Os pigmentos naturais, além de fornecerem cor e beleza aos diferentes organismos, desempenham importantes funções e ações biológicas, como as funções vitais de fotossíntese, respiração celular e a ação antioxidante. Assim, o presente estudo investigou os potenciais citotóxicos, genotóxicos e protetores dos pigmentos naturais clorofila b (CLb) e luteína (LT), isolados e em combinação, em doses normalmente consumidas na dieta. Para isso foram utilizados o teste do micronúcleo em células da medula óssea e do sangue periférico e o ensaio cometa em células do sangue periférico, rim e fígado de camundongos. Também foram avaliados parâmetros bioquímicos de estresse oxidativo, glutationa e substâncias reativas ao ácido tiobarbitúrico no rim e no fígado, além de glutationa e catalase no sangue periférico, a fim de investigar o papel antioxidante desses pigmentos. A capacidade da LT de alterar a expressão de genes de resposta ao estresse oxidativo e defesa antioxidante foi avaliada no tecido hepático dos camundongos utilizando a técnica de PCR em tempo real (RT-qPCR) array. Para a verificação da atividade protetora dos pigmentos, a cisplatina (cDDP) foi utilizada como indutor de danos oxidativos e ao DNA. Adicionalmente, foram avaliados os potenciais citotóxicos, genotóxicos e antioxidantes da LT em cultura de células de hepatocarcinoma humano por meio do teste do MTT [3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio], ensaio cometa e avaliação de parâmetros bioquímicos de estresse oxidativo, a fim de se estabelecer comparações entre resultados in vitro e in vivo, bem como propor mecanismos de ação para os efeitos antigenotóxicos da LT. Nossos resultados mostraram que os tratamentos com os pigmentos, tanto a LT quanto a CLb, isolados ou em combinação não causaram qualquer dano ao material genético nos testes empregados e ofereceram proteção frente aos danos induzidos no DNA pela cDDP tanto in vitro como in vivo. Efeitos antioxidantes para ambos pigmentos foram observados no sangue periférico e nos tecidos renais e hepáticos, e a LT também melhorou os parâmetros de estresse oxidativo avaliados in vitro. Na avaliação da expressão de genes de resposta ao estresse oxidativo em células do fígado de camundongos, a cDDP diminuiu a expressão 16 genes, entre eles, importantes genes responsáveis pela manutenção do estado redox da célula. Além disso, a LT mostrou que pode atuar como antioxidante não só agindo diretamente no seqüestro de radicais livres, mas também, induzindo a expressão de 11 dos 84 genes avaliados, e de 15 genes quando associada à cDDP. Em resumo, nossos resultados mostraram que a LT e a CLb, isoladas ou em combinação, nas doses consumidas normalmente na dieta, podem contribuir para a promoção da saúde considerando seus efeitos antigenotóxicos e antioxidantes. / The natural pigments, in addition to providing color and beauty to the different organisms, play important biological role, including the vital functions of photosynthesis, cellular respiration and antioxidant action. Thus, this study investigated the genotoxic and protective potential of natural pigments, alone and in combination, chlorophyll b (CLb) and lutein (LT), in concentrations usually consumed in the diet. For this purpose, we used the micronucleus test in bone marrow and peripheral blood cells and the comet assay in peripheral blood, kidney and liver of mice. Biochemical parameters of oxidative stress were also evaluated, such as glutathione and thiobarbituric acid reactive substances in the kidney and liver and catalase and glutathione in peripheral blood in order to investigate the antioxidant properties of these pigments. The ability of lutein to alter the expression of genes involved in oxidative stress response and antioxidant defense was evaluated in liver tissue of mice using the technique of real-time PCR (RT-qPCR) array. To verify the protective activity of the pigments, cisplatin (cDDP) was used as an inducer of oxidative stress and DNA damage. Additionally, we assessed the genotoxic and antioxidant potential of LT in cell cultures of human hepatocellular carcinoma using the test of MTT [3 - (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium], comet assay and assessment of biochemical parameters of oxidative stress, in order to make comparisons between results in vitro and in vivo, as well to propose mechanisms to antigenotoxic effects of LT. Our results showed that treatment with the pigments, both the LT and the CLb, alone or in combination, did not cause any DNA damage in the tests employed and offered protection against DNA damage induced by cDDP in both in vitro and in vivo. Antioxidant effects were observed for both pigments in peripheral blood, kidney and liver, and LT also improved the oxidative stress parameters measured in vitro. In the evaluation of the expression of genes involved in response to oxidative stress in liver cells of mice, cDDP decreased the expression of 16 genes, among them, important genes responsible for maintaining the redox status of the cell. Moreover, LT showed that it can act as an antioxidant not only acting directly in the scavenging of free radicals, but also by inducing the expression of 11 of the 84 genes evaluated and 15 when LT was associated to cDDP. In summary, our results showed that the LT and CLb, alone or in combination, at concentrations usually consumed in the diet can contribute to health promotion considering their antigenotoxic and antioxidant effects.

Neste trabalho foram realizados estudos de uma rota sintética comum para a obtenção de duas lactonas sesquiterpênicas, conocenolido A e conocenolido B. Estes compostos, por apresentarem um novo esqueleto carbônico, fazem parte de uma classe de produtos naturais sesquiterpênicos denominados tremulanos, dos quais se destacam por apresentarem a ruptura de uma ligação C-C do esqueleto básico, de onde vem a denominação 5,6-seco-tremulanos. Para fins de estudos sintéticos podemos destacar, na molécula desses tremulanos, as seguintes unidades estruturais: a) um ciclopentano substituído, b) uma ligação dupla tetrassubstituída e c) um anel de lactona (uma -lactona presente em conocenolido A e uma -lactona em conocenolido B). Em nossa proposta inicial, a estrutura do anel de ciclopentano seria preparada por contração de anel de uma epoxiciclo-hexanona. Ao contrário, porém, do esperado com base nos exemplos da literatura, esta reação não produziu nada de 2-formilciclopentanona. A estrutura do anel de ciclopentano desejada foi, então, preparada por um caminho alternativo partindo da cetona metílica e vinílica. Nos estudos relacionados à preparação da ligação dupla tetrassubstituída foram empregadas as reações clássicas de olefinação (Wittig e HWE) e reações de condensação (Stobbe e Reformatsky). O trabalho com fosforanas (Reagente de Wittig) envolveu um estudo de dinâmica rotacional destes compostos por RMN. Os estudos da reação tipo Reformatsky foram realizados tanto no contexto de preparação de olefinas quanto na obtenção de lactonas (derivados do ácido paracônico). Um estudo detalhado das estruturas destas lactonas foi realizado por meio da determinação estrutural dos derivados do ácido paracônico. / In this work were realized studies towards a common synthetic route for the preparation of two sesquiterpenes lactones, conocenolide A and conocenolide B. These compounds belong to a class of sesquiterpenoids known as tremulanes presenting a novel carbon skeleton. A special structural feature of both lactones that set them apart from class is the lack of a carbon-carbon bond in the basic skeleton, so their denomination 5,6-seco-tremulanes. For the purpose of synthetic studies, we highlight in the molecule of these tremulanes the following structural units: a) one substituted cyclopentane, b) one tetrasubstitued double bond and c) one lactone ring ( one -lactone ring is present in conocenolide A and one -lactone ring is present in conocenolide B). In our initial proposal, the structure of the cyclopentane ring would be prepared by ring contraction of an epoxycyclohexanone. However, in spite of some literature examples, the ring contraction to provide the 2-formyl-cyclopentanone did not occur. The desired cyclopentane ring was prepared by an alternative route from methyl vinyl ketone. The studies related to the preparation of tetrasubstituted double bond was based on classical olefination reactions (Wittig and Horner-Emmons) and condensation reactions (Stobbe and Reformatsky). In addition, a NMR study dealing with the rotational dynamics of phosphoranes was realized. Studies exploring the versatility of a Reformatsky-type reaction were carried out both in the context of olefin preparation as well as for the preparation of lactones (paraconic acid derivatives). A detailed study of the structures of these lactones was accomplished through structural determination of paraconic acid derivatives.

Lam Kit. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgment --- p.I / Table of Content --- p.II / List of Tables --- p.VII / List of Figures --- p.IX / Abbreviations --- p.XIV / Abstract --- p.XVI / 論文摘要 --- p.XIX / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatitis B --- p.1 / Chapter 1.1.1 --- Brief Introduction of HBV --- p.1 / Chapter 1.1.2 --- History of Hepatitis B Virus --- p.2 / Chapter 1.1.3 --- Hepatitis B Virus Infection around the World --- p.4 / Chapter 1.1.4 --- Hepatitis B Virus Infection in Hong Kong --- p.5 / Chapter 1.1.5 --- Hepatitis B Virus Infection in China --- p.7 / Chapter 1.1.5.1 --- Update of HBV Infection in China --- p.7 / Chapter 1.1.5.2 --- Problems in China --- p.7 / Chapter 1.2 --- Hepatitis B Virology --- p.8 / Chapter 1.2.1 --- Hepadnaviridae Family --- p.8 / Chapter 1.2.2 --- HBV Particles Types --- p.9 / Chapter 1.2.3 --- The HBV Genome --- p.10 / Chapter 1.2.4 --- The Life Cycle of HBV --- p.12 / Chapter 1.2.5 --- Hepatitis B Surface Antigen (HBsAg) --- p.17 / Chapter 1.3 --- HBV Transmission --- p.19 / Chapter 1.4 --- HBV Therapy --- p.19 / Chapter 1.5 --- Phyllanthus Species --- p.22 / Chapter 1.6 --- Alexander Cells --- p.26 / Chapter 1.7 --- Objectives --- p.29 / Chapter CHAPTER 2 --- COMPARISONS OF AQUEOUS AND ORGANIC EXTRACTS OF THREE PHYLLANTHUS SPECIES OF THEIR IN VITRO ANTI-HBV EFFECTS --- p.30 / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.30 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.1.1 --- Phyllanthus species --- p.30 / Chapter 2.2.1.2 --- "Chemicals, Antibodies and Instrument" --- p.31 / Chapter 2.2.2 --- Extraction Methods --- p.32 / Chapter 2.2.2.1 --- Aqueous Extraction --- p.33 / Chapter 2.2.2.2 --- Organic Extraction --- p.33 / Chapter 2.2.3 --- Cell line --- p.33 / Chapter 2.2.4 --- Toxicity of Extracts --- p.34 / Chapter 2.2.5 --- IMx Assay --- p.34 / Chapter 2.2.6 --- Semi-quantitative RT-PCR --- p.35 / Chapter 2.2.6.1 --- RNA Extraction --- p.35 / Chapter 2.2.6.2 --- RT-PCR --- p.36 / Chapter 2.2.7 --- Western Blotting --- p.37 / Chapter 2.2.7.1 --- Preparation of Protein Samples --- p.37 / Chapter 2.2.7.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.2.7.3 --- Protein Transfer --- p.38 / Chapter 2.2.7.4 --- Immumnoblotting --- p.39 / Chapter 2.2.7.5 --- Protein Assay --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Toxicity of the Extracts --- p.40 / Chapter 2.3.2 --- Effects on HBsAg Secretion and Viral Gene Expression --- p.45 / Chapter 2.3.2 --- Analysis of Intracellular Viral Proteins --- p.58 / Chapter 2.4 --- Discussion --- p.63 / Chapter CHAPTER 3 --- ISOLATION AND CHARACTERIZATION OF ACTIVE COMPONIENT FROM AN ORGANIC EXTRACT OF PHYLLANTHUS URINARIA (GUANGDONG) --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.2 --- Materials and Methods --- p.69 / Chapter 3.2.1 --- Materials --- p.69 / Chapter 3.2.2 --- Methods --- p.70 / Chapter 3.2.2.1 --- Ethanol Extraction --- p.70 / Chapter 3.2.2.2 --- Partitions --- p.70 / Chapter 3.2.2.3 --- Column Purification --- p.71 / Chapter 3.2.2.4 --- Analytical Thin Layer Chromatographic (TLC) --- p.71 / Chapter 3.2.2.5 --- Crystallization --- p.71 / Chapter 3.3 --- Results --- p.72 / Chapter 3.3.1 --- Analysis of Four Fractions after Partitions --- p.72 / Chapter 3.3.2 --- Screening of the Active Fraction after Column Chromatography of Fraction B --- p.76 / Chapter 3.3.3 --- Screening of the Active Fraction after Column Chromatography of Fraction6 --- p.79 / Chapter 3.3.4 --- Crystallization and Identification of the Isolated component --- p.82 / Chapter 3.3.5 --- Study Anti-HBV effects of pheophorbide a --- p.91 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- STUDY OF PRE S I PROMOTER ACTIVITY OF HBV --- p.103 / Chapter 4.1 --- Introduction --- p.103 / Chapter 4.2 --- Materials and Methods --- p.108 / Chapter 4.2.1 --- Materials --- p.108 / Chapter 4.2.2 --- Methods --- p.109 / Chapter 4.2.2.1 --- Cell line --- p.109 / Chapter 4.2.2.2 --- Clonning of Pre SI Promoter from HBV Genome --- p.109 / Chapter 4.2.2.3 --- Gene Clean --- p.110 / Chapter 4.2.2.4 --- Restriction Enzyme Digestion --- p.111 / Chapter 4.2.2.5 --- Synthesis of T-Overhang EcoR V Cut pBluescript® II KS (-) --- p.111 / Chapter 4.2.2.6 --- Ligation --- p.112 / Chapter 4.2.2.7 --- DH5α Competent Cells Preparation --- p.112 / Chapter 4.2.2.8 --- Transformation --- p.113 / Chapter 4.2.2.9 --- Plasmid Purification --- p.113 / Chapter 4.2.2.10 --- Transfection --- p.114 / Chapter 4.2.2.11 --- Luciferase Assay --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- Cloning of the Pre S I Promoter --- p.116 / Chapter 4.3.2 --- Sequences of the Pre S I Promoter --- p.121 / Chapter 4.3.3 --- Pre S I Promoter Activities in Hep 3B Cell Line --- p.123 / Chapter 4.3.4 --- Effects of Herbal Extracts on Pre S I Promoter --- p.126 / Chapter 4.4 --- Discussion --- p.130 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.134 / REFERENCES --- p.141

Hung Wah Johnson. / Thesis submitted in: August 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 116-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.1 / Chapter 1.2 --- Problem Statement --- p.3 / Chapter 1.3 --- Outline of the thesis --- p.6 / Chapter 2 --- Background --- p.8 / Chapter 2.1 --- Biological Background --- p.8 / Chapter 2.2 --- Sequence Alignments --- p.9 / Chapter 2.2.1 --- Pairwise Sequences Alignment --- p.11 / Chapter 2.2.2 --- Multiple Sequences Alignment --- p.15 / Chapter 2.3 --- Neighbor Joining Tree --- p.16 / Chapter 2.4 --- Marker Extractions --- p.18 / Chapter 2.5 --- Neural Network --- p.19 / Chapter 2.6 --- Conclusion --- p.22 / Chapter 3 --- Related Work --- p.23 / Chapter 3.1 --- FASTA --- p.23 / Chapter 3.2 --- Suffix Tree --- p.25 / Chapter 4 --- Sub-Sequence Segmentation Tree --- p.28 / Chapter 4.1 --- Introduction --- p.28 / Chapter 4.2 --- Problem Statement --- p.29 / Chapter 4.3 --- Design --- p.33 / Chapter 4.4 --- Time and space complexity analysis --- p.38 / Chapter 4.4.1 --- Performance Evaluation --- p.40 / Chapter 4.5 --- Summary --- p.48 / Chapter 5 --- Applications: Global Sequences Alignment --- p.51 / Chapter 5.1 --- Introduction --- p.51 / Chapter 5.2 --- Problem Statement --- p.53 / Chapter 5.3 --- Pairwise Alignment --- p.53 / Chapter 5.3.1 --- Algorithm --- p.53 / Chapter 5.3.2 --- Time and Space Complexity Analysis --- p.64 / Chapter 5.4 --- Multiple Sequences Alignment --- p.67 / Chapter 5.4.1 --- The Clustalw Algorithm --- p.68 / Chapter 5.4.2 --- MSA Using SSST --- p.70 / Chapter 5.4.3 --- Time and Space Complexity Analysis --- p.70 / Chapter 5.5 --- Experiments --- p.71 / Chapter 5.5.1 --- Experiment Setting --- p.72 / Chapter 5.5.2 --- Experimental Results --- p.72 / Chapter 5.6 --- Summary --- p.80 / Chapter 6 --- Applications: Marker Extractions --- p.81 / Chapter 6.1 --- Introduction --- p.81 / Chapter 6.2 --- Problem Statement --- p.82 / Chapter 6.3 --- The Multiple Sequence Alignment Approach --- p.85 / Chapter 6.3.1 --- Design --- p.85 / Chapter 6.4 --- Reference Sequence Alignment Approach --- p.88 / Chapter 6.4.1 --- Design --- p.90 / Chapter 6.5 --- Time and Space Complexity Analysis --- p.95 / Chapter 6.6 --- Experiments --- p.95 / Chapter 6.7 --- Summary --- p.99 / Chapter 7 --- HBV Application Framework --- p.101 / Chapter 7.1 --- Motivations --- p.101 / Chapter 7.2 --- The Procedure Flow of the Application --- p.102 / Chapter 7.2.1 --- Markers Extractions --- p.103 / Chapter 7.2.2 --- Rules Training and Prediction --- p.103 / Chapter 7.3 --- Results --- p.105 / Chapter 7.3.1 --- Clustering --- p.106 / Chapter 7.3.2 --- Classification --- p.107 / Chapter 7.4 --- Summary --- p.110 / Chapter 8 --- Conclusions --- p.112 / Chapter 8.1 --- Contributions --- p.112 / Chapter 8.2 --- Future Works --- p.114 / Chapter 8.2.1 --- HMM Learning --- p.114 / Chapter 8.2.2 --- Splice Sites Learning --- p.114 / Chapter 8.2.3 --- Faster Algorithm for Multiple Sequences Alignment --- p.115 / Bibliography --- p.121

A number of recent research studies have been done on different species of the plants of the genus Phyllanthus. The plants are widely distributed in most tropical and subtropical countries and have long been used for the treatment of liver diseases in China and India. / Hepatitis B virus (HBV) is a major pathogen of human viral hepatitis. It has been estimated that 350 million people are chronic carriers of HBV throughout the world. Increasing evidence indicates that persistent viral infection of the liver is associated with cirrhosis and hepatocellular carcinoma. Hepatitis B virus belongs to a family of DNA viruses called hepadnaviruses. The current treatments of HBV infection with interferon or lamivudine have several disadvantages, and there appears to be much room for improvement in terms of medical treatment. / My project research focuses on two poorly characterized Indian Phyllanthus species called Phyllanthus nanus ("PN") and Phyllanthus niruri ("PI"). In my studies, random amplified polymorphic DNA ("RAPD") technique and high performance liquid chromatography ("HPLC") fingerprinting were used to authenticate different species of Phyllanthus. Both aqueous and ethanolic extracts of PN and PI were prepared to study their cytotoxicity in hepatoma cell lines. The effect of these extracts on hepatitis B virus was also examined in the HBV-genome integrated cell lines - PLC/PRF/5 (Alexander) and HepG2 2.2.15. Microparticle enzyme immunoassay ("MEIA") and enzyme-linked immunosorbent assay were used to measure the amount of hepatitis B surface antigen ("HBsAg") and hepatitis B e antigen ("HBeAg") secretion from the cell lines. RT-PCR was used to detect the change in HBsAg mRNA's expression level in the drug-treated cell lines. Real-time PCR was also employed to examine the effect of drug treatment on the level of HBV DNA replication and the amount of virions secreted into the medium. The experimental results showed that both the aqueous and ethanolic extracts of PN and PI exerted suppressive effect on HBsAg secretion and HBsAg mRNA level. The PN and PI ethanolic extracts also showed mild suppression of viral replication in vitro. The ethanolic extract of PN seemed to be more potent in suppressing HBV than the other extracts tested. (Abstract shortened by UMI.) / Lam Wai Yip. / "June 2005." / Advisers: Mary Waye; Vincent Ooi. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 217-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. / Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection. / Li, Man Shan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Copolímeros, macromoléculas orgânicas ou inorgânicas com alta massa molar, consistem de unidades monômericas repetidas, unidas por ligações covalentes e que apresentam mais de um tipo de monômero. Neste trabalho estudamos três copolímeros constituídos por dois blocos distintos, isto é copolímeros dibloco, onde um dos blocos é constituído pelo polimetacrilato de metila, PMMA e o outro pelo polimetacrilato de N,N dimetilamino, PDMAEMA, e o homopolímero de PDMAEMA. A fórmula geral dos copolímeros dibloco estudados é PMMAm-b-PDMAEMAn onde o número de monômeros de cada bloco é representado pelos índices m e n. Os copolímeros PMMA1-b-PDMAEMA6,3 (MM, 54.200), PMMA1-b-PDMAEMA3,0 (MM 27.558), o PMMA1-b-PDMAEMA1,1 (MM 25.555) e o homopolímero PDMAEMA (MM 41.614,9) tiveram suas massas molares e a razão entre os monômeros de cada bloco caracterizados por cromatografia de gel permeação, GPC, e NMR. Determinamos a concentração micelar crítica (CMC) dos copolímeros e verificamos, por RMN e fluorescência, o efeito da concentração e do pH na sua agregação. A interação dos copolímeros com vesículas unilamelares grandes, LUVs, preparadas com misturas de fosfatidilcolina, PC, e fosfatidilglicerol, PG, foi estudada em diferentes condições de pH e força iônica. A interação dos polímeros com as LUVs também foi estudada medindo-se o diâmetro hidrodinâmico e potencial zeta das LUVs na presença dos polímeros. Observou-se que, quando a razão polímero/lipídio é alta, os polímeros se ligam às LUVs, neutralizando completamente a carga das vesículas. Quando a concentração de LUVs é próxima à dos polímeros, forma-se uma rede que conduz à agregação e precipitação dos complexos. Estudamos o efeito dos polímeros na permeabilização de LUVs e vesículas unilamelares gigantes (GUVs) preparadas com misturas de PC:PG. Os copolímeros permeabilizaram as LUVs, dependendo da razão polímero/lipídio, do pH, força iônica e das características hidrofóbicas e hidrofílicas de cada copolímero. Quanto maior a porcentagem de PG nas LUVs maior a interação com os copolímeros. A ligação dos copolímeros às LUVs deve produzir segregação dos fosfolipídios negativos na bicamada das LUVs (i.e., separação lateral de fases) facilitando a permeabilização das vesículas. Demonstramos que os copolímeros se ligam à superfície das GUVs modificando a sua forma e levando ao rompimento das vesículas. Estes efeitos foram modulados pelo pH e a força iônica do meio. O efeito de permeabilização dos copolímeros foi correlacionado com as razões entre os blocos hidrofóbicos e hidrofílicos. Quando o copolímero tem uma maior fração de PDMAEMA, o bloco mais hidrofílico do copolímero, este efeito é mais evidente. A porcentagem de permeabilização, após um tempo definido, para copolímeros de mesma massa molar, como o PMMA1-b-PDMAEMA3,0 (MM 27.558) e o PMMA1-b-PDMAEMA1,1 (MM 25.555), é muito maior com o copolímero com maior porção hidrofílica, o PMMA1-b-PDMAEMA3,0. O homopolímero DMAEMA também se mostrou eficiente na interação com as LUVs, porém menos que o PMMA1-b-PDMAEMA6,3.que possui maior numero de unidades DMAEMA e uma sequencia de monômeros de MMA. Demonstramos, nesta Tese, que o efeito de copolímeros sintéticos contendo regiões hidrofóbicas e hidrofílicas são bons modelos de peptídeos e proteínas permitindo avaliar quantitativamente o efeito dessas interações em modelos de membranas. / Copolymers are organic or inorganic macromolecules with high molecular weight, consisting of repeated monomer units joined by covalent bonds and exhibit more than one type of monomer. Here, we studied three copolymers consisting of two different blocks, i.e. diblock copolymers, where one block is constituted by methyl polymethacrylate, PMMA and other by polymethacrylate N, N-dimethylamino, PDMAEMA and a homopolymer PDMAEMA. The general formula of the diblock copolymers studied was PMMAm-b-PDMAEMAn where the number of monomers of each block is represented by indexes m and n. The PMMA1-b-PDMAEMA6,3 copolymers (MW, 54,200), PMMA1 PDMAEMA3,0-b (MW 27,558), the PMMA1 PDMAEMA1,1-b (MW 25,555) and PDMAEMA homopolymer (MW 41614.9) had their molecular weights and the ratio between the monomers of each block characterized by gel permeation chromatography, GPC, and NMR. The critical micelle concentration (CMC) of the copolymers was determined by fluorescence and the aggregation of the copolymers verified by NMR. The effect pH on the copolymers CMC was also determined. The interaction of copolymers with large unilamellar vesicles, LUV, prepared with mixtures of phosphatidylcholine, PC, and phosphatidylglycerol, PG, was studied under different conditions of pH and ionic strength. The interaction of the polymers with the LUVs was also studied measuring the hydrodynamic diameter and zeta potential of the LUVs in the presence of the polymers. It was observed that when the ratios polymer / lipid are high, the polymers bind to the LUVs, completely neutralizing the charge of the vesicles. When the concentration of LUVs is close to that of the polymers, it forms a network that leads to aggregation and precipitation of the complexes. We studied the effect of polymers in permeabilization LUVs and giant unilamellar vesicles (GUVs) prepared with PC:PG. The copolymers permeabilized the LUVs, depending on the ratio polymer / lipid, pH, ionic strength and the hydrophobic and hydrophilic characteristics of each copolymer. The higher the percentage PG LUVs lead to greater interaction with the copolymers. The binding of the copolymers to the LUVs with negative charge induced phase separation of phospholipids in the bilayer of LUVs (ie, lateral phase separation) facilitating the permeability of the vesicles. We demonstrate that copolymers bind to the surface of GUVs changing its shape and leading to rupture of vesicles. These effects are modulated by pH and ionic strength of the media. The permeabilization effect of the copolymers was correlated with the ratios of the hydrophobic and hydrophilic blocks. When the copolymer has a larger fraction of PDMAEMA, the more hydrophilic block copolymer, this effect is more evident. The percentage of permeabilization, after a set time, for the same molar mass copolymers such as b-PDMAEMA3,0 PMMA1 (MW 27,558) and PMMA1 PDMAEMA1,1-b (MW 25,555), is much more efficient with copolymer with greater hydrophilic portion, the b-PMMA1 PDMAEMA3,0. The DMAEMA homopolymer also proved efficient in interacting with the LUVs. We demonstrated that synthetic copolymers containing hydrophobic and hydrophilic regions are good models of peptides and proteins allowing quantitatively evaluate the effects of these interactions on model membranes