Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 160-170). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Multiple myeloma (MM) is a plasma cell neoplasm that remains incurable despite utilization of aggressive protocols that combine conventional and novel chemotherapy drugs. Recent studies show that drug resistance can be attributable to genetic and developmental heterogeneity in MM. One theory for the high rates of relapse and refractory disease is the existence of a pool of cancer stem cells in MM. We predict that identifying metabolic differences in the MM stem-like cells could offer novel pathways to create combination therapies that would improve the clinical outcome for patients. Utilizing human MM cell lines, we characterized the stem-like population under both normoxic and hypoxic conditions. Our results show that the MM stem-like cells have decreased basal reactive oxygen species (ROS) and increased glucose uptake. Additionally, growth under hypoxia increases the stem-like population and the expression of microRNA-210, a master regulator of hypoxia. Notably, decreasing miR-210 expression alters the rate of cellular metabolism under hypoxia. By combining therapies that increase mitochondrial ROS levels and inhibit glucose metabolism, we attained clonogenic death of MM cells under normoxic conditions. Our studies provide the rationale to further investigate drug combinations that disrupt cellular redox homeostasis to cause oxidative stress-induced cell death to improve therapies for MM patients.

Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. In the vascular system, redox reactions help regulate key physiological responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathological states, altered redox balance can cause vascular cell dysfunction and contribute to disease. It is well known that vascular diseases are associated with increased generation of reactive oxygen species (ROS). However, little is known about the molecular mechanisms connecting elevated vascular ROS and disease pathogenesis. A growing number of vascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are post-translationally oxidized to methionine sulfoxide (MetO). Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases (MSR). The work presented in this thesis focuses on the mechanistic role of reversible protein methionine oxidation in vascular disease. Chapter 1 discusses our current understanding of the role of ROS and redox reactions in vascular disease, highlighting the potential role of protein methionine oxidation. Chapter 2 investigates the significance of protein methionine oxidation in the prothrombotic phenotype of vascular disease. We focused primarily on thrombomodulin (TM), an endothelial surface protein critically involved in anticoagulation, and tested the hypothesis that oxidation of TM Met388 is a reversible process that contributes to vascular thrombosis. We found that methionine oxidation can inhibit TM anticoagulant function and contribute to thrombotic vascular disease although this process was not physiologically reversed by MSR. Overall, these results support a role of methionine oxidation as a redox regulator of thrombotic vascular disease. Chapter 3 examines the pathophysiologic role of protein methionine oxidation in the proinflammatory mechanisms of acute ischemic stroke. Both inflammation and oxidative pathways are activated during cerebral ischemia/reperfusion. We tested the hypothesis that protein methionine oxidation potentiates neurovascular inflammation and contributes to cerebral ischemia/reperfusion injury. We found that deficiency of methionine sulfoxide reductase A (MsrA) exacerbates cerebral ischemia/reperfusion injury through activation of redox-regulated proinflammatory pathways. These results support a novel role of methionine oxidation as a reversible redox regulator of acute ischemic stroke. An overall discussion is presented in Chapter 4 and highlights potential applications and future directions of this thesis project. Also discussed are additional targets of protein methionine oxidation and the increasing number of tools being developed to identify and quantify MetO in proteins. The work presented in this thesis contributes to the growing body of research showing the pathophysiologic role of reversible methionine oxidation. These data also advance our understanding of the mechanisms regulating vascular disease.

No description available.

Bibliography: leaves 59-64. / One of the contributors of the widespread interest the yeast Saccharomyces cerevisiae has received is its ability to yield and tolerate high levels of ethanol. S. cerevisiae is able to grow and remain viable in growth media containing ethanol concentrations as high as 19.8% (w/v), a level that is toxic to many other microorganisms. Since production of ethanol is a normal event in the growth cycyle of S. cerevisiae, this organisms has evolved a number of mechanisms to cope with deleterious effects of ethanol. These include induction of heat shock proteins (HSPs). Among these, HSP30 is particularly interesting in that it is the only stress-induced protein known to be instrinsically bound to the yeast plasma membrane. Another ethanol induced HSP; HSP12 has previously been shown to have a peripheral plasma membrane localisation. It has further been shown that HSP12 protects liposomes against damage by ethanol. This study was initially aimed at investigating whether there is co-operation between HSP30 and HSP12 in this membrane protection role.

Includes abstract. / Includes bibliographical references (leaves 121-158). / A recent study from the Hapgood laboratory demonstrated the presence of a novel crosstalk mechanism between the glucocorticoid receptor (GR) and gonadotropin-releasing hormone receptor (GnRHR), indicating an additional direct mechanism for the effects of stress on reproduction. The present study investigated whether this crosstalk between the GR and GnRHR involves the co-localization of these receptors to lipid rafts, providing a specialized distinct region where the receptors can be in close proximity and reciprocally modulate each other’s signaling pathways.

Bibliography: leaves 125-146. / Corynebacterium glutamicum is extensively used for the commercial production of a host of amino acids including lysine, glutamate, and threonine. Consequently, much research has been directed at analyzing nitrogen metabolism in this bacterium. In particular, our research focused on investigating the regulation of nitrogen assimilation. Initially, we searched for homologs of the Streptomyces glnR, glnII, and glnE genes in C. glutamicum. These studies, however, were met with limited success, and we therefore decided to use promoter probe vectors in order to identify nitrogen-responsive promoters.

Includes bibliographical references (leaves 149-180). / Reports on the natural and laboratory infection of cereals by rhizobium provided the impetus to embark on research using African landraces of sorghum and millet to study their interaction with rhizobia. Seven strains of root-nodule bacteria (namely Rhizobium GHR2, Bradyrhizobium japonicum Tal 110, Sinorhizobium meliloti strain 1, Rhizobium leguminosarum bv. viceae Cn6, R. leguminosarum bv. viceae strain 30, Rhizobium NGR234 and Azorhizobium caulinodans ORSS71, hereafter referred to as ""rhizobia"") that fix N2, were used to study rhizobial effects on sorghum and millet seedlings grown aseptically in Leonard jars with Yz strength Hoagland nutrient solution containing 1 mM KN03.

Includes abstract. / Includes bibliographical references (leaves 80-90). / Polyphenols have been shown to exhibit anti-viral activity in vitro, making them a promising starting point for the development of HIV treatment drugs. The main objective of this thesis was to assess the inhibitory effect of polyphenols extracted from Myrothamnus flabellifolia (Welw.) on M-MLV and HIV-I reverse transcriptases. The first part of the thesis was an attempt to isolate 3,4,5 tri-O-galloylquinic acid, the major polyphenol found in Namibian Myrothamnus flabellifolia plants. This polyphenol was successfully purified by column chromatography (Sephadex LH-20) and its purity was confirmed by HPLC and MALDI-TOF mass spectrometry. The second part of this thesis involved the development of a polymerase enzyme activity assay based on ethidium bromide fluorescence. A calibration curve for quantification of DNA was therefore prepared from the ethidium bromide fluorescence of Calf Thymus DNA. Results demonstrated that Calf Thymus DNA was a good standard for estimating the amount of cDNA synthesised during reverse transcription, thus enabling the monitoring of both M-ML V and HIV -1 reverse transcriptase activity. The reverse transcriptase activity assay was optimised using a poly (rA) template, an oligo (dTb primer and dTTP as a substrate. It was observed that the rate of catalysis for M-ML V and HIV -1 RTs decreased with increase in the concentration of dTTP, which suggested substrate inhibition. A decrease in M-MLV RT activity with increased substrate concentration was found to be due to depletion of Mg2+ ions by dTTP. True substrate inhibition was however observed for HIV-I RT, and analysis of the observed kinetics suggested the formation of an ineffective enzyme substrate complex with two substrate molecules binding to HIV -1 reverse transcriptase. A Hill coefficient of one was obtained at low dTTP concentration and less than one at high dTTP concentration, suggesting zero and negative cooperativity respectively. The final part of this thesis tested the inhibitory effect of pure and crude polyphenol fractions on the activity of M-MLV and HIV-1 RTs. Results showed that all polyphenol fractions inhibited M-ML V and HIV -I reverse transcriptase activity, with the highest inhibitory activity demonstrated by the fraction that contained pure 3,4,5 tri-O-galloylquinic acid. The 50 % inhibitory concentrations of 3,4,5 tri-O-galloylquinic acid was 0.5 μM for M-MLV RT and 34 μM for HIV-I RT. Lineweaver-Burk plots showed that 3,4,5 tri-O-galloylquinic acid inhibited both enzymes non-competitively. Pure non-competitive inhibition was observed for M-MLV RT and mixed non-competitive inhibition for HIV-I RT. Results showed that the binding of 3,4,5 tri-O-galloylquinic acid to M-MLV RT was irreversible, suggesting strong binding under the conditions tested. 3,4,5 Tri-O-galloylquinic acid, however, bound to HIV-I RT reversibly. A comparison of catalytic efficiencies showed that M-MLV RT was more efficient than HIV -1 RT under saturating substrate concentrations with Kcat (min-¹) values of II ± 3 and 1.31 ± 0.02 respectively. M-MLV RT and HIV-¹ RT were, however, equally efficient under limiting substrate concentrations with Kcat/Km (min-¹M-¹) values of 1.1 ± 0.3 x 10⁴ and 1.2 ± 0.2 x 10⁴ respectively.

Word processed copy. / Includes bibliographical references (leaves 117-132). / Since 1990, the yeast Saccharomyces cerevisiae small heat shock protein Hsp12p, has continuously appeared in data associated with stress responses in this organism. Hsp12p is expressed abundantly in response to a large variety of different stresses, but for many years has eluded researchers as to its function, primarily because the viability of yeast strains lacking HSP12 are unaffected by osmotic stress and heat shock. Subsequent studies indicated that Hsp12p played a role in the adaptation of the cell wall of Saccharomyces cerevisiae to conditions of stress. However, the exact in vivo localization, specific function and mediation of function of Hsp12p had yet to be elucidated. The localization of Hsp12p was determined by fusion to the green fluorescent reporter protein, Gfp2p and a combination of epifluorescent microscopy and confocal imagery. Chemical extraction revealed that Hsp12p was present in the cell wall while fluorescent imagery was not conclusive. This fluorescent Hsp12p construct was later employed in a novel application to sense the stress status of yeast, which bears future promise for use in an industrial setting.