In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). <br /><br /> Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules. <br /><br /> Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

Ethylene has long been considered as the main plant growth regulator that plays a key role in the regulation of leaf senescence. In sweet potato, ethephon, an ethylene releasing compound, promoted leaf senescence and H2O2 elevation. These ethephon-mediated effects were alleviated or attenuated by exogenous reduced glutathione and ascorbic acid. Ethephon treatment gradually increased endogenous total and reduced glutathione and ascorbic acid levels in sweet potato detached leaves 3 days after treatment. The H2O2 amount, however, was also increased at 72 h after treatment. Sweet potato detached leaves pretreated with reduced glutathione did significantly increased endogenous total and reduced glutathione levels at 24 h and remarkably decreased H2O2 amount at 72 h after ethephon application compared to that of ethephon alone control. Ethephon caused quick elevation of a small H2O2 peak at about 4 h after application, and the enhancement was eliminated by reduced glutathione pretreatment in treated sweet potato leaves. Pretreatment of diphenylene iodonium (DPI), an NADPH oxidase inhibitor, also repressed leaf senescence and H2O2 elevation at day 3 after ethephon treatment in sweet potato detached leaves, and the attenuation was effective within the first 4 h after ethephon treatment. For senescence-associated gene expression, ethephon and L-buthionine sulfoximine (BSO), an endogenous glutathione synthase inhibitor, did induced asparaginyl endopeptidase (SPAE) and cysteine proteases (SPCP1, SPCP2 and SPCP3) gene expression and the activation was repressed by reduced glutathione pretreatment. Based on these data we conclude that ethephon treatment may cause quick elevation of a small H2O2 peak likely via the NADPH oxidase, which may function as a signal component leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato detached leaves. The rate of endogenous antioxidant such as reduced glutathione elevation is also important and affects leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato leaves.

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In 2015, American Heart Association (AHA) reported that 1 in 9 deaths are attributed to Heart failure (HF), the number one killer in the world. While advancements in interventional cardiology in conjunction with pharmacotherapies have significantly reduced the rate of mortality following MI, there has been a corresponding rise in chronic heart failure (CHF) in surviving patients, largely attributed to the limited regenerative capacity of the heart and the inadequate healing response. Myocardial ischemic injury triggers an exuberant local and systemic inflammation, and the extent and quality of the cardiac wound healing process is intricately tied to the delicate equilibrium of this inflammatory response. While cardiac regeneration is an important goal, it is imperative in the meantime to explore therapeutic strategies that target these inflammatory mediators of early cardiac repair. These interventions to influence and improve cardiac wound healing can represent a new therapeutic window to halt the progression of heart failure between the few hours that may be used to limit infarct size by reperfusion and an irreversible non-contractile cardiac scar. This dissertation examines three therapeutic delivery strategies aimed at modulating the immune response to enhance cardiac repair in rodent models MI: 1) Polyketal nanoparticles as siRNA delivery vehicles for antioxidant therapy; 2) Spherical nucleic acid particles for anti-inflammatory therapy and; 3) Bioactive PEG (polyethyleneglycol)-based hydrogel for immunomodulation. The work presented here applies novel nucleic acid delivery strategies for cardiac gene silencing and has contributed to new knowledge with regard to modulating the immune response following MI.

CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.

Recent studies support the association between metabolic syndrome (MetS), a cluster of cardiovascular risk factors, and diastolic dysfunction. Disproportionate collagen accumulation, particularly cross-linking of collagen, plays a key role in translating interstitial fibrosis into mechanical chamber stiffness and diastolic dysfunction. Characteristic changes in the expression and activity of myocardial lysyl oxidase (LOX), a matrix modifying enzyme that catalyzes cross-linked collagen, are unclear in MetS. We established a diet-induced MetS model to study diastolic dysfunction by treating male C57BL/6 mice a high-fat high-simple carbohydrate (HFHSC) diet for 6 months. Despite blunted gene expression of LOX isoforms, MetS mice demonstrated significant increase in the ratio of protein expression of mature to proenzyme LOX, enhanced LOX activity, and increased cardiac cross-linked collagen compared with controls. This fibrotic response coincided with marked increase in left ventricular end-diastolic pressure and stiffness and impaired diastolic filling pattern. Our data demonstrate that diet-induced MetS alters the remodeling enzyme LOX, thereby increasing the amount of crosslinking and inducing diastolic dysfunction.Furthermore we examined the role of T-lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice which are devoid of functional T-lymphocytes and C57BL/6 mice were treated with HFHSC diet for 12 months. Similar to male C67BL/6, female HFHSC-fed C57BL/6 mice demonstrated significant increase in maturation and catalytic activity of myocardial LOX, cross-linking, ventricular stiffness and diastolic dysfunction. Whereas induction of LOX protein was minimal in SCID mice compared with wild-type counterparts. Correspondingly fibrillar cross-linked collagen formation and diastolic dysfunction were less prominent in SCID mice. Our results suggest a potential role of T-lymphocytes in induction of myocardial stiffness and diastolic dysfunction through modulation of LOX-dependent collagen maturation.Moreover we studied the role of leptin, an adipokine over-produced in MetS with fibrotic effects in non-cardiac tissues, as a key mediator of profibrogenic responses in the heart by administrating leptin to C57BL/6 and leptin-deficient ob/ob mice. With exogenous leptin administration ob/ob mice displayed passive diastolic filling dysfunction that coincided with increase in myocardial collagen compared with ob/ob controls. Our findings suggest profibrotic effects of leptin in the heart, primarily through predominance of collagen synthesis over degradation.

Copper metallothionein (Cu-MT) was extracted from the induced biomass of Aspergillus niger. The crude extract (FI), obtained by cell homogenization, was partially purified by heat treatment (FII) and ultrafiltration (FIII). Further purification of the Cu-MT extract by affinity chromatography resulted in three major fractions, FIVa, FIVb and FIVc, of which fraction FIVc was considered to be the Cu-MT extract fraction. Fraction FIVc was re-chromatography on affinity chromatography and the eluted fraction showed a single peak (FIVc'). Spectrophotometric analysis of fraction FIVc' demonstrated a maximum absorption peak at 268 nm. Native and denatured electrophoretic analysis of fraction FIVc ' showed the presence of a single band with an estimated molecular weight of 9.5 and 10.0 kDa, respectively. Inhibition of mushroom tyrosinase (PPO) by the Cu-MT extracts was investigated, using selected phenolic substrates, including catechin, chlorogenic acid, catechol, 4-methylcatechol, caffeic acid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3-( p-hydroxyphenyl) propionic acid, p-hydroxyphenylpyruvic acid, p- and m-cresol. The results showed that the inhibitory effect of the Cu-MT extract increased with the degree of purification. The results revealed that the Cu-MT extracts were effective inhibitors of PPO activity and the best inhibitory effect was demonstrated with catechin as substrate; however, PPO activity was not inhibited by the Cu-MT extract when p-hydroxyphenylpyruvic acid and p- and m-cresol were used as substrates. The results also showed that the Cu-MT extracts exhibited different types of inhibition, including mixed, competitive and uncompetitive on PPO activity. In addition, the experimental findings indicated that the nature and degree of enzymatic inhibitions by the Cu-MT extracts were dependent upon the structural nature of the substrates as well as the methods including, spectrophotometer and polarograph, used for the detection of enzyme

Two natural enzymatic browning inhibitors, copper-metallothionein (Cu-MT) and polyphenol esterase (PPE), were obtained from A. niger and investigated. Reflectance measurements, expressed as L (lightness variable) and a (red to green degree of color) were used to compare, over extended periods of time, the relative inhibitory effectiveness of Cu-MT and PPE to those observed with the use of selected chemicals including ascorbic acid (AA), citric acid (CA), ethylenediaminetetraacetic acid (EDTA), sodium bisulfite (NaHSO3) and 4-hexylresorcinol (4HR), in the prevention of browning on the cut surfaces of selected food products such as apple and potato slices as well as freshly prepared apple juice. Treatment of each food product required an optimum concentration of the selected inhibitor for the inhibition of browning. (Abstract shortened by UMI.)

Two projects were undertaken to study the non-energy conserving alternative pathway present in the plant mitochondrial ETC. In the first project, a tobacco AOX2 gene was cloned and characterized. AOX2 showed tissue specificity in expression and could not be induced by common stresses. In the second project I carried out a physiological characterization of transgenic tobacco plants with increased or decreased expression of AOX1 subjected to cold stress. Under non-stress condition, a strong inverse relationship between levels of AOX1 and levels of oxidative damage was observed, while after cold treatment AOX1 transgenic lines and WT showed more complicated and differential responses in aspects of oxidative damage and the capacity of antioxidant system. I also discovered that the pool sizes of monosaccharides after temperature shift were proportional to AOX1 levels. These results indicated that AOX1 might have crucial but complex impacts on ROS balance and carbon metabolism during cold stress.

Two projects were undertaken to study the non-energy conserving alternative pathway present in the plant mitochondrial ETC. In the first project, a tobacco AOX2 gene was cloned and characterized. AOX2 showed tissue specificity in expression and could not be induced by common stresses. In the second project I carried out a physiological characterization of transgenic tobacco plants with increased or decreased expression of AOX1 subjected to cold stress. Under non-stress condition, a strong inverse relationship between levels of AOX1 and levels of oxidative damage was observed, while after cold treatment AOX1 transgenic lines and WT showed more complicated and differential responses in aspects of oxidative damage and the capacity of antioxidant system. I also discovered that the pool sizes of monosaccharides after temperature shift were proportional to AOX1 levels. These results indicated that AOX1 might have crucial but complex impacts on ROS balance and carbon metabolism during cold stress.