Thesis (Ph.D.)--University of Western Sydney, 2008. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 77-87).

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 181-212). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-86).

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

<p>Multidrug tolerance (MDT) is the ability of pathogenic bacteria to survive killing from exposure to multiple antibiotics, and is a major obstacle in the treatment of infectious disease. A small population of bacteria (0.0001%) termed persisters is the culprit that causes MDT and allows these cells to persist. In <italic>Escherichia coli</italic>, the HipBA toxin&ndash;antitoxin pair plays a role in multidrug tolerance. HipA, a 50 kDa serine protein kinase, is the more stable toxin and abrogates cell growth in the absence of the more labile antitoxin HipB. HipB is a transcription repressor that binds to the four conserved (TATCCN₈GGATA) operator sites of the <italic>hipBA</italic> promoter to autoregulate expression of the <italic>hipBA</italic> operon. Delineation of the molecular mechanism of HipB&ndash;<italic>hipBA</italic>operator binding is critical to understand fully the regulation of persistence by HipB. Thus, we determined the equilibrium dissociation constants (K_d) of HipB for each of the four <italic>hipBA</italic> operators and the paired operator sites <italic>O₁O₂</italic> and <italic>O₃O₄</italic>. We found that the affinity of HipB for binding the <italic>O₁</italic> and <italic>O₃</italic> operators is seven to eight times higher than for the <italic>O₂</italic> and <italic>O₄</italic> operators. In addition, the affinity of HipB for the <italic>O₁O₂</italic> and <italic>O₃O₄</italic> operators is at least four times higher than the <italic>O₁</italic> and <italic>O₃</italic> operators. The HipB&ndash;operator complex structures reveal that HipB makes the same key contacts to the conserved TATCC motifs and bends each operator DNA by the same extent between 50&deg; to 70&deg; implying thus the affinity differences are attributed to indirect readout of the 8 base pair spacer (N₈). Mutational studies on residues involved in HipB&ndash;DNA interaction revealed the contribution of a series of selected residues to binding affinity with residues K38 and Q39 contributing greatly to affinity whereas other base contacting residues S29 and A40 contribute less to affinity. Surprisingly residue S43, which is involved in a hydrogen bond to the DNA phosphate backbone contributes more than expected because S43 forms a hydrogen bond network with nearby water molecules. </p><p>HipA was the first described <italic>bona fide</italic> persistence factor. The <italic>hip</italic> locus was discovered through a mutagenesis screen whereby <italic>hipA7</italic> was isolated. Described herein, biochemical and structure&ndash;function studies on HipA7, the gene product of the high persistent mutant allele having two point mutations G22S and D291A, revealed that the D291A mutation weakens the binding affinity for HipB by 3 to 4 fold. The HipA7 structure revealed the conformational heterogeneity of the P&ndash;loop motif (the ATP binding motif), which suggests a dynamic role of the loop in regulation of the kinase activity of HipA. To identify <italic>in vivo</italic> HipA substrates, we developed a mass spectrometry (MS)&ndash;based kinase assay, which led to identification of a novel phosphorylation site (residue S348) on HipA and a proposed consensus phosphorylation motif +&#981;S, where +, &#966; and S designate a positive, hydrophobic and serine amino acid residue, respectively. Phosphorylation of peptides with this consensus motif, including the S150 (EENDF<bold><underline>RIS</underline></bold>VAGAQEK), S348 (TGI<bold><underline>HIS</underline></bold>DLK) and GltX (GK<bold><underline>KLS</underline></bold>KRH), was confirmed subsequently by the MS&ndash;based kinase assay. Further analysis of the HipA7 structure suggested that HipA might undergo pyrophosphorylation on residue S150, and the MS&ndash;based kinase assay confirmed pyrophosphorylation of HipA. </p><p>Thus, our data support that HipA is a persistence factor via its kinase activity and precise <italic>hipBA</italic> gene regulation through HipB binding tightly to <italic>O₁</italic> and <italic>O₃</italic> is critical for the survival of bacteria in the presence of antibiotics. In addition, we propose a consensus motif for HipA substrates.</p> / Dissertation

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy