The ability to sense and rapidly respond to oxygen availability is crucial to the survival and physiology of facultative anaerobes. In many gram negative bacteria such as Escherichia coli, this process is primarily controlled by the dimeric, [4Fe〖-4S]〗^(2+) containing global transcription factor FNR, which regulates transcription of genes necessary for the anaerobic metabolism. Activity of FNR is directly regulated by the presence of oxygen, which inactivates FNR by oxidizing the [4Fe〖-4S]〗^(2+) cluster and causing the dissociation of the FNR dimer. Although the biological function of FNR has been well established, structural and biochemical characterization of the FNR dimer has been limited due to its extreme lability to oxygen. In the current study, I conduct molecular engineering on FNR protein and obtain oxygen stable variants that are suitable for in vitro biochemical studies. By combining several approaches including covalently linking two FNR monomers using a flexible peptide linker, amino acid substitutions to promote dimerization, and removal of protease recognition sites to prevent proteolysis, a series of FNR variants which are potentially active in the presence of oxygen are constructed. Various in vivo and in vitro assays led to the identification of the construct (FNRD154A)2 which covalently links two copies of FNRD154A, an FNR variant that has greater dimerization capability, in tandem displays significantly improved transcription regulation and DNA binding to various FNR regulated promoters in the presence of O2. Circular Dichroism analysis showed that this variant maintains a similar secondary structure as that of native FNRD154A and in vivo transcription assay demonstrated that this protein retains other properties of the native FNR dimer including [4Fe〖-4S]〗^(2+) cluster binding, oxygen sensing, and capability to support the anaerobic growth of E. coli. All these together led the conclusion that an FNR variant that retains structural and functional properties of native FNR has been constructed, but with significantly improved O2 stability. Thus, it has the potential to be widely used in various biochemical and structural studies of FNR in the presence of oxygen. In addition to the major project of molecular engineering of FNR protein, in this thesis, I also initiated the study of using metabolomics approaches to identify the cellular substrates of the multidrug efflux pump MdtEF. MdtEF is an important efflux pump in E. coli and its expression has been shown to be induced under a number of stressed conditions. It is thus proposed to have a general detoxification function in E. coli, but the cellular substrates it expels have not been identified. In this study we established and applied metabolite profiling on the wild type and ΔmdtEF E. coli strains and confirmed that indole red, a metabolic by-product formed during anaerobic respiration of nitrate, is one of the cellular substrates of MdtEF under anaerobic conditions. This study provides a general methodology to identify endogenous substrates of efflux pumps and contributes to the understanding of the physiological roles of multidrug efflux pumps in bacteria. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

The abundance of tumour initiating cells (TIC) has been suggested to be an important prognostic indicator in cancers. Both SOX2 and ALDH have been individually reported to be putative TIC markers but their combined status is unclear and their usefulness in the prognostication of non-small cell lung cancer (NSCLC)has not been reported. This study investigated the patterns of ALDH and SOX2 protein expression in NSCLC using immunohistochemistry. Expression was graded using semi-automated signal capturing and image analysis software. ALDH and SOX2 were expressed in 41% and 43% of all NSCLC, respectively. ALDH was expressed in 36% of adenocarcinomas (AD)and 65% of squamous cell carcinomas (SCC), while SOX2 was expressed in 36% of AD and 80% of SCC., respectively. Taking all cases into consideration, the expression of ALDH and SOX2 significantly correlated with each other (p=0.003). No prognostic value of the abundance of ALDH and SOX2-expressing cancer cells was found with regard to all NSCLC or in AD. In contrast, for SCC, a significantly better prognosis with longer cancer-specific survival (CSS) and disease-free survival was found in tumours with higher ALDH expression, while a longer CSS was found in those with higher SOX2 expression. Contrary to the hypothesis that a high TIC content indicated by high combined ALDH and SOX2 expression would predict poor patient outcome, amongst all NSCLC, the combined phenotype of SOX2+/ALDH-was associated with the worst prognosis compared with the SOX2+/ALDH+(p=0.026) and SOX-/ALDH-(p=0.048),while no significant difference was observed with the SOX-/ALDH+ phenotypes. In view of the tight correlation between ALDH and SOX2 protein levels, in vitro studies were performed to investigate whether ALDH could be an upstream regulator of SOX2 expression. Pharmacological inhibition of ALDH enzyme function led to down-regulation of SOX2 mRNA and nuclear protein expression in lung cancer cell lines, indicating a regulatory role of ALDH on the SOX2 stemness pathway in lung cancer. In summary, the findings implicate complex factors are likely to be involved in determining the expression levels of ALDH and SOX2 in clinical lung cancers and their mechanisms affecting patient survival remain to be clarified. Further investigations on the specificity of ALDH/SOX2 as TIC marker, TIC interaction with the tumour micro-environment, and potential complex antagonistic functions of ALDH in TIC maintenance are required. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Objectives To evaluate the association between exposures to the main chemical contaminants released by the industry in freshwater and the rise in cancer cases among the population in China. Methods A systematic review was undertaken of the scientific literature compiled in the MEDLINE (via PubMed©), Google scholar, Web of Knowledge. The descriptors used were "cancer", "water pollution”, “industry” and “chemical", limited to studies that relevant to the research questions. Articles selected were of any type in English, from the inception of the indexing of the primary source until July 28th of 2013. With the quantitative data, Health impact assessment formulas are developed and then applied to subsequent data to make estimate. Results The search generated 306 articles, from which 10 were selected after applying the inclusion and exclusion criteria. The analysis of freshwater contaminants that attributed to industry in this review included aromatic amine, vinyl chloride, benzene, hexavalent Chromium, dioxin, and others of industrial origin. The majority of the studies find a significant link between exposure to drinking water contaminants and the increase in cancer cases, especially in the rural areas. In some of the studied populations a significant dose-response relationship was observed. Discussion After reviewing the included studies and the estimation of health impact assessment, I concluded that the association between cancer risks and industrial contamination in freshwater resources in China does indeed exist. While there are several other factors that interact the cancer risks, such as agriculture related water pollution and rapid growth of population. Taking into account that most of the articles were located in western countries, more Chinese studies are required in order to know the effect of freshwater contamination on cancer risks, in particular among those who lived in rural industry area. Conclusion This study provides the first estimated health impacts based on the relationship between industrial freshwater pollution and cancer risks, supporting decision makers to formulate public health recommendations to ensure a safer and healthier environment in the future. However, further study is critically needed for the prevention of this form of contamination. / published_or_final_version / Public Health / Master / Master of Public Health

A facultative anaerobe such as Escherichia coli is able to switch between the aerobic and anaerobic modes of metabolism in response to O2 availability. This adaptation is primarily controlled by a global transcription regulator called FNR (fumarate nitrate reduction). The key property that allows FNR to act as an O2 responsive transcription factor is its capability to dimerize and being activated upon binding of an O2 labile [4Fe-4S]2+ cluster. Previous functional studies have largely focused on the regions of FNR analogous to CRP (cAMP receptor protein), a prototype CRP/FNR family protein which X-ray crystal structure has been resolved. However, E. coli FNR contains extra N- and C-terminal regions that are conserved among various FNR orthologs but are absent in CRP. The functions of these two regions have not been resolved. In this study, their functions and physiological significance to the O2 sensing capacity of FNR were systematically investigated. A three-alanine (3-Ala) scanning library on amino acid 2-19 and 236-250 of FNR was constructed and selective 3-Ala substitution mutants exhibited variable defects. These defects were found to be due to their impairment of intracellular FNR protein levels which was unique only among FNR mutations in these two regions. Introduction of 3-Ala substitution at the residues 239-244, resulting in LAQ239-241A3 and LAG242-244A3 respectively, caused an especially accelerated degradation and decrease of intracellular FNR proteins. These variants were found to be degraded by the ClpXP protease. Sequence alignment of FNR orthologs revealed a highly conserved “L239XXL242XG244” motif, and my experimental data further revealed that L239 and L242 were important residues and were responsible for the defects of LAQ239-241A3 and LAG242-244A3, respectively. Circular dichroism analysis revealed that introduction of LAQ239-241A3 caused conformational changes with a significant loss of secondary structures in FNR. These studies taken together suggest that the N- and C-terminal regions of FNR play an important role in mediating the intracellular protein level of FNR. My studies also specified the ClpXP signals as the N-terminal RR9-10 and C-terminal VA249-250, and indicated that VA249-250 is a more important site than RR9-10 in targeting FNR to proteolysis. The second topic of the thesis involves exploration of the regulatory mechanism of an anaerobically activated multidrug efflux pump MdtEF in E. coli. MdtEF is an important multidrug efflux pump that causes antibiotic resistance upon overexpression. Previous studies revealed that expression of MdtEF was significantly upregulated under anaerobic conditions, but its regulatory mechanism was unknown. In the current study, systematic analyses on the unusually long promoter region of the gadE-mdtEF operon which drives the expression of MdtEF were performed. It was found that unlike FNR, mdtEF was not regulated at post-translational level by proteolysis, but at transcriptional level through the promoter region of gadE. My study showed that anaerobic activation of mdtEF was mediated by the anaerobic regulator ArcA and nitrate responsive regulators NarL and NarP. Important promoter regions P3 and P1 were also identified. This study provides essential molecular basis for the upregulation of MdtEF in a host and clinically relevant conditions. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Background: Although some classic risk factors of NPC such as salted fish, tobacco and vegetable consumption were established a few decades ago, no convincing evidence that the decreasing trend in NPC incidence and mortality rates seen in most parts of the world could be explained by the changes of these consumption. As different histological types and age groups may have distinct risk factor profile in NPC development, it is important to look at incidence and mortality trends across different jurisdictions before any further individual studies are carried out worldwide. Objectives: With the focus on both high and low risk areas, this study aimed to 1) examine the descriptive epidemiology of NPC, including the secular trends of age-standardised incidence rate (ASIR), age-standardised mortality rate (ASMR) and age-specific incidence and mortality rate by sex; 2) perform an ecologic analysis between ASIR, ASMR and classical exposures. This included a multi-jurisdiction comparison between ASIR, ASMR and salted fish, cigarette and vegetable consumption per capita and 3) investigate the secular trend of ASIR and ASMR by sex, age and histologic subtype. Methods: NPC ASMR was obtained from the WHO cancer mortality database and ASIR, age-subtype specific incidence and mortality rates were provided by various cancer registries. All age and subtype specific rates were plotted in every 5 calendar years. Per capita consumption of salted fish, tobacco and vegetables in 8 regions (Hong Kong, China, Finland, Japan, Portugal, Singapore, United Kingdom and United States) were obtained from the Food and Agriculture Organization of the United Nation (FAO) and from different corresponding governmental departments. Pearson correlation coefficients and multivariate regression analysis were performed to examine both crude and adjusted associations. Results: There were markedly decreasing trends of NPC ASIR and ASMR in most of the high risk areas over the past three decades, while only some declines in incidence and mortality rates was observed in low risk areas. No association was found between salted fish, vegetable consumption and ASMR or ASIR in any region, except in Hong Kong where lag year cigarette consumption in males was correlated with ASIR (Pearson r for 10 lag year = 0.680; 15 lag year = 0.739 and 20 lag year = 0.747, all p<0.05). Multivariate regression analysis did not show association with any of the consumption in Japan, Portugal, the US and the UK. An earlier age of onset around 45-50 was observed with non-keratinizing carcinoma as the dominant subtype in high risk areas, while in low risk area the peak age was not seen until after 60 years old with most of the cases being keratinizing NPC. Conclusions: There were distinct differences in risk profile between NPC age-standardised and age-subtype specific rates between high and low risk areas. With the general secular trends of NPC incidence and mortality rates by age and tumour type being revealed in this study, further exploration in other jurisdictions with different potential risk or protective factor is warranted. / published_or_final_version / Public Health / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer related death worldwide. Chemo-therapy has been commonly used to treat unresectable HCC but with limited efficacy. Therefore, there is an urgent demand for the development of better therapeutic approaches. Granulin-epithelin precursor (GEP) is a novel growth factor with over-expression in more than 70% of HCCs and has been demonstrated as potential therapeutic target. The aims of this study are to examine the role of GEP in chemo-resistance and the therapeutic potential of GEP antibody therapy in combination with chemo-therapy in HCC. The role of GEP in HCC chemo-resistance has been examined by HCC in vitro models in the first part of the study and by in vivo human HCC xenograft models in immunocompromised mice in the second part of the study. It was shown that the chemo-therapeutic agents selected HCC cells in vitro and in vivo resulted in increased cellular expression of GEP, ABCB5, hepatic cancer stem cell (CSC) marker CD133/EpCAM positive populations and demonstrated enhanced CSCs properties including colony formation ability and chemo-resistance. Over-expression and knockdown of GEP expressions respectively demonstrated that GEP levels were important in conferring resistance to the chemo-therapeutic agents and the drug-induced apoptosis. GEP antibody therapy not only sensitized the parental HCC populations but also the chemo-resistant subpopulations to chemo-therapy induced apoptosis. Importantly, combination of GEP antibody therapy with chemo-therapy inhibited the chemo-therapy induced GEP, ABCB5 and heaptic CSCs marker over-expression through neutralization of the secretary GEP levels in the culture supernatant, and the serum GEP levels in the HCC orthotopic mice model. In human HCC xenograft models, GEP antibody treatment alone is consistently able to inhibit the tumor growth, but is unable to eliminate the established intrahepatic tumor. Cisplatin treatment, low and high dose respectively, was only able to eradicate a fraction of the intrahepatic tumor and the residual tumors grew aggressively after chemo-drug withdrawal. Combination of GEP antibody with low dose of cisplatin resulted in significant proliferation inhibition and apoptosis induction respectively. Importantly, combination of GEP antibody with high dose of cisplatin resulted in eradication of all established intrahepatic tumor. In addition, chemo-therapy induced the Akt/PKB and MEK/ERK prosurvival pathways, disturbed the balanced between the ratio of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) member through the induction of Bcl-2. Nonetheless, combination GEP antibody therapy suppressed the chemo-therapy induced phosphorylation of PDK1, Akt, MEK, ERK, and Bcl-2 levels. It was shown that Wortmannin, the PI3K/Akt inhibitor, suppressed the expression of ABCB5 and Bcl-2 induced by chemo-therapy but showed no effect on GEP expression levels. In summary, the study demonstrated the chemo-therapy treatment alone induced the expression of growth factor GEP, drug transporter ABCB5, hepatic cancer stem cell markers expressions, and the residual cancer cells showed enhanced CSCs properties. Combination treatment with GEP antibody reversed the signaling and cancer stem cell properties induced by chemo-therapy alone. Therefore, further investigations of this combination treatment approach may lead to the development of novel therapeutic approach for the clinical treatment of chemo-resistant HCC. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

Choriocarcinoma is a malignant form of gestational trophoblastic disease arising from the trophoblastic epithelium. It is characterized by the presence of a mixed population of mononuclear cytotrophoblasts and multinucleated syncytotrophoblasts surrounded by hemorrhage and necrosis. Clinically, it is difficult to distinguish postmolar choriocarcinoma from an invasive mole. They also share similar histopathological features and are only distinguishable from invasive moles by the absence of chorionic villi. Since choriocarcinoma is an aggressive tumor with a high tendency to metastasize, it is better to have a definitive diagnosis to detect the disease at an earlier stage in order to tackle the problem before it becomes too advanced. It is thus necessary to investigate new potential markers which could help in making diagnosis at the early stage of the disease. MicroRNAs are recognized as a new class of non-coding RNAs that regulate gene expressions post-transcriptionally through translational repression or degradation of the target messenger RNAs. They are involved in almost every biological process, including cell proliferation, differentiation as well as apoptosis. MiR-125 is one of the most widely investigated microRNAs in recent years, particularly in cancers. It is a highly conserved sequence that expresses ubiquitously in multiple human organs in a tissue-specific manner. The deregulation of miR-125 was commonly found in various types of cancers. Depending on the target messenger RNAs, miR-125 exerts either tumor suppressive or oncogenic effects. There are three homologues of miR-125, including miR-125a, miR-125b-1 and miR125b-2. Since all three homologues have very similar sequence and have the same seed region, they have common mRNA targets and similar functions but may express differentially in different tissues. Factor that binds to inducer of short transcript-1(FBI-1) is a transcription factor that is involved in cell cycle arrest and terminal differentiation in different tissues. The deregulation of FBI-1 was associated with oncogenesis and the overexpression of FBI-1 was frequently demonstrated in multiple human cancers. However, the connection between miR-125 and FBI-1 in choriocarcinoma has not been reported. In this study, an inverse relationship between miR-125, including both miR-125a and miR-125b, and FBI-1 was demonstrated. Higher expression levels of miR-125a and miR-125b were demonstrated in the first-trimester extravillous trophoblasts,TEV-1, than in the JAR and JEG-3 choriocarcinoma cells, whereas the protein and mRNA expression levels of FBI-1 were significantly higher in JAR and JEG-3 cells than in TEV-1 cells. Moreover, the overexpression of miR-125 down-regulated the FBI-1 expression level in both JAR and JEG-3 cells, suggesting that miR-125 may regulate FBI-1 through a direct interaction. By treating JEG-3 cells with a histone deacetylase inhibitor, trichostatin A (TSA), the expression levelsof miR-125a and miR-125b were up-regulated while the FBI-1 was down-regulated, suggesting the possible transcriptional silencing effect on miR-125 through histone deacetylation. Altogether, miR-125 affects FBI-1 expression and may serve as a new marker to differentiate malignant tumors from the benign GTD as well as a therapeutic target. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Bacterial dehalogenase is a key enzyme involved in bioremediation of halogenated organic compounds. A dehalogenase, Deh4a, was isolated from the Gram-negative bacterium Burkholderia caribensis MBA4, which can utilize haloacetic acids as carbon source. The haloacid operon in MBA4 was identified and characterized. It is composed of the structural genes forDeh4a and a transporter Deh4p. Transcription of this operon is negatively regulated, but the mechanism and the relevant regulator are still poorly understood. In this study, magnetic DNA affinity chromatography and Tn5transposon mutagenesis were employed to explore the regulatory factors that affected the expression of this haloacid operon. A process that uses lysates from glycolate-grown cells, magnetic DNA affinity chromatography and LC-MS/MS has identified a TetR family transcriptional regulator, TetR8620, which binds to the promoter region of deh4a. Disruption of the TetR8620 gene in mutant Ins8620 abolished the formation of a slow migrating complex in electrophoretic mobility shift assay (EMSA) using lysates from glycolate-grown cells. Moreover, expressions of deh4a were enhanced in bothglycolate- and MCA- grown Ins8620. The addition of recombinant histidine-tagged TetR8620 to lysates of Ins8620 resumed the formation of a retardation complex, but different from that using purified His-tagged TetR8620.This suggested that TetR8620 is responsible for formation of retardation complexes, and an additional protein might be involved. To investigate other putative factors that interact with TetR8620, purified His-tagged TetR8620 was immobilized with Ni-NTA agarose and used for isolation of interacting proteins. Chemical cross-linking of the purified fraction with BS3established that TetR8620 interacts with a proteinof30 kDa. Separation of the cross-linked complex in SDS-PAGE gel also showed that a protein with similar MW was specifically pulled down. These results suggest that TetR8620 was interacting with a ~30 kDa protein. Protein identification using mass spectrometry assay proposed that this protein is probably a universal stress protein UspA encoding by peg.3485 or acetyl-glutamate kinase (EC 2.7.2.8) encoding by peg.714 in MBA4. Tn5transposon mutagenesis was also employed to explore the factors that regulate the haloacid operon ofMBA4. A derivative of MBA4, MK06, which contains a kanamycin resistant gene (kan) with a deh4apromoter was constructed. Kanamycin resistancy of this derivative was MCA inducible. Transposon mutagenesis was conducted on this derivative, and Tn-containing mutants were isolated as tetracycline resistant colonies on pyruvate plates. These colonies were further selected on their resistance tokanamycin in pyruvate plates. Gene peg.6589 encoding a putative transcriptional regulator, DehR1, was disrupted by Tn insertion. While the production of dehalogenase was still MCA-inducible, this mutant has partially relieved the repression of the haloacid operon in media containing pyruvate. Moreover, constitutive production of DehR1 in MBA4 decreased the transcript levels of deh4ain medium containing pyruvate or MCA. This study has identified two transcription factors, TetR8620 and DehR1, which regulate the expression of Deh4a negatively. TetR8620 is a DNA-binding protein that interacts with the deh4apromoter. Results from this study imply that the regulation of the haloacid operon in MBA4 is likely to be under the control of multiple factors. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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