Graduation date: 1995

Introduction: To evaluate the effectiveness of smoking cessation intervention, reliable outcome is essential. Exhaled nitric oxide (ENO) is decreased in smokers, tends to normalize after cessation and might be a good tool to evaluate abstinence. Objective: To evaluate changes in ENO after smoking abstinence of 7 or more days. Methods: 58 smokers in a cessation attempt and 12 non-smokers were recruited: 7 visits for smokers and 2 for non-smokers. Carbon monoxide and cotinine were used to detect smoking status. Results: ENO is decreased in smokers compared to non-smokers (10.8 vs. 20.1 ppb, p<0.001). There was no significant difference in ENO pre and post quitting (p=0.080) although there was a trend to increase as early as 3 days after abstinence (10.78 vs. 15.11, p>0.05). There were no differences in nasal NO measurements (p=0.278). Conclusion: ENO doesn’t seem to be a reliable marker of short-term abstinence.

Introduction: To evaluate the effectiveness of smoking cessation intervention, reliable outcome is essential. Exhaled nitric oxide (ENO) is decreased in smokers, tends to normalize after cessation and might be a good tool to evaluate abstinence. Objective: To evaluate changes in ENO after smoking abstinence of 7 or more days. Methods: 58 smokers in a cessation attempt and 12 non-smokers were recruited: 7 visits for smokers and 2 for non-smokers. Carbon monoxide and cotinine were used to detect smoking status. Results: ENO is decreased in smokers compared to non-smokers (10.8 vs. 20.1 ppb, p<0.001). There was no significant difference in ENO pre and post quitting (p=0.080) although there was a trend to increase as early as 3 days after abstinence (10.78 vs. 15.11, p>0.05). There were no differences in nasal NO measurements (p=0.278). Conclusion: ENO doesn’t seem to be a reliable marker of short-term abstinence.

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 53-67).

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 113-129).

Thesis (M.S.)--Mississippi State University. Department of Chemical Engineering. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.