The increase of calcium in the cell can induce cellular functions such as fertilization, cell division and cell communication. Calcium (Ca2+) carries out these processes through proteins called calcium sensors. An important calcium modulator is calmodulin. Calmodulin has four possible Ca2+ binding sites that have the characteristic helix-loop-helix (EF hand) motif. When the EF hands bind to Ca2+, methionine rich hydrophobic patches are exposed allowing for CaM to interact with target proteins. However, there are proteins that can interact with CaM at low levels of Ca2+ or in the absence of Ca2+. An enzyme that is activated by CaM is nitric oxide synthase (NOS), which converts L-arginine to L-citrulline and nitric oxide (•NO), where •NO is used to carry out important cellular functions. There are three isoforms of the enzyme; endothelial, neuronal and inducible NOS. The first two isoforms are activated by Ca2+-bound CaM when there is an influx of Ca2+ and are therefore Ca2+-dependent whereas inducible NOS (iNOS) is activated and binds tightly to CaM regardless of the Ca2+ concentration and is therefore Ca2+-independent. Of particular interest is the iNOS enzyme, since no three-dimensional structures of the reductase domain or the CaM-binding region have been solved. All three isoforms of NOS exist as homodimers, where each monomer consisting of a reductase domain and an oxygenase domain separated by a CaM-binding region. The reductase domain contains binding sites for NADPH and the flavins, FAD and FMN, which facilitate electron transfer from the NADPH to the catalytic heme in the oxygenase domain of the opposite monomer. The transfer of electrons from the FAD to the heme is carried out by the FMN domain which is proposed to swing between the two docking points since the distance between the two points is too large for electron transfer. This electron transfer point is under the control of CaM, which is essential for NOS activation. This dynamic process and the direct role of CaM have yet to be observed structurally. A method to monitor dynamics structurally is through the use of nuclear magnetic resonance (NMR) spectroscopy. Therefore as the first step to determine the NMR structure of the FMN domain with the CaM-binding region, the structure of the iNOS CaM-binding region bound to CaM will be determined. The structure will allow for further characterization and identification of important interactions between the iNOS CaM-binding region and CaM which contribute to the unique properties of iNOS.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 338-340). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 338-340). Also available online.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 126-139). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--School of Textile and Fiber Engineering, Georgia Institute of Technology, 2004. Directed Haskell W. Beckham. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 1989.

Thesis (Ph. D.)--University of Hong Kong, 1988.

Magnetic Resonance Spectroscopy (MRS) was used to study the relationship between brain regional concentrations of metabolites and normal aging in Chinese. Our goal in this study is to create a database of normal aging and hence enhance further understanding on the degenerative process leading to dementia and related neurodegenerative diseases. Thirty cognitively normal healthy volunteers of age 22-82 years were recruited and the bias on gender effect in data sampling was minimized by recruiting 15 females and 15 males. In the first part of the study, 1H MRS was obtained using single-voxel-spectroscopy (SVS). Offline software java-based version of Magnetic Resonance User Interface (jMRUI) was employed for data analysis. Cerebrospinal fluid was normalized using software voxel based morphormetry (VBM). Brain morphometry data was also analyzed. Brain metabolites choline (Cho), creatine (Cr) and N-acetyl aspartate (NAA) were quantified using internal water as reference. It was found that brain metabolite concentrations of Cr, Cho and NAA increase significantly with age. Gender effect on metabolite concentrations were also discovered, being higher in the female group. For brain morphometry, white matter and grey matter volumes and fractions all reveal a siginificant negative correlation with age, whereas CSF volume and fraction show a significant positive correlation with age. Gender effect was found on grey matter, white matter and intracranial volume, being higher in the male group. In the second part of the study, 31P SVS MRS was performed on the same population of volunteers. jMRUI was also employed for data analysis. Metabolic ratios were obtained. Similar to the 1H MRS study, apart from creating a database in studying normal aging, an additional aim of this 31P MRS study is to correlate with 1H MRS and assist in interpreting the corresponding metabolic activity. Brain metabolite concentrations were found to increase significantly with age. The increase of PCr (phosphocreatine)/Ptot (total phosphorus content) in posterior cingulate suggests lower metabolic activity throughout the course of aging. The strong evidence of PDE (phosphodiester) increase with age in left hippocampus proposes the fact that phospholipid membrane breakdown will be enhanced by aging. In conclusion, MRS can act as a non-invasive tool to study aging at molecular level. Metabolite levels are significant means to investigate the metabolic change in the human brain during the process of aging as the variations in metabolite levels are believed to be footprints of biochemical changes. / published_or_final_version / Diagnostic Radiology / Master / Master of Philosophy

Influenza infections continue to be a global health concern that causing both seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses, which are important for their host cell sialic acid (Sia) receptor binding and cleaving activities. Although numerous methods have been developed to study the HA and NA interactions with sialic acid, x-ray crystallography remained the only method to provide detailed information at atomic resolution. The aim of this study is to develop and evaluate a novel strategy for the investigation of influenza virus-receptor interactions, which is able to provide information about an interaction down to atomic resolution. Influenza virus-like particles (VLPs) containing HA and NA separately were developed and it was reported here for the first time that sole expression of NA in mammalian cell led to VLP formation. Characterization of these VLPs demonstrated that they are non-infectious, but morphologically and biochemically mimic the native viruses. Therefore the VLPs can be regarded as an ideal research model to study the HA-Sia interaction without the interference of NA, or vice versa. Saturation transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to determine how a binding-ligand interacts with its target protein. Modification of STD-NMR methodology was performed to adapt the technique to influenza VLP system. HA-Sia interaction was investigated in great detail and group epitope mapping of the interacting ligands was performed by analyzing the STD-NMR spectra. The data obtained are in a good agreement with the well established crystallography technique, reflecting the reliability of the STD-NMR technology. Regarding the NA-Sia interaction, my data demonstrated that substrate-hydrolysis specificity of NA is dependent on the binding of NA to those ligands. In addition, using competition experiments with NA inhibitor, a secondary sialic acid binding site was detected. It is the first direct experimental evidence that confirms avian, seasonal human and human pandemic swine-origin influenza virus N1 neuraminidases exhibit a distinct secondary binding site. In conclusion, here I presented a novel interdisciplinary strategy using VLP and NMR technology to study the interaction of influenza virus with its receptor. This method is unique in its ability to provide detailed information on the HA and NA interactions with sialic acid leading to group epitope mapping of the binding ligands, which will help us not only to understand the virus tropism but also to define new therapeutic targets. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Influenza virus has long been a major threat to public health worldwide. The virus can be highly deadly because of antigenic shift. Since the H5N1 outbreak in Hong Kong in 1997, avian flu is regarded as the next pandemic threat. For combating the disease, it is essential to investigate more on the influenza virus, in particular H5N1. Nucleoprotein (NP) is a major component of the ribonucleoprotein complex (RNP) in the influenza virus. NP exhibits both structural and functional roles for influenza virus assembly and propagation and is involved in mediating the transcription-replication process. The NP of the virus binds the RNA genome and acts as a key adapter between the virus and the host cell. It therefore plays important roles and represents an attractive drug target. Recently, the X-ray structure of H5N1 NP was solved to a resolution of 3.3 Å , which provides valuable clues on how NP carries out its functions. However, the N-terminal 1-20 residues were not resolved in the H5N1 NP crystal structure. This N-terminal region is thought to contain a nuclear localization signal (NLS), a cellular splicing factor BAT1/UAP56 binding site, and a nuclear export signal. It has been suggested that the N-terminal NLS binds to importin (a cytosolic protein) for the nuclear import of NP. In the present study, the solution structure of H5N1 NP N-terminal peptide (NP20) in membrane mimetic solvent condition was determined using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies. The CD results show that NP20 adopted an α-helical conformation. The NMR data indicate that NP20 formed a single α-helix spanning from residues Gly5 to Gly16. Surface electrostatic potentials further showed that the NP20 peptide is amphipathic in nature, which may be important for its binding with importin. NMR titration experiments have been carried out between NP20 and importin. Addition of importin into the solution of NP20 peptide caused significant broadening of the NMR signals of NP20 and progressive changes of the chemical shifts of NOE cross-peaks at increasing importin concentration confirm that NP20 could bind with importin. Therefore, the present study supports that NP20 region is the binding site of importin mediating the import of NP into the host cell nucleus. In conclusion, the knowledge gained from this study provides a better understanding on the structure of NP20 and its interaction with the host importin protein, and may serve as a template for the development of novel antiviral drug targeting NP with improved therapeutic index. / published_or_final_version / Chemistry / Master / Master of Philosophy