Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 169-192). Also available in print.

Thesis (Ph. D.)--University of Wyoming, 2008. / Title from PDF title page (viewed Feb. 3, 2010). Includes bibliographical references.

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2004. / Includes bibliographical references.

Thesis (M.S.)--Marshall University, 2003. / Title from document title page. Document formatted into pages; contains xv, 148 p. including illustrations. Includes bibliographical references (p. 131-148).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p.113-118).

Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions. In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506. In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection. MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding. The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers. / published_or_final_version / Pathology / Master / Master of Philosophy

Epitope tagging in metazoans is an important tool for biochemical analyses and is generally accomplished by using trans-genes with in-frame epitope tags. However, protein levels from trans-genes are rarely representative of native levels. To overcome the shortcomings using trans-genes, epitope tags were introduced by homologous recombination technology, termed CLEP tagging (Chromosomal Locus EPitope tagging), immediately upstream of the stop codon of targeted genes in chicken B cell line DT40 and mouse embryonic stem (ES) cells. I first demonstrated the feasibility and promise of this technique in DT40 cells by purifying low abundance polypeptides and factors loosely associated with the SmD3 protein, a core protein participating in pre-mRNA splicing and mRNA turnover, with a TAP (tandem affinity purification) tag. Glycerol gradient separation was performed to further characterize the SmD3-associated protein complexes from the 200S fractions, corresponding to the supraspliceosomes. The purification included all five spliceosomal snRNAs. Most known snRNP-associated proteins, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs, and other RNA binding proteins were identified from the protein components. Intriguingly, the purified supraspliceosomes also contained a number of structural proteins, nucleoporins, chromatin remodeling factors, and several novel proteins that were absent from splicing complexes assembled in vitro. I showed that the in vivo analyses provide a more comprehensive list of polypeptides associated with pre-mRNA splicing apparatus as well as those that coupled transcription to the pre-mRNA processing steps. With similar techniques, the TAP tag was inserted into the chromosomal locus of a pre-mRNA splicing factor component, mSART-1 in live mice. Surprisingly, a profound autoimmune response was induced in homozygous-modified mice, due likely to an inappropriate stimulation of the immune system. I believe these mice will serve as a valuable tool for the studies of mammalian autoimmune diseases, especially those resulting from the generation of autoantibodies against RNP components. / text

Systematic identification of protein-protein interactions (PPIs) on a genome scale has become an important focus of biology, as the majority of cellular functions are mediated by these interactions. Several high throughput experimental techniques have emerged as effective tools for querying the protein-protein interactome and can be broadly categorized into those that detect direct, physical protein-protein interactions and those that yield information on the composition of protein complexes. Tandem affinity purification followed by mass spectrometry (TAP/MS) is an example of the latter that identifies proteins that co-purify with a given tagged query (bait) protein. Though TAP/MS enables these co-complexed associations to be identified on a proteome scale, the amount of data generated by the systematic querying of thousands of proteins can be extremely large. Data from multiple purifications are combined to form a very large network of proteins linked by edges whenever the corresponding pairs might form an association. Only a fraction of these pairwise associations correspond to physical interactions, however, and further computational analysis is necessary to filter out non-specific associations. This thesis examines how differing computational procedures for the analysis of TAP/MS data can affect the final PPI network, and outlines a procedure to accurately identify protein complexes from data consolidated from multiple proteome-scale TAP/MS experiments in the budding yeast \textit{Saccharomyces cerevisiae}. In collaboration with the Greenblatt and Emili laboratories at the University of Toronto, this methodology was extended to yeast membrane proteins to derive a comprehensive network of 13,343 PPIs and 720 protein complexes spanning both membrane and non-membrane proteins.

Spontaneously hypertensive rats (SHR) are a well accepted model of primary hypertension. Among other features common to human hypertension, these rats exhibit sympathetic hyperactivity. The neurons of the superior mesenteric ganglion (SMG) from SHR display enhanced collateral sprouting, higher firing rates and hyperinnervation of the mesenteric arteries compared to the SMG neurons from age-matched, normotensive Wistar-Kyoto (WKY) rats. Furthermore, SMG neurons in SHR are exposed to different conditions than are SMG neurons from WKY rats, including enhanced oxidative stress, increased afferent stimulation, and an altered hormonal environment. In order to identify proteins with potential involvement in the establishment or maintenance of peripheral sympathetic hyperactivity in SHR, we used proteomic techniques to search for differences in protein expression between the SMG of SHR and the SMG of WKY rats at 16 and 22 weeks of age. We found an upregulation of predominantly fetally expressed T1 domain and haptoglobin and a downregulation of serine protease inhibitor 2.1 in SHR relative to WKY rats at 16 and 22 weeks; Apolipoprotein-A1 was also found to be upregulated in 22 week SHR SMGs compared to age-matched WKY SMGs. These identifications improve our understanding of the ganglionic microenvironment in SHR and represent targets for the development of novel therapies to treat primary hypertension. Hypertension is one of the defining components of the metabolic syndrome, together with insulin resistance, visceral adiposity and hyperlipidemia. Non-alcoholic fatty liver disease (NAFLD) is also a common feature of the metabolic disorder, and thus primary hypertension and NAFLD are common comorbidities. Despite these clinical connections, very little is known about the effects of primary hypertension on hepatic physiology. We used proteomic techniques to search for evidence of significant involvement of the liver in SHR phenotype at the molecular level. We detected changes in the expression of several proteins involved in the regulation of oxidative stress and lipid metabolism which together show that the liver is strongly involved in the pathologies associated with hypertension. Our results suggest several novel mechanisms for the initiation of oxidative stress in SHR which could contribute to new advances in the treatment of metabolic abnormalities associated with hypertension. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-10-27 10:11:05.052

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