Дисертації з теми "CCCDCC"

L'infection par le virus de l'hépatite B (VHB) est un problème de santé publique majeur avec plus de 257 millions de porteurs chroniques dans le monde ayant un risque important de développer une cirrhose et/ou un hépatocarcinome. L'histoire naturelle de l'infection est très différente selon l'âge auquel l'infection est contractée. Alors que chez l'adulte l'infection est spontanément résolutive dans la majorité des cas, la contamination materno-infantile ou en bas âge aboutit le plus souvent à une infection chronique. Le cccDNA est la forme de persistance du génome viral dans les hépatocytes infectés et la base de transcription de tous les ARN viraux. La protéine virale HBx joue un rôle crucial dans le recrutement des facteurs épigénétiques sur le cccDNA et favorise son activité transcriptionnelle. Les traitements actuels à base d'interféron et d'analogues nucléos(t)idiques ne permettent pas l'éradication du cccDNA et leur interruption est presque toujours suivie d'une réactivation de la réplication du virus. De nouvelles molécules thérapeutiques ciblant le cccDNA sont donc nécessaires pour espérer obtenir une cure fonctionnelle chez les patients chroniquement infectés. Il existe des liens étroits entre l'infection par le VHB et le métabolisme des acides biliaires (AB). Ainsi, notre équipe a précédemment montré que le récepteur nucléaire des acides biliaires, le farnesoid X receptor alpha (FXRalpha) se fixe sur deux éléments de réponse présents dans la région Enhancer II - promoteur de Core du génome viral et module son activité transcriptionnelle. De plus, le VHB et les AB entrent en compétition pour le même récepteur d'entrée hépatocytaire NTCP, modifiant la concentration cellulaire des AB avec des conséquences sur la fonction et l'expression de FXRalpha. Enfin, HBx interagit avec FXRalphaet modifie son activité. Au cours de cette thèse nous avons dans un premier temps identifié une régulation réciproque existante entre la réplication du VHB et FXRalpha. Puis nous avons montré in vitro, dans des cellules HepaRG différenciées et des hépatocytes primaires humains, que FXRalphaest un facteur proviral pour le VHB et que les agonistes de FXRalpha inhibent l'expression de l'ensemble des marqueurs viraux de manière dépendante ou indépendante de la protéine virale HBx. Enfin, dans un modèle in vivo de souris C3H/HeN transduites par un vecteur recombinant AAV2/8-VHB, nous avons obtenu l'effet inhibiteur des agonistes de FXRalphamais uniquement chez les souris adultes et pas chez les souris jeunes. Compte tenu de l'évolution de la flore intestinale avec l'âge et de son importance dans le métabolisme des AB, ces résultats suggèrent que le fort taux d'évolution chronique chez les jeunes enfants pourrait être lié à l'immaturité du métabolisme des AB. La mise en évidence d'un lien entre microbiote, métabolisme des AB et infection par le VHB contribuerait grandement à une meilleure compréhension de l'histoire naturelle de cette infection. De plus, l'identification de FXRalphacomme un facteur de l'hôte favorisant l'infection et l'existence de molécules capables de moduler l'activité de FXRalphasuggèrent que FXRalphapourrait constituer une cible thérapeutique intéressante ciblant le cccDNA et permettant d'améliorer le traitement des patients infectés par le VHB
Hepatitis B virus (HBV) infection is a major global health problem with more than 257 million chronic carriers worldwide that remain at significant risk for developing cirrhosis and/or hepatocellular carcinoma. The natural history of infection is very different depending on the age at which the infection is contracted. Whereas in adults most HBV infections spontaneously resolve, in infants and young children they usually result in chronic infection. cccDNA is the molecular form of viral persistence in infected hepatocytes and serves as a transcription template for all viral RNAs. The viral protein HBx plays a crucial role in the recruitment of epigenetic factors to the cccDNA and promotes its transcriptional activity. Currently, interferon and nucleot(s)ide analogues are the first-line agents in the treatment of chronic hepatitis B without allowing eradication of cccDNA and their interruption are almost always followed by a reactivation of the replication of the virus. New therapeutic molecules targeting cccDNA are therefore needed to hope for a functional cure in chronically infected patients. HBV infection and bile acid (BA) metabolism are tightly linked. Therefore, our team has previously shown that the bile acid nuclear receptor, the farnesoid X receptor alpha (FXRalpha) bind to two response elements present in the Enhancer II - Core promoter region of HBV genome and modulate its transcriptional activity. Moreover, HBV and BA compete for the same entry receptor of hepatocytes NTCP and modify BA cell concentration with consequences on the function and expression of FXRalpha. Finally, HBx interacts with FXRalpha and modify its activity. During my PhD. we have first identified a reciprocal regulation between HBV replication and FXRalpha. Second, we have showed in vitro, in HepaRG differentiated cells and in primary human hepatocytes, that FXRalpha is a proviral factor for HBV and that FXRalpha agonists inhibit the expression of all HBV markers in a dependent or independent manner of the viral protein HBx. Finally, in an in vivo model of C3H/HeN mice transduced with a recombinant AAV2/8-HBV vector, we obtained the inhibitory effect of FXRalpha agonists but only in adult and not in young mice. Considering the evolution of the gut flora with age and its importance in the metabolism of BA, these results suggest that the high rate of chronic progression in young children might be related to the immaturity of BA metabolism. The identification of a link between BA metabolism, gut microbiome composition and evolution of HBV infection will represent a big step toward the understanding of HBV natural history. Moreover, the identification of FXRalpha as a proviral factor for HBV and the capacity of FXRalpha ligands to modulate the transcriptional activity of cccDNA suggest that FXR ligands might represent a new class of molecules with the aim to obtain functional cure for HBV infected patients

CTCF is an evolutionally conserved 11-zinc finger protein. It controls multiple cellular functions and is itself partly regulated via poly (ADP-ribosyl)ation (PARylation). Recent evidence suggested that the hypo PARylated isoform was exclusively expressed in breast tumours and low expression levels was associated with worse prognostic features. It also possessed proliferative activity. The exact mechanism by which CTCF exerted its effects in breast cancer is not known. In order to define the interaction between CTCF and proliferation markers, Ki67 and PCNA, colocalisation studies were performed in a panel of five breast cancer cell lines which possessed different hormone receptor / invasive phenotypes. Following co-localisation, co-immunoprecipitation assays and mass spectrometry were carried out to determine whether CTCF was physically bound to either Ki67 or PCNA. To determine whether CTCF directly regulated ERα activity, CTCF plasmid overexpression and siRNA knockdown assays were performed in the hormone receptor positive MCF7 breast cancer cell line. Changes in endogenous expression of ERα were monitored by quantitative polymerase chain reaction (QPCR). CTCF, Ki67 and PCNA were all found to be strongly expressed in breast cancer cell lines though the strength of this expression for CTCF and Ki67 was antibody dependent. CTCF and Ki67 were shown to colocalise in the nucleoli of all breast cancer cell lines while CTCF and PCNA demonstrated nucleolar colocalisation only in the weakly invasive, hormone receptor positive cell lines. Despite colocalisation, there was no physical interaction detected between CTCF and Ki67 / PCNA on coimmunoprecipitation and mass spectrometry in all the cell lines studied suggesting that the proteins did not exist as a functional complex. Three novel CTCF - interacting protein partners (general transcriptional factor 2, glucose regulated protein 78 and the huntingtin interacting protein-1 related) were however discovered. These new protein partners, known to function at least in part via epidermal growth factor receptor (EGFR) signalling in cancer formation, were discovered only in ER positive breast cancer cell lines. Further investigation did not detect a direct regulatory effect of CTCF on ERα expression suggesting that the effect of CTCF on EGFR signalling in breast cancer cell lines did not involve an indirect action on ERα expression.

CTCF is an evolutionally conserved 11-zinc finger protein. It controls multiple cellular functions and is itself partly regulated via poly (ADP-ribosyl)ation (PARylation). Recent evidence suggested that the hypo PARylated isoform was exclusively expressed in breast tumours and low expression levels was associated with worse prognostic features. It also possessed proliferative activity. The exact mechanism by which CTCF exerted its effects in breast cancer is not known. In order to define the interaction between CTCF and proliferation markers, Ki67 and PCNA, colocalisation studies were performed in a panel of five breast cancer cell lines which possessed different hormone receptor / invasive phenotypes. Following co-localisation, co-immunoprecipitation assays and mass spectrometry were carried out to determine whether CTCF was physically bound to either Ki67 or PCNA. To determine whether CTCF directly regulated ERα activity, CTCF plasmid overexpression and siRNA knockdown assays were performed in the hormone receptor positive MCF7 breast cancer cell line. Changes in endogenous expression of ERα were monitored by quantitative polymerase chain reaction (QPCR). CTCF, Ki67 and PCNA were all found to be strongly expressed in breast cancer cell lines though the strength of this expression for CTCF and Ki67 was antibody dependent. CTCF and Ki67 were shown to colocalise in the nucleoli of all breast cancer cell lines while CTCF and PCNA demonstrated nucleolar colocalisation only in the weakly invasive, hormone receptor positive cell lines. Despite colocalisation, there was no physical interaction detected between CTCF and Ki67 / PCNA on coimmunoprecipitation and mass spectrometry in all the cell lines studied suggesting that the proteins did not exist as a functional complex. Three novel CTCF - interacting protein partners (general transcriptional factor 2, glucose regulated protein 78 and the huntingtin interacting protein-1 related) were however discovered. These new protein partners, known to function at least in part via epidermal growth factor receptor (EGFR) signalling in cancer formation, were discovered only in ER positive breast cancer cell lines. Further investigation did not detect a direct regulatory effect of CTCF on ERα expression suggesting that the effect of CTCF on EGFR signalling in breast cancer cell lines did not involve an indirect action on ERα expression.

Introduzione: la riattivazione del virus HBV dopo trapianto di fegato è legata alla persistenza del virus sotto forma di DNA covalente chiuso circolare (cccDNA). Abbiamo dunque esaminato la sicurezza della sospensione completa della profilassi anti-HBV in una coorte di pazienti trapiantati negativi per cccDNA intraepatico. Metodi: 30 pazienti HBsAg positivi, HBeAg e HBV-DNA negativi al momento del trapianto, con cccDNA e HBV-DNA intraepatico negativi hanno sospeso le immunoglobuline anti-HBs, continuando la lamivudina, con controlli clinici mensili. Dopo 6 mesi, tutti i pazienti sono stati sottoposti ad una seconda biopsia epatica: solo i pazienti nuovamente negativi per cccDNA e HBV-DNA intraepatico hanno sospeso anche la lamivudina e sono stati seguiti per ulteriori 6 mesi, prima di sottoporsi a nuova biopsia. I pazienti negativi per tutti i marcatori virologici e sierologici di infezione da HBV, su siero e tessuto epatico, sono stati seguiti per un periodo complessivo di 2 anni. Risultati: 25 pazienti sono risultati negativi per cccDNA e HBV-DNA intraepatico in tutte le biopsie epatiche eseguite durante lo studio, in assenza di alcun segno di riattivazione di HBV dopo un follow-up mediano di 25.2 mesi. Cinque pazienti sono diventati HBsAg positivi, 1 dopo la sospensione delle immunoglobuline, 4 dopo la sospensione della lamivudina. Nessuno di questi 5 pazienti ha mostrato eventi clinici rilevanti. Nel primo pazienti, le immunoglobuline sono state prontamente reintrodotte e il paziente è tornato ad essere HBsAg negativo. Degli altri 4 pazienti, solo 1 è rimasto HBsAg positivo, HBV-DNA positivo, con transaminasi moderatamente levate, per cui ha iniziato tenofovir. Gli altri 3 pazienti hanno mostrato invece un HBsAg positività transitoria, seguita in realtà dalla comparsa di un titolo spontaneo anti-HBs, in assenza di alcun trattamento antivirale. Conclusioni: i pazienti con HBV-DNA negativo al momento del trapianto, risultati cccDNA e HBV-DNA negativi su tessuto epatico, possono cautelativamente sospendere la profilassi anti-HBV. Solo una minoranza di questi in realtà ha mostrato ripositivizzazione dell’HBsAg, in assenza di eventi clinicamente rilevanti, andando incontro alcuni a spontaneo sviluppo di anticorpi anti-HBs.
Background and Aim: HBV reactivation after liver transplantation is due to the persistence of covalently closed circular (ccc) DNA. We investigated the safety of HBV prophylaxis withdrawal in transplanted patients negative for intrahepatic total and ccc-DNA. Methods: thirty HBsAg-positive, HBeAg and HBV-DNA-negative patients at transplant, with undetectable intrahepatic total and ccc-DNA underwent HBIG withdrawal and were continued on lamivudine with monthly monitoring. After 6 months, a second liver biopsy was obtained: patients with confirmed total and ccc-DNA negativity also underwent lamivudine withdrawal and were monitored for additional 6 months, when a third biopsy was obtained. Patients negative for all virological assays were followed-up without prophylaxis for up to 2 years. Results: Twenty-five patients had undetectable total and ccc-DNA in all sequential biopsies and did not exhibit HBV reinfection after a median follow-up of 25.2 months following lamivudine withdrawal. Five patients became HBsAg-positive: one early, after HBIG withdrawal,the other 4 after lamivudine withdrawal. None of these had clinically relevant events. In the first patient HBIG were reinstituted with prompt HBsAg negativization. Of the other 4, only one remained HBsAg-positive with detectable HBV-DNA and mild ALT elevation, and was given tenofovir. In the remaining 3, HBsAg positivity was transient and followed by anti-HBs seroconversion. No antiviral treatment was given to these patients. Conclusions: Patients with undetectable HBV viremia at transplant and undetectable intrahepatic total and cccDNA may undergo cautious weaning of prophylaxis. A minority of them experience transient HBsAg positivization, without clinical or virological events, and some undergo spontaneous anti-HBs seroconversion.

La particularité de ce virus de l'hépatite B est la synthèse d'un ADN circulaire clos covalent (ADNccc) qui est la forme de persistance du virus dans la cellule. Cet ADN est maintenu à une copie par cellule en moyenne chez l'homme grâce à un recyclage des nucléocapsides dans le noyau. En effet, durant le cycle viral, les nucléocapsides sont soit redirigées dans le noyau pour former de l'ADNccc, soit enveloppées puis sécrétées pour former de nouveaux virions. Du fait de son maintien au sein de l'hépatocyte, la formation et la régulation de l'ADNccc restent des éléments clés du traitement antiviral. Il a été montré in vitro que l'accumulation de cet ADN était régulée par les protéines d'enveloppe. Lors de l'étude du taux d'ADNccc dans des biopsies de foie de patients coinfectés HIV-HBV chronique, il a été découvert un patient présentant 300 fois plus d'ADNccc intrahépatique que la moyenne de la cohorte. L'objectif de ma thèse a été de comprendre quel était le mécanisme conduisant à cette accumulation d'ADNccc in vivo. Cela nous a permis de mettre en évidence le rôle du gène core dans l'accumulation de l'ADNccc
The feature of hepatitis B virus is the synthesis of a covalently closed circular DNA (cccDNA) which is the persistence form of the virus in cell. cccDNA is maintained to 1 copy per human cell thanks to the recycling of capsids into the nucleus. Indeed, during the viral cycle, capsids are either transported into the nucleus to form cccDNA or enveloped and secreted to form new infectious virions. Because of its maintenance in the hepatocyte, cccDNA formation and regulation are still key elements of antiviral treatment. It has been shown that, in vitro, cccDNA accumulation was regulated by envelope proteins. Upon the study of cccDNA levels in liver biopsies of HIV-HBV co-infected patients, an individual with a cccDNA level 300 fold higher than the average of the cohort was identified. My thesis objective was to understand which is the mechanism leading to the cccDNA accumulation observed in vivo. This allowed us to highlight the role of core gene in cccDNA accumulation

Thesis (M.A.)--Florida State University, 2009.
Advisor: Kathleen Yancey, Florida State University, College of Arts and Sciences, Dept. of Englsih. Title and description from dissertation home page (viewed on Oct. 28, 2009). Document formatted into pages; contains viii, 100 pages. Includes bibliographical references.

Le virus de l'hépatite B (HBV) infecte de manière chronique 240 millions de personnes dans le monde, et est la principale cause de carcinome hépatocellulaire. Actuellement, les traitements standards permettent une suppression virale à long terme, mais ne sont pas capables d'éliminer complètement le virus, en raison de la persistance de l'ADN circulaire et clos de façon covalente (ADNccc). Ce minichromosome viral réside dans le noyau des hépatocytes infectés, grâce à sa structure chromatinienne. En effet, lors de l'infection d'un hépatocyte, l'ADN viral partiellement double brin (ADN relâché circulaire (rc)) est libéré dans le noyau, où il est réparé et enveloppé par des protéines histones, afin de former une structure d'épisome chromatinisé. Les mécanismes conduisant à la formation ainsi qu'à la chromatinisation de l'ADNccc sont encore largement inconnus, et leur élucidation constituerait une première étape vers l'identification de nouvelles cibles thérapeutiques, susceptibles d'altérer la persistance de l'ADNccc. Dans ce but, nous avons étudié le rôle des facteurs hôtes de réparation de l'ADN, et des voies d'assemblage des nucléosomes, dans la formation de l'ADNccc, à des stades précoces (entre 30 minutes et 72 heures) de l'infection, dans des lignées cellulaires d'HepG2-NTCP, ainsi que dans des hépatocytes primaires humains. Nous nous sommes particulièrement concentrés sur la protéine chaperone d'histones, HIRA, qui est connue pour déposer le variant d'histone 3.3 (H3.3) sur l'ADN cellulaire d'une manière indépendante de la réplication et en association avec le remaniement des nucléosomes pendant la transcription et la réparation de l'ADN. Nous avons été capables de détecter l'ADNccc dans la fraction nucléaire des hépatocytes dès 30 minutes et 24 heures post-infection, par qPCR et Southern Blotting (SB), respectivement. L'extinction de HIRA par ARN interférent (siARN) avant l'inoculation du virus, a conduit à une forte diminution de l'accumulation de l'ADNccc (à la fois par qPCR et Southern Blot), qui était indépendante de la protéine HBx (en utilisant un virus HBx-défectueux). Les niveaux d'ADNrc sont restés stables, indiquant soit une éventuelle transition de l'ADNrc en ADNccc incomplète, ou retardée. L'analyse par immunoprécipitation de la chromatine a montré que HIRA était liée à l'ADNccc dès 30 minutes après infection, et que son recrutement était concomitant avec le dépôt de l'histone H3.3, ainsi que la liaison de la protéine de capside du HBV (HBc). Après 24 heures d'infection, une augmentation de la liaison de H3.3 et de l'ARN polymérase II sur l'ADNccc a été observée, en corrélation avec l'initiation de la transcription de l'ARN viral de 3.5 kb. Par des expériences de co-immunoprécipitation et de test de proximité entre protéines (PLA), nous avons montré que HIRA était capable d'interagir avec HBc dans des hépatocytes infectés et dans une lignée cellulaire HepaRG exprimant HBc de manière inductible. En conclusion, nos résultats suggèrent que la chromatinisation de l'ADN viral entrant est un événement très précoce, nécessitant l'histone chaperone HIRA. Bien que HBx ne soit pas requis pour ce processus, HBc pourrait jouer un rôle majeur, suggérant que l'interaction entre HIRA et HBc pourrait représenter une nouvelle cible thérapeutique à étudier
Hepatitis B virus (HBV) chronically infects 240 million people worldwide and is the major cause of hepatocellular carcinoma (HCC). Currently standard-of-care treatments can achieve longterm viral suppression, but are not able to completely eliminate the virus, due to the persistence of the covalently closed circular DNA (cccDNA). cccDNA, the viral minichromosome, resides in the nucleus of infected hepatocytes by virtue of its chromatin structure. Indeed, upon entry into hepatocytes, the partially double stranded viral DNA (relaxed circular (rc)DNA) is released into the nucleus, where it is repaired and wrapped by histones to form an episomal chromatinized structure. The mechanisms leading to cccDNA formation and chromatinization are still largely unknown and their elucidation would be a first step toward the identification of new therapeutic targets to impair cccDNA persistence. To this aim, we investigated the role of host factors belonging to DNA repair and nucleosome assembly pathways in cccDNA formation at early time points (i.e. between 30 minutes and 72 hours) post-infection in both HepG2-NTCP cell line and Primary Human Hepatocytes (PHH). We particularly focused on the histone chaperone Hira, which is known to deposit histone variant 3.3 (H3.3) onto cellular DNA in a replication-independent manner and in association to nucleosome reshuffling during transcription and DNA repair. We were able to detect cccDNA in the nuclear fraction of hepatocytes as early as 30 minutes and 24h post-infection, by qPCR and Southern Blotting (SB), respectively. Knock-down of Hira by RNA interference before virus inoculation led to a strong decrease in cccDNA accumulation (both in qPCR and SB) which was independent from HBx protein expression (using an HBx defective virus). rcDNA levels remained stable, indicating either a possible incomplete or delayed rcDNA to cccDNA transition. Chromatin Immunoprecipitation analysis showed that Hira was bound to cccDNA already at 2 hours post-infection and that its recruitment was concomitant with the deposition of histone H3.3 and the binding of HBV capsid protein (HBc). After 24 hours of infection, an increase of H3.3 and Pol2 binding on cccDNA was observed, correlating with the initiation of the transcription of the 3.5 kb RNA. By Co-Immunoprecipitation and Proximity Ligation Assay experiments, we showed that Hira was able to interact with HBc in infected hepatocytes and in a HepaRG cell line expressing HBc in an inducible manner. Altogether, our results suggest that chromatinization of incoming viral DNA is a very early event, requiring the histone chaperone Hira. While HBx is not required for this process, HBc could play a major role, suggesting that the interaction between Hira and HBc could represent a new therapeutic target to be investigated

Objectives: To use the Common Cognitive Complaints Checklist to provide base-rate data of common cognitive complaints in non-clinical individuals; and to identify common cognitive complaints that discriminate between three populations: non-clinical, mental health, mixed-neurological. Methods: 133 volunteers, recruited from three populations (non-clinical, mental health, mixed-neurological), completed measures of psychological distress, cognitive complaints and intellectual functioning. Results: The mental health group reported significantly higher levels of distress, and individuals with higher levels of distress tended to report more cognitive complaints. Base-rate data was established by calculating patterns of endorsement in the non-clinical group, providing a profile of ‘normal’ reporting. Three discriminant function analyses were applied, which performed excellently, revealing 26 items that maximally discriminated between the groups. Conclusions: The base-rate data revealed that it was unusual for individuals in the non-clinical group to report cognitive complaints occurring very frequently. These data could help clinicians determine whether or not the frequency of any complaint is ‘normal’. The calculated discriminant functions for the 26 identified items could be used to plot probabilities of responses falling within each of the three populations, helping clinicians determine the population in which their patients’ responses are likely to fall. Strengths and limitations are discussed along with suggestions for future research.

A substantial amount of data is available to illustrate just how few films have women in key creative positions, and how little the situation is changing. Critical sociological studies of work in creative industries have revealed a reestablishment of inequality along traditional lines such as gender but reasons for this continued inequality has only recently become a focus of creative labour studies. This thesis examines the dynamics of socialized recruitment processes that rely on subjective judgments of creative talent and ability. I identify the rhetorical work done in the UK film industry whereby these practices have become accepted as natural and unproblematic even by those they seemingly disadvantage. It is informed by key thinking from the fields of creative industries, cultural studies and gender and work, and introduces new empirical data from interviews with screenwriters and their employers, to examine how inequality of opportunity is sustained through structural and subjective mechanisms that are not held accountable through equal opportunities policies. Using a Bourdieusian framework I will consider how success in creative work is less attributable to qualifications and experience than to social, economic and particularly cultural capital and the right habitus. I contend that symbolic violence is used to suppress the very discourse of the experience of women by disallowing their voices to be heard in sufficient variety as authors of feature film screenplays. My study of screenwriting labour offers a more complex explanation than is provided by the usual justifications for the lack of women in key creative roles. In this way I contribute to a more nuanced understanding of the mechanisms that perpetuate gender inequality in creative professions.

Les patients infectés chroniquement par le VHB ont un risque élevé de développer des maladies graves du foie telles que le carcinome hépatocellulaire. Dans les cellules infectées, le virus persiste sous la forme d’un ADN circulaire covalemment clos (ADNccc) qui reste stable même chez les patients qui suivent un traitement. La protéine virale HBc, en plus de son rôle dans la formation des capsides, est retrouvée dans le noyau et est recrutée sur l’ADNccc et modifie sa structure. Cependant son rôle dans ce contexte n’est pas encore compris. Afin de mieux comprendre le rôle nucléaire d’HBc notamment sur la biologie de l’ADNccc et l’expression des ARN viraux, nous avons construit plusieurs mutants déficients pour l’expression d’HBc. Nous avons tout d’abord construit un mutant qui possède deux codons stop après le codon 27 d’HBc suivi d’une substitution d’une partie du gène HBc par une séquence codant différents épitopes. Lors de l’infection, ce virus possède un fort défaut d’expression des ARN viraux, qui ne peut être restauré par la ré-expression d’HBc. La quantification des ARN naissants montre que le défaut semble être post-transcriptionnel mais est présent rapidement après la transcription (<2h). Ces résultats suggèrent que le défaut observé est indépendant d’HBc et que la séquence délétée de HBV HBc-Flag27* pourrait être impliquée dans un mécanisme de régulation post-transcriptionnelle. Grâce à ce virus, nous avons pu mettre en évidence que la protéine HBc apportée avec la capside lors de l’infection est capable de se réassocier sur l’ADNccc dans le noyau. Nous avons ensuite étudié des mutants ayant une séquence plus proche de la souche sauvage, sans cette substitution. Lorsqu’ils sont exprimés à partir d’un plasmide exprimant à la fois le génome et la protéine HBc sous le contrôle d’un promoteur SV40, nous observons un défaut d’expression de l’ARNpg pour des mutants possédant les codons stop après le 27e ou le 38e codon du gène HBc, mais pas lorsqu’il est placé après le 67e codon. Ces résultats suggèrent que le déplacement du codon stop d’HBc induit la diminution de l’expression de l’ARNpg et que le codon stop naturel d’HBc pourrait être protégé des voies de surveillance des ARN viraux comme la nonsense-mediated decay (NMD) qui reconnait les ARN ayant un codon stop prématuré. Lors des infections par ces virus, et donc en absence d’HBc, nous observons un défaut accru pour les virus ayant des codons stop aux codons 27 et 38 et un défaut apparait pour le virus ayant le codon stop après la position 67. Dans ce contexte, en absence d’HBc, nous avons pu voir que les ARN codants pour les protéines de surface sont également impactés et que l’expression des ARN peut être partiellement restaurée par l’expression d’HBc. Par des techniques de chromosome conformation capture nous avons pu mettre en évidence que le virus HBV HBc-27* n’est plus exclu des régions réprimées contactant des lamines, indiquant que la protéine HBc pourrait être impliquée dans la l’adressage de l’ADNccc vers des régions favorables pour la transcription. Afin de comprendre les mécanismes impliqués dans la régulation par HBc, nous avons isolé les partenaires nucléaires de la protéine et mis en évidence de nombreux facteurs impliqués dans la régulation de l’ADN et de la transcription ainsi que dans la réparation des dommages à l’ADN.Dans l’ensemble, nos résultats permettent de mieux comprendre les mécanismes de régulation de la biologie des ARN du VHB
Chronic HBV carriers (CHB) are at high risk of developing hepatocellular carcinoma. Because covalently closed circular DNA (cccDNA) persists in infected cells through RNA expression, deciphering the mechanisms involved in RNA transcription and stability is a crucial step to identify new antiviral targets. In addition to its role in capsid formation, HBV core protein (HBc) has been shown to be associated with the cccDNA and to modulate its structure, yet the impact of this modification on HBV transcription is not fully understood. To better understand the role of HBc in this context we constructed several mutants deficient for HBc expression. The first mutant has two stop codons after codon 27 in HBc followed by a substitution of the HBc sequence by a sequence encoding different epitopes. During infection, this virus shows a strong decrease in expression of viral RNA, which cannot be rescued by re-expression of HBc. The quantification of nascent RNAs shows that the defect appears to be post-transcriptional and is present as early as 2h after transcription. These results suggest that the observed defect is independent of HBc and that the deleted sequence in the HBV genome of this mutant could be involved in a post-transcriptional regulatory mechanism. With this mutant, we have been able to demonstrate that the HBc protein provided by the capsid during infection is able to re-associate onto the cccDNA in the nucleus. We then studied HBc mutants generated in the context of the the wild-type virus sequence, without the substitution. When expressed from a plasmid expressing both the genome and the HBc protein under the control of SV40 promoter, we observe a decrease of the pgRNA expression for mutants having the stop codons after the 27th or 38th codon of HBc, but not when the stop codon is located after the 67th codon. These results suggest that displacement of the HBc stop codon induces a decrease in pgRNA expression, independently of HBc protein, and that the natural stop codon of HBc could be protected from viral RNA surveillance pathways such as nonsense-mediated decay (NMD) that recognizes RNAs with a premature stop codon. During infections with these viruses, and therefore in the absence of HBc, we observed an increased in the defect for viruses having stop codons at position 27 and 38 and a defect appears for a virus having the stop codon at position 67. In this context, in the absence of HBc, we have seen that RNAs encoding surface proteins are also impacted and that RNA expression can be partially restored by HBc expression. By chromosome conformation capture techniques we were able to observe that the HBc-27* HBV virus is no longer excluded from repressed regions associated with lamins, indicating that the HBc protein could be involved in the localization of the cccDNA at active chromatin regions favorable for transcription.In order to understand the mechanisms involved in HBc regulation, we have isolated the nuclear partners of HBc and highlighted many factors involved in the RNA and DNA regulation, in the DNA damage repair and RNA processing.Overall, our results shed light on the regulatory mechanisms of HBV RNA biology

This dissertation discusses the interaction between fuel cells and the DC-DC converter just connected across its terminals. For that, it presents a modified algorithm to represent the dynamic behavior of fuel cell stacks as much the current ripple effects from power converters on such cells. PEM type fuel cells (Proton Exchange Membrane) are studied to get a reasonable model of the electrochemical and electric characteristics of the involved phenomena in the generation of electric energy from fuel cells. A T filter is used as interface between the fuel cell and the DC-DC converter. This converter must prevent large current ripples going through the cell terminals as well as keeping constant the power flow between the FC and the link DC to prevent itself of possible current transient. To connect it to the grid it was used a three-phase DC-AC converter to inject or to absorb current from the grid at reduced ripple distortion and to stabilize the DC link voltage. DC current controllers were designed to curb the link DC and AC currents within reasonable limits. Validation of the model was carried out through computer simulation in order to have an evaluation of the control system behavior both for the stack and for the injection and/or absorption of energy. Also, theoretical results with practical data from a fuel stack were compared with respect to the load variation across its terminals.
Esta dissertação discute a interação entre as células de combustível e o conversor de potência conectado aos seus terminais. Para isto é apresentado um algoritmo modificado para a modelagem dinâmica de conversores chaveados tanto quanto um estudo sobre os efeitos das ondulações de corrente sobre as células. Células do tipo PEM (Proton Exchange Membrane) são estudadas, obtendo-se um modelo razoável para análise das características eletroquímicas e elétricas dos fenômenos envolvidos na geração de energia elétrica com células de combustível. Como interface utiliza-se um conversor CC-CC associado a um filtro do tipo T . Este conversor tem a função de evitar a grande ondulação de corrente absorvida pela célula bem como, manter constante o fluxo de potência entre a FC e o barramento CC evitando-se assim, possíveis transitórios nos terminais da célula. O projeto do conversor de potência prevê a conexão à rede da concessionária através de um conversor CC-CA trifásico para injetar ou absorver corrente da rede com reduzida distorção harmônica e estabilização da tensão do barramento CC. Foram projetados controladores para a corrente CC, a tensão CC e a corrente CA. Como formas de validação foram realizadas simulações para avaliar o comportamento dos sistemas de controle para a pilha e para a injeção e/ou absorção de energia. Também foram comparados os resultados teóricos com os dados práticos de uma pilha submetida à variação de carga em seus terminais.

Malgré l’existence d’un vaccin préventif efficace, l’infection chronique par le virus de l’hépatite B (HBV) demeure un problème majeur de santé publique. La persistance de l’infection par HBV étant clairement associée à des réponses immunitaires insuffisantes, l’immunothérapie par le vaccin à base d’ADN nu, visant à stimuler les réponses humorales et cellulaires, apparaît comme particulièrement pertinente pour la thérapie des hépatites B chroniques. Toutefois, l’efficacité thérapeutique d’une telle stratégie reste limitée chez l’homme, d’où la nécessité d’optimiser cette approche vaccinale pour une utilisation ultérieure en clinique. Ainsi, l’objectif général de ce travail de thèse était d’explorer, avec le modèle du DHBV (« Duck Hepatitis B Virus »), étroitement apparenté au HBV humain, si l’administration du vaccin à ADN par électroporation (EP) pouvait davantage améliorer son efficacité prophylactique et thérapeutique. Nous avons montré, dans un 1er temps chez des canards naïfs, que l’administration du vaccin à ADN par EP permet de potentialiser le pouvoir neutralisant et d’élargir le répertoire épitopique de la réponse humorale dirigée contre la protéine d’enveloppe du DHBV, même avec des doses d’ADN relativement faibles. Dans un 2ème temps, nous avons montré chez des animaux chroniquement infectés par le DHBV, que l’administration par EP du vaccin à ADN ciblant les protéines structurales du DHBV et le DuIFN-γ améliore considérablement l’efficacité thérapeutique du vaccin, notamment au regard de la séroconversion et de la clairance virale. Les résultats ainsi obtenus confirment l’intérêt majeur de cette approche vaccinale pour la thérapie des hépatites B chroniques
Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Since persistence of HBV infection is mostly associated with insufficient immune responses, therefore DNA vaccination capable of activating both humoral and cellular immune responses appears as a pertinent strategy for chronic hepatitis B therapy. However, the efficacy of such therapeutic approach remains limited in humans. Improvement of DNA vaccine efficacy is therefore needed for future therapeutic applications in clinic. The main objective of this thesis was to investigate in the duck hepatitis B virus (DHBV) model, whether the protective and therapeutic efficacy of DNA vaccine can be enhanced using EP-based delivery system. Firstly, we showed in naïve ducks that EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response even with relatively low DNA doses, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose. Secondly, we showed in chronic DHBV-carriers that in vivo EP is able to dramatically enhance the therapeutic potency of DNA vaccine targeting hepadnaviral proteins. Indeed, this approach was able to consistently restore humoral immune response and to sustainably decrease and even clear viral infection. Thus, these data strongly support the use of this approach for chronic hepatitis B therapy in humans

Rôle des hélicases DDX5 et DDX17 dans la régulation transcriptionnelle et la maturation des ARN du Virus de l'hépatite B. La chronicité du virus de l'hépatite B (VHB) repose sur la persistance de l'ADN circulaire et clos de manière covalente (ADNccc) dans le noyau des hépatocytes infectés. Le génome viral présente une structure chromatinisée sujette à des régulations épigénétiques impactant son activité biologique à différents niveaux. Une meilleure connaissance des facteurs cellulaires orchestrant la régulation transcriptionnelle et post-transcriptionnelle de l'ADNccc est fondamentale dans la compréhension des mécanismes à l'origine de la persistance du VHB. Afin d'identifier les acteurs cellulaires impliqués dans la biologie de l'ADNccc, un ambitieux projet de protéomique de l'ADNccc (ChROP) a été initié par le Dr. Barbara Testoni. Parmi les candidats identifiés, les hélicases à ARN DDX5 et DDX17 ont particulièrement attiré notre attention. DDX5 et DDX17 jouent un rôle crucial dans la régulation de la transcription et le métabolisme des ARN. Nous avons donc évalué leur rôle dans la régulation transcriptionnelle de l'ADNccc et le métabolisme des ARN viraux. Afin d'étudier de manière précise les différents transcrits du VHB, une technique de 5' RACE a été mise au point au laboratoire par le Dr. Bernd Stadelmayer, et a fait l'objet d'une publication. Cette technique a permis l'étude des ARN du VHB dans un contexte de déplétion des hélicases DDX5 et DDX17. Par ailleurs, DDX5/17 appartiennent au complexe insulateur de CCCTC-binding protein (CTCF). Nous avons donc en parallèle étudié le rôle de CTCF dans la biologie de l'ADNccc et le métabolisme des ARN viraux. Dans des cellules HepG2-NTCP et des hépatocytes primaires humains infectés par le VHB, la répression de DDX5/17 entraine un raccourcissement de tous les ARN viraux. Des séquençages de dernière et troisième génération ont permis l'identification de variants d’épissage alternatif ainsi qu’une utilisation différentielle du site de polyadénylation lors de la transcription des transcrits viraux. Des expériences d'immunoprécipitation de l'ARN ont montré que DDX5 et DDX17 s'associent directement aux ARN viraux et recrutent CPSF6 et NUDT21, deux facteurs impliqués dans le choix du site de polyadénylation. Par ailleurs, nous avons identifié des sites de liaison de CTCF sur le génome du VHB et par mutagénèse dirigé, nous avons mis en évidence que la mutation de ces sites impacte le recrutement de CTCF et de DDX5/17 sur l’ADNccc et affecte métabolisme des ARN viraux. L'ensemble de ces données met donc en lumière un rôle essentiel de DDX5 et DDX17 dans la maturation des ARN viraux, en complexe avec la protéine insulatrice CTCF et des facteurs de temrination, à l'interface entre l'ADNccc et les transcripts du VHB
Role of the DEAD-box helicases DDX5 and DDX17 in HBV transcriptional regulation and RNA processingChronicity of hepatitis B virus (HBV) infection hinges on the persistence of covalently-closed-circular DNA (cccDNA) in the nucleus of infected hepatocytes. The viral genome associates with histones and non-histone proteins to build a chromatin structure that is subjected to epigenetic regulation translating into different levels of biological activity. A better understanding of the host factors orchestrating HBV minichromosome transcriptional regulation and RNA processing is fundamental for deciphering the mechanisms at the basis of HBV persistence and reactivation. In order to identify the cellular factors regulating cccDNA biology, an ambitious project of cccDNA proteomics (ChroP) has been initiated by Dr. Barbara Testoni. Among the identified cccDNA-associated proteins, the DEAD-box RNA helicases DDX5 and DDX17 particularly interested us for their driving role in mammalian transcriptional regulation and RNA metabolism. Thus, we investigated their role in cccDNA transcriptional activity regulation and HBV RNA processing. Precise characterization of HBV transcripts was performed with a 5' RACE approach set up and published in our lab by Dr. Bernd Stadelmayer. This technique was applied to study viral transcript in a context of DDX5/17 depletion. Furthermore, DDX5/17 belong to the insulator complex CCCTC-binding protein (CTCF). We therefore investigated the role of CTCF in cccDNA biology and viral RNA metabolism. In HBV infected HepG2-NTCP and Primary Human Hepatocytes, siRNA knockdown of DDX5/17 led to a shortening of all the viral transcripts, together with an increase in viral transcript levels and viral particles accumulation in the cytoplasm, without affecting the global level of cccDNA. Next and third generation sequencing allowed the identification of alternative splicing of pgRNA-derived spliced variants and differential usage of polyadenylation site during HBV RNA transcription. Moreover, RNA immunoprecipitation of DDX5 and DDX17 revealed that both of these proteins are directly associated to the viral transcripts and recruit two factors, CPSF6 and NUDT21, involved in alternative polyadenylation site choice. Moreover, we identified CTCF binding sites on HBV genome and by site directed mutagenesis we showed that mutations in CTCF binding sites affect CTCF and DDX5/17 recruitment to cccDNA and subsequently impact HBV RNA processing. Altogether, our data highlight an essential role of DDX5 and DDX17 in the fine tuning of HBV RNA processing, in complex with the insulator protein CTCF and termination factors at the interface between cccDNA and HBV transcripts

In modern electronic devices, there is an essential requirement to decrease the power consumption and minimize the voltage supply to meet the demand of portable devices. This ever - increasing demand of low voltage and low power application has arisen the necessity to design analog circuit working at low supply voltage. DTMOS is one of the non conventional techniques to design the circuit with low leakage current with low supply voltage. In this thesis, a novel circuit design of CCCDCC that utilizes DTMOS technique has been proposed. This technique enhances the performance of the circuit design at low supply voltage. To show its applicability, the low pass and high pass filters have been simulated by this technique. The simulations are carried out using PSPICE software and technology used is TSMC 180nm.

碩士
玄奘大學
中國語文學系碩士在職專班
104
Abstract "The Gods"from Xu Xuan is a collection of Zhiguai XiaoShuowhich is about spirits. It brought down the root of Six Dynasties and Chinese, while opening for the trend of sketch-novel after Song Dynasty. This thesis will study the representation of different contexts phenomenon. This paper first discoursed the writer of "The Gods", his family story, evaluation and formation of the book. To understand the course of compiling "The Gods" by finding out the growth background of Xu Xuan. He was a simple, lenient man with few desires. As born in a queasy time between Five Dynasties and Song Dynasty, therefore he had been demoted for three times in his lifetime. Since the status of demoted minister, he was always trembling. Secondly, research about dreams in "The Gods" would be discoursed as well. Dreams, are mysterious, unpredictable and inseparable with human kinds since long time ago. From the research of dreams in "The Gods", it was believed that dream was the communication bridge which connected human and gods. Besides, fame and fortune were controlled by gods while living time of human was already fixed. After that, the book discussed the supernatural faith in Five Dynasties from "The Gods". This chapter discussed the relation of spirits and religion, as spirits were usually used by religion to promote doctrine. Both Taoism and Buddhism believed in Karma and Buddhist has even developed‘The Six Samsara’ cogitation. By the spirits and ghosts in "The Gods",it has an alternative beauty experience. "The Gods" recorded rare but real animals. By the fantastic and eccentric stories in "The Gods",it presents the earthling chasing for wealth. Finally, the conclusion is that the "The Gods" written by Xuan Xu having deep religious meanings and being a classic of the Supernatural Stories in the early Song Dynasty. It impacts the later novels of gods and spirits, and has an important place in the development history of the novels.

碩士
國立陽明大學
微生物暨免疫學研究所
89
Chronic hepatitis B (CHB) is one of the most serious viral infections in humans worldwide. Virus persistence in CHB patient relies on maintaining a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes, which presumably is the template of HBV gene expression. (-)-2’, 3’-thiacytidine (lamivudine; 3TC), an approved oral drug, is a nucleoside analogue which effectively inhibits the replication of HBV in CHB patients. However, it has been showed that cessation of drug treatment resulted in rapid rebound of HBV DNA in the patients’ sera. In vitro studies suggested that the cccDNA might be responsible for the reappearance of HBV DNA. To get insight into the understanding of the persistence of the HBV cccDNA, two classes of HBV producing lines, based on the RT-PCR analysis were established by transfecting BclI-bearing 1.3-fold HBV genome into HepG2 cells (The BclI site would present on the 3’end of all HBV mRNAs if their transcribed from cccDNA but not from introduced DNA template). Class one cell (clone 1.3.ES11) is characterized by containing BclI site at the 3’end at all HBV mRNAs. In the class two cell (clone 1.3.ES2 and 1.3.ES8), the produced mRNAs are mixture products in which the 3’end of HBV mRNAs either contained BclI site or did not. Southern blot analysis of the genomic DNA suggested that all clones contained intact 1.3-fold HBV genome with one integrated site (clone 1.3.ES2 and 1.3.ES11) or two sites (1.3.ES8). However, BclI restriction and Southern analysis of 1.3.ES11 genomic DNA generated an HBV-containing fragment with size approximately 3.2 kb, suggesting that the 3’end of the 1.3-fold HBV genome contain a BclI site. This finding may account for the fact that the 3’ end of all HBV mRNAs derived from 1.3.ES11 cells contains the BclI site. Southern analysis of Hirt supernatants prepared from 1.3.ES2 and 1.3.ES8 cells clearly indicated that this two cell lines produced cccDNA, as evidenced by band shifting after digesting with EcoRI and resistant to heat denaturation. We have established HBV cccDNA-producing lines. Class two cells are being used to assess the effects of proliferating or arrested cells treated with or without lamivudine on the maintenance of cccDNA. In the next, the more detailed study of cccDNA might be accomplished in this cell culture system.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2017.
The hepatitis B virus (HBV) continues to be a global health threat as chronic infection may lead to cirrhosis and hepatocellular carcinoma (HCC). Current treatments are limited in efficacy and do not target the stable HBV covalently closed circular DNA (cccDNA) minichromosome which forms the template for viral replication and is responsible for persistence of the infection. Using gene editing technologies to disable cccDNA presents a potential approach for treating HBV infection. Transcription activator-like effector (TALE) proteins provide specific and adaptable DNA binding modules, which can be used to generate engineered proteins capable of modifying DNA. Transcription activator-like effector nuclease (TALEN) mediated cleavage of cccDNA has been shown to effectively inhibit HBV replication. However, the approach to transcriptionally silence cccDNA, instead of cleaving it, may overcome the risk of unwanted host DNA cleavage. Repressor transcription activator-like effectors (rTALEs), which target and transcriptionally silence genes, have shown potential as antiviral agents. Here we generated Krüppel-associated box (KRAB)-based rTALEs targeted to the surface open reading frame (ORF) and HBx promoter region of HBV cccDNA to inhibit transcription. The rTALEs were placed under the transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, to restrict activity to hepatocytes thereby reducing the potential for off-target activity. In vitro the mTTR-driven rTALEs were shown to tissue specifically decrease secreted HBV surface antigen (HBsAg) levels by 63 - 92 %. Additionally, the mTTR-driven rTALEs were shown to tissue specifically decrease surface mRNA levels by 65 – 81 % and pregenomic RNA levels by 60 - 76 %. These results indicate that the KRAB domain was able to effectively suppress transcription from the basic core, Pre-S1 and/or vi Pre-S2 promoters which otherwise regulates HBV transcription. Furthermore, the observed inhibition was not associated with cytotoxicity or off-target effects. The work presented here is a proof-of-concept study demonstrating that highly specific transcriptional repressors designed to target and inhibit HBV viral replication without altering the genetic sequence or causing mutations in the host genome may be a promising antiviral approach. The capabilities of this technology to directly target cccDNA and inhibit its transcription, could contribute to addressing a global health problem.
LG2018

Hepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.

Indiana University-Purdue University Indianapolis (IUPUI)
The function(s) of the intracellular form of HBeAg, previously reported as the preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we propose to elucidate the translocation of p22 during its formation from endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have identified that a portion of p22, after the cleavage of its signal peptide in ER, is released back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except for the extended 10 amino acid precore region at the N-terminus, we observed that p22 wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not the secreted HBeAg significantly reduced interferon stimulated response element (ISRE) activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN- α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1, indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus evade host innate immune response and cause resistance to IFN therapy in patients. Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results indicate that there is a novel ER sorting mechanism for the distribution of the intracellular and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism.

Zhang, Bin.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 154-187).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

博士
國立臺灣大學
生化科學研究所
94
The molecular basis of mammalian sperm capacitation, the process to acquire the ability to fertilize the oocyte in the female genital tract in a time-dependent manner, either in vivo in the female reproductive tract or in vitro, is poorly understood. It is well known that sperm capacitation is associated with a cyclic AMP-dependent increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by two-dimensional gel electrophoresis. Among the protein targets, one tyrosine-phosphorylated 130-kDa protein spot was trypsin-digested, and six oligopeptide sequences were further established from the trypsin digests by mass spectral analysis. These data were completely confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved nuclear factor. Although we were unable to determine the exact site of phosphorylation of CTCF for the time being, we did demonstrate, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated mouse sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5’-GGCGGCGCCGCTAGGGGTCTCTCT-3’ found in the promoter of the amyloid

The Hepatitis B virus (HBV) chronically infects 350 million individuals worldwide, leading to mortality by end-stage liver disease, liver cirrhosis, and hepatocellular carcinoma. The vaccine to prevent HBV infection is highly effective but is not extensively available in endemic areas, resulting in high infection rates. Nucleoside analogue treatment of HBV has allowed for higher rates of viral clearance in infected individuals, but most patients must remain on therapy long term and viral resistance to the drugs is growing. The HBV viral genome is an episome in the nucleus of infected hepatocytes. It is called covalently closed circular (ccc) DNA and is highly stable, has a long half-life, and is the template for all viral transcription and progeny production. Nucleoside analogues do not directly target cccDNA, therefore many patients experience rebound when antiviral therapy is stopped. I have designed novel DNA binding proteins called zinc finger proteins (ZFPs) to specifically bind to the cccDNA in infected cells and inhibit viral transcription. Seven ZFPs targeting the model duck HBV (DHBV) and ten ZFPs targeting HBV were developed. Kinetic analyses of the purified ZFPs were performed, characterizing their specificity and binding properties. Using the DHBV tissue culture model system, I have demonstrated that the DHBV-specific ZFPs can specifically inhibit transcription from the viral template, resulting in reduced viral RNA, protein products and progeny virions. The DHBV-specific ZFPs were tested in primary duck hepatocytes (PDH) and in vivo in the Pekin duck model. ZFPs failed to express in PDH transduced by baculovirus vectors when DHBV was present in the cells. In vivo gene delivery of the ZFPs was carried out by portal vein injection of chitosan-based nanospheres. Unfortunately, non-specific reductions in viral levels masked any direct effect by the ZFPs. Testing of the HBV-specific ZFPs in tissue culture was hindered by a lack of transfectable cell culture model. A number of different transfection methods were tested to express the HBV-specific ZFPs, all without success. Further work is being carried out using baculovirus vectors to deliver the HBV-specific ZFPs to HBV-harbouring cell lines and HBV-infected scid-Alb/uPA chimeric mice with human liver cells.
Virology

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy

博士
國立陽明大學
微生物及免疫學研究所
93
In the first part of this thesis, we have evaluated the transcriptional efficiency of covalently closed circular DNA by using a RT-PCR combined with restriction enzyme digestion method. Virus persistence in chronic hepatitis B patient is manifested by sustaining a pool of covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocytes that presumably serves as a template for HBV genes expression. We used a modified 1.3-fold HBV genome, with a BclI-genetic marker embedded in the 5’-end redundancy region, to examine the transcriptional activity of cccDNA and the regulatory effect of HBx protein on the transcriptional activity. After harvesting total RNA from transfected cells or stable lines, we specifically monitored the transcription ability of cccDNA by using a RT-PCR combined with restriction enzyme digestion method. With this approach, we have found that (1) RT-PCR combined with digestion by BclI marker is highly specific and can distinguish transcripts originating from cccDNA from those originating from an integrated viral genome; (2) the transcriptional ability of cccDNA was shown to be less efficient than that from integrated viral genome; (3) the transcriptional activity of the cccDNA was significantly regulated by the HBx protein, a potential transcription activator. Accordingly, this information may provide a meam to further examine the transcriptional regulation of cccDNA and possibly, a very useful tool in the development of a cure for patients with a chronic HBV infection. In the second part of this thesis, we focused on the elucidation of the anti-viral effect of Transforming Growth Factor-beta1 on HBV replication. The replication of hepatitis B virus (HBV) has been reported to be suppressed by several cytokines, such as alpha/beta interferon (IFN-□/□), gamma interferon (IFN-□), and tumor necrosis factor alpha (TNF-□). Here we described that transforming growth factor beta 1 (TGF-□1), one of the cytokines elevated in hepatitis, has potential antiviral activities to decrease HBV replication in vitro. Using a virion-producing cell line derived from HepG2, we showed that TGF-□1 could suppress HBV replication as demonstrated by dramatically reduction of extracelluar and intracellular nucleocapsid. Concurrently, the cytosolic viral replicative intermediates were significantly diminished after TGF-□1 treatment. Furthermore, we also have evidence suggests that TGF-□1 can suppress HBV particle assembly by interfering with pgRNA transcription and HBc translation. RNase protection analysis revealed that TGF-□1 inhibited viral replication primarily by downregulating the expression of pregenomic RNA (pgRNA), as well as hepatocyte nuclear factor(s). Following the attenuation of viral transcripts by TGF-□1, we found that the HBc protein synthetic rate was also reduced due to the failure of ribosome engagement. Taking together, our data demonstrated that the antiviral effect of TGF-□1 was primarily due to both the transcriptional and translational controls, and suggest that elevated TGF-□1 may play a critical role in suppressing viral replication in chronic hepatitis.

This is a study of the fifteenth-century, “Storie of Asneth,” a late-medieval English translation of a Jewish Hellenistic romance about the Patriarch, Joseph, and his Egyptian wife, Asneth (also spelled Aseneth, Asenath). Belonging to the collection of stories known as The Old Testament Pseudepigrapha and derived from Jewish Midrash, the story was widely read among medieval religious in England in Latin before being translated into the vernacular for devotional purposes. Part of this study considers and identifies the aristocratic female patron (Elizabeth Berkeley) and author (John Walton) of the fifteenth-century Middle English text, based on literary, historical, and manuscript evidence from the sole surviving copy of the text in Huntington Library EL.26.A.13, a manuscript once owned by John Shirley. Also explored is the ritualistic pattern of events in the text (original to its Hellenistic origins) that coincides with ancient female initiation rites as we understand them from recent studies of Greek mythology. Centred in the narrative, culminating Asneth’s liminal seclusion, is her sacred marriage with a heavenly being. The argument suggests that in the Middle Ages this sacred consummation would have been interpreted as the union of God with the soul, similar to the love union in the Song of Songs. In the Christian tradition it is referred to as mystical marriage. Early Christian exegesis supports that Joseph was considered a prefigurement of Christ in the Middle Ages. In her role as divine consort and Joseph’s wife, Asneth would also have been identified as a type of Ecclesia in the Middle Ages—the symbolic bride of Christ. Patterns of female initiation in the story are also reflected in the hagiographical accounts of female saints, female mystics, and the ritual consecration of nuns to their orders, especially where they focus on marriage to Christ. The similarity of Asneth with Ecclesia, and therefore Asneth’s identity as a type of the church in the Middle Ages, is then explored in the context of the theology of the twelfth-century Cistercian prophet, Joachim of Fiore. The thirteenth-century Canterbury manuscript, Cambridge Corpus Christi College MS 288 (CCCC MS 288), which holds a Latin copy of Asneth also contains one of the earliest Joachite prophecies in England, known as Fata Monent. The study suggests Asneth may have held theological currency for early followers of Joachim of Fiore in England.
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