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

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published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

No description available.

In efforts to expand the limited amount of functional groups available for anion recognition, a series of highly acidic, strongly hydrogen bond-donating groups were envisaged as suitable candidates. These included the thoroughly studied N-aryl sulfonamides along with the less utilized N-acyl sulfonamides and tetrazoles. These groups were affixed to a well-understood supramolecular platform in calix[4]arene and their binding affinities for various halides and oxyanions probed. It was found that although in its least energetically favourable conformation that is orthogonal to the aryl group to which it was bound, the tetrazole proved a superior anion-binding element. Noting that tetrazoles prefer co-planarity with aryl neighbours, a series of pyrrolyl-tetrazole anion binding compounds were prepared, first a simple bidentate pyrrolyl-tetrazole which when tested for anion binding affinity demonstrated some of the strongest binding with anions for a bidentate compound ever observed, especially chloride. It was then conceived to hybridize this new binding motif with the well-known amidopyrrole moiety and two new tetrazolyl-amidopyrroles were constructed. When compared to an ester-functionalized pyrrolyl-tetrazole, binding strength with halides was not much different, leading to the postulation that the amide N-H may just be a spectator in the binding event, and the electron-withdrawing nature of the adjacent carbonyl was what led to the binding potency. Nonetheless, a new class of diversifiable anion binders with superior strength to analogous amidopyrroles has been constructed and could perhaps be used in a variety of functional applications. / Graduate

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.

Enantiopure A and A lanthanide complexes, bearing a phenanthridinium or a dipyridoquinoxaline chromophore as a sensidser, have been designed with the aim of developing structural and reactive probes for nucleic acids. Their interaction with DNA was studied using various spectroscopic techniques. A certain degree of stereoselectivity in DNA binding was discerned. The A enantiomers of the Eu tetramide and of the EuPh(_3)dpq complexes interacted with nucleic acids in a predominantly intercalative binding mode, by inserting their planar aromatic chromophore between the base-pairs. The former showed a preference for C-G sites, while the latter bound preferentially to A-T base-pairs. A rather different binding mode, probably involving the minor groove, was revealed in the interaction of the A enantiomer of the EuPh(_3)dpq complex with nucleic acids, with a higher affinity for C and G bases. In the presence of nucleic acid, a charge transfer process occurred in each case, which quenched the singlet excited state of the phenanthridinium moiety or the lanthanide excited state (in Ph(_3)dpq complexes). In the unique case of the EuNp(_3)dpq complexes, the interaction resulted in an increase in the metal emission intensity and lifetime, as a consequence of the protection of the molecule, probably accommodated in the DNA minor groove, from a quenching process. This light switch' effect can be exploited in the development of spectroscopic probes. The TbNp(_3)dpq, on the other hand, was found to generate singlet oxygen efficiently and could therefore act as a reactive probe. Moreover, the EuPh(_3)dpq and TbPh(_3)dpq complexes showed extraordinarily high emission quantum yields in aqueous media, due to the favourable photophysical properties of the dpq antenna as well as the nonadentate nature of the Ph(_3)dpq ligand. This makes them valuable luminescent probes.