We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Kruppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 mu M), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

El aumento de la actividad minera en el Perú hace necesario el desarrollo de tecnologías rápidas y económicas de detección de contaminantes para su monitoreo y control. Implementando conocimientos de biología molecular y de la regulación génica podemos construir un circuito genético sintético que posibilite el monitoreo de sustancia toxicas que generen estrés oxidativo como son los compuestos cianurados. El objetivo de esta investigación es desarrollar un circuito genético sintético conformado por el promotor de la proteína del shock por fagos (psp) de Escherichia coli y las secuencia codificante del gen de la proteína verde fluorescente (GFP). La construcción de dicho circuito se logró usando estrategias de clonamiento por topoisomerasas y clonamiento clásico con enzimas de restricción, se usó la reacción en cadena de la polimerasa (PCR) para confirmar que todos los segmentos del circuito estén presentes en el vector. Los estudios preliminares de la actividad del nuevo circuito se realizaron transformando genéticamente células competentes de E. coli. La observación de dichas bacterias muestra una expresión de GFP continua, lo que indica que el circuito sintético está siendo activado sin estar en presencia de agentes de estrés oxidativo, lo que suponía una posible interacción con otros sistemas de regulación de estrés en la célula. Due to the increase of mining activity in Peru new technologies that can detect and monitor hazardous pollutants in a faster and cheaper way must be developed. Implementing molecular biology knowledge about genetic regulation we are able to construct a synthetic genetic circuit that can allow the monitoring of toxic substances that generate oxidative stress such us cyanide compounds. The objective of this research is to develop a synthetic genetic circuit from the promoter of the phage shock protein operon from E. coli and the complementary DNA of the green fluorescent gene (GFP). The construction of the circuit was achieved using classic cloning strategies with restriction enzymes and also more advanced strategies such us topoisomerase cloning, the polymerase chain reaction (PCR) was used to confirm the presence of all the desire segments in the vector. Preliminary studies of the circuit activity were carried out by genetically transforming competent E. coli cells. The observation of the bacteria shows a continuous expression of GFP without any inducer, this indicates that the synthetic circuit is being activated through a possible interaction with other stress response pathway.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

L’oestradiol joue un rôle important dans la reproduction en général, particulièrement dans la croissance folliculaire chez la vache. La production de l’œstradiol nécessite l’expression du gène CYP19A1 suite à la stimulation des cellules de granulosa par l’hormone folliculostimulante (FSH) ou le facteur de croissance insulinique de type 1 (IGF-1). Chez la vache, il existe six promoteurs (1.1 ; 1.2 ; 1.3 ; 1.4 ; 1.5 et 2) qui dirigent la transcription du gène CYP19A1 dans les cellules de la granulosa. Le principal promoteur qui dirige la transcription au niveau de l’ovaire (cellules de granulosa) est le promoteur 2 (P2). Cependant, l’effet de la FSH et de l’IGF-1 sur l’activation de ces promoteurs d’aromatase demeure mal connu. De plus, la demi-vie du transcrit CYP19A1 est très courte avec une région 3’UTR relativement longue. L’analyse de la séquence 3’UTR montre la présence des motifs ARE (séquence riche en AU), des études antérieur montrent que ces séquences impliquent dans la régulation de la stabilité ou la dégradation de l’ARNm, ce qui est fort probable que la courte demi-vie de l’ARNm CYP19A1 est sous le contrôle post-transcriptionel. L’objectif de la thèse visait à étudier la régulation de l’expression du gène CYP19A1 chez la vache. Il y a deux thèmes soit étude de la régulation transcriptionnelle ciblant le promoteur et soit étude de la régulation post-transcriptionnelle impliquant la région 3’non traduite (3’UTR). Le premier objectif vise à étudier la régulation transcriptionnelle du gène CYP19A1. Nous avons étudié l'activité du promoteur ovarien bovin dans deux modèles de cellules de la granulosa, les cellules lutéinisées et nonlutéinisées in vitro, suite à une stimulation des cellules par la FSH ou IGF-1. Nous avons également évalué la voie de signalisation impliquée dans la régulation des différents promoteurs en utilisant un RT-PCR et un gène rapporteur (les différents promoteurs d’aromatase ont été insérés dans le vecteur pGL3promoter en amont du gène exprimant la luciférase). Les résultats de RT-PCR démontrent que la FSH et l’IGF-1 augmentent les concentrations d’ARNm provenant des deux promoteurs 2 et 1.1 dans les cellules de la granulosa non lutéinisées. Des expériences subséquentes ont montré que la FSH stimule le promoteur 2 via la voie PKA tandis que l'IGF-1 stimule le promoteur 2 via la voie PKC. La FSH et l’IGF-1 stimulent l’expression du promoteur 1.1 via la voie PI3K. L’analyse de l’activité luciférase démontre que dans les cellules de granulosa lutéinisées, la FSH stimule le promoteur 1.1 de façon dose dépendante et ne semble y avoir aucun effet significatif sur le promoteur 2. Nous avons donc comparé l’activité du promoteur PII/P2 humain, du rat, de la chèvre et de la vache dans les cellules de granulosa bovine lutéinisées. Le résultat le plus significatif est que le promoteur 2 bovine (et caprine) dépend de plusieurs facteurs de transcription (NR5A2, FOXL2) comparé au promoteur PII humain et celui du promoteur proximal du rat qui dépendent principalement de l'AMPc. En effet, nos résultats ont démontré une expression raisonnablement robuste du P2 bovine lorsque les cellules sont traitées à la forskoline, NR5A2 et FOXL2. Le facteur FOXL2 semble déterminer l'activité du promoteur 2 chez le ruminant. Le deuxième objectif vise à étudier la régulation post-transcriptionnelle du gène CYP19A1. Pour ce faire, nous avons déterminé la séquence minimale de l'ARNm CYP19A1 requise pour la régulation de sa demi-vie. Différents séquences de la région 3’UTR ont été insérés dans le vecteur pGL3promoter en aval du gène exprimant la luciférase ou soit dans le vecteur pGEMTeasy. Le vecteur pGL3promoter a été transfecté dans les cellules de granulosa lutéinisées pour évaluer l'impact de la séquence 3'UTR sur l'expression du gène rapporteur de la luciférase, alors que le vecteur pGEMTeasy a été utilisé pour la transcription in vitro afin de générer de l’ARNm. Ce dernier sera utilisé en réaction croisée au UV avec des extraits protéiques pour démontrer l’association du complexe ARNm/protéine. L’analyse de l’activité luciférase a permis d’identifier une séquence de 200 pb située entre 926 et 1134 pb de la région 3'UTR de l’ARNm CYP19A1 qui a réduit significativement l’activité de la luciférase. Selon les analyses de la réaction croisée au UV, une ou plusieurs protéines de 66 et 80 kDA se lient spécifiquement à la séquence de 200 pb qui réduit l’activité de luciférase. Cette protéine s'exprime dans les cellules de granulosa, mais n’a pas été détectée dans d'autres tissus comme le foie et le cœur. Par ailleurs, l’utilisation du gène rapporteur sensible à la FSH a suscité l’intérêt d'une compagnie pharmaceutique qui vend de l’equine chorionic gonadotropin (eCG) pour lui permettre de distinguer facilement l’eCG ayant une forte activité FSH et donc, avoir un produit commercial plus efficace et de meilleure qualité. Dans cette étude, nous avons développé un système de bioessai à la FSH basé sur la transfection des cellules avec un récepteur à la FSH et un gène rapporteur colorimètrique qui permet d’estimer l’activité de la FSH dans le sérum de la jument et qui pourrait être applicable au niveau de la ferme/industrie. / Oestradiol plays an important role in reproduction in general, particularly during follicular growth. Production of estradiol requires the expression of CYP19A1 following stimulation of granulosa cells by follicle-stimulating hormone (FSH) and insulin like growth factor-1 (IGF-1). In cows, there are six promoters (1.1, 1.2, 1.3, 1.4 and 1.5 and 2) that direct transcription of CYP19A1, and promoter 2 (P2) is the major promoter used in granulosa cells. However the effect of FSH and IGF-1 on the activation of these promoters of aromatase remains unclear. Further, the CYP19A1 gene has a very short half-life and a long 3' non-translated region (3'UTR) that suggests post-transcriptional as well as transcriptional regulation. The aim of my PhD project is to study the regulation of the CYP19A1 gene in the cow. This summary is divided into two parts, the transcriptional regulation involving the promoter region and the post-transcriptional regulation involving the 3'UTR. The first part of my project was to study the transcriptional regulation of CYP19A1 gene; we measured the expression of the different promoters in luteinized or nonluteinized bovine granulosa cells following stimulation of cells with FSH or IGF-1. The results of RT-PCR showed that FSH and IGF-1 increases mRNA levels from both promoters 2 and 1.1 in non luteinized granulosa cells. Subsequent experiments showed that FSH stimulates the promoter 2 via the PKA pathway and IGF-1 stimulated promoter 2 via the PKC pathway. FSH and IGF-1 stimulate the expression of 1.1 via the PI3K pathway. In subsequent studies in luteinized cells with luciferase reporter genes driven by the specific CYP19A1 promoters, FSH stimulated promoter 1.1 in a dose dependent manner but that promoter 2 was weakly activated and not responsive to FSH. We then compared the activity of human, rat, goat and bovine promoters in luteinised bovine granulosa cells. The most significant result is that the bovine (and caprine) P2 depends on several transcription factors (NR5A2, FOXL2) whereas the human and rat promoters largely depend on cAMP. In fact, these data demonstrate a reasonably robust expression of the bovine P2 when treated with forskolin, NR5A2 and FOXL2. FOXL2 appears to be a determinant of promoter activity in ruminants. The second part of my project was to study the post-transcriptional regulation of the CYP19A1 gene. The objective was to identify the elements required for the regulation of the half-life of CYP19A1 mRNA. To do so, we generated and inserted different fragments of the 3'UTR region of CYP19A1 mRNA in the pGL3promoter vector downstream of the luciferase gene, which was then transfected into luteinized granulosa cells to assess the impact of the 3'UTR sequence on the expression of luciferase reporter gene. We identified a sequence of 200 bp between 926 and 1134 bp of the 3'UTR region of CYP19A1 mRNA that significantly reduced luciferase activity. The same fragments were inserted into the pGEMTeasy vector for in vitro transcription and the generation of mRNA for UV crosslinking with protein extracts to demonstrate the presence of mRNA/protein complexes. We detected protein complexes of 66 and 80KDA that specifically bound to the 200 pb probe. This protein is expressed in granulosa cells but not in other tissues such as the liver and heart. The use of reporter gene attracted the interest of a company producing equine chorionic gonadotropin (eCG), and an interest was expessed in developing this system to measure the FSH-like bioactivity in eCG, and therefore have a more effective commercial product. In this study, we developed a FSH bioassay system based on the transfection of cells with an the FSH receptor and a colorimetric reporter gene to estimate the activity of FSH in the serum of the mare ; these results may be applicable at the farm / industry.

During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows. We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity. CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin. Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP. Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...

Ce travail porte sur la mise en scène et mise en visibilité des problèmes publics et débats de société dans les séries et feuilletons télévisés français. Le terrain mobilise un corpus de quatre séries françaises, deux de service public et deux de la chaîne payante Canal+. Au travers d'une double approche, nous envisageons le rôle de ces productions dans la sphère publique française ainsi que les enjeux différenciés qu'elles revêtent. D'une part, certaines endossent une fonction de lien social et de consolidation des valeurs de la société française, en élaborant des mises en scène traitant de débats de société ou de questions de moeurs et en offrant au téléspectateur-citoyen l'accès à ces problématiques grâce aux potentialités spécifiques du genre sériel. Il s'agit également d'étudier les stratégies de communication publique qui, en s'inscrivant dans le cadre de ces feuilletons ou séries, renouvellent leur champ d'action et bénéficient des atouts de la fiction. D'autre part certaines de ces productions peuvent être envisagées comme une tribune alternative permettant à des acteurs engagés - ou issus de sphères professionnelles antérieures – de relayer des revendications et d'accéder à la visibilité de la scène médiatisée. La série s'érige alors en « opérateur de la visibilité » au service d'entrepreneurs de cause et devient partie intégrante des processus de construction des problèmes publics dans les arènes publiques françaises. / This work focuses on the staging and public visibility of social problems and social debates in French television series and soap operas. The field mobilises a corpus of four French series, two from public service and two from the pay-TV channel Canal +. Through a double approach, we consider the role of these productions in the French public sphere as well as the differentiated stakes they take. One the one hand, some endorses a function of social bonding and consolidation of values of the French society by elaborating stagings regards with societal debates or morality issues and by offering the citizen-viewer access to these problematic thought the specific potential of the serial genre. It is also a question of studying the strategies of public communication which, by being part of these soap operas or series, renew their field of action and benefit from the assets of the fiction. On the other hand, some of these productions can be considered as an alternative platform allowing actors involved – or from previous professional spheres – to relay claims and gain access to visibility of the media scene. The series then become a “visibility promoter” in theof entrepreneurs' cause and become part of the process of building social problems in the French public arena.

Devido as suas características funcionais, as proteínas vêm sendo cada vez mais utilizadas em diferentes áreas e atividades da sociedade humana como: alimentícia, indústria, têxtil, papel e celulose, química e médica. A produção industrial de proteínas de valor comercial para diversas aplicações, vem sendo cada vez mais conduzida através do desenvolvimento de sistemas de expressão em diferentes hospedeiros, como Saccharomyces cerevisiae. O presente trabalho teve por objetivo desenhar um sistema de expressão metabolicamente independente induzido pelo hormônio estradiol que, em S. cerevisiae, seja capaz de produzir e secretar com eficiência proteínas homólogas e/ou heterólogas de interesse industrial. Para tanto, foi desenhado e construído um plasmídeo regulador (que expressa o fator de transcrição quimérico c-mycGal4(DBD)hER&#945;(LBD)VP16(AD) e um plasmídeo de expressão (que contém o promotor quimérico p5xUASGAL-UARcb1, o qual será induzido pelo fator de transcrição quimérico, regulando a expressão da proteína repórter celobiohidrolase I - cbh1 de Trichoderma reesei). Ambos plasmídeos foram utilizados para transformar a cepa ScW303-1A/pdr5&#916;(construída neste trabalho) e induzido com diferentes concentrações de estradiol. Para analisar a habilidade do promotor em dirigir a expressão da proteína repórter cbh1, foi feito o teste de DNS, com os transformantes selecionados, utilizando carboximetilcelulose 1% como substrato. A maior atividade enzimática ocorre na indução do sistema com 5 &#956;M de 17-&#946;-estradiol e DES (dietilestilbestrol). Os resultados mostram que o sistema de expressão induzido por 17-&#946;-estradiol e DES, funciona de forma eficiente em S. cerevisiae e que o mesmo pode ser utilizado na produção biotecnológica de outras proteínas de interesse. / Due to its functional characteristics, the proteins are being increasingly used in different areas and activities of human society: food, industry, textile, pulp and paper, chemical and medical. The industrial production of commercially valuable proteins for various applications is increasingly being conducted through the development of systems of expression in different hosts such as Saccharomyces cerevisiae. This study aimed to design a metabolically independent expression system induced by estradiol hormone in S. cerevisiae is able to produce and secrete effectively homologous proteins and / or heterologous of industrial interest. Thus, it was designed and built a regulatory plasmid (expressing the chimeric transcription factor c-myc-Gal4 (DBD) -hER&#945; (LBD) - VP16 (AD) and an expression plasmid (which contains the chimeric promoter 5xUASGAL- UARcb1, which is induced by the chimeric transcription factor, regulating the expression of the reporter protein cellobiohydrolase I - cbh1 of Trichoderma reesei). Both plasmids were used to transform ScW303-1A / pdr5&#916; strain (constructed in this work) and induced with different concentrations of estradiol. To analyze the ability of the promoter to direct the expression of the reporter protein cbh1, the DNS testing, with the selected transformants was done using 1% carboxymethylcellulose as a substrate. The highest enzymatic activity occurs in the induction system with 5 &#956;M of 17-&#946;-estradiol and DES (diethylstilbestrol). The results show that the expression system induced by 17-&#946;-estradiol and DES operates efficiently in S. cerevisiae and that it can be used for the biotechnological production of other proteins of interest.

Angiopoietin-2 (Ang-2) wirkt gefäßdestabilisierend und ist Voraussetzung für das Aussprossen von Gefäßen am Anfang der angiogenen Kaskade. Die Expression des Antagonisten der endothelialen Rezeptor-Tyrosin-Kinase Tie-2 ist streng gewebsspezifisch reguliert. Trotz des Zusammenhangs von Ang-2 und pathologischer Angiogenese sind die molekularen Mechanismen der ang-2 Regulation noch unverstanden. Mittels Microarray wurden die genomweiten Expressionsänderungen in endothelialen Zellen nach Behandlung mit dem demethylierenden 5-Aza-2’-deoxycytidine (5-Aza-dC) untersucht. Unter den induzierten Genen wurde ang-2 mit dem Fokus auf angiogeneserelevante Gene identifiziert. Obwohl die Endothelzellen unter Kontrollbedingungen ang-2 exprimieren, wurde die Expression durch Demethylierung weiter gesteigert. Es wurden jedoch keine potentiellen CpG-Inseln in unmittelbarer Nähe des Transkriptionsstarts identifiziert. Diese Daten lassen auf einen methylierungsunabhängigen Effekt von 5-Aza-dC auf die ang-2 Expression schließen. Zur molekularen Untersuchung der ang-2 Expression wurden 3kb der 5´-flankierenden Sequenz des humanen ang-2 Gens kloniert und der Transkriptionsstartpunkt (TS) bestimmt. Durch funktionelle 5´-Deletionsanalyse und zielgerichtete Mutagenese wurden regulatorische Promotorelemente identifiziert. Die Promotorregion -105 bis +51 relativ zum TS war ausreichend für die Vermittlung der basalen ang-2 Expression. Mittels Bindungsstudien wurden die Transkriptionsfaktoren Sp1 und Sp3 als Proteine, die primär an den ang-2 Minimalpromotor binden, identifiziert. Die Basen -78 bis -74 relativ zum TS sind eine essentielle Sp-Bindestelle für die Regulation der ang-2 Expression. Durch Mutation von potentiellen Bindungsstellen für Proteine der ETS-Familie wurde die ang-2 Promotoraktivität signifikant reduziert. Jedoch konnte die Spezifität von ETS-Proteinen in Bindungsstudien nicht bestätigt werden. Die Ergebnisse dieser Arbeit haben neue Einblicke in die ang-2 Regulation offenbart und zeigen, dass die Sp1/Sp3-abhängige Aktivierung des proximalen Promotorbereichs (-105/-56) entscheidend für die transkriptionelle ang-2 Regulation in Endothelzellen ist. / Angiopoitein-2 (Ang-2) acts destabilizing on blood vessels and is mandatory for the onset of the angiogenic cascade. The expression of the antagonistic ligand of the endothelial cell tyrosine kinase receptor Tie-2 is tightly regulated. Despite the accumulating evidence confirming the involvement of Ang-2 in pathologic angiogenesis, the molecular mechanisms controlling ang-2 expression are still unclear. Using microarray analysis, the global changes of gene expression were investigated after treatment of endothelial cells with the demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC). Focusing on angiogenesis related genes, ang-2 was identified among the upregulated ones. Although endothelial cells expressed ang-2 under control conditions already the expression was further increased by drug-induced demethylation. A screen for CpG-islands revealed no putative islands surrounding the transcription initiation site. These data indicate a methylation-independent effect of 5-aza-dC on the ang-2 expression. To elucidate underlying molecular mechanisms of ang-2 expression, 3kb of the human ang-2 gene were cloned and the transcription start site (TS) determined. Regulatory promoter elements were identified by functional 5’-deletion analysis and site-directed mutagenesis. The promoter region -105 to +51 relative to TS was recognized as sufficient and necessary for the ang-2 gene transcription. Electrophoretic Mobility Shift Assays revealed Sp1 and Sp3 as dominant nuclear proteins binding to the ang-2 promoter. The region spanning -78/-74 was identified as essential Sp1/3 site regulating ang-2 expression. The mutation of potential ETS-binding sites resulted in a significant decrease of ang-2 promoter activity. However, the binding of ETS-proteins could not be confirmed by means of EMSA. The results of this thesis revealed new insights of ang-2 regulation and strongly suggest that Sp1/Sp3-dependent activation of an upstream enhancer at -105 to -56 is crucial for the regulation of ang-2 expression in endothelial cells.

Chan Ka Ping Sophie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 140-144). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.viii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter 1 --- Introduction / Chapter 1.1 --- Pathogenesis of HBV Infection --- p.1 / Chapter 1.2 --- Classification and Structure --- p.2 / Chapter 1.3 --- HBV Genome --- p.4 / Chapter 1.4 --- Replication Cycle --- p.7 / Chapter 1.5 --- HBV Genotypes and Nomenclature --- p.9 / Chapter 1.5.1 --- Asian prevalent genotypes --- p.9 / Chapter 1.5.2 --- Numbering system --- p.9 / Chapter 1.6 --- Identification of Markers in HBV Genome for HCC Development --- p.11 / Chapter 1.7 --- Project Objective --- p.13 / Chapter 1.8 --- Promoters of HBV --- p.14 / Chapter 1.8.1 --- Pre-S1 promoter --- p.14 / Chapter 1.8.2 --- X promoter and enhancer I --- p.14 / Chapter 1.8.3 --- Core promoter and enhancer II --- p.15 / Chapter 1.8.4 --- Pair of mutations at BCP --- p.17 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of pGL3-promoter Plasmids --- p.18 / Chapter 2.1.1 --- Templates selection --- p.18 / Chapter 2.1.2 --- Amplification of promoters --- p.19 / Chapter 2.1.3 --- Cloning into pGL3-basic vector --- p.21 / Chapter 2.1.4 --- Screening and plasmid preparation --- p.21 / Chapter 2.2 --- Construction of Mutant Promoter Clones --- p.23 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.23 / Chapter 2.2.2 --- pPreS 1 /2712C mutant clone --- p.24 / Chapter 2.3 --- Cloning of Full-length HBV Genomes --- p.26 / Chapter 2.3.1 --- Replication-competent HBV clone --- p.26 / Chapter 2.3.2 --- Amplification of full-length HBV genome --- p.28 / Chapter 2.3.3 --- Cloning into pUC19 vector --- p.28 / Chapter 2.3.4 --- Screening for insert and sequence confirmation --- p.29 / Chapter 2.3.5 --- Excision of full-length HBV from plasmid --- p.29 / Chapter 2.4 --- Re-construction into a 1.3-fold HBV Clone --- p.32 / Chapter 2.4.1 --- Cloning of HBV fragment nucleotide 979-2617 (nt 979-2617) --- p.32 / Chapter 2.4.2 --- Screening for insert and sequence confirmation --- p.33 / Chapter 2.4.3 --- Cloning of HBV fragment (nt 905-2000) --- p.33 / Chapter 2.4.4 --- Construction of a 1.3-fold HBV genotype Cs clone --- p.34 / Chapter 2.5 --- Cell Culture --- p.37 / Chapter 2.5.1 --- Cell culture maintenance --- p.37 / Chapter 2.5.2 --- Transient transfection of promoter clones --- p.37 / Chapter 2.5.3 --- Transient transfection of HBV genomes --- p.38 / Chapter 2.6 --- Dual-Luciferase® Reporter Assay System --- p.40 / Chapter 2.6.1 --- Principle of the assay --- p.40 / Chapter 2.6.2 --- Cell harvest --- p.43 / Chapter 2.6.3 --- Luciferase assay --- p.43 / Chapter 2.7 --- Data Analysis --- p.44 / Chapter 2.8 --- Extraction of HBV DNA from Intracellular Cores --- p.45 / Chapter 2.8.1 --- Harvest of intracellular cores --- p.45 / Chapter 2.8.2 --- Phenol/chloroform extraction --- p.45 / Chapter 2.9 --- Southern Blotting --- p.47 / Chapter 2.9.1 --- Transfer of DNA to membrane --- p.47 / Chapter 2.9.2 --- Preparation of probes --- p.47 / Chapter 2.9.3 --- Hybridization with radiolabeled probes --- p.48 / Chapter 2.10 --- Detection of HBeAg and HBsAg --- p.50 / Chapter 2.10.1 --- HBsAg assays --- p.50 / Chapter 2.10.2 --- HBeAg assays --- p.51 / Chapter 2.11 --- SEAP Reporter Gene Assay --- p.52 / Chapter 3 --- Results / Chapter 3.1 --- Templates Selected --- p.53 / Chapter 3.2 --- Results of Luciferase Assays --- p.58 / Chapter 3.2.1. --- BCP mutation of genotype A as control --- p.58 / Chapter 3.2.2. --- Effect of C1165T mutation on Xpro/enhI activity of HBV genotype B --- p.60 / Chapter 3.2.3. --- Effect ofT2712C mutation on pre-S1 promoter activity of HBV Genotype B --- p.60 / Chapter 3.2.4. --- Effect of G1613A mutation on core pro/enhII activity of HBV Genotype Cs --- p.64 / Chapter 3.2.5. --- G1613A and BCP mutation --- p.67 / Chapter 3.3 --- Full-length HBV Genome Clones --- p.70 / Chapter 3.3.1. --- Construction of replication-competent full-length HBV genome clones --- p.70 / Chapter 3.3.2. --- Drawbacks of the system --- p.78 / Chapter 3.4 --- Construction of a Replication-competent 1.3-fold HBV Clone --- p.82 / Chapter 3.4.1. --- Construction of the HBV (nt 979-2617) clone --- p.82 / Chapter 3.4.2. --- Construction of the HBV (nt 905-2000) clone --- p.86 / Chapter 3.4.3. --- Construction of 1.3-fold genotype Cs HB V clone --- p.89 / Chapter 3.4.4. --- Test for replication competency --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- BCP Mutation as Control of the Luciferase Assay --- p.94 / Chapter 4.2 --- Promoter Activities Not Altered by T2712C and C1165T --- p.96 / Chapter 4.3 --- Mutation G1613A of Core pro/enhll --- p.98 / Chapter 4.3.1 --- Mutation resides in negative regulatory element of core promoter --- p.98 / Chapter 4.3.2 --- NRE and NRE-binding protein --- p.98 / Chapter 4.3.3 --- Relationship with BCP mutation --- p.101 / Chapter 4.4 --- HBV Constructs --- p.103 / Chapter 4.4.1 --- Rationale in re-construction of 1.3-fold HB V clone --- p.103 / Chapter 4.4.2 --- Replication competency --- p.104 / Chapter 4.5 --- Conclusion --- p.106 / Chapter 4.6 --- Future Work --- p.107 / Appendix --- p.108 / References --- p.140