Chiu Hoi Shan Clarissa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 110-126). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vi / List of Figures --- p.viii / Introduction and Objectives / Hepatocellular Carcinoma / Epidemiology --- p.1 / Sex and Age --- p.1 / Geographic distibution --- p.2 / Risk factors of HCC --- p.2 / Mortality from liver cancer --- p.4 / Treatment for HCC --- p.4 / Tumor markers --- p.5 / Alpha-fetoprotein / Structure --- p.5 / Physiological Functions of AFP --- p.7 / Re-expression of AFP in Adult --- p.7 / Re-expression of AFP in HCC --- p.8 / Isoforms of AFP --- p.8 / Specific AFP isoform in HCC --- p.9 / Sialic Acid --- p.11 / Sialyltransferase / "Galβ 1,4GlcnAc α2,6-sialyitransferase" --- p.12 / "Characterization of ST2,6Gal I" --- p.12 / "Expression of ST2,6Gal I" --- p.12 / "General features of ST2,6Gal I Activity" --- p.15 / Relationship Between AFP isoforms and ST2，6Gal in Fetal Mouse Model --- p.15 / "Change in ST2,6GaI I Activity in Transgenic Mouse Models of HCC" --- p.16 / "Study of Activity of ST2,6Gal I in Colon Carcinoma" --- p.16 / Objective of the Project --- p.18 / Chapter Chapter 1 --- Formation of Monosialyated AFP by Hepatoma Cells / Chapter 1.1 --- Introduction --- p.21 / Chapter 1.2 --- Materials and Methods --- p.23 / Chapter 1.3 --- Results / Chapter 1.3.1 --- AFP obtained from cell culture --- p.34 / Chapter 1.3.2 --- IEF for AFP in cell culture medium --- p.34 / Chapter 1.3.3 --- SDS-PAGE analysis of AFP --- p.34 / Chapter 1.3.4 --- Stability of AFP isoforms after secreted to cell culture medium --- p.39 / Chapter 1.3.5 --- Comparison of the AFP isoforms between liver tissues and serum --- p.38 / Chapter 1.4 --- Discussion / Chapter 1.4.1 --- Origin of the extracellular msAFP - in vitro Model --- p.43 / Chapter 1.4.2 --- Origin of circulating msAFP - in vivo Model --- p.44 / Chapter 1.5 --- Conclusion --- p.46 / Chapter Chapter 2 --- "Presence of msAFP in the serum of HCC patient is a Consequence of Decrease in the Activity of ST2, / Chapter 2.1 --- Introduction --- p.47 / Chapter 2.2 --- Materials and Methods --- p.49 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Semi-quantitation of msAFP --- p.61 / Chapter 2.3.2 --- Evaluation of the ST2，6Gal I Assay --- p.65 / Chapter 2.3.3 --- "Measurements and comparisons of the activity of ST2,6Gal I in non-tumor and tumor tissue" --- p.65 / Chapter 2.3.4 --- "Evaluation of the RT-PCR ELISA for semi-quantitation and comparisons of ST2,6Gal I mRNA levels" --- p.75 / Chapter 2.3.5 --- "Semi-quantitation and comparisons of ST2,6Gal I mRNA levels in the non-tumor and tumor tissues" --- p.84 / Chapter 2.3.6 --- Correlations between the markers --- p.87 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- "Overproduction of AFP, a possible cause for increased msAFP formation" --- p.99 / Chapter 2.4.2 --- "Decrease of ST2,Gal I activity, a possible cause for increased msAFP formation" --- p.100 / Chapter 2.4.3 --- "ST2,6Gal I activity in tumor is not regulated at transcriptional level" --- p.102 / General Discussion / Origin of blood stream msAFP --- p.103 / Physiological Mechanism for the formation of msAFP in HCC --- p.104 / Regulation of ST2，6Gal I activity in HCC --- p.105 / "Comparison between the ST2,6GaI I activities in human HCC and Colon Cancer" --- p.107 / Conclusion and Future studies / Conclusion --- p.108 / Future studies --- p.109 / References --- p.110

Sze, Siu Ching. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 109-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

He, Mian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 165-188). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

Magnetic resonance imaging is utilized as an important tool in radiation oncology for delineation of healthy and cancerous tissues, and evaluating the functionality of those tissues, structures, and organs. Currently, the clinical imaging protocol at Virginia Commonwealth University includes anatomical imaging for tissue and structure delineation, and to observe treatment induced changes. Diffusion weighted imaging (DWI) is also acquired for calculation of apparent diffusion coefficient (ADC) values to provide quantitative information on tissue diffusivity and microstructure. However, anatomical images and ADC values may not display the true extent of changes in tissue. This work seeks to further utilize the capabilities of MRI and expand its role in treatment response monitoring for liver cancer patients treated with stereotactic body radiotherapy (SBRT). To do so, an imaging protocol and image analysis methodology to evaluate treatment changes on pre- and post-treatment image sets was developed. An extension of DWI, termed intravoxel incoherent motion (IVIM) imaging, was utilized to quantitatively assess levels of perfusion and diffusion within the liver and tumor. Acquisition of high-quality diffusion weighted images of the liver necessitated the development of an MR safe respiratory motion management device, which was designed, constructed and evaluated in this work. An imaging protocol was developed providing anatomical and functional images of the liver, acquired under breath hold, utilizing the respiratory motion management device. An IVIM parameter calculation and texture analysis workflow was developed using MATLAB, and applied to acquired data sets from multiple studies, including past clinical cases, investigator, healthy volunteer, and liver cancer patient . Differences in IVIM and texture analysis parameters were investigated for healthy and diseased tissue, and for select dose regions from pre- and post-treatment imaging sessions. Significant differences, at a voxel level, were found between healthy and diseased tissue, and pre- and post-treatment volumes, for multiple parameters, including apparent diffusion coefficient, pure diffusion, and perfusion, as well as for various texture features. Overall, this study showed the potential of IVIM and texture analysis to be used for discriminating between healthy and diseased tissues in the liver, and for indication of treatment response.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, especially in Asia and Africa countries. The distribution pattern shows geographical variation and pathogesis in multifactors as environment, infection, nutrition, metabolism, and endocrine contribute to hepatocarcinogenesis. Alpha fetal protein (AFP), the major tumor marker used at present accounts only 50% HCC diagnostic efficiency. This study aims to identify potential tumor markers or suppressor genes for further application in early HCC diagnoses and treatments. Therefore we utilized available cDNA microarray databases in conjunction with other bioinformatic resources to identify our candidate genes related to HCCs. cDNA microarray technology and bioinformatic resources which enable investigators to obtain comprehensive data with respect to gene-expression profiles, is progressing rapidly. The Okabe¡¦s and Stanford¡¦s HCC database were our major data-mining material. A total of 85 potential tumor markers and 106 potential tumor suppressors were found via preliminary in silico datamining. We furthermore narrowed down to 14 candidate tumor markers and 7 candidate tumor suppressor genes by the way of quantitative RT-PCR technologies were applied in various HCC cancer cell lines and 21 patient¡¦s in pair, tumor/non-tumor tissues to confirm gene expression profile. The results revealed that 6 genes (PRO2000, PYGB, STMN1, AFM, C8FW, NNMT) conformed to our data-mining studies.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Hepatocarcinogenesis is considered a multifactorial and mulitstep process that involves the activation of oncogenes or the inactivation of tumor suppressor genes. Tumor suppressor gene PTEN (also known as MMAC or TEP1) is located on human chromosome 10q23. The 403¡Vamino acid PTEN protein encodes dual specificity protein phosphatases. Mutation of the PTEN is a common event in advanced stage of diverse human cancers. In our previous studies, immunohistochemical analysis indicated that reduced PTEN expression was found in nearly 40% of HCC specimens. Furthermore, restored PTEN expression by adenovirus gene delivery effectively inhibited the in vitro and in vivo tumorigenicity of Mahlavu cells, a human HCC cell line with PTEN inactivation. In the present study, we further characterize whether PTEN gene delivery still suppressed the oncogenic potential in HCC cell lines with functional PTEN. By expression and sequencing analysis, we identified human SK-Hep-1 cells as the hepatoma cell line with functional PTEN expression. The optimal condition for adenovirus vector to infect SK-Hep-1 cells was determined at the multiplicity of infection (MOI) of 50-100. Tough SK-Hep-1 cells were effectively transduced with exogenous PTEN gene, the enhanced PTEN expression by adenovirus gene delivery did not alter the phosphoryation extent of Akt in SK-Hep1 cells. Nevertheless, PTEN gene delivery reduced the proliferation of SK-Hep-1 cells by ~20%. In addition, the motility of PTEN-transduced SK-Hep-1 cells significantly decreased comparing to cells of control groups. Western blot analysis suggested the decreased cell motility might be attributed to the reduced phosphorylation of focal adhesion kinase (FAK) by PTEN gene delivery. Above all, PTEN gene delivery profoundly reduced the colony formation of SK-Hep-1 cells in soft-agar. However, PTEN gene delivery did not affect the secretion of matrix metallo-proteinases (MMPs) release. Animal studies will be carried out in the future to validate the present in vitro findings. In summary, PTEN gene delivery holds promise for treatment of HCC even when the hepatoma cells possess functional PTEN gene.

PURPOSE: HCC is a complicated disease with high mortality rates and limited treatment options. No universal clinical or molecular classification established to inform better treatment options. There has been very limited success in determining a molecular profile that represent valid drivers in HCC patients and thus no targeted agents have obtained marketing approval. However, emerging data suggest the FGF19-pathway as a HCC driver and a potential therapeutic target. This research study aims to investigate whether the HCC prognostic risk factor, serum AFP, is predictive of FGF19 protein expression as assessed by immunohistochemistry in advanced HCC patients. METHODS: A cross-sectional analysis was performed from baseline data collected in a Phase 1 study conducted at various centers across the US, EU, and Asia. Only advanced HCC patients with adequate liver function were eligible for enrollment. Demographic data, detailed history of HCC, and any prior treatments or surgeries were recorded. Baseline laboratory values and prognostic factors including performance status (ECOG), lab values (i.e. bilirubin, albumin), and the number, size and biomarker status of the tumor(s) were collected. Differences between groups were assessed by t test, or Chi-square test, as appropriate. Multivariate logistic stepwise regression analyses were performed including all parameters with highly significant correlations in the multivariate analysis. RESULTS: Only AFP, metastatic disease, and prior surgery met the criteria to be incorporated into the final model. Results indicated that high AFP had a statistically significant (p-value = .01) positive association (Wald chi-square statistic = 6.601) with positive FGF19 IHC status. The odds ratio for being FGF19 IHC+ was 12.216 among the high AFP subjects as compared to low AFP subjects, and also statistically significant but had a very wide 95% confidence interval (1.811, 82.79). CONCLUSIONS: The results indicated that HCC patients with high serum AFP levels have a twelve fold higher chance of having a positive FGF19 IHC status than those with low AFP levels. Further studies are warranted in order to replicate the data in a larger sample size to understand future clinical implications once treatment options become available for FGF19 IHC positive patients.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.

Orientador: Cecilia Amelia Fazzio Escanhoela / Dissertação (mestrado) - Universidade Estadual de Campínas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T12:26:43Z (GMT). No. of bitstreams: 1 Segatelli_Vanderlei_M.pdf: 1173808 bytes, checksum: efbd82ac6962165e42a166dfef483327 (MD5) Previous issue date: 2010 / Resumo: O carcinoma hepatocelular (CHC) destaca-se como uma das formas mais comuns de câncer no mundo, correspondendo a aproximadamente 90% das neoplasias primárias do fígado, sendo a cirrose seu mais importante fator de risco. A sua progressão, no entanto, depende de inúmeros fatores e, entre estes, a angiogênese assume papel fundamental, principalmente por ser o CHC um tumor altamente vascularizado. É sabido que tumores sólidos, ao atingirem volume de 1 a 2mm3, passam a ter seu crescimento dependente de neovascularização e no fígado esta se faz através de capilarização sinusoidal, processo pelo qual ocorrem alterações morfológicas nos sinusóides hepáticos, com perda das fenestrações e deposição de membrana basal. Neste estudo retrospectivo avaliamos a angiogênese, através dos marcadores imuno-histoquímicos CD34 e CD105 (Endoglina), com determinação da densidade microvascular (DMV) em 44 produtos de hepatectomia total, compreendendo 44 nódulos neoplásicos (CHC), 44 nódulos regenerativos (NR) e 15 nódulos com características microscópicas de lesões pré-malignas (ou Nódulos Displásicos - ND), sendo 8 de baixo grau e 7 de alto grau. A avaliação compreendeu a determinação da DMV no centro e na periferia de todos os nódulos. Para análise estatística foi utilizada análise descritiva com apresentação de medidas de posição e dispersão para variáveis contínuas; para a comparação dos grupos foi utilizada a ANOVA, considerando-se p<0,05 estatisticamente significante. O CD34 demonstrou positividade difusa no CHC, com valores médios da DMV de 38,3±12,6 e 34,2±11,2 no centro e periferia, respectivamente. Já o CD105 apresentou expressão sinusoidal predominantemente periférica no CHC, com valores médios da DMV de 6,2±4,1 e 10,7±4,4, com diferenças significativas entre os grupos (p<0,0001). Também foram observadas diferenças na imunoexpressão entre os marcadores em NR (p<0,0001) e ND (p<0,0019). Concluímos que existem diferenças na imunoexpressão do CD34 e CD105 entre os nódulos benignos e malignos, com gradual aumento da neovascularização, bem caracterizado pelo CD34, devido ao processo de capilarização sinusoidal. O CD105, expresso em "subset" de microvasos neoformados, pode ser um indicador de diferenciação diagnóstica entre NR e CHC / Abstract: Hepatocellular carcinoma (HCC) is one of the most common forms of cancer worldwide, accounting for about 90% of primary tumors of the liver. Cirrhosis is the most important risk factor for HCC. However, the progression of hepatocellular carcinoma is dependent on a number of factors. Among these factors, angiogenesis plays a major role, mainly because HCC is a highly vascular tumor. It is known that when solid tumors reach a volume of 1 to 2mm3 their growth depends on neovascularization. In the liver, this occurs through sinusoidal capillarization, a process by which morphologic alterations occur in the hepatic sinusoids, with loss of fenestrations and basement membrane depositions. In this retrospective study, we evaluated angiogenesis by using immunohistochemical markers CD34 and CD105 (Endoglin), with determination of tumor microvessel density (MVD) in 44 products of total hepatectomy, including 44 malignant nodules (HCC), 44 regenerative nodules (RN) and 15 nodules with microscopic characteristics of preneoplastic lesions (or Dysplastic Nodules-DN), 8 being low-grade and 7 high-grade. The evaluation encompassed measurement of MVD in the center and periphery of all nodules. For statistical analysis, a descriptive analysis with measurements of position and dispersion for continuous variables was used. ANOVA was used for group comparison, considering p<0.05 as statistically significant. CD34 demonstrated a diffuse positivity in HCC, with median MVD values of 38.3±12.6 and 34.2±11.2 in the center and periphery, respectively. On the other hand, CD105 exhibited sinusoidal expression predominantly in the periphery of the HCC, with median MVD values of 6.2±4.1 and 10.7±4.4, with significant differences between groups (p<0.0001). Differences in the immunoexpression between markers in RN (p<0.0001) and DN (p<0.0019) were also observed. In conclusion, there are differences in the immunoexpression of CD34 and CD105 between benign and malignant nodules, with a gradual increase in neovessel formation, well-characterized by CD34, due to a process of sinusoidal capillarization. CD105, expressed in a "subset" of newly formed microvessels may be an indicator of the diagnostic differentiation between RN and HCC / Mestrado / Anatomia Patologica / Mestre em Ciências Médicas

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