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ABSTRACT Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Taiwan. The development of hepatocellular carcinoma is a multi-step process associated with alterations in genes expression such as activation of oncogenes and inactivation of tumor suppressor genes. Mutation/deletion of tumor suppressor gene p53 occurs in 40-50% HCC. Moreover, patients with p53 inactivation have significantly shorter survival after surgery. Inactivation of p53 leads to chromosome instability and may alter expression of its downstream target genes including 14-3-3s for cell cycle arrest or PUMA for apoptosis induction. In this thesis study, we employed five human hepatoma cell lines and ten surgical HCC samples containing paired normal and tumor tissues to investigate 14-3-3s and PUMA expression during liver carcinogenesis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that 14-3-3s mRNA expression was detected in well and poorly differentiated hepatoma cells except Mahlavu cells. Western blot analysis further validated such finding that 14-3-3s protein is not detectable in Mahlavu cell. In human surgical HCC tissues, qRT-PCR showed that 14-3-3s mRNA was elevated in 90% of HCC tissues. Western blot analysis indicated that 14-3-3s protein level was increased in 60% of HCC tissues. Finally, immunohistochemical analysis revealed that 14-3-3s was up-regulated in 50% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that 14-3-3s expression was up-regulated in HCC. qRT-PCR and western blot analysis indicated that PUMA mRNA and protein levels were decreased in human and rat hepatoma cells. In human surgical HCC tissues, qRT-PCR showed that PUMA mRNA was reduced in 60% of HCC tissues. Western blot analysis indicated that PUMA protein level was decreased in 100 of HCC tissues. Finally, immunohistochemical analysis revealed that PUMA was down-regulated in 70% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that PUMA expression was down-regulated in HCC. In the future, large-scale analysis using more HCC samples will be required to delineate the correlation of 14-3-3s/PUMA expression with clinical parameters of HCC.

The association between extent of chronic cannabis use (CCU-extent) and cognitive impairment among adolescents has been the subject of controversial debate. Linking DNA methylation to CCU-extent could help to understand cannabis associated changes in cognitive performance. We analyzed cognitive task performances, CpG methylation in peripheral whole-blood samples and self-reported past-year CCU-extent of n = 18 adolescents (n = 9 psychiatric outpatients with chronic cannabis use (CCU), n = 9 without) who were matched for age, gender and psychiatric disorders. Patients with CCU were at least 24 h abstinent when cognitive tasks were performed. A Principal Component Analysis (PCA) was carried out to identify group differences in whole genome DNA methylation. Mediation analyses were performed between CCU-extent associated CpG sites and CCU-extent associated variables of cognitive tasks. PCA results indicated large differences in whole genome DNA methylation levels between the groups that did not reach statistical significance. Six CpG sites revealed reduced methylation associated with CCU-extent. Furthermore, CCU-extent was associated with lower scores in verbal learning. All six CpG sites mediated the effects between CCU-extent and verbal learning free recall. Our results indicate that CCU is associated with certain patterns in the methylome. Furthermore, CCU-extent associated impairments in memory function are mediated via differential methylation of the six CCU-associated CpG sits. Six identified CpG are located in genes previously described in the context of neurodegeneration, hippocampus-dependent learning and neurogenesis. However, these results have to be carefully interpreted due to a small sample size. Replication studies are warranted.