Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / The HIV-­‐1 pandemic continues unabated. Although treatments exist that can substantially alleviate the morbidity and mortality associated with HIV, there is still a need for improved anti-­‐HIV treatments that reduce toxicities and administration frequency and mediate sustained inhibition of viral replication. Given the high mutability and variability of the virus, a strategy that is garnering increasing focus is the targeting of host factors that the virus requires to replicate, so-­‐called HIV-­‐dependency factors (HDFs). It is hoped this will reduce the emergence of viral drug resistance. A number of genome-­‐wide screens have been performed to identify HDFs, although many remain to be validated, particularly in relevant cells lines. An objective of this thesis was to validate three host factors as HDFs, in both TZM-­‐bl reporter and T cell-­‐derived cell lines, and to examine their potential as anti-­‐HIV-­‐1 therapeutic targets through exploitation of the cellular gene silencing pathway, RNA interference (RNAi). These were HIV-­‐1 Tat specific factor 1 (HTATSF1), DEAD (Asp-­‐Glu-­‐Ala-­‐Asp) box polypeptide 3, X-­‐ linked (DDX3X) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1), selected because they had been previously implicated in HIV-­‐ 1 pathogenesis. The well-­‐characterised HDF, PC4 and SFRS1 interacting protein 1 (PSIP1)/lens epithelium-­‐derived growth factor (LEDGF)/p75, was included in the study as a positive control. Cassettes expressing short hairpin RNAs (shRNAs) targeting the four host proteins were generated, although shRNAs did not suppress endogenous ddx3x mRNA levels. The ability of shRNAs to inhibit HIV-­‐1 replication in the reporter cell line, TZM-­‐bl, was examined. These HeLa-­‐ derived cells are permissive for R5-­‐tropic HIV-­‐1 infection and contain an integrated luciferase gene driven by the viral promoter. shRNAs mediated a dose-­‐dependent inhibition of luciferase activity in cells infected with a HIV-­‐1 subtype B molecular clone and, although production of the viral protein p24 was unaltered, infectious particle production was decreased in cells treated with a shRNA suppressing HTATSF1. Little effect was observed with a shRNA targeting SMARCB1, suggesting that this may not function as an HDF under these conditions. No effect on infectious particle production was seen with the shRNA targeting PSIP1, which was a result of the long half-­‐ life of this protein, highlighting a limitation of using such reporter systems for HDF validation. Importantly, shRNAs were not associated with any cytotoxic effects in TZM-­‐bl cells. Whether HTATSF1 is a potential therapeutic target was interrogated further in the more relevant T cell-­‐derived SupT1 cell line. Lentiviruses were used to generate populations where >90% had one copy of the integrated shRNA expression cassette. Replication of the subtype B molecular clone p81A-­‐4 was significantly inhibited in the shH1-­‐expressing SupT1 cell line, which targets HTATSF1, for over 14 days post-­‐infection, although inhibition was not as pronounced asthat observed in the shP1-­‐expressing SupT1 cell line, which targets PSIP1. In contrast to a previous report, no change in the ratio of unspliced to singly-­‐ or multiply-­‐spliced HIV-­‐1 transcripts were detected in shH1-­‐expressing SupT1 cells, suggesting that HTATSF1 does not function as a splicing cofactor in this system. A slight rebound in p24 levels at 14 days post-­‐infection was accompanied by increased HTATSF1 expression and a decrease in the percentage of cells with transgene expression in the population. In addition, there was a slight decrease in shH1-­‐derived guide strand expression, but no change in transcription rates of the htatsf1 gene, suggesting that cells within the population with shH1 expression and HTATSF1 suppression may have a growth disadvantage. Thus, although this work demonstrates for the first time that HTATSF1 functions as an HDF in T cell-­‐derived SupT1 cells, it may not constitute a viable therapeutic target. A second objective of this thesis was to examine the feasibility of transcriptional gene silencing (TGS) of HDFs as an anti-­‐HIV strategy. TGS is a small RNA-­‐induced gene silencing pathway that operates through chromatin remodelling with the potential to mediate long-­‐term silencing of gene expression. Thus, its application may reduce the frequency of drug administration and associated toxicities. Short interfering RNAs (siRNAs) targeting the htatsf1 promoter were able to reduce target mRNA expression, which was accompanied by decreased htatsf1 transcription rates in HEK293T cells, suggesting silencing via a TGS mechanism. The htatsf1 silencing inhibited infectious HIV-­‐1 particle production from TZM-­‐bl cells. This work provides proof of principle that TGS induction at a HDF may inhibit HIV-­‐1 replication. siRNAs targeting the ddx3x promoter did not induce TGS. To examine whether gene susceptibility to TGS may be influenced by promoter architectures, 49 promoter features were examined for enrichment in genes at which small RNA-­‐induced TGS has been reported. Initially, the TGS group was compared to a random set of 2,000 promoters and then all other promoters in the genome. To control for gene activation, two further analyses were performed comparing the TGS group features to those from promoters active in the THP-­‐1 cell line and housekeeping genes. Whilst difficult to ascribe differences between the TGS group and the control groups to anything beyond a variation in the proportion of active genes within each group, there was enrichment for certain promoter features that are independent of activity; the TGS group was characterised by broad transcription start regions, high CpG content and a single expression profile. Moreover, the fraction of promoters with reported non-­‐coding RNA overlap was greater in the TGS group than the control groups. Thus, there is some evidence that a number of promoter features are associated with TGS susceptibility. It is hoped this novel analysis will facilitate selection of future TGS targets, including HDFs. In summary, the work presented in this thesis paves the way for development of improved anti-­‐HIV therapies involving HDF-­‐targeted TGS-­‐based gene therapies that mediate sustained inhibition of the virus.

by Chuen Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-166). / Abstracts in English and Chinese. / ACKNOWLDEGEMENT --- p.ii / PUBLICATIONS --- p.iii-iv / ABBREVIATIONS --- p.v-viii / INDEX FOR FIGURES --- p.ix xii / INDEX FOR TABLES --- p.xiii / ABSTRACT (Chinese and English) --- p.xiv-xvi / TABLE OF CONTENT --- p.xvii / Chapter 1. --- INTRODUCTION --- p.1-37 / Chapter 2. --- OBJECTIVES --- p.38-40 / Chapter 3. --- METHODS AND MATERIALS --- p.41 -70 / Chapter 4. --- RESULTS AND DISCUSSION --- p.71-130 / Chapter 5. --- GENERAL DISCUSSION AND CONCLUSION --- p.131-133 / BIBLIOGRAPHY --- p.134-166

A Biologia hoje é a mais comentada ciência e a que ocupa maior espaço na mídia. No entanto, isso não significa que os conceitos biológicos sejam de conhecimento público, nem tampouco dos estudantes de Biologia. Para analisar o conhecimento de conceitos fundamentais de Genética, Biologia Molecular e Biologia celular apresentado por graduandos do último ano de Cursos Superiores de Ciências Biológicas é que realizamos esse trabalho de pesquisa. Iniciamos o trabalho com a seleção das questões do Exame Nacional de Cursos de Biologia (Provão) do ano de 2002 que abordavam conceitos de Genética e disciplinas correlatas, analisamos o relatório do ENC-BIO/2002 expedido pelo MEC, aplicamos questionários com as questões analisadas do ENC-BIO/2002 em 72 estudantes do último ano dos cursos de Ciências Biológicas, realizamos 33 entrevistas semi-estruturadas com estudantes do último ano dos cursos de Ciências Biológicas e, por fim, analisamos os resultados apresentados pelo relatório do MEC e das entrevistas realizadas. Concluímos que os estudantes não chegam a alcançar os objetivos que a ele são atribuídos pelo MEC, no que diz respeito à Genética e disciplinas correlatas e a maioria apresenta aprendizagem mecânica. / Biology today is the most talked about science and also the one which gets the largest media coverage. However, that doesn\'t mean that the biological concepts are of public knowledge, neither are they of knowledge of Biology students. The main objective of this research work is to analyze the knowledge of fundamental concepts of Genetics, Molecular Biology and Cellular Biology of undergraduate students, who were majoring in Biological Sciences, in their senior year. We began our work with some selected questions from the National Exam of Biology Courses (?Provão?) for 2002, we chose the ones which involved concepts of Genetics and other correlated disciplines, we analyzed the ENC-BIO/2002 report prepared by the MEC, we prepared questionnaires with the analyzed questions from the ENC-BIO/2002 and distributed them to 72 senior students who were majoring in Biological Sciences, we conducted 33 semi-structured interviews with senior students majoring in Biological Sciences and, finally, we analyzed the results presented by the MEC report and by the interviews. We concluded that the students did not achieve their goals, which were established by the MEC. These goals were related to Genetics and other correlated disciplines and most of them demonstrated only a mechanical learning.

Reverse transcription in HIV-1 (human immunodeficiency virus type 1) is initiated from a tRNA, tRNALys3, that is annealed to the primer binding site (PBS) in the 5' region of viral RNA. This tRNA, along with the other major tRNALys isoacceptors, tRNALys1,2 , is selectively packaged into HIV-1 during its assembly. The formation of a tRNALys packaging/annealing complex is believed to involve the interaction between a Gag/GagPol/viral complex with a lysyl-tRNA synthetase (LysRS)/tRNALys complex, with Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In fact, Gag particles alone will package LysRS, but GagPol, which binds tRNA Lys, is also required for incorporation of the tRNALys. / The model we propose for the tRNALys packaging/annealing complex predicts a possible interaction between LysRS and Pol sequences in GagPol, which might facilitate transfer of tRNALys3 from LysRS to the reverse transcriptase (RT) thumb domain where tRNALys3 binds. In this work, we demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. Since these RT domains are not required for tRNALys packaging into HIV-1, the LysRS/Pol interaction is probably not involved in the transfer of tRNALys3 to RT. The LysRS/Pol interaction may instead be involved in tRNALys3 annealing since the connection domain in RT has been found to be required for this process. Also, since an interaction has been reported between Gag and Pol sequences in GagPol, we also investigated whether the Gag/LysRS/Pol interaction played an important role in stabilizing the Gag/Pol interaction, and found, using siRNA to LysRS, that it did not. / tRNALys3 annealing to viral RNA is promoted by nucleocapsid sequences in Gag and by mature NCp7, and we have examined the roles of Gag and NCp7 in this process. Gag- and NC-facilitated tRNALys3 annealing to HIV-1 RNA were measured both in vivo and in vitro, indirectly by the ability of annealed tRNALys3 to prime reverse transcription, and directly by measuring the occupancy of the PBS by tRNALys3. While tRNALys3 annealing can be carried out by both Gag and NCp7, exposure (in vivo or in vitro) of the tRNALys3/viral RNA complex to NCp7 is required for optimum placement of the tRNALys3. This is indicated by 1) tRNALys3's reduced ability to incorporate the first dNTP, dCTP, and 2) its more ready displacement from the PBS by DNA synthesized from a downstream primer. / It has been previously demonstrated that APOBEC3G (A3G) can inhibit tRNA Lys3 annealing to viral RNA, and we have used A3G to further dissect the roles of Gag and NCp7 in annealing, both in vitro and in vivo. Experiments studying how APOBEC3G (A3G) inhibits tRNA Lys3 annealing indicate that in protease-positive viruses, Gag-facilitated tRNALys3 annealing may only playa minor role. In vivo and in vitro, A3G only inhibits NCp7-facilitated annealing, and not Gag-facilitated annealing. Nevertheless, while Gag is able to show 70-80% of the annealing efficiency of NCp7 in a protease-negative virus, A3G can reduce annealing efficiency in protease-positive viruses to 40%. This appears to be due to the fact that, in vitro, the presence of NCp7 makes prior Gag-facilitated annealing susceptible to A3G. This suggests that in wild type viruses, any Gag-facilitated annealing of tRNALys3 to viral RNA that does occur is altered through an A3G-susceptible re-annealing by NCp7.

A Biologia hoje é a mais comentada ciência e a que ocupa maior espaço na mídia. No entanto, isso não significa que os conceitos biológicos sejam de conhecimento público, nem tampouco dos estudantes de Biologia. Para analisar o conhecimento de conceitos fundamentais de Genética, Biologia Molecular e Biologia celular apresentado por graduandos do último ano de Cursos Superiores de Ciências Biológicas é que realizamos esse trabalho de pesquisa. Iniciamos o trabalho com a seleção das questões do Exame Nacional de Cursos de Biologia (Provão) do ano de 2002 que abordavam conceitos de Genética e disciplinas correlatas, analisamos o relatório do ENC-BIO/2002 expedido pelo MEC, aplicamos questionários com as questões analisadas do ENC-BIO/2002 em 72 estudantes do último ano dos cursos de Ciências Biológicas, realizamos 33 entrevistas semi-estruturadas com estudantes do último ano dos cursos de Ciências Biológicas e, por fim, analisamos os resultados apresentados pelo relatório do MEC e das entrevistas realizadas. Concluímos que os estudantes não chegam a alcançar os objetivos que a ele são atribuídos pelo MEC, no que diz respeito à Genética e disciplinas correlatas e a maioria apresenta aprendizagem mecânica. / Biology today is the most talked about science and also the one which gets the largest media coverage. However, that doesn\'t mean that the biological concepts are of public knowledge, neither are they of knowledge of Biology students. The main objective of this research work is to analyze the knowledge of fundamental concepts of Genetics, Molecular Biology and Cellular Biology of undergraduate students, who were majoring in Biological Sciences, in their senior year. We began our work with some selected questions from the National Exam of Biology Courses (?Provão?) for 2002, we chose the ones which involved concepts of Genetics and other correlated disciplines, we analyzed the ENC-BIO/2002 report prepared by the MEC, we prepared questionnaires with the analyzed questions from the ENC-BIO/2002 and distributed them to 72 senior students who were majoring in Biological Sciences, we conducted 33 semi-structured interviews with senior students majoring in Biological Sciences and, finally, we analyzed the results presented by the MEC report and by the interviews. We concluded that the students did not achieve their goals, which were established by the MEC. These goals were related to Genetics and other correlated disciplines and most of them demonstrated only a mechanical learning.

The overall aim of this project was to investigate HIV-1 genetic diversity in South Afri ca and to characterise the immune response in mice to a South African subtype C gp120. To investigate the relationship between subtype and mode of transmission, samples were collected from individuals infected by heterosexual and male homosexual transmission from patients attending local HIV/AIDS clinics in Cape Town (n=49) and Bloemfontein (n=4). Isolates were subtyped using heteroduplex mobility assay (HMA) based on the V3-V5 region of the env geneusing reference plasmids (2 B, 2 C and 1 D) representative of local subtypes. HMA identified four env subtypes: A, B, C and D. Subtype B viruses were found in 92.9% (26/28) of the male homosexual/bisexual group and subtype C viruses in 77.2% (17 /22) of the heterosexual group. Subtype B viruses were also identified in two heterosexual patients, one patient infected by blood transfusion and in two patients with. unknown mode of transmission. Subtype D viruses were found in one male homosexual patient and one heterosexual patient and a husband and wife couple were infected with subtype A viruses. A significant association between subtype and mode of transmission (p=<0.0001) was identified, confirming two independent epidemics. To determine the subtype distribution of HIV within urban heterosexual populations throughout South Africa, samples were collected from women attending antenatal clinics in Johannesburg (n=34), Pretoria (n=S) and Durban (n=20). Samples from Bloemfontein (n=24) were taken from individuals attending an HIV/AIDS clinic. All eighty-three samples were subtyped by HMA in the env region as before. The predominant subtype circulating within the urban heterosexual population throughout South Africa was identified as subtype C (92.8%) although subtype B was also detected (7.2%). It may thus be beneficial if a HIV vaccine for South Africa is based on a subtype C model. In addition, a rapid method for identification of HIV-1 gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400bp (p17) or 650bp (p17 and 5' p24) long PCR fragments. This strategy was appl i ed to eighty-six samples (Cape Town n=47, Johannesburg n=20, Bloemfontein n=17 and Durban n=2) previously subtyped by either sequence analysis of the gag p17 region (n=31), heteroduplex mobility assay (HMA) based on the env gene (n=76), or both (n=21). RFLP analysis identified two subtype A, twenty-five subtype B, fifty-eight subtype C and one subtype D isolates. There were no discrepancies between RFLP and sequence gag subtypes, demonstrating the reliability of this method and no discordance between gag RFLP subtypes and env HMA subtypes, indicating no recombinant viruses in the genomic regions analysed.

No description available.

Lam Wai-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves [64]-[73]). / Abstracts in English and Chinese. / Abstract (in English) --- p.i-ii / Abstract (in Chinese) --- p.iii -iv / Acknowledgements --- p.v / Table of contents --- p.vi -ix / List of Figures --- p.x / List of Tables --- p.x / Abbreviations --- p.xi -xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.1-2 / Chapter 1.2 --- Major risk factors of HCC / Chapter (1) --- Hepatitis B Virus (HBV) --- p.2-4 / Chapter (2) --- Hepatitis C Virus (HCV) --- p.5-6 / Chapter (3) --- Cirrhosis --- p.6 / Chapter (4) --- Dietary alfatoxin B1 (AFB1) --- p.6 -7 / Chapter (5) --- Alcoholic consumption --- p.7 / Chapter (6) --- Iron overload --- p.8 / Chapter 1.3 --- Genetic aberrations in HCC --- p.8-9 / Chapter (1) --- Chromosomal loss --- p.10-13 / Chapter (2) --- Chromosomal gains --- p.13-15 / Chapter 1.4 --- roposed study --- p.15 / Chapter (1) --- Hypomethylation of heterochromatin in chromosome 1q copy number gain. --- p.16 / Chapter (2) --- ositional mapping on 1q21 - q22 by interphase cytogenetics. --- p.16-17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation. --- p.18-19 / Chapter 2.1.2 --- ositional Mapping by Interphase Cytogenetics. --- p.19 -24 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter (1) --- Extraction of high molecular weight DNA --- p.25 / Chapter (2) --- DNA digestion with methyl-sensitive restriction enzyme --- p.25 -26 / Chapter (3) --- Control for the complete DNA digestion. --- p.26 / Chapter (4) --- Southern Blotting. --- p.26 -27 / Chapter 2.2.2 --- ositional Mapping by Interphase Cytogenetics / Chapter (1) --- Yeast Artificial Chromosome (YAC) --- p.28 -29 / Chapter (i) --- YAC culturing --- p.29 -30 / Chapter (ii) --- YAC DNA extraction --- p.30 -31 / Chapter (iii) --- Inter-Alu-Polymerase Chain Reaction --- p.32 -33 / Chapter (2) --- -1 derived Bacterial Artificial Chromosome (PAC) --- p.34 / Chapter (i) --- AC culturing and DNA extraction --- p.34 -35 / Chapter (3) --- FISHrobe labeling by nick translation. --- p.35 / Chapter (4) --- FISHrobereparation --- p.36 / Chapter (5) --- Dot-blot analysis. --- p.36 -37 / Chapter (6) --- Verification of the YAC andACrobes by metaphase FISH --- p.37 / Chapter (7) --- Hybridization efficiency test --- p.38 / Chapter Chapter 3 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter 3.1 --- Introduction --- p.39 -40 / Chapter 3.2 --- Materials and Methods / Chapter (1) --- atients --- p.41 / Chapter (2) --- Mathyl-sensitive restriction enzyme digestion. --- p.42 / Chapter (3) --- Classical satellite 2 DNArobe labeling and hybridization. --- p.42 -43 / Chapter (4) --- Membrane washing and signal detection. --- p.43 / Chapter (5) --- Signal detection and reference ratio determination. --- p.43 -44 / Chapter (6) --- Comparative Genomic Hybridization (CGH) --- p.44 -45 / Chapter 3.3 --- Results / Chapter (1) --- Heterochromatin hypomethylation and 1q12 breakpoint. --- p.45 / Chapter (2) --- Heterochromatin hypomethylation in adjacent hepatitis Infected liver tissue. --- p.46 / Chapter 3.4 --- Discussion --- p.47-51 / Chapter Chapter4 --- ositional Mapping of 1q21 - q22 by Interphase Cytogenetics / Chapter 4.1 --- Introduction --- p.52-53 / Chapter 4.2 --- Materials and Methods / Chapter (1) --- atients --- p.53 / Chapter (2) --- YAC clones --- p.53 -54 / Chapter (3) --- AC clones --- p.55 / Chapter (4) --- Formalin-fixedaraffin-embedded tissue sections pretreatment. --- p.55 / Chapter (5) --- Hybridization --- p.56 / Chapter (6) --- Signal detection --- p.56 -57 / Chapter 4.3 --- Results / Chapter (1) --- Relative copy number gain on YAC examined. --- p.57 -59 / Chapter (2) --- AC findings --- p.60 / Chapter 4.4 --- Discussion --- p.60 -63 / References

Leung Ho-yin. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Acknowlegements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / "Table of Contents," --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviation --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 . --- Cancer Incidences in Hong Kong --- p.2 / Chapter 1.2. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.3. --- "Etiological Risk Factors," --- p.7 / Chapter 1.3.1. --- Liver Cirrhosis / Chapter 1.3.2. --- Chronic Viral Hepatitis / Chapter 1.3.2.1. --- Hepatitis B Virus (HBV) / Chapter 1.3.2.2. --- Hepatitis C Virus (HCV) / Chapter 1.3.3. --- Dietary Aflatoxin B1 exposure / Chapter 1.3.4. --- Heavy Alcohol Consumption / Chapter 1.3.5. --- Hemochromatosis / Chapter 1.4. --- Genetic Aberration in HCC --- p.12 / Chapter 1.4.1. --- Chromosomal Gains / Chapter 1.4.2. --- Chromosome Losses / Chapter 1.5. --- Epigenetic Changes --- p.18 / Chapter 1.6. --- Aims of Thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1. --- Materials --- p.23 / Chapter 2.1.1. --- Culture of Cell Lines / Chapter 2.1.2. --- Preparation of Normal Human Metaphase / Chapter 2.1.3. --- DNA Extraction from Cell Lines / Chapter 2.1.4. --- DNA Extraction from Tissues / Chapter 2.1.5. --- DNA Extraction from Blood / Chapter 2.1.6. --- Nick Translation / Chapter 2.1.7. --- Dot Blot / Chapter 2.1.8. --- Probe Preparation / Chapter 2.1.9. --- Fluorochrome-conjugated antibodies / Chapter 2.1.10. --- Fluorescence Microscopy and Image Analysis / Chapter 2.1.11. --- Primer Labeling / Chapter 2.1.12. --- Polymerase Chain Reaction / Chapter 2.1.13. --- Gel Preparation / Chapter 2.1.14. --- Gel Electrophoresis / Chapter 2.2. --- Sample --- p.28 / Chapter 2.2.1. --- Patients / Chapter 2.2.2. --- Cell Lines / Chapter 2.3. --- Comparative Genomic Hybridization --- p.30 / Chapter 2.3.1. --- Method / Chapter 2.3.1.1. --- Preparation of Normal Human Metaphase / Chapter 2.3.1.2. --- DNA Extraction / Chapter 2.3.1.3. --- Nick Translation / Chapter 2.3.1.4. --- Labeling Efficiency / Chapter 2.3.1.5. --- Probe Preparation / Chapter 2.3.1.6. --- Slide Preparation / Chapter 2.3.1.7. --- Hybridization / Chapter 2.3.1.8. --- Post Hybridization Wash / Chapter 2.3.1.9. --- Image Capturing and Analysis / Chapter 2.3.1.10. --- Control Experiment / Chapter 2.4. --- Microsatellite Analysis --- p.46 / Chapter 2.4.1. --- Method / Chapter 2.4.1.1. --- Fluorescent-Labeled Polymorphic Markers / Chapter 2.4.1.1.1. --- Polymerase Chain Reaction / Chapter 2.4.1.1.2. --- Gel Preparation / Chapter 2.4.1.1.3. --- Gel Electrophoresis / Chapter 2.4.1.1.4. --- Data Analysis / Chapter 2.4.1.2. --- Radioisotope-Labeled Polymorphic Markers / Chapter 2.4.1.2.1. --- Primer Labeling / Chapter 2.4.1.2.2. --- Polymerase Chain Reaction / Chapter 2.4.1.2.3. --- Gel Preparation / Chapter 2.4.1.2.4. --- Gel Electrophoresis / Chapter 2.4.1.2.5. --- Autoradiography and Data Analysis / Chapter 3. --- Chapter 3 Genetic Imbalances on Chromosome 1 --- p.55 / Chapter 3.1. --- Introduction --- p.56 / Chapter 3.2. --- Methods --- p.57 / Chapter 3.2.1. --- Patients and Cell Lines / Chapter 3.2.2. --- CGH / Chapter 3.2.3. --- MSA with Fluorescent-labeled Polymorphic Markers / Chapter 3.2.4. --- Refinement of lp36 loss / Chapter 3.2.5. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1. --- CGH / Chapter 3.3.2. --- MSA on Primary HCC Cases / Chapter 3.3.3. --- Refinement of lp36 loss / Chapter 3.3.4. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3.5. --- CGH vs MSA / Chapter 3.4. --- Discussion --- p.74 / Chapter 4. --- Chapter 4 Genetic Imbalances on Chromosome 4q --- p.78 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Methods --- p.82 / Chapter 4.2.1. --- Patients and Cell Lines / Chapter 4.2.2. --- CGH / Chapter 4.2.3. --- MSA with Radioisotope-labeled Polymorphic Markers / Chapter 4.3. --- Results --- p.86 / Chapter 4.3.1. --- CGH / Chapter 4.3.2. --- MSA / Chapter 4.3.2.1. --- MSA on Primary HCC cases / Chapter 4.3.2.2. --- MSA on In-house developed HCC cell lines / Chapter 4.3.2.3. --- Combined MSA Results / Chapter 4.4. --- Discussion --- p.94 / Chapter 5. --- Chapter 5 Proposed Future Studies --- p.99 / Chapter 5.1. --- "Microarray Analysis," --- p.101 / Chapter 5.2. --- Functional Studies --- p.102 / Chapter 6. --- Bibliography --- p.104