published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Previous genetic evidence suggested that RecA was required in SOS mutagenesis for its regulatory role and perhaps some other nonregulatory role (Mount, 1977; Blanco et al., 1982). I undertook a genetic study which confirmed the above studies and provided further evidence that RecA protein appeared to have a dual "role in mutagenesis; first, the cleavage of LexA repressor for the derepression of specific SOS genes and second, one or more additional role(s). For these studies a new phage mutagenesis assay was developed which allows rapid scoring of SOS mutagenesis in a large number of host mutants. I next conducted a genetic analysis to determine if the newly defined RecA mutagenesis function was separable by mutation from the numerous other phenotypes which are known to be influenced by RecA protein. From the study of recA mutants it appears that the RecA mutagenesis function(s) is genetically separable from the following RecA phenotypes: LexA cleavage, lambda cI repressor cleavage, UV resistance and homologous recombination. In addition, I discovered that the LexA cleavage function and lambda cI cleavage function is also separable. I also studied in some detail the novel genetic properties that I uncovered for recA432 mutant strains. recA432 was defined as a mutagenesis defective allele (Kato and Shinoura, 1977). LexA cleavage in recA432 cells was more easily induced that in recA⁺ cells, causing lethal filamentation of these mutant cells even at very low UV doses. I concluded that the basis for the Mut⁻ phenotype was this strain's propensity to lethally filament, which complicated the detection of mutant cells. In another set of experiments, I examined the regulatory requirements for SOS mutagenesis and Weigle phage-reactivation; I wanted to determine which SOS operons must be derepressed for this process. lexA(Ind⁻) mutant cells are defective in mutagenesis because they cannot derepress specific SOS genes required in this process. I found that the selective derepression of umuDC was sufficient to restore mutagenesis to these lexA(Ind⁻) mutants; however, derepression of umuDC and recA was required for phage reactivation.

This report describes the development and use of an expression vector system based on the single-stranded DNA bacteriophage M13. A derivative of M13mp8, designated M13mp8/P, was prepared in which the promoter and N-terminal codons of bacterial genes may be fused to a portion of β-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes, and to determine the mutational changes by dideoxy sequence analysis. The feasibility of this method was demonstrated by identification of a large number of mutations in the regulatory regions of two genes, recA and lexA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. My results suggest that this method will be generally useful for mutational analysis of transcriptional and translational regulatory elements. The mutants that were isolated by the above approach were used to investigate the specificity of LexA protein binding by quantifying the repressibility of a several mutant recA and lexA operator/promoter regions fused to the E. coli galactokinase (galK) gene. The results of this analysis indicated that two sets of four nucleotides (terminal nucleotide contacts), one set at each extreme end of the operator, are most critical for repressor binding. In addition, our results indicate that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator had comparable effects on repressibility. The inferred symmetry of the interaction justified the reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus: (5') CTGTATAT. Although the first four positions of this half-site sequence have the greatest effect on LexA repressor binding, the last four are well conserved among binding sites and appear to modulate repressor affinity. The role of the terminal nucleotide contacts and the mechanism by which the internal sequences affect repressor binding is discussed.

In the tissue distribution study, the expression of GHR2 is significantly higher than GHR1 in many tissues of the seabream including the gonad, kidney, muscle, pituitary and spleen. In vivo hormone treatment data indicated that cortisol and testosterone have differential expression regulation between GHR1 and GHR2. On the other hand, hepatic expression of both GHR1 and GHR2 in seabream was decreased by estradiol treatment. In primary cultures of seabream hepatocytes, the expression patterns after treatment by the various concentrations of hormones were consistent with the in vivo results. / To study the actions of environmental estrogens on the somatotropic axis, a transgenic yeast system was developed for estrogenicity screening. The fish estrogen receptor (gfER) and a reporter vector containing the estrogen responsive element (ERE) were expressed in yeast cells as a means to identify potential estrogens. Using this system, more than fifty chemicals including pesticides, herbicides, industrial chemicals and phytoestrogens were screened. Ten compounds including dibutyl phthalate (DBP) and bisphenol A (BPA) were demonstrated to exhibit estrogenic activities. And a compound (malachite green, MG) with novel anti-estrogenenic activities was identified. Then BPA and MG were focused to explore the disrupting effects of environmental estrogens on the two GHRs. Through the method of real-time PCR, both compounds could attenuate the gene expression level of GHRs in seabream hepatocytes. Using the method of luciferase assay, the signal transduction of the two GHRs was found to be desensitized by both BPA and MG. / Two genomic contigs of putative growth hormone receptor (GHR) were identified in fugu and zebrafish genomes by in silico analysis, suggesting the presence of two GHR subtypes in a single teleost species. This hypothesis was tested by cloning the full-length cDNA sequence of a second GHR subtype from the black seabream in which the first GHR subtype has been previously reported. Phylogenetic analysis of known GHR sequences from various vertebrates revealed that fish GHRs cluster into two distinct clades, viz. GHR1 and GHR2. The biological activities of both GHR subtypes from seabream had been examined using the reporter transcription assays in cultured eukaryotic cells. It was demonstrated that both of them have differential signal transduction upon Spi 2.1, beta-casein and c-fos promoter activities. / by Jiao, Baowei. / "December 2006." / Adviser: Christopher H. K. Cheng. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5662. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 150-180). / 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. / Abstract in English and Chinese. / School code: 1307.

Neuroblastoma is a heterogeneous pediatric malignancy originating from the developing sympathetic nervous system, with poor long-term survival for high-risk patients (~40%). About half of advanced neuroblastomas harbor high-level amplification of the MYCN gene, and these tumors show few, if any, additional driver lesions. Despite significant increase in the body of knowledge of genetics in neuroblastoma, all the high-risk patients follow similar therapeutic procedures and little advancement has been made on molecular target based therapies. The major challenge is to dissect the complexity and heterogeneity of these tumors to find driver genes and activated pathways that are essential for the survival of these cancer cells. We used an integrated systems biology approach to define the core regulatory machinery responsible for maintenance of an aggressive neuroblastoma phenotypic state. In the first part of the thesis, I will discuss our computational approach to decipher the tumor heterogeneity by subtype classification, followed by identification of master regulator protein modules for three distinct molecular subtypes of high-risk neuroblastomas, which were validated in a large independent cohort of cases. We propose that such modules are responsible for integrating the effect of mutations in upstream pathways and for regulating the genetic programs and pathways necessary for tumor state implementation and maintenance. The second part of the thesis is focused on experimental validation of putative master regulators in the subtype of neuroblastomas associated with MYCN amplification. By using RNAi screening followed by experimental and computational analyses to elucidate the interdependencies between the top master regulators, we identified TEAD4-MYCN positive feedback loop as a major tumor maintenance mechanism in this subtype. While MYCN regulates TEAD4 transcriptionally, TEAD4 regulates MYCN through transcriptional and post-translational mechanisms. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally orchestrate 70% of the subtype-specific gene expression signature. TEAD4 gene expression was associated with neuroblastoma patient survival independently of age, tumor stage and MYCN status (P=2.1e-02). In cellular assays, MYCN promoted growth and repressed differentiation, while TEAD4 activated proliferation and DNA damage repair programs, the signature hallmarks of MYCN-amplified neuroblastoma cells. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk neuroblastoma pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. The critical role of the core master regulator module in controlling tumor cell viability, both in vitro and in vivo, and its clinical relevance as a prognostic factor highlights TEAD4 as a novel and highly effective candidate target for therapeutic intervention. In this thesis, we demonstrate that interrogation of tumor specific regulatory networks with patient-derived gene expression signatures can effectively elucidate molecular subtypes as well as the core transcriptional machinery driving subtype specific hallmarks. This approach enables identification of oncogenic and non-oncogenic dependencies of high-risk neuroblastoma and is applicable to other tumor subtypes.