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Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Throughout recent history scientists have struggled to elucidate the biochemical and biophysical mechanisms that guide the assembly of macromolecular structures. The early models of "sub-assembly" or "self assembly" attempted to explain how individual components could interact in a precisely regulated manner to form higher-ordered complex biological structures. Subsequent studies, using viral systems as assembly models, demonstrated how protein-protein and protein-nucleic acid interactions assist in lowering the thermodynamic barriers that typically disfavor assembly.Due to their simplicity, viruses provide an ideal system to investigate the biophysical mechanisms that drive the assembly of complex biological structures. Proper virion assembly requires numerous macromolecular interactions that proceed along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. Since the identification of scaffolding proteins in the bacteriophage P22, homologues have been discovered in many systems. Scaffolding proteins play multiple roles during morphogenesis by inducing protein conformational switches and lowering the thermodynamic barriers to promote virion assembly, while ensuring the efficiency and fidelity of the final product.

Gel filtration coupled with radioimmunoassay of fractions has demonstrated the existence of an 8000 dalton immunoreactive form of GIP (glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide), which may be a precursor in the biosynthetic pathway. A monoclonal antibody to GIP has been shown to have highly suitable characteristics for affinity purification of different species of IR-GIP. An enzyme-linked immunosorbent assay (ELISA) was developed for GIP, employing the monoclonal antibody and was used for screening fractions for peptides with the same antigenic determinant i.e. IR-foras of GIP. Classical strategy used in peptide purification may result in loss of related peptides if they are sensitive to the pH or temperature conditions used. Tissue from hog duodenal and jejunal mucosa was boiled and extracted into acetic acid. Peptides were then adsorbed to alginic acid, eluted with 200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted with methanol. The methanol insoluble fraction demonstrated the highest content of IR-GIP₈₀₀₀⋅ The overall acidic charge on the larger IR-GIP oUUU moiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP₅₀₀₀ was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A µBondapak C₁₈ column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The ability to suppress a prepotent response is a crucial component of cognition that begins to develop during infancy and peeks during preschool. As part of understanding how one develops inhibitory control, learning about what conditions may help or hurt task performance is of great interest. The purpose of this project was to study the effects of familiarity and novelty on inhibitory control. Thirty-five preschoolers between two and five years of age were tested in four different versions of the Dimensional Change Card Sort (DCCS). Performance was no different among standard, 2D-familiar, and 3D-familiar conditions. When comparing novel with the standard condition, children performed worse (37% and 68% respectively). Findings support the attentional inertia hypothesis. An exploratory analysis on temperament was also investigated. Children who scored higher in effortful control performed better in the 2D-familiar condition. / Master of Science

In metozoans, most of the key events that lead to cell activation and inhibition are controlled by tyrosine phosphorylation. Extracellular signals are transmitted by membrane bound receptors, which have intrinsic kinase activity or themselves recruit intracellular kinases to specialised inhibitory or activating phosphorylation motifs. In this way, the pattern of kinase activation creates its own turnover and can rapidly generate amplified signals by positive feedback, or recruit inhibitory proteins to counteract the signals. This process of inhibition is also constitutive since it requires continuous counter-inhibition by phosphatases at the cell surface and intracellularly even in the absence of ligands. The absence of phosphatase activity results in unbridled protein phosphorylation and form this and other data it has been proposed that the triggering of the T cell receptor and other co-receptors may result simply by physical exclusion of the large phosphatases such as CD45 from the vicinity of the receptors. Superagonist monoclonal antibodies may work in a similar way, by binding receptors close to the plasma membrane and excluding extracellular phosphatases. The work described in this thesis seeks to discover if antibody superagonists can be generated against the T cell inhibitory cell surface receptor PD-1 and test if this approach can attenuate the immune response. Using in vitro assays of lymphocyte activation and a mouse model expressing human PD-1, this study characterises a series of anti-PD-1 antibodies and shows how patterns of inhibitory activity varying according to binding sites. The inhibitory effects of the anti-PD1 antibodies are seen in the humoral, cellular and transplant immune responses. Agonistic anti-PD1 antibodies induce regulatory T cells and may have role in suppression of autoimmune disease. The thesis suggests that superagonism may be harnessed clinically to dampen the immune response, through activation of inhibitory receptors.

Class of 2017 Abstract / Objectives: Evaluate thymoquinone (TQ) and thymol (THY) inhibitory activities against α-glucosidase enzyme by using an in vitro assay and determine the IC50 (concentration of TQ/THY to inhibit 50% of maximum enzyme activity). Methods: Various concentrations of thymoquinone and thymol were incubated, separately, with one concentration of the substrate - p-nitrophenol-α-D-glucopyranoside (PNPG) (<<Km) in presence of α-glucosidase enzyme. A positive and a negative control consisting of acarbose, and buffer, respectively, were included in the incubation as well. The incubation time was set at 30 min in a 37 °C controlled water bath. The enzyme activity was determined by detecting and quantitating the levels of p-nitrophenol using a spectrophotometer set at 405 nm. The percent inhibition exhibited by any studied drug was calculated as shown in equation 1. % inhibition = Absorbance Substrate Alone – Absorbance of Substrate + Inhibitor Absorbance Substrate Alone Results: Results of the in vitro incubation of thymoquinone, thymol and acarbose revealed “statistically” significant inhibition of -glucosidase (p<0.05). At 400 g/ml, thymoquinone inhibited the enzyme activity by ~52 % whereas the enzyme inhibition by thymol and acarbose were calculated to be ~84% and 57%, respectively. IC50 were tentatively determined although the maxima inhibitions of the inhibitors were not reached fully. IC50s were calculated as 234 μg/ml, 304 μg/ml and 157 μg/ml for each of thymoquinone, thymol and acarbose, respectively. Conclusions: Thymoquinone and thymol do exhibit antagonistic pharmacological activity against α-glucosidase.