Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 313-321). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 179-229). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1992. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references (leaves 166-205). Also available via World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 87-97).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 107-121). Also available in electronic version.

The motivation of this dissertation is to provide insight towards primary ionization mechanisms within MALDI mass spectrometry. Albeit MALDI-MS is an extensively used analytical technique, the mechanism in which primary ions are created is still under scrutiny. Two current models of primary ionization exist which claim to elucidate the ion formation mechanisms within MALDI. In this work, excited state dynamics of MALDI matrices are shown to play an important role in the ionization mechanism. Upon inspection of the thermodynamic properties of commonly used MALDI matrices, no correlation was observed when plotted against their respective analyte ion yields. However, the excited state singlet lifetimes of these matrices seem to correlate well with their respective analyte ion yields. In the broadest sense, this correlation further supports the fact that photophysical properties of the matrix should be included in current UV-MALDI models. Investigation of a claim which stated singlet energy pooling reactions were absent in the MALDI matrix 2,4,6-trihydroxyacetophenone (THAP) resulted in the discovery of a new energy pooling mechanisms. Characteristic of aromatic ketones such as THAP, intersystem crossing is an efficient process in solution, which gives way to fluorescence in the solid state. Triplet pooling mechanisms from two neighboring THAP molecules are proposed and appear to be dependent on the preparation solvent used. These triplet pooling reactions are thought to play an important role in the primary ion formation mechanism within MALDI. To further investigate the theory of triplet species playing a vital role in MALDI ionization, the internal heavy-atom effect was employed to determine the effect of the triplet species. MALDI mass spectra and excited state decays of these heavy-atom substituted matrices were collected to demonstrate the relationship between triplet species and analyte ionization efficiency. Gas-phase thermodynamics and absorption at 337 nm were also examined to determine if these properties affected the analyte ion signal observed in the MALDI mass spectrum. Using the information collected from the previous study, an advanced MALDI matrix is synthesized. Addition of covalently bound iodine to the gold standard matrix, α-cyano-hydroxycinnamic acid, should drastically improve the performance of the non-substituted matrix due to the increase in triplet species present for pooling reactions. Sample preparation methods in MALDI are examined as are the effects of crystal morphology on the overall signal observed in the mass spectrum. Exciton hopping and pooling rates are highly dependent on intermolecular interactions, so it is expected that crystal packing will affect MALDI. As noted for THAP, preparation solvent plays a significant role in not only crystal morphology, but also the excited state dynamics for all matrices studied.

Due to the diverse functions of Fc eRII, such as its roles in cellular adhesion, growth and differentiation of B and T lymphocytes, rescue of B cells from apoptosis and release of cytotoxic mediators, it is clear why it is believed to be a central molecule in allergic response. Because of its important role in the regulation of IgE production, FceRII may be the primary cause of certain allergic conditions. This study attempted to express and purify a recombinant human soluble FceRII to test its effect on a B-lymphoblastoid (RPMI 8866) and a monocytic (U937) cell line. The protein was expressed in Escherichia coli inclusion bodies, before being refolded and purified in a single gel chromatography step. This pure protein was then tested for biological activity by testing its IgE binding func tion. Once proven functional, it was used to test its effect on the cell lines at three concentrations for its apoptotic rescue properties and its cytokine effects. The recombinant protein did not seem to have any significant effect on the apoptotic rescue of either cell line. While the recombinant sFceRII appeared to have a slight effect on the stimulation of IL-1ß and TNFa in the RPMI 8866 cells, there was no apparent effect on the production of NF?B. In U937 cells, the protein did not seem to have any effect on the stimulation of IL-1ß, TNFa or NF?B. However, the cytokine effects of the recombinant protein were tested on isolated PBMCs from a healthy individual and a hyper-IgE syndrome patient. The recombinant protein was able to stimulate the production of cytokines in both individuals’ PBMCs, proving that it has the same effect as the natural protein. The upregulation of these cytokines indicates that the recombinant protein is able to stimulate the immune system. Therefore, this recombinant soluble FceRII protein could possibly be used for immune therapy.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) causes a tremendous economic loss in swine industry worldwide. The capabilities to evade host immune responses and to establish persistent infection are the two hallmark features of PRRSV infection. In this dissertation, the research was mainly focused on investigating the novel mechanisms underlying PRRSV transmission and persistence. In chapter 2, the research was focused on an alternative pathway of PRRSV intercellular transmission. Our data showed that intercellular nanotube connections can be utilized for cell-to-cell spreading the core infectious viral machinery (viral RNA, certain replicases and structural proteins) of PRRSV. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2 in a receptor-independent manner. The cytoskeleton proteins F-actin and myosin-IIA were identified as co-precipitates with PRRSV nanotube associated proteins. Drugs inhibiting actin polymerization or myosin-IIA activation prevented nanotube formations and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. In chapter 3, we further showed that PRRSV infection could induce the formation of nanotubes between infected and uninfected cells following a ROS-dependent nanotube formation model. Co-culturing PRRSV-infected cells with uninfected cells rescued PRRSV-induced cell death. Mitochondrion was observed transferring from uninfected to PRRSV-infected cells. Importantly, impaired formation of nanotube or defective mitochondrion was unable to rescue infected cells from apoptosis/necrosis. Certain PRRSV proteins were detected to associate with mitochondria and transport from infected to uninfected cells through TNTs. Our results suggest that TNTs-transfer of functional mitochondria rescued PRRSV-infected cells from apoptosis/necrosis in the early stage of infection. On the other hand, mitochondria could be utilized as a cargo to transport viral materials for spreading the infection. In chapter 4, a novel mechanism s of PRRSV persistent infection has been studied. In this study, a cellular model of persistent infection was established. Strand-specific quantitative RT-PCR and RNase I treatment analysis showed that double-stranded RNA (dsRNA) conformation existed in persistently infected cells. This data has been further confirmed in vivo by performing two independent PRRSV persistence studies. Immunohistochemistry analysis showed that viral dsRNAs were detected aggregating inside the germinal centers of tonsils and lymph nodes from PRRSV persistence pigs, but RNA array analysis further showed that dsRNA in lymphoid tissues had limited ability to stimulate host antiviral responses during persistent infection stage. These results suggest that the PRRSV dsRNA functions as a mediator for viral persistence. The viral dsRNA persistence in germinal centers of lymphoid tissues may reveal a novel mechanism for PRRSV to escape antiviral immune responses. In summary, this study investigated two novel pathogenic mechanisms of PRRSV infection, which could provide insights on the development of effective control strategies.

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