Master of Science / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.

G-protein coupled receptors (GPCRs) are integral membrane proteins which represent primary cellular targets for intracellular signalling. Many of these receptors are altered in disease states and hence are the target for over 50% of marketed drugs. Despite their physiological importance, high-throughput, cell-free assays which measure functional or signalling activity are only recently being investigated. The current approach by the pharmaceutical industry to initially screen compounds for functionality is to use heterologous cell-based assay formats. The aim of this work was to reconstitute a cell-free GPCR signalling system on an appropriate platform (surface) as a prototype for future rapid drug screening and other applications. The proof-of-concept approach involved using the �2A-adrenergic receptor (�2A-AR) containing cell membrane preparations as the model GPCR, reconstituted with a set of heterotrimeric G-proteins; G�i1 and �1�2 (the signal transducing complex being termed a �transductosome�). However, other receptors and G-proteins were also investigated. Receptors were initially obtained from natural (tissue) sources, however in the later stages they were expressed in a heterologous system (insect or mammalian expression system). G-proteins were expressed in Spodoptera frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor expression was verified by radioligand binding assays and endogenous G-proteins were removed from membrane preparations using the chaotropic agent urea to allow for reconstitution with purified G-proteins. Signal transduction through the transductosome was measured using the [35S]GTP�S binding assay. Receptor activated [35S]GTP�S binding was used to determine functional reconstitution and to validate that the system was working in the normal physiological manner both on and off a surface (with surface attachment being via histidine attachment on the G�i1 (6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent �2-AR antagonists), Prazosin (potent �1- AR antagonist) and Propranolol (�-AR antagonist) displayed the appropriate rank order for this class of receptor. This cell-free, surface-attached signalling complex prototype may have use in the future development of drug screening and discovery assay technologies as well as other applications as an alternative to cell-based assays which are not readily amendable to miniaturisation, long term storage and therefore stable robust microarray formats.

Fecal specimens from 130 different mares were collected from an endemic farm for 2 consecutive years at 4 different times pre- and post-foaling (41 mares contributed data in both years). A modified NANAT agar medium was used to quantitatively culture 1-g aliquots of the mare feces without inhibition of growth of Rhodococcus equi. Once the R. equi in the mare feces were quantified and the total concentrations of R. equi determined, a colony immunoblot procedure was performed to detect the presence of the virulence-associated protein antigen on the isolates. This allowed for the proportion and concentration of virulent R. equi to be determined. Foals that were found to have ultrasonographic evidence of peripheral pulmonary abscessation or consolidation underwent aseptic trans-cutaneous tracheobronchial aspiration. Positive results of TBA were used to categorize foals as affected with R. equi pneumonia. R. equi pneumonia developed in 31% of the foals. Shedding of virulent R. equi was observed in at least 1 sampling period for every mare examined, and >33% were culture-positive during all sampling periods. However, significant differences were not observed in either the fecal concentrations of total or virulent R. equi from dams of affected foals compared to dams of unaffected foals. No significant temporal changes in the fecal concentrations of R. equi were observed. It was concluded that dams of affected foals do not shed more R. equi in feces than do dams of unaffected foals, indicating that heavier shedding by particular mares does not explain infection in their foals. However, the finding that virulent R. equi were excreted in the feces of all sampled mares indicates that mares are likely an important source of R. equi for their surrounding environment.

Phosphohistidine phosphatase 1 (PHPT1) is a small protein, consisting of 125 amino acids, that catalyzes the dephosphorylation of histidine but does not have any activity towards other phosphorylated amino acids. PHPT1 was identified in 2002, and is so far the only mammalian histidine phosphatase known, but still little is known about its physiological role. No mammalian histidine kinases have hitherto been identified. Phosphorylation is one of the most important ways in which the structure and activity of a protein may be changed after translation. Proteins are phosphorylated on the side chain of amino acid residues. When a hydroxyl is phosphorylated the result is a phosphoester and when a nitrogen is phosphorylated the result is a phosphoamidate. Histidine may be phosphorylated on either of the two nitrogens of the imidazole ring of the side chain. The resulting phosphoamidate bond is labile and rich in energy, which makes histidine phosphorylation highly reversible and flexible. However, histidine phosphorylation is less studied than that of the phosphoesters due to the acid lability of the phosphoamidate bond. The work described in this thesis was focused on further elucidating the physiological role of PHPT1. Amino acid residues of importance for the activity of PHPT1 were identified, and mutants with decreased phosphatase activity were produced. These mutants have been used in studies on the function of PHPT1. By using immunohistochemical methodology the localization of PHPT1 in both mouse and human tissues was determined, with mainly similar results. A general finding was that expression of PHPT1 was high in epithelial cells with short turnover time, indicating that PHPT1 may have an important role in proliferating cells. We have also developed a comparatively fast and simple screening method for determination of PHPT1 activity. Since research in this field has been hampered by the lack of efficient and practical methodology, hopefully this new method will be an asset in search of inhibitors for PHPT1, which in turn may be used for detection of the elusive mammalian histidine kinases, the finding of which may give major breakthroughs in the field.

Fecal specimens from 130 different mares were collected from an endemic farm for 2 consecutive years at 4 different times pre- and post-foaling (41 mares contributed data in both years). A modified NANAT agar medium was used to quantitatively culture 1-g aliquots of the mare feces without inhibition of growth of Rhodococcus equi. Once the R. equi in the mare feces were quantified and the total concentrations of R. equi determined, a colony immunoblot procedure was performed to detect the presence of the virulence-associated protein antigen on the isolates. This allowed for the proportion and concentration of virulent R. equi to be determined. Foals that were found to have ultrasonographic evidence of peripheral pulmonary abscessation or consolidation underwent aseptic trans-cutaneous tracheobronchial aspiration. Positive results of TBA were used to categorize foals as affected with R. equi pneumonia. R. equi pneumonia developed in 31% of the foals. Shedding of virulent R. equi was observed in at least 1 sampling period for every mare examined, and >33% were culture-positive during all sampling periods. However, significant differences were not observed in either the fecal concentrations of total or virulent R. equi from dams of affected foals compared to dams of unaffected foals. No significant temporal changes in the fecal concentrations of R. equi were observed. It was concluded that dams of affected foals do not shed more R. equi in feces than do dams of unaffected foals, indicating that heavier shedding by particular mares does not explain infection in their foals. However, the finding that virulent R. equi were excreted in the feces of all sampled mares indicates that mares are likely an important source of R. equi for their surrounding environment.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 33-36).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 85-89). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 85-89). Also available on the Internet.

Thesis--Ph. D., University of Hong Kong, 1981.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 108-114).