Thesis (M. Phil.)--University of Hong Kong, 1988.

Apoptosis is a biologically essential phenomenon executed in large part by caspases. Members of the caspase family are activated at different points during apoptosis to proteolyze specific substrates. Given that both excessive and insufficient apoptosis is related to the pathogenesis of various diseases, proper regulation of caspases and apoptosis is necessary for the health of living organisms. Inhibitor of apoptosis (IAP) proteins are endogenous inhibitors of caspases, and since XIAP, the prototypical IAP, binds to and inhibits caspases, all IAPs have been speculated to engage in similar inhibition mechanisms. However, in this dissertation, I demonstrate that cIAP1 binds to the effector caspases-3 and -7, through distinct mechanisms. cIAP1 readily binds to and ubiquitinates, but dos not directly inhibit the activity of fully mature caspase-7. By contrast, cIAP1 does not bind to caspase-3. cIAP1 binding to caspase-7 is mediated primarily by the N-terminus of the large subunit of caspase-7. An AKPD motif located on the N-terminus of caspase-7 is involved in the proteasome-mediated degradation of caspase-7 in cells, thereby decreasing the sensitivity of these cells to apoptosis. Thus, I demonstrate for the first time that cIAP1 is capable of inhibiting caspase-dependent apoptosis through indirect regulation of caspase activity. / text

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

Not available / text

Not available / text

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Movements of calcium (Ca) across the maternal and fetal aspects of the human placenta were investigated using the isolated placental lobule dually perfused <I>in vitro.</I> Tissues uptakes and releases of calcium were measured and the effects on calcium movements by calcium-protein binding in the perfusion fluids, (associated with extracellular pathways and non-uniform perfusion), evaluated. The effects of ouabain, dinitrophenol (DNP), and cooling on calcium movements were measured and compared to movements of Na⁺ and K⁺. These indicated the presence of active transport of calcium but no evidence was obtained for Ca²⁺/Na⁺ exchange. Cyclic adenosine 3', 5' -monophosphate (cAMP) levels in dually perfused tissue were measured following microwave fixation. This technique was used to measure changes in tissue cAMP production following exposure to forskolin, 3-iso-butyl-l-ethyl-xanthine (IBMX), and various fragments of both bovine parathyroid hormone (bPTH) and human parathyroid hormone-related peptide (hPTHrP). Rises in cAMP were produced by exposure to bPTH(1-34) but not hPTHrP(1-34), hPTHrP(67-86) or hPTHrP (107-138). It is concluded that calcium is actively transported across the placenta but there is no major contribution via a Ca²⁺/Na⁺ exchanger. The patterns of calcium uptake as a function of perfusate calcium concentration support the evidence of other workers that extracellular pathways are present in the syncytiotrophoblast. A significant amount of passive movement of calcium may therefore take place across the perfused placenta. The 1 to 34 region of the PTH molecule stimulates the production of cAMP by the trophoblast, but there is no indication that this has any effect on transplacental Ca transport.

xxi, 126 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1978

Thesis (D.Sc.) -- University of Adelaide, Dept. of Physical and Inorganic Chemistry, 1974.