High pressure infrared and micro-Raman vibrational spectroscopy has been used to study the following organoplatium(II) complexes: Zeise's salt, K (Pt($ eta sp2$-C$ sb2$H$ sb4$)Cl$ sb3$); Zeise's dimer, (Pt($ eta sp2$-C$ sb2$H$ sb4) sb2$Cl$ sb2 rbrack sb2$; and dichloro(1,5-cyclooctadiene)platinum(II), Pt(COD)Cl$ sb2$. Pressure dependences of the vibrational modes have been used in the assignments of the $ upsilon$(C=C) modes and the ethylene and cyclic diene C-H stretching vibrations. Pressure increases the extent of $ pi$-backbonding, as shown by the negative pressure dependence of the $ upsilon$(C=C) stretching frequencies.

A thickness-shear mode (TSM) acoustic wave sensor system has been constructed and used to examine binding phenomena that occur in the liquid phase. The work is broadly divided into two sections. / The first section examines the use of cholestyramine as a sensor coating for detection of bile acids. A 10-MHz piezoelectric crystal is coated on one side with cholestyramine resin and mounted in a batch-mode sensor block which exposes the coating to solution. After sample injection, the binding process is observed in real time as a drop in frequency as the bile salt binds to the coating, reaching >90% completion within 10 minutes with most of the binding occurring within the first minute. Linear calibration curves are generated with sensitivity increasing in the order cholate &ap; glycocholate < taurocholate << taurodeoxycholate. Detection limits in water are in the range 0.2--9 nmol and are better than those observed in phosphate buffer. A multi-step regeneration protocol allows the coating to be reused more than 400 times over a period of several months. Precision for replicate injections is about 10% RSD and depends on the reproducibility of the regeneration and injection steps. / In terms of the binding process, hydrophobic interactions are observed to be of importance in the ability of bile salts to displace other counterions. However, anions with greater charge density also appear to compete effectively for binding sites on the resin. In particular, at equimolar concentrations of citrate and bile salt, the trivalent citrate anion reduces the amount of bile salt binding by about 40%. This suggests that the efficiency of cholestyramine-based bile salt sequestering drugs used in the reduction of hypercholesterolemia may be improved by eliminating citric acid as an excipient and avoiding the use of fruit juices during ingestion. / In the second section, a selection of compounds were examined for their potential as stable and reactive liquid-phase coatings with the primary amine group as the functional group of interest. Based on dry TSM sensor frequency changes, all coating candidates exhibited some degree of stable coating formation after incubation and rinsing. Reactivity of the coatings was then assessed via reaction with succinic anhydride under anhydrous conditions. With pyridine- and thiol-based coatings, the frequency change is negligible or is positive, which may indicate some loss of coating material upon reaction of the amine group. A silane-based coating was stable but exhibited only small frequency decreases for the reaction step. In terms of stability, reproducibility and magnitude of the frequency changes observed, poly(ethyleneimine) (PEI) is seen to be the best choice for providing analytically useful signals where covalent reaction with surface-confined amino groups in the liquid phase is desired. / Reaction of PEI with succinic anhydride also serves as a method for fabrication of sensor coatings with the carboxylic acid group as the reactive functional group of interest. The formation of reactive COOH groups was tested with a carbodiimide-activated covalent immobilization of dopamine. TSM sensor frequency changes, ellipsometry, and x-ray photoelectron spectroscopy provided support for all steps involved. The coating surface structure is uneven and is likely highly branched and three-dimensional. The method is quick and simple, and the films are also sufficiently robust for liquid-phase acoustic wave sensor applications.

Researchers have linked resin and fatty acids (RFAs) to acute toxicity, especially in thermomechanical and chemithermomechanical pulp and paper mill effluents. Thus, the measurement of total RFAs may be a rapid, inexpensive alternative to standard methods of toxicity monitoring, such as 96-hour rainbow trout testing. Current procedures for the analysis of RFAs typically involve solvent extraction and concentration, derivatization, and analysis by GC, HPLC, or TLC. These procedures are far too expensive, complicated, and time-consuming for implementation at mill sites. / This thesis reports the development of a rapid, colourimetric assay based on the dye methylene blue (MB) for the quantification of resin and fatty acids in pulp and paper mill effluents. This MB assay uses the complexation of the cationic organic dye molecule to the carboxylic acid groups of RFAs to form a measurable chromophore. The electrically neutral, blue-coloured complexes are then extracted into a poorly polar organic solvent, dichloromethane. The measured absorbance at 655 nm is directly related to the total RFA concentration in the effluent sample. / The methylene blue assay is inexpensive and simple to use. It has a method detection limit of 0.589 mg/L total RFA. There are good correlations between the results obtained using the methylene blue assay and a well-established GC assay, and between RFA concentrations measured by the MB assay and acute toxicity measured by Microtox. The assay is sufficiently simple and rapid to be practical for routine in-mill monitoring.

The investigation of the interaction of light with tissue has many applications in the field of diagnostic medicine. The objective of the project was to obtain quantitative measurements of constituents in a medium in which the absorption and scattering optical properties vary. Initially the project was divided into two separate experiments, namely multiwavelength steady state and time resolved measurements. Both methods are applicable to complex media, providing information related to absorption and scattering. / The first phase was the study of Near Infrared diffuse reflectance for the determination of pulmonary edema. Non-contact pulmonary edema measurements were made on in vitro isolated perfused animal lungs. Multispectral analysis with Partial Least Squares (PLS) depicted three distinct changes associated with fluid increase: an increase in the O-H absorption, a decrease in the C-H absorption, and a decrease in the spectral baseline or scattering contribution. PLS estimates of relative water content had an R2 of 0.973 with a standard error 10.6 +/- 2.9% relative to the weight of lung. / The behavior of light in a highly scattering medium is a complex phenomenon. A method is described using analytical descriptors for the separate determination of the absorption and scattering properties from time resolved photon distributions. Estimates with the analytical descriptors were tested using simulated data generated from the time dependent diffusion equation. Objective selections of the descriptors were made using a Stepwise Multi-Linear Regression technique to independently estimate the absorption and scattering coefficients. In a medium where scattering and absorption vary simultaneously multiple parameters are required for quantitative estimates. Relative standard errors of less than 0.5% were obtained with the analytical descriptors estimates. / Estimates using the analytical descriptors were tested experimentally with the use of a photon time-of-flight instrument. Different light collection geometries and their relation to the descriptors were investigated in both transmittance and reflectance configuration. Absorption and scattering estimations of less than 9% error were attained for both collection geometries. / Information on both the absorption and scattering properties in highly variable media can assist in quantitative multispectral measurement. A method was developed whereby a multiwavelength scatter correction was made using optical coefficients determined from time resolved distributions. Optical coefficient corrected estimates produced a 70% improvement in the error over non-corrected spectra. The study suggests a practical method to correct and quantitate constituent measurements in variable media.

<p> This dissertation describes metabolic engineering of cyanobacterium <i> Synechococcus elongatus</i> PCC7942 as a photosynthetic host for the conversion of CO₂ into 2,3-butanediol. Current advances in pathway design, genetic tool development, and yield improvement are described (Chapter 1). A pathway for the synthesis of 2,3-butanediol is designed based on collective concepts of pathway strength, robustness, and irreversibility, and extensively tested through the generation of mutants (Chapter 2). This pathway is then optimized through modulation of translation by combinatorial mixing of ribosome binding sites (Chapter 3). Finally, photosynthetic productivity is investigated through expression of an exogenous pathway targeting every step between fixation and product (Chapter 4). All materials and methods are given separately for easy reference (Chapter 5).</p>

<p> Heavy metal pollution is a growing concern as growing worldwide population and industrial processes increase pollution levels in most environments. High metal concentrations throughout ecosystems pose a serious threat to wild-life and human health. Methods to monitor rising threat levels of metals are a primary concern for monitoring overall ecosystem health. Mechanisms which spread pollution must be intimately understood because of the persistence of heavy metals. Heavy metal contamination in the Tar Creek superfund site provides a great case study to selectively observe differences in heavy metals concentrations both upstream and downstream of mining activity. Thus, research is able to identify natural and man-made point sources of pollution. </p><p> The abilities of bivalves to filter-feed and sediment-feed provide a unique monitoring tool for analyzing heavy metals. Mussels are constantly filtering the environment around them. A mussel's seasonal and annual growth layers provide an excellent sample media for obtaining historical records of environmental data. Many species of mussels are found in most freshwater ecosystems throughout the United States. Mussels have low migration rates, live for a suitable amount of time, and leave relic shells. These features make mussels very practical for monitoring heavy metal pollution. </p><p> Various studies were conducted to obtain insight into developing methods for using LA-ICP-MS as a tool for monitoring heavy metals in mussel shells. Surface laser ablations, compared at additional depths, resulted in a more than 20% increase in signal intensity. Theoretical and experimental designs show signal changes as a function of depth. Mussel tissue and shell digestions were found to be best when using approximately 1.0 mL of hydrogen peroxide and 1.0 mL of nitric acid for each 0.1 grams of sample. Mussel tissue was found to have greater heavy metal concentrations than shells. Shells were found to average a 96% weight of calcium carbonate; however, the organic layers contained the greatest concentrations of heavy metals per weight. </p>

<p> G-quadruplex is the most widely known four-stranded nucleic acid structure which has shown to alter gene regulation both in vitro and in vivo. Under certain conditions, another four-stranded structure, i-motif, is also formed in the strand complementary to the G-quadruplex forming sequence. Recent studies suggest gene regulatory roles for the i-motif structure as well. Although there is substantial understanding on the folding topology of G-quadruplex and i-motif structures, their mechanical stability which determines the interaction with motor proteins, such as DNA/RNA polymerases, are poorly studied. Since DNA exists as a double stranded form in vivo, the investigation of i-motif becomes highly important to fully understand the biological functions of G-quadruplexes. Using laser tweezers based single-molecule study, we investigated the mechanical stability of an i-motif structure in the predominant variant of human ILPR fragment (5'-TGTC4ACAC4TGTC4ACAC4TGT). In addition, we have shown that a partially folded structure composed of only three tandem C-rich repeats coexists with the i-motif. Both structures share similar unfolding forces of 22-26 pN. Discovery of stable structures in less than four C-rich repeats suggested that the structure can serve as an intermediate during the i-motif folding/unfolding pathway. Using chemical footprinting and single-molecule approaches, we show that a dsDNA fragment in ILPR, 5'-(ACAG4TGTG4ACAG4TGTG4ACA), can fold into G-quadruplex or i-motif structure under specific conditions. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif, changes in free energy of unfolding provided compelling evidence that only one species is present in each dsDNA. Based on this observation, we propose that G-quadruplex and i-motif are mutually exclusive in human ILPR. Furthermore, we show that these two species have an unfolding force >17 pN. From mechanical perspective, this could justify the regulatory role a DNA tetraplex may play in the expression of human insulin inside cells in which dsDNA is the predominate form.</p>

<p> The objective in this thesis is to study the structure of peptides using molecular spectroscopy. Molecular spectroscopy, both vibrational and electronic, can be used as a sensitive tool to study molecular structure. Since it is an inherently low resolution method, theoretical calculations are essential for a complete understanding of vibrational and electronic spectra. The first part of this thesis contains quantum chemical calculations of the molecular spectra of several small peptide systems with different secondary structures. Optical trapping is a method that allows for the manipulation of sub-micron scale objects using tightly focused laser light. Raman spectroscopy, which is sensitive to molecular vibrations also requires intense laser light. Combined with optical tweezing, Raman spectroscopy can prove to be a very powerful tool to study small sample volumes and probe single living cells. In the second part of this thesis, I detail the construction an such an instrument, an optical trapping Raman spectrometer (OTRS). Our OTRS can measure Raman spectra from sub micron systems while at the same time quantifying the mechanical forces that are acting upon them. Thus the OTRS can give insight into the relationship between mechanical forces acting upon cells and their molecular structure. </p>

<p> We studied the interactions of glyoxylic acid, pyruvic acid and oxalic acid with ammonium and corresponding sodium salts in aqueous solutions simulating a dark and radical free atmospheric aqueous aerosol condition. Cleavage of a carbon-carbon bond in pyruvic acid and glyoxylic acid leading to the decarboxylation was observed in the presence of ammo&not;nium salts but was not observed from oxalic acid. At the beginning of the reaction, the decarboxylation appeared to proceeding slower compare to the later stage of reaction. The empirical rate constants for decarboxylation in the reaction solutions were estimated using a 'quasi-steady state' model: (i) glyoxylic acid and ammonium sulfate was 3.3 (&plusmn; 0.7)&times;10^-8 M^-1 s^-1; (ii) glyoxylic acid and ammonium nitrate was 1.4 (&plusmn; 0.3)&times;10^-8 M^-1 s^-1; (ii) glyoxylic acid and ammonium chloride was 1.9 (&plusmn; 0.2)&times;10^-8 M^-1 s^-1; and (iii) pyruvic acid and ammonium sulfate was 15.8 (&plusmn; 0.4)&times;10^-8 M^-1 s^-1. Negligible CO₂ was observed in the experiments with the corresponding sodium salts indicating the ammonium ion or ammonia is facilitating the carbon-carbon bond cleavage leading to carboxyl fragmentation of the &agr;-oxo carboxylic acids. It was observed that pyruvic acid undergoes decarboxylation at least four times faster than that of glyoxylic acid under similar reaction conditions. This indicates that the structure of the acid plays an important role in the decarboxylation. In the case of pyruvic acid, the reaction is likely faster because of the inhibited hydration of the carbonyl moiety due to the inductive effect of the adjacent methyl group. A tentative set of reaction mechanisms is proposed involving nucleophilic attack by ammonia on the carbonyl carbon leading to fragmentation of the carbon-carbon bond between the carbonyl and carboxyl carbons. Similar carbon-carbon bond cleavage is anticipated for &agr;-dicarbonyl compounds, which are structurally similar to the &agr;-oxo carboxylic acids. In the absence of photolysis and under limited availability of OH radicals, the decay of pyruvic acid can be dominated by the reaction with ammonium sulfate and can be an order of magnitude higher than the loss via reaction with the OH radical. Under similar conditions the reactions with ammonium salts are likely be a major sink for &agr;-oxo carboxylic acids in the atmospheric aqueous phase.</p>

<p> Radical chemistry has attracted a large amount of research interest over the last few decades and radical reactions have recently been recognized as powerful tools for organic synthesis. The synthetic applications of radicals have been demonstrated in many fields, including in the synthesis of complex natural products. Radical reactions have a number of inherent synthetic advantages over their ionic counterparts. For example, they typically proceed at fast reaction rates under mild and neutral conditions in a broad spectrum of solvents and show significantly greater functional group tolerance. Furthermore, radical processes have the capability of performing in a cascade fashion, allowing for the rapid construction of complex molecular structures with multiple stereogenic centers. To further enhance the synthetic applications of radical reactions, current efforts are devoted toward the development of effective approaches for the regioselective control of their reactivity as well as stereoselectivity, especially enantioselectivity, a challenging issue that is intrinsically challenged by the "free" nature of radical chemistry. </p><p> This research has identified a fundamentally new approach to radical reactions based on the concept of metalloradical catalysis (MRC) for controlling the stereoselectivity of both C- and N-centered radical reactions. Cobalt(II) porphyrins [Co(Por)], are stable metalloradicals, and have been shown to enable the activation of diazo reagents and azides to cleanly generate C- and N-centered radicals, respectively, with N₂ as the only byproduct in a controlled and catalytic manner. In addition to the radical nature of [Co(Por)], the low bond dissociation energy of Co-C/Co-N bonds plays a key role in the successful turnover of the Co(II)-based catalytic carbene and nitrene transfers. Through the support of porphyrin ligands with tunable electronic, steric, and chiral environments, this general concept of Co(II)-based metalloradical catalysis (Co-MRC) has been successfully applied to the development of various radical processes that enable stereoselective carbene and nitrene transfers. </p>