Theses and Dissertations at Montana State University (MSU)
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Item The role of hemoglobin A1c testing in undiagnosed diabetes and myocardial infarction in emergency and intensive care settings(Montana State University - Bozeman, College of Nursing, 2018) Nicola, Marcus; Chairperson, Graduate Committee: Teresa SerightStatement of the Problem: The disease of diabetes is insidious. Its complications can be devastating and, if left untreated, often leads to early mortality. With the effects of diabetes being so extensive within body systems, complications like myocardial infarction are all too common. To complicate the matter further, a large portion of the population of diabetics is undiagnosed. Having no knowledge of this disease process allows the disease to progress unfettered for an indeterminate amount of time. If diabetic status is unknown, an increased risk of mortality from MI exists. Systematic Hgb A1C testing for myocardial infarction patients may provide prognostic data for undiagnosed diabetics and increase our ability, as providers, to develop treatment plans to address the increased risk of mortality posed to these individuals. Methods: All MI patients admitted to ED and ICU charts were screened for hemoglobin A1C testing and diabetic care planning as evidenced in their discharge summary. Results: This project found that testing with Hgb A1C only occurred in 40% of MI patients. Of those tested, 8% were found to be new diabetics. This project also found that 16.3% of the patients tested were pre-diabetic and that this was only noted in 1.8% of these patients' discharge summaries. Conclusions: Currently, Hgb A1C testing is underutilized in MI patients. Identification of new diabetics in this population allows providers to address this diagnosis in a manner that can prevent the complications all too common to diabetics with heart disease.Item Structural investigation of IsdB, the hemoglobin receptor of Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2014) Fonner, Brittany Anne; Chairperson, Graduate Committee: Valerie CopieStaphylococcus aureus is an opportunistic pathogen which when left unchallenged can cause severe toxicity and death in mammals. Critical to S. aureus growth is the ability to scavenge iron from hemoglobin (Hb). To acquire iron S. aureus has evolved a sophisticated protein-mediated heme acquisition pathway, which comprises nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport and degradation. A key protein of the acquisition pathway is the hemoglobin receptor protein IsdB, which comprises two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the mechanisms and molecular basis of the IsdB-mediated heme acquisition process is not well understood. In order to provide more insights into the mode of function of IsdB, structural studies via nuclear magnetic resonance (NMR) spectroscopy were employed on different domains of IsdB. The three-dimensional solution structure of IsdBN1 revealed an immunoglobulin-like fold that is consistent with other NEAT domain proteins. Site directed mutagenesis studies revealed two key aromatic residues, F164 and Y167, involved in methemoglobin (metHb) interactions with IsdB. The protein variant F164D did not bind to metHb under NMR conditions. In heme transfer studies between metHb and IsdB constructs containing the two NEAT domains and the linker region, the amino acid substitution of F164D diminished but did not knock out the ability of IsdB to remove heme from metHb. A double amino acid substation of F164D and Y167D did abolish heme transfer from metHb to IsdB, therefore identifying key residues of IsdBN1 interaction with metHb. Studies of the linker region revealed an overall alpha-helical propensity and an interaction between the linker region and the second NEAT domain, IsdBN2. Solving the apo-IsdBN2 structure revealed slight differences in the heme-binding pocket, specifically in beta-strands 7 and 8 that interact with the heme moiety, when compared to the published crystal structure of holo-IsdBN2. The findings in this thesis provide a structural role for IsdBN1 enhancing the rate of extraction of heme from metHb by IsdBN2 and interactions between the domains of IsdB.Item Function of non-heme-binding domains of the Staphylococcus aureus IsdB protein in heme assimilation from methemoglobin(Montana State University - Bozeman, College of Agriculture, 2014) Li, Dengfeng; Chairperson, Graduate Committee: Benfang LeiAs a hemoglobin acceptor, IsdB rapidly and efficiently acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. The pathway of heme assimilation in S. aureus involving IsdB has been established; however, the mechanism of rapid and efficient heme assimilation of metHb heme by IsdB remains unclear. IsdB consists of five major domains: the N-terminal (ND), NEAr Transporter 1 (N1), middle (MD), heme binding NEAr Transporter 2 (N2), and C-terminal (CD) domains. The goal of this study is to elucidate the roles of these IsdB domains in the metHb-to-IsdB heme transfer reaction. Deletion of the CD region does not alter the kinetics and equilibrium of the reaction. Sequential deletions of ND and N1 of ND-N1-MD-N2 progressively reduce heme transfer rates but have no effect on the reaction equilibrium. Further deletion of MD decreases the efficiency of heme transfer from metHb to N2. The MD domain reduces heme dissociation from holo-N2 and drives the metHb/N2 reaction to the formation of holo-N2. ND-N1-MD and N2 fragments, but not ND-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction, indicating an MD/N2 interaction. Analyses of MD, N2, and MD-N2 mixture by size exclusion chromatography support an interaction between MD and N2. These results indicate that ND-N1 and MD domains critically contribute to the kinetics and equilibrium of the metHb-to-IsdB heme transfer reaction, respectively. The results also suggest that CD functions as a spacer to position IsdB in the cell wall envelope for heme relay through the cell wall. These findings support a mechanism of direct extraction of metHb heme by IsdB that involves the four structural domains of IsdB.Item Chemical interaction of nitric oxide and human hemoglobin(Montana State University - Bozeman, College of Letters & Science, 2004) Luchsinger, Benjamin Peter; Chairperson, Graduate Committee: David J. Singel; Patrik R. Callis (co-chair)In the past decade an entirely new areas of biomedical science have emerged with the recognition of the critical roles played by nitric oxide (NO) in biology. The discoveries of the role of NO in the cardiovascular systems, where it participates in regulating the tone of blood vessels and the flow of blood, raised an intriguing biochemical problem about the chemical interaction of NO and hemoglobin in blood. Historically, investigation of the interaction of NO and hemoglobin have suggested the formation of heme-Fe(II)NO hemoglobin and nitrate. Neither of these products is considered to exhibit NO-related bioactivity, thus the reaction of NO with hemoglobin was thought to quench the bioactive potential of NO. In this thesis we explore the idea that these are not the only two fates of NO in its interaction with hemoglobin. Product formation following encounters of NO and hemoglobin under various conditions of oxygenation, oxidation, and NO/heme ratios are analyzed by UV/Vis optical spectroscopy, EPR (electron paramagnetic resonance) spectroscopy, and colorimetric chemical analyses. This battery of methods enables quantification of the hemoglobin species: heme-Fe(II)NO (with distribution over the hemoglobin subunits) heme-Fe(III)NO, oxyhemoglobin, methemoglobin, deoxyhemoglobin, S-nitrosohemoglobin (SNO-hemoglobin). We present results that establish a previously unsuspected favoring of protein nitrosylation at heme and thiol positions in the reaction of oxyhemoglobin with NO. We demonstrate coupling of heme reduction/oxidation activity with thiol reactivity. We provide evidence for transfer of the NO group from heme to thiol and vice versa. We demonstrate unprecedented subunit selectivity of interactions of NO with hemoglobin. Finally, we present results that highlight the nitrite reductase activity of hemoglobin whereby nitrite, which has a significant concentration in blood, is converted to bioavailable forms of NO. Overall, the in vitro chemistry reported here provides for a complete basis for rationalizing the in vivo biochemistry required for the hemoglobin/NO system to serve as regulator of blood flow.Item Oxidation of human nitrosylhemoglobin monitored by UV-Vis and EPR spectroscopies : detection of products and intermediates(Montana State University - Bozeman, College of Letters & Science, 2005) Williams, Elizabeth Mary; Chairperson, Graduate Committee: David J. SingelOnce viewed only as a toxic free radical, nitric oxide (NO) has been established as an essential and ubiquitous signaling and regulatory molecule in biological systems. Notably, NO was identified as the endothelium-derived relaxing factor (EDRF) in the blood. NO is capable of complex redox chemistry and interaction with a host of protein families. Among these proteins is hemoglobin (Hb) which can interact with NO at the level of the heme and can bind NO at Cys93 on its â subunit to form S-nitrosylated Hb (SNO-Hb). NO bound as SNO-Hb is chemically labile and thus preserves bioavailability of NO. However, when NO reacts with oxyHb or deoxyHb NO bioavailability is quenched by conversion to nitrate or by tightly binding the heme, respectively. Therefore, the question is raised as to how NO can be EDRF in the presence of such high Hb concentrations in the blood. One way NO availability can be preserved is by exploiting the redox chemistries of both Hb and NO. Human Hb(NO)4 oxidation by K3Fe(CN)6 was studied and products and intermediates were identified by UV-Vis and EPR spectroscopies. Periodically, samples were withdrawn from the reaction mixture for nitrosylation product and/or EPR analysis. Reaction spectra converted to heme species concentration vs. time plots through leastsquares fitting of five basis spectra. These data were then utilized to generate a de minimis model of the oxidation reaction. We demonstrate that the oxidation of Hb(NO)4 by K3Fe(CN)6 1) leads to the production of SNO-Hb, 2) occurs preferentially at the â heme, and 3) proceeds through an HbFeIIINO intermediate.Item Electron paramagnetic resonance investigations of adducts of human hemoglobin(Montana State University - Bozeman, College of Letters & Science, 2010) Schwab, David Earl; Chairperson, Graduate Committee: David J. SingelHemoglobin transports oxygen to the tissues of the body. The delivery of oxygen to tissues by hemoglobin is dependent on blood flow, which is determined by vessel tension regulated by local oxygen gradients. Dilation of the blood vessels in the microcirculation of tissues under high metabolic demand is induced by the endothelium-derived relaxation factor, nitric oxide, in a process known as hypoxic vasodilation. Although the means of nitric oxide bioactivity preservation and transportation in the blood are disputed, it is clear that S-nitrosohemoglobin, a nitrosylated variant of hemoglobin, plays a pivotal role. The details surrounding S-nitrosohemoglobin formation in vivo, however, remain uncertain. Using electron paramagnetic resonance (EPR) spectroscopy, in conjunction with detailed spectral simulation and least-squares fitting, various hemoglobin species which possibly participate in the formation of S-nitrosohemoglobin were characterized. The EPR spectrum of methemoglobin-nitrite, a purported precursor to S-nitrosohemoglobin formation, was determined to be a composite spectrum arising from the presence of two species, the origin of which is proposed to lie in the differences between the distal heme pockets and histidine residues of the alpha- and beta-subunits of hemoglobin. By direct measurement of methemoglobin-nitrite by EPR spectroscopy, the weak affinity of methemoglobin for nitrite was confirmed, precluding nitrite-methemoglobin from having a direct role in physiological hypoxic vasodilation. Furthermore, the temperature dependence of the EPR spectra of the various species of neat methemoglobin was determined, as was the temperature dependence of the nitrosyl-hemoglobin (Hb(NO) 4) spectrum at high frequency. The high frequency spectrum of Hb(NO) 4 provided additional resolution of the axial and rhombic components of the spectrum, but revealed no evidence of distinct subunit spectra. Finally, synthetic routes to generate Fe(II)NO/Fe(III)-Hb hybrids have been presented, which, among other things, demonstrated that bolus addition of nitric oxide can produce similar results as the addition of time and condition dependent nitric oxide donors. Overall, this work expands the understanding of hemoglobin, specifically with regard to hemoglobin species with possible involvement in S-nitrosohemoglobin formation.Item Exogenous ligand effects on S-nitrosohemoglobin formation in reactions of methemoglobin with nitric oxide(Montana State University - Bozeman, College of Letters & Science, 2009) Mellmann, Lisa Jean; Chairperson, Graduate Committee: David J. SingelBlood's function of carrying oxygen to tissues is regulated by physiological oxygen gradients that are coupled to vasoconstriction and vasodilation by nitric oxide bioactivity. However, the mechanism by which local oxygen tension utilizes nitric oxide signaling in order to regulate blood flow remains a major unanswered question in biology. Hemoglobin in red blood cells appears to be an ideal sensor, but lack of knowledge about hemoglobin's chemistry with nitric oxide creates a problem for understanding how hemoglobin induces vasodilation. A central focus of this work was to illuminate complexity and response of nitric oxide interactions with hemoglobin while showing how S-nitrosohemoglobin plays a pivotal role in this response. Electronic absorption and electron paramagnetic resonance spectroscopy were employed to observe reactions of low-spin methemoglobin species with nitric oxide. Models were developed and tested to establish the chemistry involved and the global hierarchy of reactions. De minimis models for all experiments are reported. Electron paramagnetic resonance spectra confirmed the formation of low-spin iron species that, upon the addition of nitric oxide, showed a decrease in low-spin methemoglobin species and the formation of nitrosyl hemoblobin with a beta-subunit preference. We report the yield of S-nitrosohemoglobin production in reactions of nitric oxide with various methemoglobins distinguished by axial ligand and iron spin state.