) un effet indésirable significatif des inhibiteurs de protéase de première génération sur la fonction rénale, (iii) une tolérance moins bonne de ces premières molécules que lors du traitement par bithérapie avec une incidence accrue d'anémie probablement liée à un surdosage en ribavirine induit par les inhibiteurs de protéase et (iv) une efficacité antivirale remarquable des AAD arrivés depuis 2014 sans impact des caractéristiques du patient ni des dosages pharmacologiques sur la réponse virologique. Dans un second temps, la problématique d'un risque de récidive de CHC accru après un traitement par AAD a également été explorée par l'analyse d'une cohorte locale, celle-ci ayant conclu à l'absence de risque accru comparé à un groupe de patients non exposés. Enfin, nos travaux basés sur la modélisation de la transmission du VHC en France dans la population coinfectée VIH-VHCont montré qu'un taux annuel de traitement par AAD de 50% était nécessaire dans la population homosexuelle ayant des pratiques à haut-risque de transmission pour contrer l'épidémie actuellement observée.Nos travaux ont donc permis d'apporter des données pour optimiser l'utilisation des nouveaux traitements anti-VHC par l'intermédiaire de diverses approches statistiques et ont apporté des éléments de réponse aux grandes problématiques actuelles. L'efficacité exemplaire et la tolérance quasi-parfaite des dernières molécules antivirales permettent une utilisation universelle de ces traitements dans toutes les populations de patients. Des études complémentaires robustes sont cependant nécessaires pour apporter des arguments à la question de la récidive du CHC. Des efforts sont également attendus concernant l'accès au traitement, la diminution des coûts associés et un dépistage renforcé du VHC pour espérer pouvoir éradiquer un jour cette maladie. / The arrival of direct-acting antivirals agents (DAAs) has spurred a rapid revolution in the treatment of hepatitis C virus (HCV), supplanting the previous standard of care, i.e. pegylated interferon and ribavirin. These new treatments are associated with an increased rate of virological response however they rapidly faced some limits more particularly at the beginning with the first generation NS3/4A protease inhibitors. From 2014 on the second wave of DAA was available for treatment of chronic HCV infection and surpassed previous encountered limits. These treatments are nowadays the gold standard for HCV treatment in high-income countries.The idea of HCV eradication recently emerged since DAA treatment are highly effective. However, their associated high cost and recent high-risk behaviors associated with an increased risk of HCV transmission (among intravenous drug users and homosexuals) have been reported. These issues need therefore to be addressed in order to achieve the objectives of the World Health Organization for 2030 of an HCV eradication. Moreover, these treatments allow a sustained virological response in almost all patients and consequently reduce the risk of liver-related complications, but a recent controversy regarding a potential increased risk of hepatocellular carcinoma after DAA treatment has been raised.Three issues will be extensively discussed in this manuscript regarding how these treatments can be used to optimize their effect on HCV natural history at the individual and population level through different statistical approaches.As regards the first issue, this project allowed us to demonstrate regarding the tolerance and efficacy of DAA treatment: (i) a lower antiviral efficacy than previously reported in the phase III trials for first generationprotease inhibitor regimen (telaprevir and boceprevir), (ii) impairment of renal function during first generation protease inhibitor treatment, (iii) an increased rate of reported side effects during first-generation protease inhibitor treatment and more particularly anemia, potentially related to an increased ribavirin biodisponibility induced by protease inhibitor intake and (iv) a remarkable antiviral efficacy of second generation DAAs without impact of patients' characteristics norpharmacology on virological response rate. The recent issue regarding a higher risk of HCC recurrence after DAA treatment was also explored through a local cohort study and no impact of DAA treatment was observed when comparing DAA-exposed vs non DAA-exposed patients. Finally, we conducted amodelling study on HCV transmission in the coinfected HIV-HCV French population and our results suggested that an annual DAA treatment coverage rate of 50% was required in the homosexual population with high-risk behaviors to counter the recent observed epidemic in this population.Our different works provide new insights on how to optimize the use of DAA treatment through several statistical approaches and bring new elements for discussion on the recent controversy. The new DAA have an excellent efficacy and tolerance profile and should be universally used in all populations without restriction. However, further studies are required to explore on a deeper level the question regarding HCC recurrence after DAA treatment. Efforts are also still needed regarding DAA treatment access, associated costs and HCV screening to reach the objective of HCV eradication

Made available in DSpace on 2016-12-23T13:49:05Z (GMT). No. of bitstreams: 1 Hepatite_B_Oculta_2012.pdf: 1487094 bytes, checksum: 098e451e5a2e2af112aa8508df956a77 (MD5) Previous issue date: 2012-02-29 / Occult hepatitis B (OHB) is defined as the presence of low levels of HBV DNA in the liver or serum of individuals testing hepatitis B surface antigen (HBsAg) negative. In most cases of OHB, sera are positive for hepatitis B core antibody. The literature contains quite a few studies on the prevalence of OHB in Brazil, as well as in the worldwide population. Such reports, often controversial, demonstrate that the OHB prevalence varies among healthy individuals or patients with diseases unrelated to the liver and patients with chronic liver disease. Despite efforts, it is necessary a better understanding of: the reasons for the persistence of low levels of HBV-DNA in the absence of detectable HBsAg, the potential risk of OHB transmission and its role in the progression and aggravation of some liver diseases. Therefore, it is interesting to know the prevalence of OHB indifferent population samples which allows de monitoring of carriers of the occult infection, followed prospectively in order to try to surprise the possible effects of the presence of low levels of HBV-DNA in these individuals. In this study we investigated the presence of Occult Hepatitis B in peripheral blood obtained from 520 healthy donors of Vitoria, Espirito Santo, with the aim of guiding policies to include or not the sensitive HBV-DNA nucleic acid amplification technique (NAT) screening in blood donations with a detection limit of 54UI/mL. In order to enable the molecular detection we had also developed a method that screens plasma samples in pools which is capable of detecting the presence of HBV in the ratio of 1:40.Through the technique of nested-PCR we found that 0,2% (1/520) had occult HBV in serum samples. Despite the low prevalence of OHB detected in the study, a considerable number of patients with occult HBV infection may not have been detected if the blood units were only tested for serological markers of HBV infection. So it is important to know the prevalence of OHB in one or more additional population groups, in order to follow up carriers of occult infection prospectively to determine possible effects of the presence of HBV-DNA in low concentrations in these individuals / A Hepatite B Oculta (HBO) é definida pela presença do DNA do VHB com carga viral geralmente baixa (<200UI/mL), no fígado ou soro de pessoas com antígeno de superfície (HBsAg) indetectável. Na maioria dos casos de HBO há positividade para o anticorpo contra o antígeno core da Hepatite B (anti-HBc). Na literatura constam poucos estudos sobre a prevalência da HBO no Brasil, bem como na população mundial. Existem relatos, muitas vezes controversos, que reportam frequências variáveis da HBO em indivíduos sem doença ou portadores de doença não relacionada com o fígado e em hepatopatas crônicos. Apesar dos esforços, faz-se necessário ainda uma melhor compreensão das razões para a persistência da baixa viremia do VHB na ausência de HBsAg detectável, do potencial risco de transmissão da HBO e do seu papel na progressão e agravamento de algumas hepatopatias. No estudo investigou-se a presença de HBO em 520 doadores de sangue de Vitória, Espírito Santo, com o objetivo de nortear políticas para incluir ou não a triagem molecular NAT (Nucleic Acid Amplification Technique ) de bolsas de sangue. Para viabilizar a detecção molecular desenvolveu-se também uma metodologia de extração em pool de amostrascapaz de detectar a presença do VHB na proporção de 1:40 com limite de detecção de 54UI/mL. Através da técnica de nested-PCR detectou-se a presença do VHB oculto em 0,2% (1/520) das amostras. Apesar da baixa prevalência de HBO detectada no estudo, um número considerável de portadores da infecção oculta podem não estar sendo detectados, se apenas a pesquisa de marcadores sorológicos da presença do VHB esta sendo realizada nas bolsas de sangue. Sendo assim é importante conhecer a prevalência da HBO em diferentes amostras da população para que se possa ter em mãos portadores da infecção oculta acompanhados prospectivamente a fim de tentar surpreender possíveis efeitos da presença do DNA do VHB em baixas concentrações nesses indivíduos

Made available in DSpace on 2016-12-23T13:56:09Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda Magri de Carvalho PDF.pdf: 4813468 bytes, checksum: f874502e73ece86d6ada6693a89b3fc4 (MD5) Previous issue date: 2009-03-19 / A incidência do carcinoma hepatocelular (CHC) apresenta grandes variações geográficas associadas a diferentes prevalências dos seus fatores etiológicos mais comuns. Embora não se conheça a sua real incidência no Espírito Santo, o CHC é freqüentemente diagnosticado no estado, associado à infecção com os vírus da hepatite B (VHB) e da hepatite C. No entanto, cerca de 40% dos casos não apresentam evidências destes fatores etiológicos. Por essa razão, buscou-se verificar se outro fator aflatoxinas poderia estar envolvido na etiologia do CHC no ES. Nas regiões tropicais, as aflatoxinas são consideradas um importante fator etiológico do CHC, especialmente quando associadas à infecção com o VHB. O objetivo da presente investigação foi estudar a prevalência da mutação específica ser-249 no gene TP53, cuja presença é um indicador indireto de contaminação alimentar com estas micotoxinas e sua relação com a infecção pelo VHB. Foram investigadas 41 amostras de CHC e 74 amostras de cirrose hepática sem CHC, utilizando-se a metodologia de PCR-RFLP e sequenciamento. O DNA do VHB foi pesquisado com iniciadores específicos para seis diferentes regiões do seu genoma. Os iniciadores utilizados foram escolhidos considerando as regiões mais conservadas do genoma viral e englobando os oito genótipos já identificados do VHB. A presença do DNA do VHB foi observada em 46% (19/41) dos casos de carcinoma hepatocelular estudados. A mutação ser-249 TP53 foi observada em 15% (6/41) e 1,5% (1/69) dos casos de CHC e de cirrose hepática sem CHC associado, respectivamente, não havendo correlação estatisticamente significativa com a presença do DNA do VHB. Foram encontradas outras quatro mutações no códon 250 do gene TP53. A prevalência total de 24% (10/41) de mutações ser-249 e mutações no códon 250 nos pacientes com carcinoma hepatocelular possivelmente também associadas à contaminação com aflatoxinas pode indicar a provável exposição alimentar às aflatoxinas no Espírito Santo / The hepatocellular carcinoma (HCC) incidence has large geographic variation, which is associated with the different prevalences of its most common etiological factors. Although its real incidence in the Espirito Santo State is not known, the HCC is frequently diagnosed in ES associated with hepatitis B (HBV) and C virus infections. However, approximately 40% of the cases do not present known etiological factors. Therefore, it was the purpose of this study to verify if another factor - aflatoxins - could be involved in the HCC etiology in ES. In tropical areas, the aflatoxins are considered an important etiological factor of CHC, especially when associated with HBV infection. The aim of this investigation was to study the prevalence of the specific ser-249 mutation in the TP53 gene was studied, that indicates indirect food contamination with aflatoxins and its association with HBV infection. Forty-one HCC samples and seventy-four hepatic cirrhosis samples were investigated, using PCRRFLP methodology and sequencing. The HBV DNA was amplified with specific primers targeting six different regions of its genome. Primers were chosen considering the most conserved regions of the viral genome and encompassing the eight known genotypes of HBV. Presence of HBV DNA was observed in 46% (19/41) of the hepatocellular carcinoma cases studied. The ser-249 TP53 mutation was observed in 15% (6/41) and 1,5% (1/69) of the HCC and cirrhosis cases respectively, not having statistical correlation with the presence of HBV DNA. Other four mutations in TP53 codon 250 were found, yielding a total mutation prevalence of 24% (10/41), considering both ser-249 and codon 250 mutations, possibly also associated with food contamination by aflatoxins, and suggesting an alimentary exposition to aflatoxins in Espirito Sant

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-15Bitstream added on 2014-06-13T21:03:48Z : No. of bitstreams: 1 provazzi_pjs_dr_sjrp.pdf: 1081709 bytes, checksum: 9a35b9ad50fc4eed266a481d830f02de (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A proteína NS3 apresenta dois domínios e é bifuncional. Apresenta três funções enzimáticas que são; 1) atividade de protease; 2) NTPase e 3) helicase. A função protease relaciona-se a tradução da proteína precursora e as funções NTPase e helicase tem grande participação na replicação do material genético viral. Trata-se de uma molécula essencial para o processamento da poliproteína precursora e também para a replicação viral e portanto, um dos principais alvos para o desenvolvimento de drogas antivirais. No domínio Protease foram evidenciadas substituições na tríade catalítica e na região de ligação ao íon zinco nos pacientes avaliados. Estas substituições, quando somadas podem explicar a resposta ao tratamento. Também foram visualizadas alterações na porção Helicase da NS3. As substituições ocorreram nos sítios de ligação ao ATP e ao RNA. Outros resíduos da Helicase relevantes para o desenvolvimento de inibidores, como R2133 e F258 e F264 não apresentaram substituições, evidenciando tratarem-se de aminoácidos conservados nessa região. Os resultados obtidos nesse trabalho fornecem informações sobre o perfil genético do vírus HCV do genótipo 3 especificamente da região codificadora da proteína NS3, permitindo o conhecimento do genoma viral e a identificação de regiões para ligação de possíveis inibidores. Este projeto certifica que a modelagem é uma ferramenta útil para a biologia estrutural e funcional, e que os modelos obtidos aqui contribuem para o desenho de novas drogas anti-virais específicas para o genótipo 3 do vírus HCV / The NS3 protein has two domains and is bifuntional. It presents three functions: 1) protease activity, 2) NTPase and 3) helicase. The protease function is related to the translation of the poliprotein precursor and functions NTPase and helicase has great participation in the replication of the viral genetic material. So. The NS3 is considered the major target for the development of antiviral drugs. In the Protease portion substitutions were evidenced in catalytic triad and the zinc ion binding sites, in the patients evaluated. These substitutions, when added up can explain the response to treatment. Also were observed changes in Helicase portion of NS3. The substitutions took place on ATP and RNA binding sites. Other residues of Helicase relevant to the development of inhibitors, as R2133 and F258 and F264, showed no substitutions, highlighting the great conservation of amino acids in this region. The results obtained in this work provide information on the genetic profile of the HCV virus genotype 3, specifically the region of NS3 protein, allowing the knowledge of the viral genome and the identification of regions for possible connection of inhibitors. This project certifies that the modeling is a useful tool for structural biology and functional, and that the models obtained here contribute to the design of new anti-viral drugs specific to the genotype 3 of HCV virus