In der Promotion wird die Entwicklung, Optimierung und Validierung einer Reversed-phase-Chromatography Methode zur Messung des Ribavirinplasmaspiegels beschrieben. Diese wurde mit einer Solid Phase Extraction zur Probenvorbereitung kombiniert. Zudem finden sich zahlreiche Auswertungen von gemessenen Patienenchromatogrammen zu ausgewählten, klinisch relevanten Fragestellungen, wie beispielsweise die Darstellung des Ribavirinplasmaspiegels im Tagesverlauf, im Verlauf der ersten sechs Therapiewochen, im Vergleich von Männern und Frauen, sowie bei einem niereninsuffizienten Patienten. Zu den erhobenen Ergebnissen wird Stellung genommen, und daraus resultierende Schlussfolgerungen bezüglich einer zukünftigen Optimierung der Hepatitis-C-Therapie kommentiert. / The doctorate program will describe the development, optimization and validation of a reversed phase chromatography method for measuring ribavirinplasma levels. This has been combined with solid phase extraction for preparing samples. In addition, numerous analyses of patients’ chromatograms are included relative to clinically relevant questions such as the depiction of ribavirinplasma levels during the course of a day, during the course of the first six weeks of therapy, in comparison to other men and women as well as patients suffering from kidney insufficiencies. The collected data will be commented on, as well as the resulting implications relative to a future optimization of hepatitis-C therapy.

In conclusion, two new methods of cccDNA quantitation were developed and validated. The two assays are complementary to each other and may be used in patients with extreme HBV DNA levels. These cccDNA assays should be further validated in larger studies and may become important tests for diagnostic, prognostic and treatment monitoring purposes. / Over 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, which leads to many cases of cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a critical intracellular replicative intermediate and cannot be eliminated during antiviral therapy. Current methods for cccDNA detection are limited by false positive detection due to the interference by HBV relaxed circular DNA (rcDNA). The tests also have limited sensitivity to detect cccDNA at low concentrations. Hence, we aimed to develop a highly sensitive and highly specific assay for cccDNA detection with wide linear range. / The modified Bowden's assay had the highest intrahepatic cccDNA detection rate (60 positive results out of 61 cases). The detection rate of the modified Bowden's assay is significantly higher than that of the Bowden's assay. On the other hand, the cccDNA detection rate in serum samples was low at 20--27% by all 3 assays. In 5 samples in which cccDNA was undetectable by the Bowden's assay but detectable by the other two assays, a point mutation in the HBV genome was found in the forward primer binding site of the Bowden's assay. This partly explained the false negative results. / The quantification result of cccDNA by the bisulfite conversion assay was significantly lower than that by the Bowden's assay assay (P=0.001) and the modified Bowden's assay (P=0.003). When the total HBV DNA was higher than 107 copies/ml, the serum cccDNA level detected by the bisulfite conversion assay was significantly lower than that detected by the Bowden's assay (P=0.008) and the modified Bowden's assay (P=0.046). When the total HBV DNA is less than 107 copies/ml, there were no significant differences. This suggests that the bisulfite conversion assay was less affected by rcDNA even in samples containing a high viral load. / With this background, two new cccDNA assays were developed and optimized. Bowden's assay was used as a standard to evaluate the performance of new assays. The first new assay (modified Bowden's assay) involved the use of new primers and probes that targeted more conserved regions in the HBV genome. The second assay adopted the bisulfite conversion method, which introduced gene sequence changes into the HBV genome and thereby enhance the specificity of the assay. Capillary sequencing was performed to find mutations in primers and probe range of different assays. / Yu, Ling. / Advisers: Vincent Wai-Sun Wang; Joseph Jao-Yiu Sung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 105-111). / 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.

Ng Wah. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-113). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF TABLES --- p.X / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Etiological Factors of Hepatocellualr Carcinoma (HCC) --- p.4 / Chapter 1.2.1 --- Dietary Aflatoxins --- p.4 / Chapter 1.2.2 --- Liver Cirrhosis --- p.5 / Chapter 1.2.3 --- Alcohol Abuse --- p.6 / Chapter 1.2.4 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3 --- Literature Review on the Investigations of HBV Integrants in HCC --- p.16 / Chapter 1.3.1 --- Affected Host Junctions --- p.17 / Chapter 1.3.2 --- Viral Junctions --- p.18 / Chapter 1.4 --- Restriction Site Polymerase Chain Reaction (RS-PCR) --- p.19 / Chapter 1.5 --- Aims of Thesis --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Chemicals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Cell Cultures --- p.24 / Chapter 2.1.4 --- Nucleic Acids --- p.24 / Chapter 2.1.5 --- Enzymes --- p.25 / Chapter 2.1.6 --- Equipment --- p.25 / Chapter 2.1.7 --- Software and Web Resources --- p.26 / Chapter 2.2 --- Methods --- p.27 / Chapter 2.2.1 --- DNA Extraction --- p.27 / Chapter 2.2.2 --- RS-PCR --- p.31 / Chapter 2.2.3 --- Sequencing --- p.37 / Chapter 2.2.4 --- Spectral Karyotyping (SKY) --- p.38 / Chapter 2.2.5 --- Fluorescence In situ hybridization --- p.39 / Chapter Chapter 3 --- Investigation of HBV Integration Sites in HCC Cell lines --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell Lines --- p.47 / Chapter 3.2.2 --- RS-PCR --- p.47 / Chapter 3.2.3 --- Spectral Karyotyping --- p.48 / Chapter 3.2.4 --- Tyramide Signal Amplification for HBV in FISH Analysis --- p.48 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Identification of HBV Integration Sites in Cell Lines --- p.51 / Chapter 3.3.2 --- Evaluation of RSO Primer Efficiency --- p.52 / Chapter 3.3.3 --- SKY and FISH Analysis --- p.53 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- HBV Insertions in HCC Cell Lines --- p.64 / Chapter 3.4.2 --- Efficacy of RSO Primers --- p.65 / Chapter 3.4.3 --- Investigation of HBV Integration on Chromosomal Rearrangement --- p.65 / Chapter Chapter 4 --- Investigation of Hepatitis B Virus Integration Sites in Primary HCC --- p.67 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and Methods --- p.69 / Chapter 4.2.1 --- Patients --- p.69 / Chapter 4.2.2 --- RS-PCR --- p.70 / Chapter 4.3 --- Results --- p.72 / Chapter 4.3.1 --- HBV Integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.72 / Chapter 4.4 --- Discussion --- p.88 / Chapter 4.4.1 --- HBV integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.88 / Chapter 4.4.2 --- Summary on HBV Integrants Identified --- p.91 / Chapter Chapter 5 --- Proposed Future Studies --- p.98 / Chapter 5.1 --- Correlation of Structural Aberrations with HBV Integrations --- p.99 / Chapter 5.2 --- Transcriptional Expression Study on the Genes Interrupted by or Located near the Virus Host Junctions --- p.100 / Chapter Chapter 6 --- References --- p.101

Made available in DSpace on 2015-02-04T21:42:14Z (GMT). No. of bitstreams: 1 Celina Soares.pdf: 585127 bytes, checksum: dbb978516e57361340a9d4e0b3953a00 (MD5) Previous issue date: 2006-04-27 / Esta pesquisa é um estudo transversal, com método quantitativo e análise de prevalência dos dados. Apresenta como objetivo estimar a soroprevalência das hepatites virais B e C em pacientes infectados pelo HIV. A justificativa deste estudo evidencia-se nas atuais doenças sexualmente transmissíveis (HIV e HBV) e de transmissão sanguínea (HIV, HBV e HCV), por protagonizar substancialmente o panorama do enfrentamento mundial em saúde pública. Santos/SP, cidade litorânea e turística, onde está localizado o maior porto marítimo da América Latina, com um grande número de homens que fazem sexo com homens, mulheres trabalhadores do sexo e usuários de drogas injetáveis, além de apresentar população flutuante de turistas, trabalhadores diretos e indiretos na atividade portuária. Neste sentido constrói um perfil humano relevante que caracteriza a elaboração do atual estudo. A coleta de dados foi realizada na população de pacientes matriculados no Centro de Referência em Aids CRAIDS, Santos, SP. A amostra foi constituída por 1438 pessoas, no período de fevereiro de 2004 a fevereiro de 2005. A análise nos prontuários foi realizada selecionando resultados de marcadores sorológicos para as hepatite B (HBsAg e anti-HBs) e hepatite C (anti-HCV), pela técnica de ELISA, além dos exames sorológicos de menor contagem de linfócitos T CD4+ e maior contagem de carga viral para o HIV, considerando as variáveis: sexo, idade, escolaridade, estado civil, forma de contaminação. Os resultados demonstraram que na população dos 1438 indivíduos HIV+, 54.6% eram do sexo masculino e 45.4% do feminino. Em relação à idade, 71% estavam entre 30 e 49 anos e 54.3% com nível de escolaridade no ensino fundamental. A contagem de linfócitos CD4+ revelou 43.7% com níveis abaixo de 200 células /mm³. Considerando a soroprevalência dos marcadores HBsAg, anti-HBs, Anti-HCV e fatores de risco para essas doenças obtivemos uma prevalência global de HBsAg de 7.4% e 23.5% para o anti-HCV. As variáveis que apresentaram associação com o HBV foram: idade, baixos níveis de células T CD4+, homossexualidade masculina e UDI; para HCV: idade, sexo, escolaridade de nível superior e UDI.

Introdução: As hepatites virais causadas pelo VHB e VHC são um importante problema de saúde pública, representando, juntas, mais de 530 milhões de indivíduos no mundo inteiro, que desenvolveram hepatites crônicas ao serem expostos a esses vírus. No entanto, grande parte da população infectada desconhece sua condição, pois frequentemente encontra-se assintomática. O diagnóstico dessas viroses muitas vezes se dá de forma ocasional, geralmente após procedimentos clínicos rotineiros ou após a realização de doações voluntárias de sangue. Entretanto, já foi demonstrado que mesmo assintomáticos, ou com a presença de sintomas mínimos, os portadores do VHB ou do VHC podem apresentar distúrbios de ordem emocional, social ou mesmo físicos, que repercutem na queda de sua QVRS. Objetivos: O principal objetivo desse estudo foi o de avaliar o impacto que a informação do diagnóstico da presença do VHB ou do VHC poderia causar na QVRS de doadores voluntários de sangue. Método: Participaram do estudo 105 doadores com sorologias alteradas ou para o VHB ou para o VHC nos exames de triagem das doações de sangue, sendo que 65 (62%) eram do sexo masculino e 40 (38%) eram do sexo feminino. Deste total, 32 (30,5%) apresentaram confirmação do diagnóstico para o VHB e 35 (33,3%) apresentaram confirmação do diagnóstico para o VHC. Entretanto, 38 doadores de sangue (36,2%), não tiveram a confirmação dessas viroses após a realização de exames confirmatórios, sendo que seus exames de triagem alterados na doação de sangue foram considerados falso-positivos. Os doadores com diagnóstico sorológico confirmado foram divididos em 2 grupos: um grupo formado por 32 doadores com diagnóstico de hepatite B e um grupo de 35 doadores com diagnóstico de hepatite C. Foram estabelecidos 2 grupos controles para as comparações. O primeiro, um grupo controle negativo, composto de doadores com sorologias negativas em doações de sangue sucessivas, pareados por sexo, cor e idade, e em igual número para cada grupo de doadores infectados. O segundo, um grupo controle falso-positivo, composto por 38 doadores que apresentaram sorologias falso-positivas na doação de sangue. As avaliações da QVRS nos indivíduos infectados foram realizadas em 3 etapas: a) 1ª Etapa: quando compareciam no banco de sangue para a repetição dos exames alterados na doação, contudo sem saberem qual alteração sorológica estava sendo investigada. b) 2ª Etapa: Quando o médico do banco de sangue informava ao doador qual era o seu diagnóstico. c) 3ª Etapa: No momento em que o doador iniciava o acompanhamento clínico ambulatorial. As comparações foram feitas seguindo-se quatro abordagens: a) Comparando-se o nível de percepção das diferenças na QVRS, conforme a evolução das etapas do estudo, através de uma análise longitudinal de cada grupo infectado. b) Comparando-se as diferenças na QVRS entre os grupos infectados com o VHB ou com o VHC e doadores do grupo controle negativo. c) Comparando-se as diferenças na QVRS entre os grupos infectados com o VHB ou com o VHC e doadores do grupo controle falsopositivo. d) Comparando-se a QVRS do grupo de doadores com diagnóstico de hepatite B com a QVRS do grupo de doadores com diagnóstico de hepatite C, no momento em que iniciaram o acompanhamento ambulatorial. As avaliações foram realizadas utilizando-se o questionário SF-36, genérico, de aplicação na população geral, e o questionário LDQOL, de aplicação específica nas doenças hepáticas. Resultados: Nas análises longitudinais, utilizando-se o questionário SF-36, somente o grupo de doadores com diagnóstico de hepatite C demonstrou diferenças estatisticamente significantes da QVRS, nos domínios Dor (p = 0,011), Estado Geral da Saúde (p < 0,001), Aspectos Sociais (p = 0,019), Saúde Mental (p = 0,033) e no Componente Físico da escala sumarizada do SF-36, à custa do aumento nos escores dos questionários. Diferentemente, o grupo de doadores com diagnóstico de hepatite B não apresentou diferenças estatisticamente significantes na análise longitudinal. Nas comparações do grupo de doadores com diagnóstico de hepatite B com seu grupo-controle negativo, utilizando-se o questionário SF-36, foram encontradas diferenças estatisticamente significantes: a) Fase 1 do estudo: nos domínios Dor (p = 0,036), Estado Geral de Saúde (p = 0,007) e no Componente Físico (p = 0,004) da escala sumarizada do SF-36. b) Fase 2 do estudo: nos domínios Capacidade Funcional (p = 0,034), Limitação por Aspectos Físicos (p = 0,048), Dor (p = 0,035), Estado Geral de Saúde (p = 0,002), Saúde Mental (p = 0,047) e no Componente Físico (p = 0,007) da escala sumarizada do SF-36. Fase 3 do estudo: nos domínios Capacidade Funcional (p = 0,028), Dor (p = 0,002), Estado Geral de Saúde (p = 0,006), Vitalidade (p = 0,046) e no Componente Físico (p = 0,006). Nas comparações do grupo de doadores com diagnóstico de hepatite B com o grupo controle falso-positivo, utilizando-se o questionário SF-36, foram encontradas diferenças estatisticamente significantes: a) Fase 1 do estudo: nos domínios Limitação por Aspectos Físicos (p = 0,032). b) Fase 2 do estudo: nos domínios Capacidade Funcional (p = 0,006), Limitação por Aspectos Físicos (p = 0,006), Dor (p = 0,006), Vitalidade (p = 0,017), Limitação por Aspectos Emocionais (p = 0,038), Saúde Mental (p = 0,030), no Componente Físico (p = 0,019) e no Componente Mental (p = 0,022) da escala sumarizada do SF-36.Nas comparações do grupo de doadores com diagnóstico de hepatite C com o seu grupo controle negativo, utilizando-se o questionário SF-36, foram encontradas diferenças estatisticamente significantes: a) Fase 1 do estudo: nos domínios Capacidade Funcional (p = 0,038), Dor (p = 0,003), Estado Geral de Saúde (p<0,011), Vitalidade (p = 0,034), Aspectos Sociais (p = 0,006), Limitação por Aspectos Emocionais (p = 0,024), Saúde Mental (p = 0,007) , no Componente Físico (p = 0,002) e no Componente Mental da escala sumarizada do SF-36. b) Fase 2 do estudo: nos domínios Limitação por Aspectos Físicos (p = 0,040), Dor (p = 0,017), Estado Geral de Saúde (p < 0,001), Vitalidade (p = 0,019), Aspectos Sociais (p = 0,005), Limitação por Aspectos Emocionais (p = 0,025), Saúde Mental (p = 0,034) e no Componente Físico (p = 0,008) da escala sumarizada do SF-36. c) Fase 3 do estudo: nos domínios Dor (p = 0,041), Estado Geral de Saúde (p = 0,003), Vitalidade (p = 0,030) e Limitação por Aspectos Emocionais (p = 0,027). Nas comparações entre o grupo de doadores com diagnóstico de hepatite C e o grupo controle falso-positivo, utilizando-se o questionário SF-36, foram encontradas diferenças estatisticamente significantes : a) Fase 1 do estudo: nos domínios Limitação por Aspectos Físicos (p = 0,040) e Limitação por Aspectos Emocionais (p = 0,042). b) Fase 2 do estudo: Limitação por Aspectos Físicos (p = 0,010), Vitalidade (p = 0,037), Aspectos Sociais (p = 0,010) e Limitação por Aspectos Emocionais (p = 0,005). Nas comparações da QVRS entre o grupo dos doadores com diagnóstico de hepatite B e o grupo dos doadores com diagnóstico de hepatite C no momento em que iniciavam o acompanhamento clínico, utilizando-se o questionário LDQOL, não foram observadas diferenças estatisticamente significantes. Conclusões: A análise longitudinal dos doadores com diagnóstico de hepatite C, as comparações com o seu grupo-controle negativo e com o grupo controle falso-positivo, demonstraram alteração na QVRS. Segundo a análise longitudinal, os doadores com o VHC, ao evoluírem nas etapas do estudo, apresentaram um aumento em seus escores, sugerindo que as informações prestadas pela equipe médica sobre a doença e a perspectiva de um atendimento clínico adequado causaram uma tranquilização que melhorou o impacto inicial do diagnóstico da doença, promovendo melhora relativa da QVRS. Ainda que houvesse melhora dos escores na análise longitudinal, os doadores com o diagnóstico de hepatite C tiveram queda de sua QVRS, demonstrada quando comparados ao seu grupo-controle negativo e com o grupo-controle falso-positivo. Nos doadores do grupo com o diagnóstico de hepatite B, ao contrário, não houve alteração da QVRS que pôde ser demonstrada na análise longitudinal. Este achado sugere a baixa QVRS nestes indivíduos mesmo antes do diagnóstico, e que não houve alteração perceptível de sua queda neste grupo de infectados durante a evolução do estudo. Entretanto, quando comparados ao grupocontrole de doadores negativos e ao grupo-controle falso-positivo, foi demonstrada queda na sua QVRS, que pôde ser associada ao impacto do diagnóstico da infecção pelo VHB / Introduction: Viral hepatitis caused by HBV and HCV are an important public health problem. Together they represent around 530 million individuals who have been exposed to these viruses all over the world. Much of that infected population is unaware of their condition, because they are often asymptomatic. Diagnosing these viral infections often comes only occasionally, usually after clinical routine procedures or after volunteer blood donations. However, it has been shown that even asymptomatic, or even in the presence of minimum symptoms, carriers living with HBV or HCV virus can show emotional, social or even physical disturbs, which echoes in the fall of their Health Related Quality of Life (HRQOL). Aims: The main goal of this study was to evaluate the impact of the diagnosis information of the HBV or HCV presence could cause on HRQOL of voluntary blood donors. Method: The study included 105 donors with altered serology to HBV or HCV tests in blood donations screening. 65 (62%) blood donors were male and 40 (38%) were female. From this total, 32 (30.5%) blood donors were submitted to HBV diagnosis confirmation and 35 (33.3%) were submitted to HCV diagnosis confirmation. However 38 (36.2%) blood donors had no confirmation of these infections after conducting confirmatory tests, and were considered as false positive result tests. Donors with a confirmed serological diagnosis were divided into 2 groups: a group of 32 blood donors with hepatitis B diagnosis and 35 blood donors with hepatitis C diagnosis. Two control groups have been established for comparisons. The first, a negative control group, composed of donors with negative serology, matched by sex, race and age, and in equal numbers for each group of infected donors. The second, a false-positive control group composed by 38 blood donors who had false-positive serology in their blood donation. Assessments of HRQOL in individuals infected were performed in 3 phases: a) Phase 1: When they went to the blood bank for collecting blood samples to new confirmatory tests, however, they werent informed about which serological test was specifically being investigated. B) Phase 2: When the blood banks doctor informed what the donors diagnosis was. C) Phase 3: At the moment the donor started his clinical follow-up. Comparisons were performed according to 4 approaches: A) Comparing the level of perceived HRQOL differences according to the evolution of the studys phases, through a longitudinal analysis of each group infected. B) Comparing the HRQOL differences between the infected donor groups with HBV or HCV and their negative control group. C) Comparing the HRQOL differences between the infected donor groups with HBV or HCV and the false-positive control group. C) Comparing the HRQOL differences between the infected groups with HBV or HCV virus with the false-positive control group. D) Comparing the HRQOL in the group of donors with diagnosis of hepatitis B and the HRQOL in the group of donors with diagnosis of hepatitis C at the time they started the ambulatory monitoring. Evaluations were performed using the SF-36, the generic form applied for the general population, and the LDQOL, specific form applied in liver diseases. Results: In the longitudinal analysis in patients with HBV or HCV using the SF-36, only the group of donors diagnosed with hepatitis C showed significant differences in HRQOL, which occurred in the domains: Bodily Pain (p = 0.011), General Health (p <0.001), Social Function (p = 0.019), Mental Health (p = 0.033) and in the Physical Component of the summarized SF-36 scale, at the costs of increasing in the questionnaire scores. On different way, the hepatitis B group did not show statistically significant differences in longitudinal analysis. Comparing the hepatitis B diagnosis donors group with their negative control group using the SF-36, were found the following statistically significant differences, in the domains: A) Phase 1 Study: Bodily Pain (p = 0.036), General Health (p = 0.007) and in the Physical Component (p = 0.004) of the summarized SF-36 scale. B) Phase 2 Study: Physical Function (p = 0.034), Role Physical (p = 0.048), Bodily Pain (p = 0.035), General Health (p = 0.002), Mental Health (p = 0.047) And in the Physical Component (p = 0.007) of the summarized SF-36 scale. C) Phase 3 Study = Physical Function (p = 0.028), Bodily Pain (p = 0.002), General Health (p = 0.006), Vitality (p = 0.046), and in the Physical Component (p = 0.006) of the summarized SF-36 scale. Comparisons between the hepatitis B diagnosis donors group and their false positive control using the SF-36, were found statistically significant differences in the domains: A) Phase 1 Study : Role Physical (p = 0.032). B) Phase 2 Study = Physical Function (p = 0.006), Role Physical (p = 0.006), Bodily Pain (p = 0.006), Vitality (p = 0.017), Role Emotional (p = 0.038), Mental Health (p = 0.030), and in the Physical Component (p = 0.019) and Mental Component (p = 0,022) of the SF-36 summarized scale. In comparisons of the group of donors with a diagnosis of hepatitis C with their negative control group, using the SF-36 were found statistically significant differences in the domains: A) Phase 1 Study: Physical Function (p = 0.038), Bodily Pain (p = 0.003), General Health (p <0.011), Vitality (p = 0.034), Social Function (p = 0.006), Role Emotional (p = 0.024), Mental Health (p = 0.007), Physical Component (P = 0.002) and Mental Component of the SF-36 summarized scale. B) Phase 2 study: Role Physical (p = 0.040), Bodily Pain (p = 0.017), General Health (p <0.001), Vitality (p = 0.019), Social Function (p = 0.005), Role Emotional (p = 0.025), Mental Health (p = 0.034) and Physical Component (p = 0.008) of the SF-36 summarized scale. C) Phase 3 Study: Bodily Pain (p = 0.041), General Health (p = 0.003), Vitality (p = 0.030) and Role Emotional (p = 0.027). Comparisons between the group of donors diagnosed with hepatitis C and the control group false-positive, using the SF-36 were statistically significant differences: A) Phase 1 Study: Role Physical (p = 0.040) and Role Emotional (p = 0.042). b) Phase 2 Study: Role Physical (p = 0.010), Vitality (p = 0.037), Social Function (p = 0.010) and Role Emotional (p = 0.005). In comparisons of HRQOL between the group of donors with a diagnosis of hepatitis B and the group of donors diagnosed with hepatitis C at the time of beginning the clinical follow-up, using the questionnaire LDQOL, there were no statistically significant differences. Conclusion: The longitudinal analysis of donors diagnosed with hepatitis C, the comparisons with their negative control group and the control group false-positive, showed changes in HRQOL. According to the longitudinal analysis, donors with HCV, when evoluting in the phases of the study showed an increase in their scores, suggesting that the information provided by the medical team about the disease and the prospect of an appropriate clinical care caused a tranquilization that has improved the initial impact diagnose of the disease, promoting a relative improvement on HRQOL. Although there were improvement in scores on the longitudinal analysis, the donors with the diagnosis of hepatitis C had a reduction of their HRQOL, as demonstrated when compared to their negative control group and the control group false-positive. In the donor group with the diagnosis of hepatitis B, in contrast, there was no change in HRQOL that could be demonstrated in longitudinal analysis. But when compared to their control groups negative and false-positive, statistically significant differences were identified. This finding suggests a poor HRQOL in these individuals even before the diagnosis, and that there was no noticeable change to its fall in this group infected during the course of the study. However, when compared to the negative control group of donors and the false-positive control group it was demonstrated decrease in HRQOL of these donors, which might be associated with the impact of the diagnosis of HBV infection

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.