http://hdl.handle.net/2374.OX/4067 2013-05-24T03:07:55Z http://hdl.handle.net/2374.OX/105007 Hydrogenase Inhibition by O2: Density Functional Theory/Molecular Mechanics Investigation Dogaru, Daniela [Fe-Fe]-hydrogenases are enzymes that reversibly catalyze the reduction of protons to molecular hydrogen, which occurs in anaerobic media. In living systems, [Fe-Fe]-hydrogenases shift the reversible reaction towards H2 formation. The [Fe-Fe]-hydrogenase H-cluster is the active site, which contains two iron atoms (Fep-Fed, i.e., proximal and distal iron). Because most experimental and theoretical investigations confirm that the structure of di-iron air inhibited species is FepII-FedII-O-O-H-, O2 has to be prevented from binding to Fed in all di-iron subcluster oxidation states in order to retain a catalytically active enzyme. By understanding the catalytic processes of metalloenzymes, researches are enabled to produce an excellent source of fuel and energy storage (H2) for the future, which is clean and highly energetic when reacted with oxygen. H-cluster oxidation in gas phase, and in aqueous enzyme phase, has been investigated by means of quantum mechanics (QM) and combined quantum mechanics-molecular mechanics (QM/MM). The inhibitory process occurs at the coordination site, distal iron (Fed), of the catalytic H-cluster. The processes involved in the H-cluster oxidative pathways are O2 binding, e- transfer, protonation, and H2O removal. We found that oxygen binding is non-spontaneous in gas phase, and spontaneous for aqueous enzyme phase where both Fe atoms have oxidation state II; however, it is spontaneous for the partially oxidized and reduced clusters in both phases. Hence, in the protein environment the O2-inhibited H-cluster is obtained by means of exergonic reaction pathways. A unifying endeavor has been carried out for the purpose of understanding the thermodynamic results vis-à-vis several other performed electronic structural methods, such as frontier molecular orbitals (FMO), natural bond orbital partial charges (NBO), and H-cluster geometrical analysis. Since hydrogenases become O2 inactivated, residue mutations were carried out in order to make them O2 resistant. Residue mutations consist of deletions and substitutions 8 �… radially outward from Fed. In order to screen the polar residues (in the 8 �… apoenzyme layer), individual residue deletions were carried out to determine what residue substitutions should be made to improve O2 inhibition. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, FepII-FedII, FepII-FedI, and FepI-FedI of [Fe-Fe]-hydrogenase. From the screened residues, two deletions (�”Thr152, and �”Ser202) were found most effective in hindering O2 binding to Fed. The two-residue deletions, �”Thr152and �”Ser202, on FepII-FedII hydrogenase, gave �”GQM/MM = +5.4 kcal/mol, which evidently hinders O2 binding. An improvement in Gibbs��™ energy (+4.4 kcal/mol) has also been found for FepI-FedI hydrogenase. Comparing the simultaneous residue deletions (�”Thr152 and �”Ser202) with the dual residue substitutions (Thr152Ala, and Ser202Ala), a small difference in Gibbs��™ energy has been found (�”GQM/MM ~ +2 kcal/mol), for O2 binding, which is attributed to an overall charge of approximately zero for alanine. The eventual propose hydrogenase mutation in molecular biology laboratory should avail researchers in using it for the full cells of the future. 2009-04-09T23:55:27Z http://hdl.handle.net/2374.OX/105005 Tenuous Legitimacy: The Administrative State, the Anti-Government Movement and Constitutional Democracy Peffer, Shelly L. The public administration literature is inundated with books and articles despairing about the legitimacy crisis in the field. There have been numerous bases proposed for legitimizing the administrative state, including expertise, virtue or public service, and leadership and vision. Yet the issue remains contested, and the lack of agreement has wide reaching implications. One under-examined implication is the role that this tenuous legitimacy has in weakening the administrative state's ability to temper anti-government sentiment. This dissertation explores the connections and patterns in the ideologies, actions, and philosophical foundations of strongly held views that the administrative state is an illegitimate democratic institution. These domestic anti-government ideologies are illuminated through case studies of the sovereign citizens movement, the modern militia movement, and the patriot movement. By studying these groups it becomes clear that the anti-government ideology is, at least partly, a result of these groups interactions with the administrative state. The implication of these cases is that the current legitimacy arguments are ineffective in countering these strongly held anti-government sentiments. This research argues that in order for citizens to, not only believe that the administrative state is legitimate, but to experience this, the administrative state must be legitimized in practice not in theory. For public administrators legitimizing the administrative state must include a more direct relationship with citizens in the practice of expertise, virtue and public service, and leadership and vision. Through this practice, not a tenuous theory, public administrators may be able to start repairing the relationship that they have with citizens and truly legitimize the administrative state. 2009-04-09T23:55:15Z http://hdl.handle.net/2374.OX/105003 ROTOR POSITION AND VIBRATION CONTROL FOR AEROSPACE FLYWHEEL ENERGY STORAGE DEVICES AND OTHER VIBRATION BASED DEVICES Alexander, BXS Flywheel energy storage has distinct advantages over conventional energy storage methods such as electrochemical batteries. Because the energy density of a flywheel rotor increases quadratically with its speed, the foremost goal in flywheel design is to achieve sustainable high speeds of the rotor. Many issues exist with the flywheel rotor operation at high and varying speeds. A prominent problem is synchronous rotor vibration, which can drastically limit the sustainable rotor speed. In a set of projects, the novel Active Disturbance Rejection Control (ADRC) is applied to various problems of flywheel rotor operation. These applications include rotor levitation, steady state rotation at high speeds and accelerating operation. Several models such as the lumped mass model and distributed three-mass models have been analyzed. In each of these applications, the ADRC has been extended to cope with disturbance, noise, and control effort optimization; it also has been compared to various industry-standard controllers such as PID and PD/observer, and is proven to be superior. The control v performance of the PID controller and the PD/observer currently used at NASA Glenn has been improved by as much as an order of magnitude. Due to the universality of the second order system, the results obtained in the rotor vibration problem can be straightforwardly extended to other vibrational systems, particularly, the MEMS gyroscope. Potential uses of a new nonlinear controller, which inherits the ease of use of the traditional PID, are also discussed. 2009-04-09T23:55:01Z http://hdl.handle.net/2374.OX/105001 MASS SPECTROMETRY-BASED HIGH THROUGHPUT APPROACH FOR IDENTIFICATION OF MOLECULAR MODIFICATION OF OXIDATIVE PROCESS IN RESPIRATORY Song, Wei Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) have been implicated in generating reactive species and promoting oxidative modifications in numerous diseases. The detection and identification of potential pathways has been proven extremely challenge due to evanescence nature of these reactive species. An alternative approach to study the involvement of oxidative modification is to detect and quantify the stable molecular fingerprints, like oxidized tyrosine species, in biological matrices. Previously reported analytical methods for quantifying oxidized amino acids have typically been limited by: low sensitivity, specificity and the failure of detection potential oxidation products generated during sample handling. Using GC/MS in combination with stable isotope dilution, a sensitive and self-quality control assay was developed. Using this isotope-dilution GC/MS and multiple allergen challenge models, we demonstrate that EPO is the major pathway for generating protein-bound 3-bromotyrosine in vivo. However, the need for chemical derivatization of samples increases the possibility that artifactual generation of oxidation products which may interfere with the detection of native levels. Requirement of unique derivatization reagent for each oxidized tyrosine results sample analysis both cumbersome and time/labor intensive. Therefore, we develop a liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) assay that simultaneously detects and quantifies multiple structurally informative protein oxidation products along with their precursor amino acids. Use of four HPLC systems in multiplex array with column switching permits on-line analyses of only relevant portions of chromatographic profiles and up to four-fold increased throughput efficiency of mass detector usage. Using this high-throughput multiplexed array-based LC-MS/MS method, we found significant correlations among individual oxidized tyrosine in sera from healthy volunteers. These correlations provide chemical evidence that activated leukocytes utilize various physiological available substrates to participate multiple oxidative modifications in vivo. In rhinosinusitis study, significantly elevated protein-bound oxidized tyrosine species were detected in healthy nasal tissue compared to that at the peripheral level. In addition, rhinosinusitis patients had more severe oxidative modification in their nasal tissue. The evidence supports elevated bromination and tyrosylation may contribute to the pathogenesis of rhinosinusitis. 2009-04-09T23:54:45Z