Glutathione (GSH) is a small antioxidant in the body and exists in large quantities compared to other antioxidants. The GSH redox state (Eh) helps developmental processes, however, when the GSH Eh is disrupted, cells may undergo significantly poor developmental pathways, possibly leading to long-lasting damages. Similarly, NADPH and Thioredoxin redox states can have a major impact on cellular function, viability, and response to both endogenous and exogenous toxicants. Subcellular, compartmentalized redox environments during normal or perturbed situations, specifically in the cytosol, mitochondria, and nucleus, are not well understood. Here, using the P19 neurogenesis model of cellular differentiation, the kinetics of subcellular H2O2 availability and GSH/GSSG and NADPH/NADP+ redox shifts were evaluated following oxidant exposure. Additionally, modified mouse embryonic fibroblasts (MEFs) were used to observe redox changes and protective mechanisms when major antioxidative pathways are inhibited, mainly those involving the GSH/GSSG and Trxred/Trxox pathways. Overall, treated undifferentiated cells showed a greater degree and duration of both H2O2 availability and GSH/GSSG and NADPH/NADP+ disruption throughout all compartments than differentiated neurons. Pretreatment with an Nrf2 inducer prevented H2O2-induced effects in all compartments of undifferentiated cells. Additionally, MEF cells without either GSH or Trx showed a greater degree and duration of GSH/GSSG and Trxred/Trxox disruption throughout the cytosol and nucleus when compared to normal functioning cells. Disruption of redox-sensitive developmental pathways is likely stage-specific, where cells that are less differentiated and/or are actively differentiating are most affected. Undifferentiated cells are more susceptible to oxidant-induced redox dysregulation but are protected through prior Nrf2 induction, which appears to preserve developmental programs and diminish the potential for poor developmental outcomes. The GSH and Trx antioxidant pathways converge to protect the cell, while cells that are missing one pathway or the other may undergo damaging developmental outcomes.

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Nicotinamide cofactor-dependent oxidoreductases have become a valuable tool for the synthesis of high value chiral compounds. The feasibility of biocatalytic processes involving these enzymes stands and falls with the efficiency of the regeneration of cofactors. In this study, we describe a novel NADPH regeneration method based on the natural thioredoxin electron delivery system. Thioredoxin 1 (Trx1) and thioredoxin reductase (TR) from Thermus thermophilus were characterized for the dithiol-dependent reduction of NADP+, revealing good catalytic activities and a particularly remarkable thermostability. The TR/Trx1 system was then coupled with two representative NADPH-dependent oxidoreductases, alcohol dehydrogenase and cyclohexanone monooxygenase. Reaction conditions for both systems were optimized for reaction yield and selectivity. The results demonstrate the feasibility of the TR/Trx1-system for its application as NADPH regeneration system.

Some bacteria produce a potent antifungal agent (pyrrolnitrin) from tryptophan using four dioxygen dependent steps to outcompete other microbes. Each step of this process is catalyzed by an oxygenase encoded by the prnABCD cassette. The first enzymatic step in pyrrolnitrin biosynthesis is the regioselective chlorination of tryptophan to form 7-chlorotryptophan. This halogenation is catalyzed by PrnA, a Flavin dependent oxygenase, which has been isolated and characterized from P. fluorescens. The pyrrolnitrin biosynthesis pathway (prnABCD) has been also observed in the Burkholderia genus. This thesis comprises my studies on the expression, purification, and characterization of PrnA from Burkholderia ambifaria. Beyond the comparative preliminary data on B. ambifaria PrnA, we report an NADPH-dependent activity assay for PrnA coupling the oxidative chlorination of tryptophan (and related substrates) with NADPH consumption. The steady-state kinetic parameters associated with PrnA of kcat, KM, and catalytic efficiency of enzymes are also reported for this system under defined experimental conditions.

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Oxidative stress plays a vital role in the pathogenesis of many chronic and acute inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two key mediators that are known to induce cellular and tissue oxidative stress. The generation of ROS and RNS is mediated by innate immune signaling processes. Lipopolysaccharide (LPS), a major inflammatory signal, is known to be a potent inducer of ROS/RNS. Thus, strategies that may block LPS-mediated generation of free radicals may hold promise in treating various inflammatory disease processes. However, the molecular mechanisms underlying LPS-mediated ROS/RNS production are not fully defined. Interleukin-1 Receptor associated kinase (IRAK-1), an intracellular kinase downstream of Toll-like Receptor 4 (TLR4) has been shown to contribute to the inflammatory cascade associated with LPS-TLR4 signaling pathway. However, its role in ROS production has not been defined. Therefore, we tested the hypothesis that IRAK-1 plays an important role in regulating ROS/RNS production. Both in vitro and in vivo studies were conducted to investigate the role of IRAK-1 in modulating free radicals as well as oxidative stress. In vitro studies demonstrate that IRAK-1 is a critical molecule involved in the induction of ROS/RNS. IRAK-1 deletion ablated free radical production following LPS challenge in a variety of cell types including macrophages, fibroblasts and microglia. Mechanistically, we observed that IRAK-1 is required for optimal expression and activity of NADPH oxidase subunits and iNOS. IRAK-1 deletion reduced LPS-triggered p47phox membrane translocation, suppressed NOX-1 expression and protein levels as well as hampered Rac1 activation. On the other hand, IRAK-1 deletion sustained antioxidative enzyme activity and levels in IRAK-1-/- macrophages and fibroblasts. In terms of the in vivo physiological consequences, IRAK-1-/- mice exhibited attenuated lipid peroxidation in vital organs, attenuated histopathological lesions in liver and kidney, and reduced endotoxemia-associated mortality. Taken together, IRAK-1 may, at least in part, serve as an important therapeutic target in the treatment of various inflammatory disease processes. / Ph. D.