published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Ovarian cancer is regarded as the most lethal gynecological malignancy around the world. Despite the advancing medical improvements in both surgery and chemotherapy, the mortality rate did not appear to be reduced. This could be account for the late diagnosis of ovarian cancer until advanced stage. Recently, p-21 activated kinase 4 (PAK4), as a potential significant prognostic marker of ovarian cancer, has been widely studied on its contribution in oncogenesis properties. It was suggested that PAK4 proteins were activated and confer chemoresistance in ovarian cancers. In this study, we hypothesized that the up-regulation of PAK4 in ovarian cancers maybe resulted from mutations and amplification in genomic DNA level. Investigations on PAK4 genetic alterations were carried out. Recurrent mutations were found in the kinase domain of PAK4 in three ovarian cancer cell lines and two clinical samples. Single mutation was found in the exon 3 of PAK4 coding for GTPase binding domain (GTB). Amplifications of PAK4 genomic DNA were also found in four ovarian cancer cell lines. On top of that, dysregulated PAK4 level in chemosensitivity ovarian cancer cell line, A2780s showed PAK4 contribution in protection against apoptosis. Meanwhile PAK4 transfected chemoresistance cell line A2780cp also showed similar effect to PAK4 transfected A2780s. Kinase-dead and constitutively active PAK4 did not show any significance contribution to the apoptosis property. This may suggest that PAK4 do not operate all kinase domains towards apoptotic function. Immortalized normal ovarian epithelial cell line, HOSE6-3 was also upregulated with PAK4 transfection. However it did not induce the oncogenesis property of cell survival. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Triptolide, a diterpene triepoxide extracted from traditional Chinese medicinal herb Tripterygium wilfordii Hook. F has been shown to have profound inhibitory effects against tumor progression, pathological angiogenesis and inflammation. However, the mechanisms by which triptolide exerts these effects remain unclear. To understand its cellular mode of action, biotinylated/desthiobiotinylated and fluorophore-labeled triptolide derivatives were used as probes to identify cellular proteins that bind to triptolide. By using two different approaches for screening drug-protein interactions, the most prominent cellular protein bound to triptolide was confirmed to be peroxiredoxin 1 (PRDX1). This result was validated by demonstrating the ability of triptolide or its conjugated probes to bind recombinant human PRDX1. Specificity of the drug-protein interaction was established by competitive inhibition of binding of fluorophore-labeled triptolide to PRDX1 by triptolide itself. Two binding sites of triptolide to PRDX1 were found, one of which being Cys173 as confirmed by orbitrap LC-MS/MS analysis. Further study by size exclusive chromatography revealed that triptolide altered the oligomeric state of PRDX1. The decameric form of PRDX1 was dissociated into lower molecular weight species in the presence of triptolide. This observation was responsible for attenuation of PRDX1’s chaperone activity upon triptolide treatment, which was supported by evidence from both light scattering and native mass spectrometry studies. Functionally, triptolide’s synergistic effect on stress-induced cell apoptosis may be mediated, at least in part, by the interaction of triptolide with PRDX1 and the consequent inhibition of its chaperone activity. Several natural products, Celastrol, Withaferin A and Radiciol were discovered as new PRDX1 inhibitors and confirmed to physically interact with PRDX1 and exert similar functional effects as triptolide. The interaction between PRDX1 and those natural products may shed light on the detailed mechanism of their biological actions and render PRDX1 a potential target for cancer therapy. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

T7 RNA polymerase is a single protein capable of driving transcription from a simple promoter in virtually any context. This has made it a powerful tool in a range of biotechnology applications. In this work, previous efforts to evolve or engineer T7 RNA polymerase are reviewed. This work is then expanded upon, first with the development of a method for the cell-free evolution of T7 RNA polymerase based on the functioning of an autogene. The autogene is a transcriptional feedback circuit in which active T7 RNA polymerase proteins transcribe their own gene, resulting in exponential amplification of their genetic information. While this system is doomed by an error catastrophe, this can be delayed by the use of in vitro compartmentalization. In response to the limits of the autogene, a novel directed evolution approach termed compartmentalized partnered replication (CPR) is presented. CPR couples the in vivo functionality of a gene to its subsequent in vitro amplification by emulsion PCR. The use of CPR to generate a panel of six versions of T7 RNA polymerase, each specific to one of six promoters, is described. Separately, a rational engineering approach, taken to facilitate the high-yield transcription of fully 2′-modified RNA, is detailed. Two sets of mutations to T7 RNA polymerase, previously known to confer thermal stability and enhance promoter clearance respectively, can be used to enhance the activity of existing T7 RNA polymerase mutants that utilize non-standard nucleotides as their substrates. Next, CPR and random mutagenesis is used to populate the functional fitness landscape of T7 RNA polymerase. This neutral drift library is then challenged to increase the processivity of T7 RNA polymerase, enabling long-range transcription. Finally, the lessons that can be learned about T7 RNA polymerase specifically and molecular evolution and protein engineering generally are discussed. / text

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Pyridoxal-5-phosphate (PLP), the active form of vitamin B6 (B6), is a co-factor for enzymes in macronutrient metabolism. Increasing protein intake may affect B6 by increasing PLP-dependent enzymes in amino acid metabolism, which may be more pronounced during moderate B6 deficiency. Decreased B6 status decreases PLP-dependent enzyme activity possibly altering macronutrient metabolism. We examined changing dietary carbohydrate: protein ratios in rats consuming recommended vs. moderately deficient intakes of pyridoxine (PN)-HCl, on plasma markers of B6 status and enzymes in intermediary metabolism. Marginal B6 deficiency decreased all plasma B6 vitamers except for pyridoxic acid. Protein intake (40% energy) significantly reduced plasma PN and tended to decrease plasma pyridoxal with no significant alterations in plasma homocysteine or cysteine. Hepatic cystathionine-γ-lyase, glycogen phosphorylase, plasma aspartate and alanine aminotransferase significantly decreased with marginal B6 deficiency and cystathionine-γ-lyase decreased with increasing protein intake. Marginal B6 deficiency significantly increased hepatic glycogen with no changes in plasma haptoglobin. / October 2015