Thesis (MScEng)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Hybrid concrete construction (HCC) is a construction technique that makes use of both in-situ and pre-fabricated concrete products by making optimum use of the advantages of both these methods in the same project. Although the advantages of this building method is well recognised throughout the world and a feasibility study illustrated that HCC is a suitable building method in South Africa, it remains under-utilised in the country. HCC encourages early involvement from the project team and provides clients with the best value projects and a "win-win" situation for the project team during the project development. The objective of this study is to identify barriers that prohibit an increased utilisation of HCC for the South African construction industry. Additionally, it provides possible solutions to overcome these barriers. These proposed solutions are similar to the programmes and methods that are implemented in other countries to improve their HCC utilisation. Furthermore, drivers are identified in this study to implement these solutions in the industry and a proposed "change model" is developed for private companies to assist with the change process. This study is unique because current South African conditions are used to establish the barriers and solutions. Furthermore, it takes into account that not all countries have such progressive construction industries as the European markets and therefore it considers solutions and drivers that are suitable for less advanced industries such as South Africa. The model that is proposed is applicable to any industry regardless of its nature or country. Although the United Kingdom is not the widest user of pre-fabricated concrete products, their HCC research is extensive, making it a suitable comparative country for the South African construction industry. During the investigation, the procurement methods in South Africa were found to be traditional and do not allow for early involvement or partnering in the project team. To obtain early involvement, it is critical to initiate methods such as design-and-build, contract management or public-private-partnerships during the procurement phase. Decision and design assistance from the whole project team and software tools are essential when HCC is considered. Because the South African construction industry does not provide such support it is advised here to incorporate contractors from an early stage and to develop software tools that can assist during the decision making and design process of HCC structures. The industry does not provide the project team with sufficient training programmes that increase knowledge of HCC. Training programmes that are cognitive stimulating must be developed for the industry. Furthermore, a database is required to illustrate the utilisation of different pre-fabricated products and in different projects within South Africa. Labour-intensive construction regulations and green building techniques (not compulsory) are promoted in South Africa. Labour-intensive programmes should consider the negative impact it has on the labourers and consider HCC as a building technique because the pre-fabricated elements are manufactured in a controlled and safe environment. It is advised that the toolkits that are used for green building accreditation, must be revised to accommodate the additional benefits such as less waste generation, that HCC offers to a project. Regardless of the country in which HCC is utilised, the environment must be acceptable for the innovation. By analysing the criteria that influences the use of HCC in a country and adding weighting functions to these criteria, the acceptability towards the innovation can be obtained. Furthermore, public clients and companies are recognised here as drivers of change. While public clients can commit to HCC for the cost, labour-intensive factories, green building methods, improved health and safety, training for labourers, private companies can utilise HCC to provide best value to a project. A model is proposed here for innovative companies that will allow them to change not only the company, but the industry towards a HCC "mindset". / AFRIKAANSE OPSOMMING: Hibriede beton konstruksie (HBK) is ’n tegniek waarby beide voorafvervaardigde betonelemente en in-situ beton in dieselfde projek gebruik word ten einde voordeel uit beide metodes te trek. Alhoewel die voordele van die konstruksietegniek wel bekend is in talle lande van die wêreld en ’n uitvoerbaarheidsanalise getoon het dat HBK ’n voordelige konstruksiemetode vir Suid-Afrika is, is dit tans onderbenut in die plaaslike sektor. HBK bied vir kliënte projekte van goeie waarde en omdat dit vroeë betrokkenheid verg vanaf die projekspan, resulteer dit ’n "wen-wen" situasie tydens die projekontwikkeling. Die doel van hierdie studie is om die hindernisse te ondersoek wat veroorsaak dat HBK nie meer gereeld in Suid-Afrika gebruik word nie. Ten einde die effekte hiervan te verminder, word moontlike oplossings vir hierdie hindernisse voorgestel. Die oplossings is gebaseer op tegnieke en metodes wat geïmplimenteer word in ander lande waar HBK gebruik word. Rolspelers wat hierdie verandering na HBK kan dryf word geïdentifiseer en ’n model om verandering binne ’n maatskappy te lei word voorgestel om die rolspelers te ondersteun tydens die proses. Hierdie studie is uniek omdat dit inligting gebruik van die huidige situasie van die Suid-Afrikaanse konstruksie-industrie om die hindernisse en oplossings te identifiseer. Omdat nie alle konstruksie-industrieë so vooruitstrewend is soos in Europese lande nie, is die oplossings en rolspelers relevant vir enige land wat ’n industrie het wat soortgelyk is aan Suid-Afrika. Die model wat voorgestel word is ook relevant tot enige industrie afgesien van die land waar nuwe tegnieke geïmplimenteer word. Alhoewel dit bevind is dat die Verenigde Koningkryk nie die grootste gebruikers van HBK is nie, is hulle navorsing in die veld omvattend. Daarom word die Verenigde Koningkryk beskou as ’n voorbeeld om Suid-Afrika se konstruksie-industrie mee te vergelyk, asook om inligting van HBK te verkry. Tydens die studie is bevind dat die projekaanstellingsmetodes van projekte in Suid-Afrika tradisioneel is, aangesien dit nie toelaat vir vroeë betrokkenheid of vennootskappe binne die projekspan nie. Om vroeë betrokkenheid te verkry, is dit noodsaaklik om van ontwerp-en-bou, kontrakbestuur of publiek-privaat-vernootskappe gebruik te maak. Tans is daar geen hulp wat aan ontwerpers verleen word ten opsigte van besluitnemingsmodelle en ontwerpsagteware wanneer HBK oorweeg word in Suid-Afrika nie. Die behoefte aan sulke bystand dui daarop dat sagtewarepakkette ontwerp moet word wat toepaslik is vir Suid-Afrika se konstruksie-industrie. Die industrie bied nie doelgerigte en genoegsame opleidingsprogramme aan om die industrie se kennis van HBK te verbreed nie. Addisionele opleiding wat kognitiewe stimulasie sal bied vir die hele projekspan word aanbeveel. ’n Databasis wat HBK projekte insluit, moet geskep word om die industrie bloot te stel aan die verskillende moonlikhede. Arbeid-intensiewe konstruksie-regulasies asook groen konstruksietegnieke (nie wetgedrewe nie) word sterk bevorder in Suid-Afrika. Dit word hier aanbeveel, dat voorafvervaardiging se addisionele voordele in ag geneem word wanneer hierdie regulasies en tegnieke relevant is. Hierdie voordele sluit onder meer in: die veiligheid wat aan werkers gebied word in arbeid-intensiewe fabrieke en die vermindering van afval in HBK projekte. Afgesien van die land waarin HBK gebruik word, moet die omgewing ontvanklik vir die tegniek wees. Deur verskillende kriteria te analiseer wat die gebruik van HBK beïnvloed binne ’n land en geweegde faktore daaraan te koppel, kan die ontvanklikheid van die innovasie bepaal word. Laastens word publieke kliënte en maatskappye as die drywers (of katalisators) wat verandering kan meebring, beskou. HKB bied onder andere die volgende voordele aan publieke kliënte: koste-effektiwiteit, arbeid-intensiewe werk, groen konstruksie, beter gesondheid, veiligheid en opleiding vir werkers. Maatskappye kan gebruik maak van HBK om waarde tot hul projekte toe te voeg. ’n Model word hier voorgestel om innoverende maatskappye in staat te stel om nie net hulself nie, maar ook die industrie as ’n geheel te help om ’n gesindheidsverandering jeens HBK te skep.

Thesis (MScEng (Civil Engineering))--Stellenbosch University, 2008. / Introduction South Africa is currently experiencing a significant increase in infrastructure investment. Forecasts by BMI-BRSCU have shown that the building and construction industry is expected to grow considerably to 2010, before languishing slightly to 2015. This growth will be driven in particular by investment in non-residential building (41% growth) and construction (73% growth) activities. Even beyond 2015 however, the demand will still be high on the construction industry to provide infrastructure for South Africa’s growing population. South Africa is also facing a serious shortage of engineers, technicians and other skilled workers in the construction industry. This places high demands on designers and contractors to provide services and to realise projects in ever-reducing time periods and at less cost. These conditions have made it increasingly difficult to maintain the required quality of construction in an industry where mistakes can lead to disastrous consequences. Recent advances in structural materials, structural systems and the way in which projects are handled, now enables a new look at the possibilities of combining pre-fabrication with on site work. This method, known as Hybrid Concrete Construction (HCC), has the potential to revolutionize the South African construction industry if applied correctly. Local research into this technique is however required and it is the aim of this thesis to draw attention to this subject. Key Findings Hybrid Concrete Construction (HCC) can be applied to any structural project, it will however not necessarily be successful. A structure needs to be adapted from the very start to suit a particular construction method. This ensures that all the advantages of the selected construction method may be achieved. Adapting a structure to a different construction method requires a mutual understanding and commitment from all project participants, including the architect, engineer, contractor and client. HCC also requires a certain degree of repetition in a project to be financially viable. A theoretical cost exercise was performed where only the material and erection costs were considered. In this exercise, HCC was found to be slightly less expensive than other construction methods for the Office Building of more than 10 storeys. HCC was also found to be significantly faster than other construction methods for the Office Building of more than 3 storeys. The time calculation was however based on the simplified time estimates from one source. Because of HCC’s shorter estimated construction period, the client can expect to earn revenue from a much earlier date. This decreases the relative cost of a HCC project. This advantage, however, needs to be quantified for chosen South African projects. On-site safety is still an important issue with HCC projects. Labourers are not accustomed to this construction method and it may be necessary to alter current skill development programs to include a crane safety course. The training of qualified riggers and crane operators should receive priority if HCC is to develop in South Africa. This preliminary investigation has shown that Hybrid Concrete Construction (HCC) can be feasible for the South African market. Further investigation is however required to determine the parameters for which HCC would be the preferred construction method. Recommendations Based on the findings and conclusions of this investigation, the following recommendations are made. The following actions should be undertaken by individual South African companies: · Develop relationships with external project partners · Train competent riggers and crane operators The South African concrete industry should invest in the following actions: · Invest in mass-producing precast concrete facilities · Develop a central database of South African projects with information on time, costs, project concepts and layouts to be used as a guideline for decision making · Develop local guidelines for the production and application of self compacting concrete · Compile guidelines for the design and construction of HCC and precast concrete construction in South Africa · Develop a local hidden corbel type connection to its full potential

Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeurs. / Glypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors.

Student Number : 9902006N - MSc(Med) Dissertation - School of Medicine - Faculty of Health Sciences / Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen, and dietary iron overload has been shown to contribute to HCC development in black africans. Both are well studied hepatotoxins. The aim of this study was to use a Wistar rat model over a 12 month period to investigate synergy and the extent thereof between AFB1 ingestion and dietary iron overload. 25ug/day of AFB1, reconstituted in DMSO, was administered by gavaging the animals, over a period of 10 days with a 2 day interval in between. The chow diet was supplemented with 0.75% (w/w) ferrocene iron. Experimental subjects were divided into 4 groups. Group 1 was fed the normal chow diet. Group 2 was fed 0.75% (w/w) ferrocene iron alone. Group 3 was gavaged 250μg AFB1 alone. Group 4 was fed the 0.75% (w/w) ferrocene iron and gavaged 250μg AFB1. A number of assays were conducted to investigate synergy. Colorimetric assays were used to measure serum iron, total-iron binding capacity, ALT, AST, GGT, nitrite production, lipid peroxidation and hydroxyproline concentrations. ELISA’s were used to determine ferritin, 8-isoprostane and 8-hydroxyguanosine concentrations. Nontransferrin bound iron was measured using an HPLC method. A chemiluminescent assay was used to measure superoxide anion production. Cytokines were measured using a suspension array system. Mutagenicity was assessed using the Ames mutagenicity assay using salmonella typhimirium strains TA97, TA98, TA100 and TA102. Iron profiling indicated that iron overloading occurred with the ingestion of the ferrocene diet. Biomarkers of oxidative stress, as illustrated by the measurement of 8-hydroxyguanosine and lipid peroxidation, showed additive synergistic effects between the two carcinogens. The anti-inflammatory interleukin-10 was shown to be markedly elevated with the co-administration of the two carcinogens, indicating the elevated inflammatory processes. Additive synergistic effects were noted in terms of the liver disease marker ALT. The salmonella typhimirium strain TA102 used in the Ames mutagenicity test showed increased colony counts with respect to the coadministration of carcinogens (P<0.05), although no synergistic effect was noted. In a few of the presented parameters, the AFB1 group was not significantly different to the control group, although significant differences between the Fe group and the Fe + AFB1 groups were noted. The implication of which is that the presence of AFB1 is increasing the activity of Fe as a carcinogen, thereby acting as a co-carcinogen. Examples of such parameters illustrating this are presented in the results section including serum ALT, serum nitrite, liver and serum lipid peroxidation, liver and serum 8-hydroxyguanosine, some of the mutagenicity assays, and interleukin-10. The conclusion of this study suggests that AFB1 acts as a co-carcinogen in the presence of iron overloading, implying that a synergistic relationship between these two toxins exists.

Hepatocellular carcinoma (HCC) is the second most common cancer in Taiwan and transcatheter arterial chemoembolization (TACE) is now the mainstay of treatment for noncurative HCC. Due to increasing medical costs yearly and financial problem of the Bureau of National Health Insurance, it is important to reduce medical resource utilization including hospital stay and medical costs. The aim is to figure out the risk factors of extended hospital stay, and increased in-hospital medical costs in hepatocellular carcinoma patients receiving transcatheter arterial chemoembolization. The result of this study should be available for further improvement of medical care quality in the limited medical resource. From January 2008 to January 2010, 162 patients (121 male and 41 female) with histologically proven hepatocellular carcinoma underwent TACE only (131 pts) or TACE followed by catheter placement for hepatic artery infusion chemotherapy (HAIC) (31pts) at district teaching hospital. The extended hospital stay (EHS) and extended post-procedure stay (EPS) are defined as stay larger than their median values (11 & 7 days respectively). Clinical demographic, disease factors, tumor factors, procedure (TACE)-related factors and complications are used to identify the univariate factors related to EHS and EPS statistically. To find out predictors of EHS, EPS and increased in-hospital medical costs, multiple linear regression analyses are used. The risk factors for EPS are procedure-related, including complications and procedure methods ( TACE + HAIC related to TACE only) (R2=.367, p<.001), while those for EHS are complications, encephalopathy, procedure methods, Child-Pugh classification C (related to classification A) and age (R2=.490, p<.001). The predictors for increased in-hospital medical costs include procedure methods, AJCC stage IV, T4 stage, hepatoencephalopathy and complications (R2=0.615, p<.001). Taking total hospital stay into consideration, the most important risk factor related to increased medical cost is total hosptial stay itself. The most powerful risk factor for EPS, EHS is procedure-related complication. The different procedure methods also affect hospital stay and medical costs. In order to reduce medical resource utilization, we should avoid post-procedure complication and pay attention to cirrhotic degree as well as American Joint Committee of Cancer (AJCC) tumor stage system. The result of this study can provide some ideas to adjust medical expense polices for the Bureau of National Health Insurance and to control medical cost for the hospitals.

La polarisation des cellules est essentielle à la division cellulaire asymétrique, la migration cellulaire, la fonction neuronale et le développement embryonnaire. Elle peut se faire par le mécanisme de localisation des ARNm dans des compartiments cellulaires spécifiques. Bien que le mécanisme de localisation des ARNm soit assez bien défini dans tous les types cellulaires, son implication dans le développement du cancer n'a pas été caractérisée. Une étude de séquençage direct de l'ARN, réalisée sur des cellules métastatiques (HCCLM3) du carcinome hépatocellulaire (CHC) a déterminé qu'au niveau de leurs protrusions, certains ARNm y sont enrichis, alors qu'aux protrusions des cellules CHC non-métastatiques SMMC7721, ces ARNm n’y sont pas enrichis. L'ARNm qui encode pour le facteur de transcription STAT3 figure parmi ceux identifiés comme étant enrichis. La technique du smiFISH a été utilisée pour valider l'enrichissement de l'ARNm STAT3 au niveau des protrusions des cellules CHC associées à un phénotype métastatique. Les résultats obtenus démontrent que l'essai de déplétion de sérum permet d'induire la migration des cellules SMMC7721, HCCLM3 et MHCC97H, qui forment des protrusions de type lamellipode. En parallèle, l'expression de l'α-actinine-GFP a permis de visualiser les régions protrusives et de les délimiter. Les expériences de smiFISH et les mesures d'expression relative par RT-qPCR ont révélé que les cellules métastatiques HCCLM3 et MHCC97H expriment peu d'ARNm STAT3, comparativement aux cellules non-métastatiques SMMC7721. De plus, les quantifications absolues de l'enrichissement démontrent que dans toutes les lignées CHC, les molécules d'ARNm STAT3 uniques localisent majoritairement au niveau des corps cellulaires, et très peu dans les protrusions. Le ratio du nombre de molécules d'ARNm STAT3 uniques localisées dans les protrusions sur le nombre de molécules uniques localisées dans les corps cellulaires est nettement plus petit pour les cellules HCCLM3 et MHCC97H que pour les cellules SMMC7721. La densité de l'ARNm STAT3 / unité de surface dans les protrusions des cellules HCCLM3 et MHCC97H est inférieure à celle des cellules SMMC7721. Ensemble, ces résultats confirment qu'il n'y a pas d'enrichissement de l'ARNm STAT3 aux protrusions des cellules CHC associées à un phénotype métastatique. / Subcellular mRNA localization regulates cell polarization. Asymmetric distribution of transcripts is important in cell division, cell migration, neuronal function and embryonic development. In all cell types, the mechanisms of mRNA localization are well defined, but their involvement in cancer development is still unknown. A recent study has identified protrusion-localized STAT3 mRNA in metastatic hepatocarcinoma cells (HCCLM3). In comparison, the STAT3 mRNA was not found to be enriched in the non-metastatic cells (SMMC7721) protrusions. The enrichment of the STAT3 mRNA at the protrusions of HCC cells associated with a metastatic phenotype was studied by smiFISH. Our results showed that the serum starvation assay induced lamellipodia-based migration in SMMC7721, HCCLM3 and MHCC97H cells. Also, expression of GFP-α-actinin allowed to mark the protrusive regions of migrating HCC cells. The smiFISH results and RT-qPCR quantification revealed that the metastatic cells (HCCLM3 and MHCC97H) express lower levels of STAT3 mRNA compared to the non-metastatic cells (SMMC7721). Absolute quantification of localization and enrichment revealed that STAT3 mRNA mainly localizes in the cell bodies of all HCC cell lines. The ratio of the number of single molecules of STAT3 mRNA localized in the protrusions on the number of single molecules localized in the cell bodies is smaller for the metastatic cells than the non-metastatic cells. In the metastatic cells protrusions, the density of STAT3 mRNA / unit of surface was found the be lower than the non-metastatic cells. Altogether, these results confirm that there is no enrichment of STAT3 mRNA at the protrusions of metastatic HCC cells.

Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.

No description available.

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.

Recent studies from our laboratory on the aromatic amine carcinogen, 4-aminobiphenyl (ABP) have shown a significantly lower prevalence of ABP-induced liver tumors in male mice lacking the N-acetyltransferases, and a dramatically lower prevalence in females than in males, but no association of tumor prevalence with strain or sex differences in levels of acute ABP-induced DNA damage. This thesis aimed to investigate the possible involvement of acute cytotoxic effects of ABP in the development of a tumor-promoting inflammatory environment. We found that wild-type male mice showed higher acute hepatotoxicity to ABP, as well as, a possible trend towards higher serum levels of the pro-inflammatory cytokine interleukin 6. This correspondence between acute ABP cytotoxicity and inflammatory response with ultimate tumor growth is consistent with a model whereby ABP not only initiates cells by damaging DNA but also promotes tumor growth in a gender-selective fashion that may be governed by gonadal hormone influences.