By means of reverse-transcription polymerase chain reaction, overexpression of ZNF146 was detected in two human HCC cell lines HepG2 and Hep3B and a clear relationship between HCC and overexpression of ZNF146 has been established. Subcellular localization of ZNF146 protein in liver cells was studied by generation and expression of a green fluorescent protein (GFP) fusion protein. The nuclear localization and the reported DNA binding ability of ZNF146 protein provided a hint that ZNF146 may carry out its function in the cell system by interacting with specific genomic DNA sequences. Recombinant ZNF146 protein was expressed using bacterial and yeast system for the genomic DNA pull down assay in the identification of potential interacting genomic DNA sequences. Several potential genomic DNA sequences that interact with ZNF146 were identified and the gene MDM2 is the one of the candidates that is directly related to human carcinogenesis. MDM2 is a negative regulator of the tumor suppresser protein p53. Deregulation of MDM2 will impair the cell's ability in cell cycle arrest, DNA repair and apoptosis upon induced DNA damage. / Hepatocellular carcinoma (HCC) is a type of primary malignant liver tumor. And is one of the most frequent malignancies worldwide. The focus of this research project is the characterization of a Kruppel zinc finger protein, zinc Finger Protein 146 (ZNF146) using HCC as a disease model. The aim of this project is to understand the functional role ZNF146 and try to explore the mechanism of how ZNF146 might be involved in the carcinogenesis of HCC. / In order to have a better understanding with the protein ZNF146, SUMOylation properties of this protein has been studied. SUMO1 modification on ZNF146 has already been reported. And in our study, experimental result demonstrated that ZNF146 is also modified by SUMO2 and SUMO3 in liver cells. Other than the SUMOylation sites for SUMO1 protein which has been reported, modification sites for SUMO2 at the K247 and K275 positions were mapped, while K191R, K219R, K247R, K256R and K275R, five positions were mapped for SUMO3 modification. A more complete picture of the SUMOylation properties of ZNF146 has been revealed. Since we hypothesized that ZNF146 is related to the p53 tumor suppressor, cell cycle control and DNA repair pathway, a cell cycle study using flow cytometry was performed for the investigation of the effect on cell cycle regulation by ZNF146 overexpression. In our study, ZNF146 overexpression promoted the G1/S transition in the cell division cycle, which indicated that liver cells were more active for the progression of cell cycle. / On the other hand, using cDNA microarray technology expression profiles of ZNF146 overexpressing and non-overexpressing liver cell lines were compared and with real-time polymerase chain reaction, six candidate genes CRLF1, IFI44, ST6GAL1, LOC441601, IL18 and RAD17 were confirmed with their deregulation induced by the overexpression of ZNF146. Four of the candidates, IFI44, LOC441601, IL18 and RAD17 were found to be related to the p53 tumor suppressor activity or DNA damage, repair response and control. This observation, together with the result of genomic DNA pull down assay, gives us a hint that ZNF146 is possibly involved in liver carcinogenesis by affecting DNA repair and cell cycle control upon induced DNA damage. / The gene ZNF146 codes for a member of the Kruppel zinc finger proteins, however ZNF146 protein is different from most members of the Kruppel zinc finger proteins subfamily. It encodes a 33 kDa protein solely composed of 10 zinc finger motifs and is devoid of any non-zinc finger regulatory domain for interactions with other proteins. ZNF146 overexpression has been reported in a number of cancers including colon cancer and pancreatic carcinoma. However, the functional role of ZNF146 overexpression in tumorigenesis is yet to be solved and not much research on how ZNF146 might be invovled in the establishment of HCC was published. / To conclude, the experimental results of this study support the hypothesis that ZNF146 overexpression may deregulating the cell division cycle and some genes differentially regulated upon over-expression of ZNF146 are related to the regulations of DNA damage response. Future research on ZNF146 can be focused on the detail regulatory pathway of ZNF146 overexpression and its interaction between the p53 tumor suppressor, DNA damage response and cell cycle regulation, and a fuller picture of how ZNF146 overexpression might induce hepatocarcinogenesis can be revealed. / Yeung, Tsz Lun. / Adviser: Miu Yee (Mary) Waye. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3329. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 287-304). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Leung, Jackie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 117-131). / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.iv / List of publications --- p.vi / List of Tables --- p.vi / List of Figures --- p.vi / Table of Contents --- p.ix / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Liver cancer --- p.1 / Chapter 1.1.1. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2. --- Lung Cancer --- p.5 / Chapter 1.3. --- Pteris semipinnata L --- p.8 / Chapter 1.4. --- Extract of PsL: 5F --- p.10 / Chapter 1.5. --- Animal models in chemotherapy researches --- p.13 / Chapter 1.5.1. --- Model of HCC --- p.13 / Chapter 1.5.2. --- Model of lung cancer --- p.15 / Chapter 1.6. --- Apoptosis: Significance of programmed cell death --- p.17 / Chapter 1.6.1. --- The extrinsic pathway --- p.18 / Chapter 1.6.2. --- The intrinsic pathway --- p.19 / Chapter 1.7. --- Apoptotic molecules related to this study --- p.22 / Chapter 1.7.1. --- Bcl-2 family --- p.22 / Chapter 1.7.1.1. --- Bax --- p.22 / Chapter 1.7.1.2. --- Bcl-2 --- p.23 / Chapter 1.7.2. --- Nuclear factor kappa B --- p.25 / Chapter 1.7.3. --- Inducible nitric oxide synthase --- p.27 / Chapter 1.8. --- Side-effects of chemotherapy --- p.29 / Chapter 1.8.1. --- Chemotherapy and liver dysfunction --- p.30 / Chapter 1.8.2. --- Nephrotoxicity of chemotherapeutic agents --- p.31 / Chapter 1.9. --- Aim of study --- p.33 / Chapter Chapter 2: --- Materials and Methodology --- p.34 / Chapter 2.1. --- Animals --- p.34 / Chapter 2.1.1. --- HCC model --- p.34 / Chapter 2.1.2. --- Lung cancer model --- p.35 / Chapter 2.2. --- Tumors induction --- p.36 / Chapter 2.2.1. --- HCC induction in C3H/HeJ mice --- p.36 / Chapter 2.2.2. --- Lung cancer induction in A/J mice --- p.37 / Chapter 2.3. --- 5F preparation --- p.38 / Chapter 2.4. --- 5F treatment --- p.39 / Chapter 2.5. --- Harvest of samples and tissues --- p.41 / Chapter 2.6. --- Tumor assessment --- p.43 / Chapter 2.7. --- Investigation of apoptosis and cell proliferation --- p.44 / Chapter 2.8. --- Immunohistochemistry --- p.47 / Chapter 2.9. --- Biochemical test --- p.51 / Chapter 2.9.1. --- Liver Function Tests (LFT) --- p.52 / Chapter 2.9.1.1. --- Aspartate aminotransferase (AST) & Alanine aminotransferase (ALT) --- p.52 / Chapter 2.9.2. --- Renal Function Test (RFT) --- p.53 / Chapter 2.9.2.1. --- Serum creatinine level (CRE) --- p.53 / Chapter 2.9.2.2. --- Blood Urea Nitrogen index (BUN) --- p.54 / Chapter 2.10. --- Statistical analysis --- p.55 / Chapter Chapter 3: --- Results --- p.56 / Chapter 3.1. --- Anti-tumor effect of 5F is dose- dependent --- p.56 / Chapter 3.2. --- 5F reduces cell proliferation and induces apoptosis in-vivo --- p.60 / Chapter 3.3. --- Effects of 5F on apoptotic signaling molecules --- p.68 / Chapter 3.3.1. --- 5F up-regulates pro-apoptotic Bax and Bak --- p.68 / Chapter 3.3.2. --- 5F down-regulates anti-apoptotic NF-kappa B and Bcl-2 --- p.76 / Chapter 3.3.3. --- 5F up-regulated iNOS in HCC but not in lung cancer --- p.88 / Chapter 3.3.4. --- Regulation on Erk1/2 was associated with treatment of 5F --- p.93 / Chapter 3.4. --- Side-effect studies of 5F --- p.97 / Chapter Chapter 4: --- Discussion --- p.105 / Chapter Chapter 5: --- Conclusion --- p.116 / Bibliography --- p.117

Chau, Kin Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 106-119). / Abstract also in Chinese. / Abstract --- p.II / 摘要 --- p.VI / Acknowledgements --- p.IX / List of figures --- p.X / List of Abbreviations --- p.XII / Table of Contents --- p.XV / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Incidence and etiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Structure of Hepatitis B Virus (HBV) --- p.2 / Chapter 1.3 --- Hepatitis B X protein (HBx) and HCC --- p.5 / Chapter 1.4 --- HBx and Apoptosis --- p.8 / Chapter 1.5 --- The role of Bcl-2 family in apoptosis and cell survival --- p.10 / Chapter 1.6 --- "Bid, the BH3-domain only protein" --- p.14 / Chapter 1.7 --- Dual Functions of Bid --- p.16 / Chapter 1.8 --- The relationship between Bid and HBx --- p.19 / Chapter 1.9 --- Hypoxia and HCC --- p.21 / Chapter 1.10 --- Hypoxia and HBx --- p.25 / Chapter 1.11 --- Hypoxia and Bid --- p.28 / Chapter 1.12 --- Aim of study --- p.29 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Confirmation of the culture of the stable cell lines --- p.30 / Chapter 2.2 --- Doxorubicin treatment to the cell lines --- p.34 / Chapter 2.3 --- Culture of the cell lines under hypoxic conditions --- p.35 / Chapter 2.4 --- Protein sample preparations --- p.37 / Chapter 2.5 --- Determination of protein samples --- p.38 / Chapter 2.6 --- Sodium dodecyl sulfate 226}0ؤ polyacrylamide gel electrophoresis (SDS- PAGE) --- p.39 / Chapter 2.7 --- Transfer of protein to nitrocellulose membranes --- p.39 / Chapter 2.8 --- Western blot analysis of proteins --- p.41 / Chapter 2.8.1. --- Antibodies --- p.41 / Chapter 2.8.2. --- Determination of expression profiles of desired proteins by immunoblotting --- p.45 / Chapter 2.9 --- "Measurement of cell viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay" --- p.46 / Chapter 2.10 --- Determination of cell proliferation by BrdU proliferation assay --- p.47 / Chapter 2.11 --- Detection of apoptosis of the cell lines by TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) --- p.50 / Chapter 2.12 --- Determination of the involvement of p38 MAPK in the generation of truncated Bid by p38 MAPK inhibitor SB203580 --- p.52 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Confirmation of plasmids and the stable cell lines --- p.53 / Chapter 3.2 --- Morphology and the basic parameters of the cells with full-length HBx or mutant HBx --- p.53 / Chapter 3.3 --- Cell viability under doxorubicin treatment with or without hypoxia --- p.59 / Chapter 3.4 --- Determination of cell proliferation under stress --- p.70 / Chapter 3.5 --- Expression profiles of various proteins in the stable cell lines under doxorubicin treatment with or without hypoxia --- p.74 / Chapter 3.5.1. --- Verification of hypoxia --- p.74 / Chapter 3.5.2. --- Pro-apoptotic proteins --- p.74 / Chapter 3.5.3. --- Anti-apoptotic proteins --- p.74 / Chapter 3.6 --- Determination of apoptosis of various cell lines under stress --- p.82 / Chapter 3.7 --- "p38 MAPK, but not Akt, was activated by doxorubicin" --- p.87 / Chapter 3.8 --- The p38 MAPK inhibitor SB203580 could attenuate the cleavage of Bid --- p.89 / Chapter Chapter 4: --- Discussion --- p.92 / Chapter Chapter 5: --- Conclusion and future prospective --- p.103 / Chapter Chapter 6: --- References --- p.106

University of Macau / Institute of Chinese Medical Sciences

High intensity focused ultrasound (HIFU) is an emerging non-invasive thermal ablative modality that can be utilised for the treatment of solid organ tumours, including liver cancer. Acoustic cavitation is a phenomenon that can occur during HIFU and its presence can enhance heating rates. One major limitation of thermal ablative techniques in general, such as radiofrequency and microwave ablation, is the heat sink effect imparted by large vasculature. Thermal advection from blood flow in vessels ≥ 3 - 4 mm in diameter has been shown to significantly reduce heating rates and peak temperatures in the target tissue, potentially leading to treatment failure. With regards to HIFU therapy, a clearer understanding is required of the effects of blood flow on heating, cavitation and thermal tissue necrosis, which is the treatment endpoint in clinical thermal ablation. Therefore, the overall aim of this thesis project was to elucidate the effects of blood flow on HIFU-induced heating, cavitation and histological assessment of thermal ablation. A unique isolated, perfused porcine liver model was used in order to provide a relevant test bed, with physiological and anatomical characteristics similar to the in vivo human liver. The normothermic liver perfusion device used in all studies presented in this work can keep an organ alive in a functional state ex vivo for in excess of 72 hours. A further advantage of the liver perfusion device was that it allowed blood flow to be stopped completely and resumed rapidly, allowing studies to be conducted under zero flow conditions. A therapeutic HIFU system was used in order to deliver HIFU therapy to regions of hepatic parenchyma adjacent (≤ 3 mm) to large (≥ 5 mm) blood vessels or away from vasculature (≥ 1 cm) at either 1.06 MHz or at 3.18 MHz. Cavitation events during HIFU therapy were spatio-temporally monitored using a previously developed passive acoustic mapping (PAM) technique. The cavitation threshold at each frequency was determined through assessment of acoustic emissions acquired through PAM during HIFU exposure at a range of acoustic pressures. Real time thermal data during HIFU therapy were obtained using an implantable 400 μm thermocouple, aligned with the HIFU focus, in order to assess the effect of large vessel blood flow on peak tissue temperatures. Thermal data were obtained at 1.06 MHz, in the presence of acoustic cavitation and at 3.18 MHz, in the absence of cavitation, both in the presence and complete absence of blood flow. Finally, histological assessment of cell viability and cell death was performed in order to determine whether any heat sink effect could be overcome, with the achievement of complete tissue necrosis in treatment regions directly adjacent to large vasculature. This work demonstrated for the first time that in perfused, functional liver tissue, the presence of large vasculature and physiological blood flow does not significantly affect ablative HIFU therapy, both in terms of peak focal tissue temperatures attained and histological evidence of complete tissue necrosis. Therefore, HIFU may be superior to other ablative modalities in treating tumours in tissue regions adjacent to major vascular structures, but further work needs to be performed to correlate the experimental findings with clinical outcomes.

Hepatocellular carcinoma is a primary liver cancer.Among treatment options for hepatocellular carcinoma, Yttrium-90 radioembolization is a promising transarterial therapy.This thesis investigates potential clinical applications of radioembolization in the treatment of the hepatocellular carcinoma (techniques of radiation segmentectomy and radiation lobectomy), and adress the problematic of the response Assessment after radioembolization. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

The hepatitis C virus (HCV) was characterised in 1989. HCV was transmitted through transfusion of blood/blood products, but injection drug use is now the most common route of transmission. The infection is usually asymptomatic but becomes chronic in about 75%, and in 20 years 15-25% develops liver cirrhosis, with a risk for liver failure and liver cancer. HCV has also been associated with lymphoproliferative disorders. The aim of this thesis was to study morbidity and mortality in a national, population-based cohort of HCV-infected individuals. The study population consisted of all persons with a diagnosed HCV-infection recorded in the national surveillance database. This file was linked to other national registers to obtain information of emigration, deaths, cancers, and inpatient care. All personal identifiers were removed before analysis. In Paper I the standardized incidence ratios (SIR) for Hodgkin’s and non-Hodgkin’s lymphoma (NHL), multiple myeloma, acute and chronic lymphatic leukaemia, and thyroid cancer were studied. In the HCV-cohort (n: 27,150) there was a doubled risk for NHL and multiple myeloma in patients infected for more than 15 years, compared with the general population (age-, sex- and calendar-year specific incidence rates). The results strengthened these earlier controversial associations. The SIR and also the absolute risk for primary liver cancer were estimated in Paper II. In the HCV-cohort (n: 36,126) the individuals infected for more than 25 years had a more than 40 times increased risk for liver cancer compared with the general population. The absolute risk of primary liver cancer was 7% within 40 years of HCV-infection. Mortality and cause of death were studied in Paper III. The standardized mortality ratio (SMR) demonstrated a 5.8 times excess mortality in the HCV-cohort (n: 34,235) compared with the general population, and a 35.5 times excess mortality from liver disease. Deaths from illicit drugs and external reasons were common in young adults. Paper IV presents a study of inpatient care. The HCV-cohort (n: 43,000) was compared with a matched reference population (n: 215,000). Cox regression was used to estimate the likelihood, a hazard ratio, for admission to hospital, and frequencies and rates to estimate the total burden. In the HCV-cohort inpatient care was high and about 50% was psychiatric, often drug-related care. The likelihood for liver-related admissions was very high, and serious liver complications increased in the 2000s, indicating that HCV-associated liver disease will increase the next decade. In the 2000s, about 1000 individuals per year were treated with HCV-combination therapy. To conclude, the risk for NHL and multiple myeloma was doubled, and liver- and drug-related morbidity and mortality was very high in the HCV-cohort. Serious liver complications increased in the 2000s and will probably increase the coming decade.

No description available.

Le carcinome hépatocellulaire (CHC) est la deuxième cause de mortalité par cancer dans le monde. Plusieurs études attestent du rôle du microenvironnement tumoral (MET) comme acteur fondamental de la carcinogenèse. A l’aide de modèles murins mimant un sous groupe de CHC fréquent, notre équipe avait identifié la molécule LECT2 comme un effecteur moléculaire important du MET dans le contrôle de l’agressivité tumorale.L'objectif de ma thèse a été d’adresser le rôle fonctionnel de LECT2 dans le microenvironnement immunitaire au cours du CHC.A l’aide de modèles murins, nous observons que l’absence de LECT2 entraine une accumulation importante de cellules myéloïdes dans le MET. Nous montrons que ces cellules myéloïdes sont immatures, arborent des capacités immunosuppressives puissantes vis-à-vis des lymphocytes T et ont un programme transcriptionnel permettant une action promotrice de tumeurs. De façon intéressante, l’accumulation de ces acteurs dans le microenvironnement est associée à l’émergence de nodules tumoraux indifférenciés exprimant des marqueurs de transition épithélio-mésenchymateuse/cellules progénitrices/métastases.D’un point de vue mécanistique, nous avons démontré une perte de différenciation plus importante des hépatocytes en absence de LECT2 dans des conditions d’activation de la signalisation β-caténine. Nous montrons également par des expériences de co-culture que les cellules myéloïdes infiltrant les tumeurs en absence de LECT2 ont une forte capacité à induire une perte de différenciation des hépatocytes.Enfin, nous avons analysé l'expression de LECT2 dans une vaste cohorte d’échantillons humains de CHC. Nous montrons que la diminution d’expression de LECT2 corrèle fortement avec 1)- la présence d’invasion vasculaire, 2)- la perte de différenciation des hépatocytes tumoraux et 3)- la présence d’infiltrats inflammatoires.L’ensemble de ces données démontre que LECT2 agit comme un régulateur essentiel dans la cancérogénèse hépatique à travers son action double sur les hépatocytes et sur la fonction des cellules myéloïdes infiltrant les tumeurs. Ainsi, ces travaux identifient LECT2 comme un biomarqueur potentiel ouvrant de nouvelles perspectives de traitement du CHC. / Hepatocellular carcinoma (HCC) is the second cause of cancer-rel ated death worldwide. Several studies highlighted the tumor microenvironment (TEM) as a key player in cancer from initiation to progression steps of tumorigenesis. Using relevant HCC mouse models, our team identified the chemokine-like LECT2 as a critical actor of liver TEM in the control of tumor aggressiveness.The aim of my thesis was to address functionally the role of LECT2 in the immune microenvironment during HCC.Using mouse models, we observed that the absence of LECT2 induces a significant accumulation of myeloid cells in the TEM. We showed that these myeloid cells were immature, harbored strong immunosuppressive capabilities on T cells and expressed a transcriptional program sustaining tumor progression. Interestingly, the accumulation of these actors in the microenvironment is associated with the emergence of poorly differentiated tumor nodules expressing epithelial-to-mesenchymal transition / progenitor / metastasis markers.Mechanistically, we demonstrated that LECT2-deficient hepatocytes in the context of β-catenin activation were able to perform EMT like WT hepatocytes do after TGF-β1 challenge. In co-culture experiments, we demonstrated that tumor-infiltrating myeloid cells in the absence of LECT2 have a strong ability to induce hepatocyte EMT.Finally, we analyzed the expression of LECT2 in a vast cohort of HCC liver samples and found that downregulation of LECT2 expression strongly correlates with 1) - the presence of vascular invasion, 2) – histological grade and 3) - the presence of inflammatory infiltrates.Altogether, our data demonstrate that LECT2 acts as a strong regulator of liver tumor aggressiveness through its dual action on hepatocytes and impact on the function of tumor infiltrating myeloid cells. This work identifies LECT2 as a new biomarker for HCC and pave the way to new therapeutic strategies.

No description available.