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

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

Not available / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.