Background: Hepatocellular carcinoma is one of the most fatal diseases worldwide. Liver transplantation dramatically improved the survival rate of HCC patients. However, tumor recurrence remains a huge threat to HCC patients without any promising curative treatment. Chemotherapy, as one of the potential treatments to recurrent HCC, did not show any significant effect either. Objective: We aim to investigate the role of interferon-gamma inducible protein 10 (IP10) in acute-phase liver graft injury induced late-phase cisplatin resistance after liver transplantation and to explore the underlying mechanism. Furthermore, a potential adjuvant therapy was expected to be identified to sensitize cisplatin treatment in HCC. Materials and methods: A rat orthotopic liver transplantation model was established with applying whole or small-for-size (50%) graft. Afterwards, a rat hepatoma cell (MH7777) was injected via portal vein to generate recurrent tumor. The expressions of genes linked to multi-drug resistance and graft injury were compared between tumors developed after liver transplantation using small and whole grafts. IP10 expression was further validated in clinical samples from two cohorts of patients including HCC patients with hepatectomy and HCC patients with liver transplantation. The extracellular and intracellular roles of IP10 were examined in vitro by using IP10 recombinant protein and IP10 stable transfectants in HCC cell lines. The correlation between IP10 expression and tumor growth was investigated in three in vivo nude mice models including a subcutaneous model, an orthotopic model and ischemia reperfusion injury model. The underlying mechanism was further explored in vitro, in vivo and in clinical samples. IP10 neutralizing antibody was employed as an adjuvant therapy to identify its effect on sensitizing cisplatin treatment in HCC. Results: The expressions of multidrug resistant genes were significantly up-regulated in liver and tumor from small-for-size group in rat liver transplantation model. IP10 was selected as the potential target for its constantly higher expression in liver and tumor tissues in small-for-size group. In clinical studies, IP10 was overexpressed in around 45% HCC patients with hepatectomy. The expression of circulating IP10 well correlated with tumor recurrence and small graft ratio in HCC patients after liver transplantation. In in vitro studies, it was demonstrated that overexpression of IP10 could significantly promote HCC cell proliferation either in short term or in long term cisplatin administration. In in vivo studies, subcutaneous and orthotopic nude mice models showed that the overexpression of IP10 have significant correlations with larger tumor volume and less tumor necrosis after cisplatin treatment. In mechanism studies, IP10 overexpression was found to be well correlated with the activation of endoplasmic reticulum (ER) stress signaling pathways in vitro and further validated in vivo models and in clinical specimens. IP10 neutralizing antibody was identified as a potential therapy which could sensitize cisplatin treatment in vitro and in vivo. Conclusions: The high expression of IP10 was identified in two cohorts of clinical samples and showed significant correlations with tumor recurrence. Graft injury induced IP10 overexpression could significantly increase cisplatin resistance after liver transplantation via ER stress signaling pathways. IP10 neutralizing antibody may be applied as an alternative treatment for recurrent HCC after liver transplantation. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Ovarian carcinoma is one of the most leading causes of cancer death among all gynaecologic malignancies worldwide. Although there are advances in cancer treatment for the last decades, the curative rate of this disease is just modestly improved. Chemoresistance is the major obstacle in clinical management of ovarian cancer nowadays. Thus, it is an urgent need for exploring effective alternative therapeutic strategies for ovarian cancer patients with advanced or recurrent disease. Emerging evidence has suggested that targeting cancer cell metabolism is the most promising molecular therapeutic approach in combating human cancers. Recently, the application of pharmaceutical AMPK activators is a plausible approach in selectively and specifically killing cancer cells without hampering normal cells. However, these pharmaceutical AMPK activators have many side-effects. Therefore, searching for replaceable reagents from nutraceuticals is a “new vista”. Bitter melon and its bioactive components are proposed to be natural activator of AMPK not only to reduce triglycerides levels in hyperlipidemic diabetic or insulin-resistant rodents but also to suppress human cancer cell growth specifically without toxicity to normal cells. In this study, the anti-cancer effect and molecular mechanism of bitter melon extract (BME) and one of its bioactive components, MAP30, on ovarian cancer cells were examined. Upon treatment of BME and MAP30, ovarian cancer cells showed a drastic reduction in cell proliferation and an increase of cell apoptosis in a dose dependent manner. Intriguingly, co-treatment of BME or MAP30 could enhance cisplatin-induced cell cytotoxicity in ovarian cancer cells. On the other hand, tumor microenvironement has been known as a key factor promoting cancer progression and chemoresistance. Results herein showed that BME or MAP30 could inhibit cell growth, cell migration and invasion of ovarian cancer cells mediated by omentum conditioned medium (OCM), as well as enhanced cisplatin-mediated cell cytotoxicity in a xenograft mouse tumour model. Mechanistic studies revealed that the inhibitory effect of BME and MAP30 was concomitantly associated with up-regulated AMPK activity but reduced expression of phospho-AKT, phospho-ERK and FOXM1. Such effects were similar to the functions of common AMPK activators e.g. AICAR, A23187, metformin or hypoxic stress, indicating that BME and MAP30 functions as natural AMPK activators in suppressing cancer cells growth through activating AMPK activity and inhibiting AKT/ERK/FOXM1 signaling cascade. Importantly, this study demonstrated that BME and MAP30 induced AMPK activation through an AMP-independent manner using a pair of isogenic HEK293 cells with overexpression of either the wild-type (WT) or R531G mutant isoform of AMPK2 subunit, implying the significance that BME and MAP30 may not affect the mitochondrial respiration and thus may be more tolerated by patients when used as anti-cancer medications. Taken together, the findings in this study suggest that the non-toxic BME and MAP30 function as natural AMPK activator in impairing ovarian cancer cell growth and enforcing cisplatin-mediated cell cytotoxicity in ovarian cancer cells through targeting cancer cell metabolism. Thus, BME or MAP30 may be used as a supplement for synergistically enhancing the efficacy of current chemotherapy regimes. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Increasing carbapenem resistance among clinical isolates of E. coli and K. pneumoniae has become a serious public health problem over the last decade. Molecular epidemiology studies have shown that there is a global dissemination of epidemic clones of carbapenem-resistant E. coli and K. pneumoniae. Besides, successful epidemic plasmids were reported to disseminate carbapenemase genes in Enterobacteriaceae. The wide spread of carbapenem-resistant E. coli and K. pneumoniae limits treatment options of the infection, poses severe challenges to clinical professionals and threatens our health. Recently, carbapenem-resistant E. coli and K. pneumoniae are increasingly reported in Hong Kong. In 2012, our group has documented the emergence of carbapenem-resistant clinical isolates in Hong Kong. The findings of the previous study showed that 26.1% of the Enterobacteriaceae isolates were confirmed to produce carbapenemase. Notably, a novel IncX3 plasmid was found to be involved in the dissemination of blaNDM-1 gene. However, the previous findings fail to explicate the carbapenem resistance mechanisms of the remaining non-carbapenemase producing isolates. Further investigation is needed to elucidate the situation. Firstly, we investigated the carbapenem resistance mechanism of carbapenem-resistant E. coli and K. pneumoniae isolates recovered from the Hong Kong West Cluster hospitals from 2010 to 2012. PCRs were used to detect carbapenemase genes (blaNDM, blaKPC, blaIMP, blaVIM and blaOXA-48), blaCTX-M ESBL genes and blaAmpC genes. SDS-PAGE was used to detect porin loss. Among the 92 isolates in this study, only nine (9.8 %) isolates were detected with carbapenemase genes. The blaCTX-M and/or blaAmpC β-lactamase genes plus porin loss were detected in 47 non-carbapenemase-producing isolates (16 E. coli and 31 K. pneumoniae). The resistance determinant profiles of these 16 E. coli included: blaCTX-M + porin loss (n= 10), blaCIT + porin loss (n = 1), blaCTX-M + blaCIT/DHA + porin loss (n = 5). The resistance determinant profiles of the 31 K. pneumoniae included: blaCTX-M + porin loss (n= 4), blaDHA + porin loss (n = 7), blaCTX-M + blaCIT/DHA + porin loss (n = 20). The results showed that apart from acquired carbapenemases, the production of AmpC β-lactamase and/or ESBLs plus porin loss played a main role in the carbapenem resistance mechanism of the carbapenem-resistant E. coli and K. pneumoniae isolates. Secondly, we accessed the clonal relatedness of the isolates. Multi-locus sequence typing results showed that 55 (77.5%) K. pneumoniae isolates fall into the clonal complex 37. Our results suggest that the CC37 K. pneumoniae are associated with the acquisition of DHA-1 β-lactamase, CTXM-1-group β-lactamase and porin alterations which could confer a high-level of resistance to carbapenems resulting in their predominance in this study. Finally, we characterized the plasmids that carry carbapenemase gene by S1-PFGE, Southern blot, plasmid replicon typing and whole plasmid sequencing. A novel IncX3 plasmid was found to carry blaKPC gene. Together with the previously reported blaNDM-1 carrying IncX3 plasmids, it shows that IncX3 plasmids might be new epidemic plasmids involved in the dissemination of carbapenemase genes. These novel IncX3 plasmids are worrisome. Nationwide surveillance and more epidemiological study of IncX3 plasmids are needed. (Word / published_or_final_version / Microbiology / Master / Master of Philosophy

Human mesenchymal stromal cells (hMSCs) are part of bone marrow micro-environment that supports hematopoiesis. However, hMSCs also enhance tumor progression and survival when they become part of the cancer micro-environment. I aimed to investigate the interaction between hMSCs and cancer cells during chemotherapy. Firstly, I studied the interaction between hMSCs and T-lineage acute lymphoblastic leukemia (T-ALL) cells under pegylated arginase I (BCT-100) treatment. Three T-ALL cell lines were sensitive to BCT-100 but not hMSCs. Conversely, hMSCs could partly protect all T-ALL cell lines from BCT-100 induced cell death under transwell co-culture condition. Concerning the possible mechanism, the intermediate metabolite L-ornithine could not rescue most T-ALL cells from BCT-100 treatment. But the downstream L-arginine precursor, L-citrulline could partly rescue all T-ALL cells from BCT-100 treatment. Ornithine transcarbamylase (OTC) converts L-ornithine into L-citrulline. OTC expression level in hMSCs remained relatively high during BCT-100 treatment but OTC expressions in T-ALL cell lines declined drastically. It suggested that hMSCs may protect T-ALL cells against BCT-100 treatment by having sustained OTC expression. Suppression of hMSCs by vincristine (VCR) disrupted the protective effect of hMSCs to most T-ALL cells during BCT-100 treatment. This suggests that by transiently suppressing hMSCs, we may abolish the protective effect of hMSCs to T-ALL cells during BCT-100 treatment. Then I studied the interaction between hMSCs and neuroblastoma under cisplatin treatment. Two neuroblastoma cell lines were used for both of them are cisplatin sensitive while hMSCs are cisplatin resistant. hMSCs could partly protect neuroblastoma cells from cisplatin induced cytotoxicity. On the other hand, exogenous IL-6 but not IL-8 could also partly rescue them from cisplatin induced cytotoxicity. IL-6 activated STAT3 phosphorylation dose-dependently and enhanced expression of detoxifying enzyme (glutathione S-transferase π, GST-π) in neuroblastoma. Such effect could be counteracted by anti-IL-6R neutralizing antibody tocilizumab (TCZ). However, TCZ failed to suppress hMSCs’ protection to neuroblastoma during cisplatin treatment. This suggests involvement of multiple factors. Up-regulation of serum GST-πin some hTertMSCs/neuroblastoma co-engrafted SCID mice compared to neuroblastoma engrafted mice provided a clue that GST-π might be a possible stromal-protection factor. Caffeic acid phenethyl ester (CAPE) is a known GST inhibitor after tyrosinase activation. Neuroblastoma cells expressed tyrosinase and CAPE enhanced cisplatin cytotoxicity on them, with or without hMSCs. Paradoxically, CAPE enhanced GST-πexpression with or without cisplatin treatment in neuroblastoma suggesting possible negative feedback to GST-π inhibition. However, such additive effect of CAPE to cisplatin cytotoxicity was not observed in vivo. Further delineation of the in vivo study design may help to verify the additive effect of CAPE to cisplatin cytotoxicity in vivo. Finally, I studied the effect of apoptotic cancer cells (AC) on the immune function of hMSCs. hMSCs could phagocytose apoptotic neuroblastoma cells with respective up-regulation of many immune-mediators including two highly-expressed cytokines IL-6 and IL-8. Up-regulation of these immune-mediators may enhance immune cells chemotaxis. Further detailed investigation on the effect of AC-engulfed hMSCs to other immune cells will help us to understand the dynamic interaction between cancer cells and stromal cells during chemotherapy. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Gliomas are the commonest type of primary malignant brain tumours of the central nervous system (CNS). The highly aggressive and infiltrative characteristics of gliomas render them one of the most lethal cancers. Amongst all, the most malignant form of glioma is glioblastoma multiforme (GBM), a World Health Organization (WHO) grade IV astrocytoma. Despite well-developed multimodal treatment including surgery, radiotherapy, and chemotherapy, the prognosis of GBM patients remains poor with median survival of just over one year. This high mortality rate is commonly the result of relentless tumour recurrence secondary to the tumour’s intrinsic resistance towards its standard chemotherapeutic agent temozolomide (TMZ). Prolyl 4-hydroxylase, beta subunit (P4HB) is an endoplasmic reticulum stress response (ERSR) chaperone protein that was previously found to be overexpressed in the chemoresistant glioma cell lines D54-MG and U87-MG. Differential expressions of numerous microRNAs (miRNAs) were also found between chemosensitive and chemoresistant glioma cell lines. As such, we surmised that the dysregulation of a P4HB-regulating miRNA may contribute to P4HB upregulation and therefore chemoresistance in glioma. MiR-210, a commonly dysregulated miRNA in various cancers, is one of the most highly downregulated miRNAs in chemoresistant glioma cells (compared to chemosensitive glioma cells), and, based on bioinformatics findings, may also regulate P4HB expression. MiR-210 was therefore selected for further investigations regarding its potential roles in glioma chemoresistance. The regulatory relationship between P4HB and miR-210 was subjected for verifications. With the use of quantitative real-time polymerase chain reaction (qPCR) and western blotting, the intrinsic expressions of P4HB and miR-210 were studied. The upregulation of P4HB in D54 and U87 chemoresistant glioma (compared to the parental) cell lines were found to correlate reciprocally with the downregulation of miR-210 in the same chemoresistant glioma cells. To delineate the potential regulatory role of miR-210, a gain of function approach was adopted. Transfection of a miR-210 mimic was performed into the D54 and U87 parental chemosensitive (D54-S and U87-S) and chemoresistant (D54-R and U87-R) cells, along with a negative control. The transfection efficiency of miR-210 as well as the subsequent P4HB expressions was verified. It was found that P4HB expression was downregulated as a result of miR-210 upregulation both at the mRNA and protein levels in glioma cells. Furthermore, the effects of miR-210 overexpression on chemoresistance in the glioma cells were tested by performing cell proliferation assay. Decrease in the half maximal inhibitory concentration (IC50) of TMZ were found in all cell lines overexpressing miR-210, suggesting that miR-210 upregulation may lead to P4HB inhibition, which would at least partially mediate an alleviation of glioma cells’ resistance towards its chemotherapeutic agent TMZ. In summary, miR-210 is downregulated in chemoresistant glioma cells in vitro. It plays a potential role in regulating P4HB expression, hence chemoresistance in GBM cells. Future investigations may focus on its mechanism of action and potentiality for therapeutic intervention. / published_or_final_version / Surgery / Master / Master of Medical Sciences

abstract / Biological Sciences / Master / Master of Philosophy

abstract / Pathology / Master / Master of Philosophy

Malaria is a preventable and treatable disease caused by infection with Plasmodium parasites. Complex socioeconomic and political factors limit access to vector control and antimalarial drugs, and an estimated 600,000 people die from malaria every year. Rising drug resistance threatens to make malaria untreatable. As for all new traits, resistance is limited by fitness, and a small number of pathways are heavily favored by evolution. These pathways are targets for drug discovery. Pairing compounds active against the wild-type and the small emerging resistant population, a strategy we termed "targeting resistance," could block the rise of competitively viable resistance.

Disease surveillance is the continuous collection, analysis, and interpretation of health-related data. Information gained from routine HIV disease surveillance is vital to national program managers deciding to implement new prevention or treatment programs. In this dissertation, we describe methods for estimation of HIV incidence and the prevalence of HIV drug resistance.

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy