Introdução: A injeção percutânea de etanol (PEI) é um método de ablação local considerado curativo para carcinomas hepatocelulares (CHC) pequenos. Não há dados sobre PEI para tratamento do CHC na América Latina. Pacientes e métodos: Incluídos 100 pacientes cirróticos submetidos à PEI como terapia primária para o CHC no Serviço de Gastroenterologia do HCFMUSP entre setembro de 1997 e dezembro de 2005. Avaliados resposta ao tratamento e taxa de sobrevida. Resultados: Resposta completa ao tratamento ocorreu em 41% dos casos. A sobrevida foi de 83% em 1 ano, 49% em 3 anos e 29% em 5 anos, mas no grupo com tumores de até 2 cm, sem invasão vascular e com resposta completa ao tratamento, atingiu 89%, 70% e 70%, respectivamente. Conclusão: PEI apresentou boa sobrevida em pacientes com tumores 2 cm, sem invasão vascular e resposta completa ao tratamento. / Introduction: Percutaneous ethanol injection (PEI) is a curative local ablative method for treatment of small hepatocellular carcinoma (HCC). There is no data about PEI for HCC treatment in Latin America. Patients and Methods: A total of 100 consecutive cirrhotic patients were enrolled. All of them had been submitted to PEI as first-line therapy for the treatment of HCC at the Service of Gastroenterology of HCFMUSP in the period of September 1997 to December 2005. Response to treatment and survival rates were assessed. Results: Complete response to treatment was obtained in 41% of the patients. Survival rates were 83% in 1 year, 49% in 3 years and 29% in 5 years, but in patients with tumors up to 2 cm, no vascular invasion and complete response to treatment, were 89%, 70% and 70%, respectively. Conclusion: PEI is associated with long-term survival in patients with tumors 2 cm, absence of vascular invasion and complete response to treatment

O câncer é um dos principais problemas de saúde pública no mundo. Dentre as neoplasias primárias que acometem o fígado, o carcinoma hepatocelular (HCC) é a mais frequente. Diversos fatores de risco predispõem ao HCC, entre eles a doença hepática gordurosa não alcóolica (NAFLD). Segundo estudos prévios do grupo, a &#946-ionona (BI), apresenta potencial quimiopreventivo na hepatocarcinogênese, promovendo redução de lesões preneoplásicas (LPN). Assim pretendeu-se investigar se a NAFLD potencializaria o desenvolvimento de LPN em ratos Wistar submetidos ao modelo do hepatócito resistente (RH) na etapa de iniciação/promoção inicial da hepatocarcinogênese e se a BI apresenta efeito quimiopreventivo nesse contexto. Para tanto, os animais foram distribuídos em 4 grupos experimentais: grupo não-tratado (NT), grupo submetido ao RH (RH), grupo submetido ao RH e ao modelo de NAFLD, que consiste na administração de emulsão hiperlipídica, (AS) e grupo AS tratado com BI (AS + BI). Em uma primeira instância, 5 animais pertencentes aos grupos NT, AS,AS + BI foram eutanasiados após 6 semanas de administração de emulsão hiperlipídica, antes da aplicação do modelo RH, para se confirmar o desenvolvimento da NAFLD. Foi possível observar que a administração de emulsão hiperlipídica durante 6 semanas foi suficiente para o desenvolvimento de esteatose hepática. Após as 6 semanas foi introduzido o modelo do RH concomitante à administração da emulsão hipercalipídica até o fim do experimento na 13a semana . Após 13 semanas o grupo AS apresentou maiores (p<0,05) valores de focos de inflamação, hepatócitos balonizados e grau de esteatose hepática em relação ao grupo AS+BI, assim como maiores (p<0,05) níveis séricos de triacilgliceróis, colesterol, HDL, LDL, VLDL, e maior (p<0,05) valor de MDA em relação ao grupo RH embora sem diferença estatística. O grupo AS também apresentou maior (p<0,05) incidência, número total e multiplicidade de nódulos, além de maior (p<0,05) número e tamanho de LPN persistentes (pLPN) e índice de proliferação quando comparado aos grupos RH e AS+BI. O grupo AS + BI, por sua vez, demonstrou menores (p<0,05) valores de escore de células ovais e menores valores de comprimentos de cometa e danos no DNA quando comparado ao grupo AS, embora sem diferença estatista para este último parâmetro. Em relação à expressão gênica, o grupo AS apresentou menores (p<0,05) valores de expressão do gene Hmgcr em relação ao grupo RH e maiores (p<0,05) valores dos genes Insig 1 e Thy 1 quando comparados ao grupo AS+BI. Portanto, no contexto de esteatose hepática associada ao modelo do RH, a administração de BI durante a etapa de iniciação/promoção em ratos Wistar resultou em atividade quimiopreventiva que se deu pela diminuição de pLNP, redução da proliferação celular e do número de células ovais, consideradas potenciais alvos para o desenvolvimento da hepatocarcinogênese, entretanto os genes analisados parecem não serem modulados pela BI. / Cancer is a major public health problem in the world. Among the primary neoplasms affecting the liver, hepatocellular carcinoma (HCC) is the most frequent. Several risk factors predispose to HCC, including the nonalcoholic fatty liver disease (NAFLD). According to previous studies of the group, the &#946-ionone (BI), has potential chemopreventive in hepatocarcinogenesis, promoting reduction of preneoplastic lesions (LPN). Thus we investigated whether NAFLD would increase the development of LPN in Wistar rats resistant hepatocyte model (RH) at the stage of initiation / promotion of hepatocarcinogenesis and if BI has chemopreventive effect in this context. Therefore, the animals were divided into 4 groups: non-treated group (NT), the group submitted to HR (HR), the group submitted to HR and NAFLD model, consisting of the fatty emulsion administration (AS) and AS group treated with BI (AS + BI). In a first point, 5 animals belonging to the groups NT, AS, AS + BI were euthanized after 6 weeks of administration of fat emulsion prior to application of the HR model, to confirm the development of NAFLD. It was observed that the administration of fatty emulsion for 6 weeks was sufficient to the development of hepatic steatosis. After 6 weeks it was introduced into the model HR the concomitant administration of fatty emulsion until the end of the experiment at 13 weeks. In the endpoint, the AS group had higher (p <0.05) of serum triacylglycerols, cholesterol, HDL, LDL, VLDL although no statistical difference inrelation to RH group, and increased (p <0.05) amount of MDA in relation to the group RH. The AS group also had higher (p <0.05) incidence, multiplicity and total number of nodes and greater (p <0.05) number and size of persistent LPN (pLPN) and proliferation index when compared to HR groups and AS + BI. AS + BI group. It was observed in AS+BI group lower (p <0.05) cell oval score values compared to AS group. In addition the AS+BI group showed lower values of the comet length and DNA damage compared to the AS group, although no statistical differences. In relation to gene expression, the AS group showed lower(p <0.05) HMGCR gene expression values in relation to HR group and higher (p <0.05) expression of Insig genes 1 and Thy 1 compared to group AS + BI.Therefore, in the context of hepatic steatosis associated with HR model BI for administration to the stage of initiation / promotion in rats resulted in chemopreventive activity was due to decrease in area of pLNP, reducing cell proliferation, and the number of oval cells, as potential targets for the development of hepatocarcinogenesis, however the genes do not seem to be modulated analyzed by BI.

A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Systemic therapies are the main treatment options for HCC patients with advanced disease (&sim; 80% of all cases). However, only very moderate clinical responses are achieved with most of the conventional therapies. Thus, more effective therapeutic strategies are much needed. The PI3K/Akt/mTOR signaling pathway, which plays a critical role in controlling cell proliferation and survival, is aberrantly activated in &sim; 45% HCC, suggesting it to be a potential target for HCC treatment. Moreover, emerging evidences indicate that activation of the PI3K/Akt/mTOR pathway may be associated with resistance to many cytotoxic chemotherapies, including microtubule targeting agents. In this study, by gene expression profiling and gene ontology analysis, "microtubule-related cellular assembly" was identified to be the major biological/functional process involved in HCC development, suggesting that microtubule is also an important therapeutic target for HCC. With these understandings, it is hypothesize in this thesis that combined targeting of a key component ofthe PI3K/Akt/mTOR pathway, namely the mammalian target of rapamycin (mTOR) and the microtubule would be an effective therapeutic strategy for HCC. The objectives of the thesis are to examine the therapeutic potential of microtubule targeting, mTOR targeting, and combined targeting of the microtubule and mTOR in both in vitro and in vivo models of HCC. / In summary, the PI3K/Akt/mTOR pathway and the microtubule represent promising therapeutic targets for HCC treatment. The findings from this thesis offer a rationale for combining mTOR inhibitors with microtubule targeting agents for effective HCC treatment. / In the second part, the effect of mTOR inhibition, either alone or in combination with an additional microtubule targeting agent (vinblastine) was investigated in HCC. Temsirolimus, an mTOR inhibitor, suppressed HCC cell proliferation in as early as 24 hrs with an IC50 of 1.27+/-0.06muM (Huh7), 8.77+/-0.76muM (HepG2), and 52.95+/-17.14muM (Hep3B). Vinblastine (1nM) alone caused 30--50% growth inhibition in 3 HCC cell lines. In these HCC cell lines, it was found that temsirolimus/vinblastine combination resulted in an additive to synergistic effect (when compared to single agents alone) with maximum growth inhibition of 80--90% as early as 24 hrs upon treatment. This marked growth inhibition was accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage (a hallmark for apoptosis). Moreover, the combination specifically caused concerted down-regulation of several important anti-apoptotic and survival proteins (survivin, Bcl-2 and Mcl-1), which was not observed in single agent treatments. It was hypothesized that inhibition of these key anti-apoptotic/survival proteins may represent a novel mechanistic action of this highly effective combination approach of dual targeting of mTOR and microtubule by temsirolimus/vinblastine in HCC cells. Indeed, transient over-expression of each of these genes (survivin, Bcl-2 or Mcl-1) in HCC cells did partially rescue the growth inhibitory effect of the temsirolimus/vinblastine combination. More importantly, this novel combination significantly suppressed the growth of HCC xenografts in nude mice (when compared with single agents alone). / In the third part, the anti-tumor effect of another mTOR inhibitor everolimus in combination with microtubule targeting agents, vinblastine and patupilone (a microtubule-stabilizing agent), was investigated in HCC cells. Everolimus/vinblastine combination resulted in an additive to synergistic effect accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage. The combination also caused concerted down-regulation of anti-apoptotic and survival proteins (survivin, Bel-2 and Mel-1) as observed with the temsirolimus/vinblastine combination. However, everolimus only moderately enhanced the sensitivity of patupilone for reasons unknown. / Taxanes are the major chemotherapeutic agents that target the microtubule. In the first part of the thesis, the anti-tumor activity of two taxanes, paclitaxel and docetaxel (which are known to stabilize microtubules) was examined and compared with doxorubicin (a DNA intercalating agent). Across all three HCC cell lines tested, it was found that the microtubule targeting agents, taxanes, were more efficacious than doxorubicin. This supports the initial finding that microtubule assembly process is functionally important in HCC. Recent studies demonstrated that using nanoparticles for drug delivery can greatly enhance therapeutic efficacy and reduce side-effects. Therefore, the nanoparticle albumin-bound (nab)-paclitaxel was employed to further evaluate the therapeutic efficacy of such a delivery strategy in HCC models. In all three HCC cell lines tested, nab-paclitaxel was found to be the most effective agent, with an average IC50 value of 0.16--10.42nM, when compared to non-conjugated taxanes (paclitaxel, docetaxel) and doxorubicin. In vitro analysis showed that nab-paclitaxel was able to induce cell cycle arrest at G2/M phase and apoptosis in HCC cells. In vivo study demonstrated that nab-paclitaxel readily inhibited the growth of HCC xenografts with lower toxicity when compared to paclitaxel, docetaxel and doxorubicin. Moreover, specific silencing of a key regulatory protein for microtubule dynamics, Stathmin 1, by siRNA significantly enhanced the effect of nab-paclitaxel in HCC cells, resulting in synergistic growth inhibition in vitro. / Zhou, Qian. / Advisers: Winnie Yeo; Vivian Lui; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / 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.

Zhang, Zhiyi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves ). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Hepatocellular carcmoma (HCC) is the third most common cause of cancer-related deaths worldwide, with a gender prevalence observed in men. Recent studies have suggested that elevated activity of the androgen axis is one major host factor underlying this disparity between genders. The androgen receptor (AR) mediates function of androgen in vital developmental and oncogenic pathways by binding to genomic androgen response elements, which influence the transcription of downstream target genes. AR is overexpressed in 60-80% of human HCCs. Genetic studies further established the pivotal role ofAR in hepatocarcinogenesis, where liver-specific knockout of AR significantly reduced tumorigenicity in carcinogen- and HBV-induced HCC mouse models. However, AR-inducedhepatocarcinogenesis is far from fully understood, in part because little is known about the identity and role of direct AR-dependent targeted genes in hepatocytes. / In this study, we used genome-wide location and functional analyses to identify a critical mediator of AR signaling, cell cycle-related kinase (CCRK), in driving beta-cateninl T-cell factor (TCF)-dependent hepatocarcinogenesis. Using chromatin immunoprecipitation followed by promoter array analysis of AR-overexpressing HCC cell lines, we found a number of cell cycle-related genes that are likely under the direct modulation of AR. Cell cycle-related kinase (CCRK), previously shown to promote glioblastoma tumorigenesis, was found to be the most significantly-bound AR target ( p&lt;0.0001). CCRK was directly up-regulated by ligand-activated AR through promoter binding and required for AR-induced G1-S cell cycle progression because (1) CCRK overexpression attenuated cell cycle blockage by AR knockdown and (2) CCRK inhibition counteracted AR-mediated cell cycle progression. Ectopic CCRK expression induced immortalized liver cell proliferation, malignant transformation and tumor formation in immunodeficient mice, whereas CCRK inhibition decreased HCC cell growth in vitro and in vivo. These functional assays demonstrated that CCRK is a potential oncogene in HCC. Mechanistically, CCRK activated beta-catenin/TCF-dependent transcription through phosphorylation of glycogen synthase kinase-3beta and induced the expressions of beta-catenin target genes, cyclin D1 (CCND1) and epidermal growth factor receptor (EGFR). Inhibition of beta-catenin/TCF signaling attenuated CCRK-induced cell cycle progression, colony formation and tumorigenicity. Conversely, HCC cell growth inhibition by CCRK knockdown was rescued by constitutively active beta-catenin or TCF. In agreement with these findings, activation of the AR/CCRK/beta-catenin axis was frequently observed in primary HCCs. More importantly, CCRK over-expression was correlated with tumor staging and poor overall survival in a cohort ofhuman HCC tissues. / Together, our data reveal a new cascade for AR function in hepatocarcinogenesis via the activation of beta-catenin/TCF signaling. This study also reveals that CCRK is a novel focal link between two prominent signaling pathways vital for HCC growth and thus represents a new therapeutic target for HCC treatment. / Feng, Hai. / Adviser: Sung Jao Yiu. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 161-177). / 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.

Amplification of chr.1 q21-22 is also an aberration frequently detected in HCC. Copy number gains of the GEF-H1 gene ranked the most frequent event from array-CGH. GEF-H1 up-regulation was significant correlated in patients with advanced HCC staging (P = 0.048), presence of micro-vascular invasion (P = 0.049) and shorter overall and disease free survival of patients (P &lt; 0.03). Similar to BOP1, functional examinations of GEF-H1 suggested profound inhibitory effects on cell motility ( P &lt; 0.035) and invasiveness (P &lt; 0.003) in cell lines studied. Upon GEF-H1 depletion, re-expression of epithelial markers (E-cadherin, cytokeratin 18, alpha-catenin and gamma-catenin) and down-regulations of mesenchymal markers (N-cadherin, fibronectin and vimentin) were also readily observed. In addition, reduced active form of GTP-RhoA together with its downstream effectors including cleaved ROCK 1 and phosphorylated MLC2 were also found in GEF-H1 depleted cells. / Array-CGH also defined candidate proto-oncogenes within 2 causal amplified regions in HCC, chr.8q24 and chr.1q21-q22. In resolving affected genes at chr.8q24, distinctive gains of BOP1 was further established in primary HCC tumors, where frequent BOP1 up-regulations in tumors compared to adjacent non-tumoral liver (P &lt; 0.0001) was identified. Increased BOP1 expression correlated with advanced HCC staging (P = 0.004), micro-vascular invasion (P = 0.006) and shorter overall and disease free survival of patients (P &lt; 0.02). siRNA-mediated suppression of BOP1 in HCC cell lines showed significant inhibition on cell invasion (P &lt; 0.003) and migration (P &lt; 0.05), whereas overexpression of BOP1 in immortalized hepatocyte cell line, L02, showed increase cellular invasiveness and cell migratory rate (P &lt; 0.0001). Evident regression of the Epithelial-to-Mesenchymal Transition (EMT) phenotype was readily identified in BOP1 knockdown cells, where re-expressions of epithelial markers (E-cadherin, cytokeratin 18 and gamma-catenin) and down-regulation of mesenchymal markers (fibronectin and vimentin) were found. It was found that BOP1 likely stimulates actin stress fibers assembly through RhoA activation. / Hepatocellular carcinoma (HCC) is a highly malignant tumor that is associated with a high incidence of cancer morbidity and mortality. Elucidation of genomic aberrations of HCC holds much importance in understanding the molecular basis that underlies the disease causation and progression. Extensive research on HCC has by now revealed a number of key genomic aberrations but, for most of these loci, the underlying cancer-related gene(s) remains unknown. / In this thesis, array-based comparative genomic hybridization (array-CGH) was deployed to define target genes within HCC-associated chromosomal regions. The first part of my study focused on mapping the homozygous deletions (HDs) in HCC. Though infrequent, HD screening has been widely utilized to define tumor suppressor genes (TSGs) in cancers. A panel of HCC cell lines was systematically examined for the presence of HDs. Array-CGH identified 6 HD regions, amongst which CRYL1 (located on chr.13q12.11) displayed most common down-regulations in primary HCC tumors. Significant associations could also be drawn between repressed CRYL1 and advanced tumor staging, increased tumor size and shorter disease-free patient survival (P &le; 0.037). Moreover, HD on CRYL1 could be detected in 36% of HCC cases with CRYL1 down-regulations. Examination of other inactivating mechanisms suggested histone deacetylation and promoter hypermethylation to be likely inactivating events as well. Re-expression of CRYL1 in SK-HEP1 cell line induced profound inhibition on cellular proliferation and cell growth (P &le; 0.002). By Annexin V staining, CRYL1 restoration readily increased pro-apoptotic cells with an induction of P ARP cleavage. Flow cytometry further revealed CRYL1 could prolong the G2-M phase, possibly through interrupting the Cdc2/cyclin B path. / The similarities in functional behaviours of BOP1 and GEF-H1 might have implications in the fundamental biology of HCC tumorigenesis. It is known that HCC is a highly aggressive tumor often associated with intra- and extra-hepatic metastasis. The finding of 2 causal changes to be closely associated with cell migration and invasiveness may have implications in the metastatic potentials of HCC cells being predisposed earlier on from genomic events. / Cheng, Kit Chong Ibis. / Adviser: Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 177-190). / 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, Chor Fung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chan, Ho Ching. / Thesis M.Phil. Chinese University of Hong Kong 2016. / Includes bibliographical references (leaves 159-166). / Abstracts also in Chinese. / Title from PDF title page (viewed on 15, September, 2016).

Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243