The reactive nitric oxide species (RNOS) nitroxyl (HNO) has exhibited both beneficial and deleterious biological effects. In particular, HNO autoxidation can lead to harmful modifications of biomolecules, yet the products of HNO/O₂ remain undetermined. A conceivable product is peroxynitrous acid (ONOOH), however a comparison of the chemistry of HNO/O₂ to synthetic peroxynitrite (ONOO⁻) determined that these RNOS have distinct reactive profiles. This study compares the reactivity of HNO and NO⁻ in the presence of O₂ to synthetic ONOO⁻ and the autoxidation of HNO at high pH (NO⁻/O₂) in an effort to shed light on the products of HNO autoxidation. All species exhibited the capacity for two-electron oxidation, but differences between ONOO⁻ and NO⁻/O₂ and HNO/O₂ were observed in terms of one-electron oxidation, hydroxylation, nitration and buffer effects. NO⁻/O₂ exhibited a reactive profile similar to ONOO⁻, suggesting that protonation of ONOO⁻leads to a unique species from the autoxidation of HNO.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP. / National Institutes of Health / Revisión por pares

In this study, the effects of bioprocess operation parameters on benzaldehyde lyase production were systematically investigated. For this purpose, the research program was carried out in mainly four parts. In the first part of the study, Escherichia coli K12 (ATCC 10798), having the highest benzaldehyde lyase production capacity, was selected as the host microorganism. Next, using the selected microorganism, the production medium was designed in terms of its carbon and nitrogen sources. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 1.8 kg m-3 and 745 U cm-3, respectively, in the medium containing 8.0 kg m-3 glucose, 5.0 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, by using the designed medium, the effects of bioreactor operation parameters, i.e., oxygen transfer and pH, were investigated in pilot scale bioreactor. Oxygen transfer effects on benzaldehyde lyase production were investigated at QO/VR=0.5 vvm / N=250, 375, 500, 625, 750 min-1 and at QO/VR=0.7 vvm, N=750 min-1 conditions. The highest cell concentration and benzaldehyde lyase activity were obtained at 0.5 vvm, 500 min-1 condition as 2.3 kg m-3 and 860 U cm-3, respectively. Finally, the effect of pH was investigated for benzaldehyde lyase production process at Qo/VR=0.5 vvm, N=500 min-1 condition, at pHC=5.0, 6.4, 6.7, 7.0, 7.2 and 7.8 values. Among the investigated pH values, the highest cell concentration and enzyme activity were obtained at pHC=7.0 condition as 2.1 kg m-3 / 775 U cm-3. However, the values obtained at this condition, were lower than the values obtained at pHUC=7.2 uncontrolled pH operation. Hence, medium oxygen transfer condition and uncontrolled pH operation are found to be favorable for benzaldehyde lyase production.

No description available.

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase.

No description available.

pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37 °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (> 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells. / pH är en viktig parameter i optimeringen av animalcellsprocesser och har sammankopplats med specifika konsumtions- och produktionsmönster rörande extracellulära metaboliter. Det extracellulära pH-värdets effekt på den intracellulära metabolismen är dock inte fullt klarlagd. Metabolisk flux analys är en matematisk metod som kan användas för att generera intracellulära fluxfördelningar i celler, exempelvis som en funktion av någon yttre parameter. Det övergripande målet i detta arbete var att utveckla ett odlingssystem och experimentellt protokoll för odling av CHO-celler som kan användas för att generera data för metabolisk flux analys där målet är att studera effekten av pH på den intracellulära cellmetabolismen. En IgG-producerande CHO-cellslinje odlades först i skakkolv för att välja ut ett akademiskt kemiskt definierat medium med känd sammansättning. Därefter följde försök att anpassa cellerna till det valda mediet. Det visade sig att ett kommersiellt medium behövde tillsättas för att ge godtagbar tillväxt och viabilitet. Effekten av den biologiska bufferten HEPES på cellernas tillväxt och viabilitet, samt pH-stabiliteten under odling, undersöktes också genom odling i skakkolv. HEPES-koncentrationer i det undersökta intervallet (7.5 – 45 mM) hade ingen större effekt på tillväxt och viabilitet. För de högre koncentrationerna var buffertkapaciteten något bättre precis vid inokulering. Dessa koncentrationer var dock ej tillräckliga för att ge stabilt pH under odlingen. Baserat på dessa resultat övergavs tanken på att använda skakkolvsodling för att odla celler vid olika pH-värden. Ett odlingssystem och ett protokoll baserat på en 100 mL Spinnerflaska med pH-reglering specialdesignades istället för projektet. I det färdiga systemet fanns lösningar för kontinuerlig övervakning av pH och DO, stabil temperatur vid 37 °C, justerbar omrörningshastighet, samt valmöjligheten att flöda in luft, O2 och CO2. Dessutom infördes möjligheten att koppla loss flaskenheten från reglersystemet för byte av medium och provtagning. För att demonstrera systemet genomfördes en odling med den anpassade IgG-producerande cellinjen enligt principen för pseudo-perfusion vid pH 7.0. Odlingen pågick under 24 dagar och optimerade reglerinställningar identifierades. Det visades att systemet kunde understödja cellkoncentrationer upp till 11 miljoner celler per milliliter, samt hög viabilitet (> 90 %). Under den senare delen av odlingen uppnåddes stabil tillväxt, vid specifika tillväxthastigheter omkring 0.7 per dygn. Den specifika konsumtions- och produktionshastigheten för metaboliterna glukos, glutamin, laktat och NH4+, samt 20 aminosyror analyserades. Majoriteten av hastigheterna stämde överens med typisk CHO-cellsmetabolism. Den förväntade konsumtionen av majoriteten av de essentiella aminosyrorna och huvudsakliga kolkällorna glukos och glutamin konfirmerades, såväl som den associerade produktionen av bi-produkterna laktat och NH4+. Odlingssystemet och det experimentella protokollet som utvecklades i detta arbete kan användas i framtida experiment för att generera data som beskriver metaboliska profiler som funktion av extracellulärt pH. Dessa data kan sedan användas i metabolisk flux analys för att dra slutsatser om pH-effekter i CHO-celler.

Ce travail concerne l'utilisation de la technique de Fracturation Hydraulique (FH) pour exploiter l'énergie géothermique des réservoirs profonds de roches sèches chaudes (HDR). La fracturation hydraulique est réalisée par injection de fluides géothermiques dans des réservoirs partiellement fracturés de faible perméabilité. Les fluides à haute pression sont destinés à faire évoluer les fissures et leur connectivité. Les valeurs de débit/pression auxquelles les fluides géothermiques doivent être pompés, ainsi que le calendrier de pompage pour initier la fracturation hydraulique, dépendent principalement des conditions géostatiques existantes (contraintes géostatiques, pression fluide et température initiales de l'HDR) ainsi que des propriétés des fissures de l'HDR (longueur, épaisseur, densité et distribution directionnelle initiales moyennes de fissures). Tous ces éléments, en sus de leurs effets sur la stabilité des forages, sont analysés dans cette recherche. Des modèles de fracturation, qui sont capables de suivre l'évolution des fissures dans toutes les orientations spatiales possibles, sont utilisés pour obtenir le tenseur anisotrope de perméabilité. Ces modèles sont intégrés dans un code domestique d'éléments finis qui est développé pour résoudre des problèmes aux limites thermo-poroélastiques. Pour supprimer/diminuer les oscillations qui accompagnent les solutions paraboliques et/ou hyperboliques lors de la convection forcée, plusieurs techniques de stabilisation ont dû être implémentées. / The application of the Hydraulic Fracturing (HF) technology to exploit geothermal energy from Hot Dry Rocks (HDR) reservoirs is addressed. HF is achieved by extensively pumping geothermal fluids to already existing fractured HDR reservoirs of low permeability. High fluid pressures are expected to drive cracks to evolve and connect. The newly created burgeoning hydraulic conduits should supposedly enhance the permeability of the existing HDR reservoirs. The flow rate/pressure values at which geothermal fluids should be pumped, as well as the pumping schedule to initiate HF, depend primarily on the existing geostatic conditions (geostatic stresses, initial HDR pressure and temperature) as well as on HDR fracture properties (initial mean fracture length, mean fracture aperture, density and orientational distribution of fractures). While these components, in addition to their effects on borehole stability, are scrutinized in this research, focus is on the evolution during circulation processes of the fracture properties. A fracturing model that is capable of tracking fracture evolution in all possible spatial orientations is used to obtain the time course of the anisotropic permeability tensor. This evolving property is integrated into a domestic finite element code which is developed to solve thermo–poroelastic BVPs: emphasis is laid on the efficiency of the doublet flow technique where a fluid gains thermal energy by circulating through the HDR reservoir from the injection well to the production well. The spurious oscillations in the hyperbolic solutions of the approximated finite element approach that are commensal with the phenomenon of forced heat convection are healed/mitigated through several stabilization approaches.