Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

Amplification and/or overexpression of the Mdm2 oncogene occurs in many human cancers. Mdm2 promotes cellular proliferation, interferes with apoptosis, and induces tumor formation through the negative regulation of the p53 tumor suppressor. More than thirty percent of human tumors overexpressing Mdm2 also present with alternatively spliced Mdm2 isoforms that cannot directly bind p53. The presence of Mdm2 isoforms in tumors correlates with a higher tumor grade and a poorer prognosis for the patient. To investigate the function of Mdm2 isoforms in tumorigenesis, we have isolated a number of Mdm2 splice forms from tumors obtained from Mdm2-transgenic mice and find that the most frequently observed splice form in human tumors, Mdm2-b, is conserved in mice. Although the Mdm2-b protein is incapable of binding to p53 and is unable to localize to the nucleus, we demonstrate that Mdm2-b promotes cell growth in NIH3T3 cells, Rb-deficient, p19-deficient, and p53-deficient primary cells. We also show that Mdm2-b inhibits apoptosis in response to serum withdrawal and restimulation, doxorubicin treatment, and TNF-alpha administration. Mdm2-b induces foci formation in vitro and directly contributes to tumor formation in GFAP-Mdm2 transgenic mice. We propose that Mdm2-b promotes tumor growth by upregulating RelA (P65) protein levels and activity in a p53-independent manner. To better understand additional functions of Mdm2 that are p53-dependent, we have generated an Mdm2 conditional mouse model. Using primary mouse embryonic fibroblasts derived from Mdm2 conditional mice, we demonstrate that p21 is required for p53-dependent apoptosis initiated by Mdm2 loss. In support of this observation, we also note that p21-loss partially rescues embryonic lethality of Mdm2 null mice. We further show that p21-loss partially rescues the embryonic lethality caused by the loss of the Mdm2 family member, Mdm4. We address the possibility that Mdm2 and Mdm4 may play redundant roles during embryonic development and find that Mdm2 overexpression fully rescues the embryonic lethality resulting from Mdm4 loss. Our findings demonstrate that both Mdm2 and Mdm4 play critical roles in modulation of the p53 tumor suppressor pathway and that their deregulation can result in tumor formation through both p53-dependent and independent pathways.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2008-2009 / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

BRCA1 est l’un des deux gènes majeur de prédisposition au cancer du sein. Les multiples partenaires protéiques de BRCA1 lui confèrent de nombreuses fonctions par lesquelles elle peut assurer la surveillance de l’intégrité cellulaire. L’équipe dans laquelle j’ai mené ma thèse a identifié un nouveau partenaire protéique de BRCA1, la protéine de liaison au poly(A) des ARN messagers PABP1, et a ainsi mis en lumière l’implication de BRCA1 dans la régulation de la traduction [1]. Durant ma thèse, j’ai étudié cette nouvelle fonction de BRCA1 et essayé de comprendre si, et comment, elle pourrait contribuer à son rôle de suppresseur de tumeur. J’ai tout d’abord montré que BRCA1 est associée avec la fraction ribosomique des cellules, et plus particulièrement à la fraction subpolysomique, ce qui pourrait indiquer sa participation à l’initiation de la traduction. De plus, nos résultats indiquent que BRCA1 pourrait participer à un complexe non canonique d’initiation de la traduction. Dans la mesure où BRCA1 n’est pas un facteur canonique d’initiation de la traduction, il est peu probable qu’elle intervienne dans la traduction de tous les ARNm. Notre hypothèse est qu’elle pourrait réguler la traduction d’ARNm spécifiques codant pour des protéines impliquées dans ses différentes fonctions de surveillance de l’intégrité cellulaire. Afin d’identifier les cibles traductionnelles de BRCA1, j’ai réalisé une analyse transcriptomique comparative du contenu des polysomes de cellules épithéliales mammaires MCF7 exprimant de façon transitoire un ARN interférent dirigé contre BRCA1 ou contrôle. Parmi les ARNm cibles de l’activité traductionnelle de BRCA1, beaucoup codent effectivement pour des protéines impliquées dans les fonctions auxquelles elle participe. Ce travail confirme que la participation de BRCA1 à la régulation de la traduction pourrait être une autre voie par laquelle BRCA1 exerce son rôle de suppresseur de tumeur. De plus, l’analyse des cibles traductionnelle de BRCA1 pourrait conduire à l’identification de nouvelles fonctions de cette protéine ainsi qu’à la découverte de marqueurs tumoraux ou de cibles thérapeutiques pour les patients porteurs d’une mutation de BRCA1 / BRCA1 is one of the two major genes of breast cancer susceptibility. The numerous binding partners of BRCA1 allow it to participate to several cellular pathways which globally contribute to its cell surveillance capacity. The team in which I performed my PhD identified a new bindind partner of BRCA1, the Poly(A)-Binding Protein 1 and, consequently, a new function of this tumor suppressor, namely, the translation regulation [1]. During my PhD, I studied this new function and try to elucidate if, and how, this function could participates to the BRCA1’s tumor suppressive activity. I first showed that BRCA1 is associated with the ribosomal fraction of the cell, and more precisely, with the subpolysomal fraction, which could indicate that BRCA1 participates to the initiation step of translation. Moreover, our results suggest that BRCA1 could participate to a non canonical initiation complex. Given that BRCA1 is not a canonical translation initiation factor, it is unlikely that BRCA1 regulates the translation of all mRNAs. Our hypothesis is that BRCA1 could regulate the translation of specific mRNAs involved in its various cell surveillance functions. In an attempt to identify the BRCA1 translational targets, we performed a microarray analysis of polysomes-bound mRNAs in MCF7 cells transiently expressing siRNA directed against BRCA1 or control siRNA. We found that, among the translational targets of BRCA1, many indeed encode proteins involved in the same main functions as BRCA1’s. Altogether, our results suggest that the involvement of BRCA1 in translation regulation could be another way by which BRCA1 exerts its tumor suppressor role. Moreover, the analysis of the BRCA1’s translational targets could lead to the identification of new functions for BRCA1 as well as to the discovery of new tumoral markers and therapeutic targets for the BRCA1 mutated patients

Os cistos e tumores odontogênicos sempre tiveram grande importância dentro da Odontologia, seja pela grande prevalência clínica seja pelo grande acometimento do indivíduo afetado pela lesão. A nova classificação da Organização Mundial de Saúde trouxe a mudança de categoria do queratocisto, que recebe agora a nômina de tumor odontogênico queratocístico, e que figura não mais na categoria de cisto odontogênico de desenvolvimento, mas sim de tumor odontogênico. Certa precipitação nessa mudança levou alguns autores a sugerirem a necessidade de estudos que esclareçam as características clínicas e histopatológicas dessa lesão para que se tenha uma classificação realmente precisa. O grande paradigma dessa lesão é: ao mesmo tempo em que apresenta características histológicas de um cisto, possui um comportamento clínico agressivo mais comumente observado nos tumores. O que realmente difere esta lesão das outras lesões que se inseriam no mesmo grupo é o padrão de crescimento diferenciado do tumor odontogênico queratocístico em relação às outras lesões císticas. Sendo assim, poderia se suspeitar que essa lesão possua um potencial proliferativo maior do que as outras que anteriormente pertenciam ao mesmo grupo, denotando uma regulação diferenciada do mecanismo proliferação-apoptose. Este estudo teve como objetivo comparar o tumor odontogênico queratocístico com uma lesão cística - o cisto dentígero - e com uma lesão tumoral - o ameloblastoma ? por meio de marcadores imunoistoquímicos para supressão tumoral e anti-apoptose. Os resultados demostraram que a maior diferença entre essas lesões está principalmente na atividade apoptótica, já que somente o resultado de bcl2 foi estatisticamente significante entre essas lesões. / Odontogenic cysts and tumors have always had a great importance in Dentistry, for its high clinical prevail and for its noticeable invasive behavior. The new classification released by WHO rearranged keratocyst, that is named now odontogenic keratocystic tumor, classifying it no longer as a development cyst, but now as odontogenic tumor. Certain haste in this change brought some authors to suggest the necessity of more studies to clarify the feautures of such lesions to determine a more accurate classification. The greatest paradigm of this lesion is that it shows cyst-like histological characteristics and simultaneuosly it has an aggressive clinical behavior, which is more commonly observed in tumors. The main difference between this lesion and the others cystic lesions is the growth pattern, which suggests that the odontogenic keratocystic tumor has higher proliferative potential than other cystic lesions. The aim of this research was to compare odontogenic keratocystic tumor with a cystic lesion ? dentigerous cyst ? and with a tumoral lesion ? ameloblastoma ? using tumor suppressor and anti-apoptosis immunohistochemical expression. Results show that the more important difference among the analysed lesions is apoptosis activity, since only bcl2 staining was significantly different among them.

Este trabalho descreve a remediação simultânea de dois genes supressores de tumor, CDKN2A e p53, em três linhagens celulares derivadas de glioblastoma multiforme: U87 (CDKN2A-/-, p53wt/wt), U251 (CDKN2A-/-, p53mut/mut) e T98G (CDKN2A-/-, p53mut/mut). A entrega gênica foi mediada por vetor adenoviral bicistrônico contendo o cassete CDKN2AIRESp53, capaz de expressar as duas proteínas simultaneamente. Vetores monocistrônicos também foram testados (AdCDKN2A e Adp53). Visando detectar apoptose, as linhagens receberam o sensor de atividade de caspase-3/7 GFP-DEVD-ssGLUC por transdução lentiviral. Este possui Gaussia luciferase (GLUC) C-terminal, que é secretada após ativação de caspases e pode ser dosada no sobrenadante. Após a marcação, realizaram-se ensaios de viabilidade celular, proliferação, formação de colônias, senescência, ciclo celular e dosagem de GLUC após remediação dos genes supressores de tumor nas linhagens GBMDEVD-GLUC. Com ensaio de viabilidade, observou-se efeito citotóxico do vetor bicistrônico AdCDKN2AIRESp53 maior que a soma dos obtidos com cada tratamento monocistrônico. No ensaio de senescência, o vetor AdCDKN2A resultou na maior indução do fenótipo senescente em todas as linhagens, seguido por Adp53, enquanto AdCDKN2AIRESp53 produziu resultados similares a um desses dois perfis em cada linhagem. Dosagem de GLUC no sobrenadante foi usada como indicador para atividade de caspase-3/7 após tratamento com os vetores supressores de tumor. O controle AdLacZ resultou em atividade de GLUC maior que nas amostras sem vírus (mock), enquanto tratamento com AdCDKN2A obteve resultados maiores que o controle em 72 h nas três linhagens. O vetor AdCDKN2AIRESp53 alcançou, inesperadamente, resultados variados em 72 h. Os dados obtidos neste trabalho indicam que a remediação simultânea de CDKN2A e p53 possui notável ação antiproliferativa tumoral, podendo levar à morte ou à senescência celular. Também é apontado que o sensor de caspase-3/7 GFP-DEVD-ssGLUC é robusto, mas detecta não apenas a indução de apoptose, mas a combinação de todos os processos ativadores de caspases em uma amostra. / This thesis describes the simultaneous remedy of two tumor suppressor genes, CDKN2A and p53, in three glioblastoma multiforme (GBM)-derived cell lines: U87 (CDKN2A-/-, p53wt/wt), U251 (CDKN2A-/-, p53mut/mut) and T98G (CDKN2A-/-, p53mut/mut). Gene delivery was mediated by a bicistronic adenoviral vector bearing the sequence CDKN2AIRESp53, which simultaneously expresses both proteins. Monocistronic vectors were also tested (AdCDKN2A and Adp53). To detect apoptosis, the GBM cell lines received caspase-3/7 sensor GFP-DEVD-ssGLUC via lentiviral transduction. This sensor has a C-terminal Gaussia luciferase (GLUC), which is secreted by the cell after caspase activation and can be measured in the supernatant. After sensorization, functional assays were carried out, including cell viability, proliferation, colony formation, cell senescence, cell cycle and GLUC measure after treatment with the tumor suppressor vectors in GBMDEVD-GLUC lineages. Viability assay with the AdCDKN2AIRESp53 vector resulted in a remarkable cytotoxic effect, greater than the sum of the effects with each monocistronic treatment. In the senescence assay, vector AdCDKN2A yielded the highest induction of cell senescence in all lineages, followed by Adp53, while AdCDKN2AIRESp53 induced results that followed one of these two profiles in each cell line. GLUC measure was an indicator of intracellular caspase-3/7 activity after treatment with the tumor supressor vectors. Control vector AdLacZ resulted in higher GLUC activity than mock treatment in all three cell lines, while AdCDKN2A treatment showed results bigger than control at 72 h in all cell lines. Bicistronic vector AdCDKN2AIRESp53 reached, unexpectedly, varied results at 72 h in all cell lines. Data obtained in this study indicate that simultaneous remedy of CDKN2A and p53 has remarkable antiproliferative activity in GBM cells, resulting in cell death or cell senescence. It is also shown that caspase-3/7 sensor GFP-DEVD-ssGLUC is a robust tool, but its results detect not only a single cellular process, such as apoptosis, but the combination of all caspase-activating processes in a cell population.

A proteína ribossomal L10 (RP L10) é uma forte candidata a ser incluída na classe de proteínas supressoras de tumor. Também denominada QM, a proteína em questão é conhecida por participar da ligação das subunidades ribossomais 60S e 40S e da tradução de mRNAs. Possui massa molecular entre 24 a 26 kDa e ponto isoelétrico (pI) 10,5. A seqüência da proteína QM é bastante conservada em mamíferos, plantas, invertebrados, insetos e leveduras indicando que esta possui funções críticas na célula. Com função supressora de tumor, a proteína RP L10 foi estudada em linhagens de tumor de Wilm (WT-1) e em células tumorais de estômago, nas quais se observou uma diminuição na quantidade de seu mRNA. Mais recentemente a RP L10 foi encontrada em baixas quantidades nos estágios iniciais de adenoma de próstata e com uma mutação em câncer de ovário, indicando uma participação no desenvolvimento destas doenças. Como proteína, já foi descrito que esta interage com as proteínas c-Jun e c-Yes, inibindo a ação ativadora de fatores de crescimento e divisão celular. Este trabalho tem um papel importante no estabelecimento da expressão desta proteína solúvel, para estudos posteriores que tenham como objetivo avaliar a ação de regiões específicas que atuam na ligação das subunidades ribossomais 60S e 40S e tradução, bem como nas regiões que se ligam a proto-oncogenes. O cDNA para proteína QM foi amplificado por PCR e clonado no vetor de expressão periplásmica p3SN8. A proteína QM foi expressa em E.coli BL21 (DE3) no citoplasma e periplasma bacteriano e na melhor condição, a expressão de QM de bactérias transformadas pelo plasmídeo recombinante p1813_QM em 25°C ou 30°C, a proteína foi obtida solúvel e com quantidad es muito pequenas de contaminantes. Os ensaios de estrutura secundária demonstraram que a proteína QM tem predominância de a-hélice, mas quando do seu desenovelmento, essa condição muda e a proteína passa a ter característica de folhas β. / The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm\'s tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribossomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813_QM at 25°C or 30°C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature.

Li Ching-wan. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 104-130). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACTS (ENGLISH/CHINESE) --- p.iii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER1 --- INTRODUCTION / Chapter 1.1. --- Liver Cancer --- p.1 / Chapter 1.2. --- Hepatocellular Carcinoma --- p.1 / Chapter 1.2.1. --- Types of Liver Cancer --- p.1 / Chapter 1.2.2. --- Epidemiology --- p.4 / Chapter 1.2.2.1. --- Geographical Distribution --- p.4 / Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8 / Chapter 1.2.3. --- Etiologic Factors --- p.9 / Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9 / Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11 / Chapter 1.2.3.3. --- Alcohol --- p.12 / Chapter 1.2.3.4. --- Summary --- p.12 / Chapter 1.3. --- HCC in Hong Kong --- p.14 / Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17 / Chapter 1.4.1. --- HBV Genome --- p.17 / Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17 / Chapter 1.4.2.1. --- HBV Integration --- p.17 / Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19 / Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20 / Chapter 1.5. --- Genetic Alterations in HCC --- p.21 / Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21 / Chapter 1.5.2. --- Proto-oncogene --- p.23 / Chapter 1.5.3. --- Genetic Studies in HCC --- p.23 / Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25 / Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26 / Chapter 1.5.3.3. --- Array CGH --- p.26 / Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27 / Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35 / Chapter CHAPTER3 --- MATERIALS AND METHODS / Chapter 3.1. --- Patients and Materials --- p.38 / Chapter 3.1.1. --- DNA Extraction --- p.40 / Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40 / Chapter 3.2.1. --- Microsatellite Markers --- p.41 / Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42 / Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42 / Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42 / Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44 / Chapter 3.2.3.1. --- Electrophoresis --- p.44 / Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45 / Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45 / Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46 / Chapter 3.3.1. --- Nick-Translation --- p.49 / Chapter 3.3.2. --- Hybridization --- p.49 / Chapter 3.3.3. --- Imaging and Data Analysis --- p.50 / Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51 / Chapter 3.3.5. --- Assessment of Data Quality --- p.51 / Chapter 3.4. --- Statistical Analysis --- p.52 / Chapter CHAPTER4 --- RESULTS / Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53 / Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54 / Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54 / Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62 / Chapter CHAPTER5 --- DISCUSSION / Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73 / Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74 / Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74 / Chapter 5.1.1.2. --- Other Neoplasia --- p.76 / Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76 / Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80 / Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80 / Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80 / Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81 / Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81 / Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81 / Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82 / Chapter 5.1.4. --- Limitation of this Study --- p.83 / Chapter 5.1.4.1. --- Markers --- p.83 / Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83 / Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83 / Chapter 5.1.4.2. --- Tissue Samples --- p.84 / Chapter 5.1.4.2.1. --- Normal Reference --- p.84 / Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85 / Chapter 5.1.5. --- Future Studies --- p.85 / Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85 / Chapter 5.1.5.2. --- Extension of the Present Study --- p.86 / Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88 / Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89 / Chapter 5.2.1.1. --- Locus of 17q23 --- p.89 / Chapter 5.2.1.1.1. --- D17S1670 --- p.89 / Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91 / Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92 / Chapter 5.2.1.2.1. --- LAMC2 --- p.92 / Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93 / Chapter 5.2.1.3.1. --- PIK3CA --- p.93 / Chapter 5.2.1.4. --- Locus of 8p22 --- p.94 / Chapter 5.2.1.4.1. --- CTSB --- p.94 / Chapter 5.2.1.5. --- Locus of 6q22 --- p.95 / Chapter 5.2.1.5.1. --- MYB --- p.95 / Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96 / Chapter 5.2.1.6.1. --- CSE1L --- p.96 / Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97 / Chapter 5.2.1.7.1. --- FGR --- p.97 / Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98 / Chapter 5.2.1.8.1. --- PGY1 --- p.98 / Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99 / Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99 / Chapter 5.2.3. --- Limitation of the Study --- p.100 / Chapter 5.2.3.1. --- Samples and Materials --- p.100 / Chapter 5.2.4. --- Further Study --- p.101 / Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101 / Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101 / Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102 / Chapter 5.2.5.1. --- Deletion Analysis --- p.102 / Chapter 5.2.5.2 --- Tissue Microarray --- p.103 / Chapter 5.2.5.3. --- cDNA Microarray --- p.103 / Chapter chapter6 --- references --- p.104