During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca�����-activated calmodulin (Ca�����-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c����� by 10 to 20 fold. To investigate the effect of the Ca�����-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c����� as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca�����-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca�����-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c����� reduction. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated reduction of cytochrome c����� suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH] indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE. / Graduation date: 2001

Graduation date: 1998

A negatively biased substrate has several advantages over a grounded substrate in CMOS technology. The on-chip generation of this negative substrate bias has made chips easier to use when only a single supply is preferred. This project demonstrates two types of charge pump circuits used to generate negative voltages not only for biasing the substrate, but in a broader sense also for other purposes in CMOS technology. One other possible use is in conjunction with 'Guard Ring Diodes for Suppressing the Substrate Noise in Mixed-Mode CMOS Circuits'. This work proposes a reasonable approach to generate the forward biasing current for the guard ring diode whose depletion capacitance and the substrate lead inductance form a resonant circuit to provide very low substrate-to-ground impedance at specific frequencies. Given this emphasis on generating a reasonably predictable current source, the generated negative voltages are regulated using a feedback loop. The amplitude of this negative voltage can be determined exclusively by transistor sizes. Simulation results support the theoretical analysis in that accurate negative voltages and current sources can be generated on-chip, although there are some limitations. / Graduation date: 1996

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.

The greenhouse gas nitrous oxide (N2O) is produced mainly by the microbial processes of nitrification and denitrification. I hypothesized that microbial community structure (composition and abundance) is linked to differences in soil N2O emissions from these two processes. Microbial community composition (type and number of nitrifier and denitrifier genotypes), abundance and N2O emission activity were determined and compared for soils from two landscapes characteristic of the North American prairie pothole region (cultivated vs. uncultivated wetlands). The landscape difference in composition of individual microbial communities was not predictive of soil N2O emissions, indicating that there is redundancy in each microbial community in relation to N2O emission activities. However, community factors influenced the pattern and distribution of N2O emission from the soils of the study site. For example, nitrification was the dominant N2O emitting process for soils of all landforms. However, neither nitrifier amoA abundance nor community composition had predictive relationships with nitrification associated N2O emissions. This lack of relationship may be a consequence of using amoA as the gene target to characterize nitrifiers. For denitrifying bacteria, there was a temporal relationship between community composition and N2O emissions. However, this may be related to the change in water-filled pore space over time. Alternatively, the presence of fungi can be linked directly to N2O emissions from water accumulating landform elements. Under hypoxic conditions, there may be two fungal pathways contributing to N2O release: fungal denitrification via P450nor and fungal heterotrophic nitrification. Results suggest that the relative importance of these two processes is linked to root exudates such as formate. It is the interaction between the seasonal fluctuations of the microbial and environmental factors that determine the level of N2O emissions from soils.

Graphene is a single layer of graphite with many unique mechanical, electrical, and optical properties. In addition, graphene is also known to adsorb wide range of biomolecules including single-stranded DNA. On the other hand, the adsorption of double-stranded DNA was much weaker. To properly disperse in water, graphene oxide (GO) is often used due to its oxygen-containing groups on the surface. Recently, it was discovered that it could efficiently quench the fluorescence of fluorophores that were adsorbed. With these properties, it is possible to prepare DNA-based optical sensors using GO. Majority of the DNA/GO-based fluorescent sensors reported so far were relied on the complete desorption of DNA probes. Even though all these reports demonstrated the sensitivity and selectivity of the system, the fundamentals of binding between DNA and GO were hardly addressed. Understanding and controlling binding between biomolecules and inorganic materials is very important in biosensor development. In this thesis, adsorption and desorption of DNA on the GO surface under different buffer conditions including ionic strength, pH, and temperature were systematically evaluated. For instance, adsorption is favored in a lower pH and a higher ionic strength buffer. It was found that once a DNA was adsorbed on the surface, little desorption occurred even in low salt buffers. Even with high pH or temperature, only small percentage of adsorbed DNA can be desorbed. To completely desorb the DNA, complementary DNA is required. The energies and activation energies associated with DNA adsorption/desorption were measured and molecular pictures of these processes were obtained. With the fundamental understanding of the DNA/GO interaction, we demonstrated that it is possible to achieve sensor regeneration without covalent immobilization. In addition, we also achieved the separation of double-stranded DNAs from single-stranded ones without using gel electrophoresis. We also studied the fluorescence property of DNA near the GO surface using covalently attached DNA probes. It was found that the fluorophore quantum yield and lifetime changed as a function of DNA length. This study is important for rational design of covalently linked DNA sensors. This study confirmed that fluorescence quenching by GO occurs in a distance-dependent manner. Energy transfer occurred between the fluorophore and GO to result in reduced quantum yield, shorter lifetime, and lower fluorescence intensity. Although fluorescent sensors based on covalently attached DNA probes on GO have not yet been reported, the study presented here clearly supported its feasibility.

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.

Riparian soils are thought to be potential hotspots for nitrous oxide (N2O) fluxes from incomplete denitrification, with soil moisture cited as a primary controller, however, because there are multiple potential pathways for N2O production in soils, each with their own environmental regulators, the timing and magnitude of N2O fluxes in difficult to predict. Often empirical observations have failed to yield consistent relationships between environmental factors in lab and field scenarios. This thesis characterizes the hydrological controls (soil moisture, water table depth, and precipitation) on N2O fluxes from different positions on the riparian landscape (dry, loamy upland, and wet, organic lowland) in the field during the growing season. Nitrous oxide and carbon dioxide (CO2) fluxes in the field, as well as environmental and climatic variables, were measured in the field. Over the three year study period N2O fluxes were consistently correlated with soil temperature during the growing season, but not with any hydrological factors. However, direct relationship between soil hydrology and N2O fluxes was more evident on an “episodic” time scales. Lab experiments were used to assess the influence of AHC on N2O production under controlled conditions. Experiment 1 employed intact soil cores collected from the upland and lowland positions of the riparian landscape and the cores were subjected to one of two contrasting moisture regimes (wet-dry-wet or dry-wet-dry). Experiment 2 used homogenized soils from the upland and lowland positions on the landscape to create a multi-factorial experiment that simultaneously altered soil moisture and soil substrate concentrations (nitrate, ammonium, organic carbon). The lab results showed that different AHC resulted in differences to the timing and magnitude of N2O fluxes, and that these patterns differed with soil type. Nitrous oxide production was often correlated with soil moisture in the lowland soils regardless of AHC. The results from Experiment 2 suggested that the upland soils were C limited, which resulted in an unpredictable relationship between soil moisture and N2O production during different AHC. The lowland soils were less affected by AHC as they were not N or C limited like the upland soils. It can be concluded from this research that the relationship between soil moisture and N2O fluxes is influenced by AHC through the influence of AHC on soil N and C dynamics. Given the differences in C and N dynamics between soils types, and the influence of AHC on soil C and N, it can be concluded that a derived relationship between soil moisture and N2O fluxes may not be directly transferable between soil types unless C and N are considered.

The greenhouse gas nitrous oxide (N2O) is produced mainly by the microbial processes of nitrification and denitrification. I hypothesized that microbial community structure (composition and abundance) is linked to differences in soil N2O emissions from these two processes. Microbial community composition (type and number of nitrifier and denitrifier genotypes), abundance and N2O emission activity were determined and compared for soils from two landscapes characteristic of the North American prairie pothole region (cultivated vs. uncultivated wetlands). The landscape difference in composition of individual microbial communities was not predictive of soil N2O emissions, indicating that there is redundancy in each microbial community in relation to N2O emission activities. However, community factors influenced the pattern and distribution of N2O emission from the soils of the study site. For example, nitrification was the dominant N2O emitting process for soils of all landforms. However, neither nitrifier amoA abundance nor community composition had predictive relationships with nitrification associated N2O emissions. This lack of relationship may be a consequence of using amoA as the gene target to characterize nitrifiers. For denitrifying bacteria, there was a temporal relationship between community composition and N2O emissions. However, this may be related to the change in water-filled pore space over time. Alternatively, the presence of fungi can be linked directly to N2O emissions from water accumulating landform elements. Under hypoxic conditions, there may be two fungal pathways contributing to N2O release: fungal denitrification via P450nor and fungal heterotrophic nitrification. Results suggest that the relative importance of these two processes is linked to root exudates such as formate. It is the interaction between the seasonal fluctuations of the microbial and environmental factors that determine the level of N2O emissions from soils.

No description available.