Enzyme dynamics occur on a wide range of length and timescales. This work is focused on understanding enzyme dynamic at the fs-ps timescale as this is the dynamic range at which bonds are typically made and broken during chemical reactions. Our work focuses on enzymes that catalyze hydride transfer between two carbon atoms - a fundamental reaction in biology. Primary kinetic isotope effects and their temperature dependence have implied that fast dynamics of the enzyme are important in facilitating hydride transfer, however these experiments do not measure any such motions directly. We make use of two-dimensional infrared spectroscopy (2D IR), a technique that interrogates the vibrations of molecules to extract dynamic information from the surrounding environment with 100 fs resolution. A model system, formate dehydrogenase (FDH), is an excellent probe of dynamics at the fs-ps timescale. Azide bound to the ternary complex of FDH offers the ability to measure dynamics of an analog structure of the reactive complex using 2D IR, while also studying the reaction directly with and KIE’s and their temperature dependence. By altering various parts of the structure of FDH via mutagenesis and other techniques, we investigate the role of structure and dynamics to determine how fast dynamics of the active site influence the the kinetics of hydride transfer. These experiments are the first means of providing a dynamic interpretation of KIEs and their temperature dependence.

This thesis presents an effort to understand the C-H-C transfer in enzymatic reactions from the comparison of different variants of enzymes that have unrelated protein sequences and structures, but catalyze the same chemical transformation. I evaluated the kinetic isotope effects (KIEs) and their temperature dependences and interpreted the findings in accordance with Marcus-like models. The enzyme system studied is dihydrofolate reductase (DHFR), which catalyzes the reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) using reduced β-nicotinamide adenine dinucleotide 2' phosphate (NADPH) as a reducing agent. H-transfer reactions in typical enzymes from three genetically unrelated families, E. coli chromosomal DHFR (cDHFR, FolA), plasmid coded R67 DHFR (FolB), and pteridine reductase 1 (PTR1, FolM) were comparatively investigated. Chapter I provides a brief introduction to the thesis. Chapter II presents optimized procedures for a one-pot, enzymatic microscale synthesis of several NADPH isotopologues used in KIE experiments. Chapter III focuses on the application of novel competitive primary H/D KIE determinations. Chapter IV compares the H-transfer reactions between primitive R67 DHFR and the chromosomal DHFR, and Chapter V describes the investigation of H-transfer reactions at high and low ionic strengths with theoretical and experimental approaches in order to understand the unusual enhancement in H-transfer rate of R67 DHFR with increasing ionic strength. Chapter VI discusses an improved PTR1 purification procedure and comparisons of steady state kinetic parameters using PTR1 and cDHFR with H2F and dihydrobiopterin (H2B) substrates. Thus, the investigation of the H-transfer reaction catalyzed by cDHFR with an unnatural substrate, H2B is described. Finally, a summary is provided and future directions are discussed in Chapter VII.

Sea surface temperature (SST) reconstructions from the low latitudes indicate the tropics/subtropics warmed significantly before glacial-interglacial decreases in global ice volume, suggesting the importance of tropical and subtropical climate in driving glacial terminations. ODP Site 625, drilled at a water depth of 889 m near De Soto Canyon in the Gulf of Mexico (GOM), provides continuous records of marine isotope stages (MIS) 1-6 sampled at a mean temporal resolution of 400 years. Age control is based on 8 AMS radiocarbon dates, marine isotope stratigraphy, and Foraminifera datum levels. Results from Globigerinoides ruber (white variety) Mg/Ca-SST indicate a rise of 4.4 °C from last glacial maximum to modern conditions and a 3.2 °C rise from the penultimate glaciation to the last interglaciation. However, model results suggest reduced thermohaline circulation (THC) causes salt and heat build-up in the Atlantic Warm Pool. Paired G. ruber Mg/Ca-SST and δ18O provide evidence of sub-millennial scale variability in GOM SST and SSS that is probably influenced by the strength of NADW production, as also observed in the Western Caribbean Sea. We test the idea that widespread abrupt climate change during the last glaciation caused by millennial scale fluctuations in the intensity of THC was modulated by Laurentide ice sheet (LIS) meltwater routed to the North Atlantic. To understand LIS melting dynamics and test the Meltwater Routing Hypothesis, we investigate the phasing of GOM SST and LIS freshwater events in relationship to high latitude climate. Estimated salinities from our multi-proxy approach suggest three freshwater events with a major freshwater influx from that occurred during Heinrich Event 2. This result confirms previous studies that suggested LIS summer melting during warmings in Antarctica. We also find a climate reversal during termination II from 130.4-128.4 ka. The initial rise in GOM SST at 132.1 ka of 2.9 °C is followed by a cold reversal of 1.5 °C at 130.4 ka for 2 ky and final increase to full interglacial warmth. The reversal in GOM SST is consonant with a pause in sea level rise and reduced NADW, suggesting a reduction in THC may have caused a global two-step deglaciation.

Bark beetle outbreaks are becoming more intense and severe when coupled with the effects of climate change. Engelmann spruce (Picea engelmannii) is one such species facing large-scale, epidemic spruce beetle outbreaks. Large-scale disturbances, such as beetle outbreaks, have major consequences for the future success of the ecosystem, thus highlighting the importance of understanding what promotes amplified outbreaks as well as their effects on future seedling establishment. Our research focused on two parts of a large-scale beetle outbreak: the mortality of spruce trees and the subsequent regeneration of seedlings. Our first study examined the timing of spruce mortality during an outbreak in order to identify the extent to which drought promotes host species mortality. Trees that are drought stressed have less resources to defend themselves against beetle attacks, however, the warmer temperatures associated with droughts also promote a more rapid population expansion of spruce beetles. We were specifically interested in determining the contribution that host drought stress plays during an epidemic outbreak. Our second study analyzed the patterns of regenerating seedlings with an aim to identify changes associated with the outbreak. Specifically, we were interested in how an epidemic outbreak changes the drivers of seedling establishment.

Stable carbon isotope ratios of soils, roots and litter along transects stretching from nearly monospecific stands of Ceratoides lanata , a species possessing c3 photosynthesis, to nearly monospecific stands of Atriplex confertifolia, a species possessing c4 photosynthesis, were analyzed to determine i f changes in the relative dominance of the shrubs have occurred in salt-desert communities. The a13c value , which reflects the proportion of 12c and 13c in a sample of plant tissue, can be used to distinguish between c3 and c4 species. Atriolex confertifolia and Ceratoides lanata have a13c values of -13.0 ofoo and - 25 ofoo, respectively. The a13c value of litter and roots was used as a measure of current community dominance. The a13c value of soil organic matter was employed as a measure of past community dominance . The differential between a13c values of roots and soils was consistently about 3.0 ofoo in Ceratoides- dominated stands. Root a13c values were always more negative than soil a13c values. The striking uniformity in the rootsoil differential l in Ceratuitles- duninateci stands is most likely the result of fractionation of carbon isotopes during decomposition. The differential between a13c values of roots and soils in Atriplexdominated stands was more variable. This most likely indicates a lack of long- term community stability in areas na The distribution of a13c values in relation to depth and among soil organic matter fractions was also studied. There was a trend for a13c values to become slightly less negative with depth . The a13c value of humic acid was most similar to the o13c value of the dominant vegetation. Fulvic acid was isotopically heavier than humic acid in all analyses. The implications of these trends are discussed.

Blooms of the red tide dinoflagellate Karenia brevis occur annually on the west Florida shelf. In the late summer/early fall months, background concentrations increase from 103 cells L-1 to excesses of 106 cells L-1. Blooms are most common between Tampa Bay and Charlotte Harbor, and may be maintained for months. The region’s hydrography may play a role in the initiation, maintenance and termination of blooms. The west Florida shelf is depauperate in inorganic nutrients. Inorganic nitrogen and phosphorus rarely exceed the limits of detection, whereas dissolved organic nitrogen is often present at concentrations of 15 to 20 µM. Because K. brevis exhibits the ability to utilize both organic nitrogen and phosphorus, the organic pool may serve as an important nutrient source. The source of nutrients for K. brevis blooms is the focus of much scientific research. Nitrogen is considered to be the limiting nutrient in marine waters and may have several sources. Potential sources of inorganic and organic nitrogen are estuarine outflow, atmospheric deposition, upwelling, dissolved organic nitrogen released from N2 fixing cyanobacteria, diatom blooms, decaying seagrasses, fish or other organic matter. The natural abundance stable isotopic signatures of particulate bloom material (δ15N and δ13C) associated with K. brevis blooms during 1998 to 2001 was analyzed and compared with known isotopic values of potential nutrient sources. Data was analyzed from blooms occurring from 1998 to 2001. Extensive analysis of the 2001 bloom showed that the δ15N of bloom material ranged from 2 0/00 to 5 0/00. δ13C of bloom material ranged from -22 0/00 and -17 0/00. Non-bloom material was considerably more variable in both δ15N and δ13C. δ13C values were higher near shore than offshore during the 2001 bloom, suggesting lower dissolved inorganic carbon levels due to high temperature and/or high biomass. δ15N of bloom material fell within the range of the δ15N values of potential nitrogen sources. It appears that K. brevis utilizes the available nitrogen sources opportunistically, and that isotopically more depleted sources are more important. More enriched sources such as upwelled nitrate or sewage nitrogen can be excluded as significant sources based on the isotopic data.

This thesis provides new evidence which contributes to a clearer understanding of the mixing history of the lunar soil, the interactions of cosmic rays with the lunar surface and any temporal and spatial variations in cosmic ray intensity at the lunar surface. The bombardment of the lunar surface by cosmic rays produces secondary neutrons which are thermalised by the lunar soil. These thermal neutrons are captured by trace elements with large neutron capture cross sections such as cadmium, gadolinium and samarium. Measurements by thermal ionisation mass spectrometry of the changes in the isotopic abundances of cadmium, gadolinium and samarium due to neutron capture are presented in this thesis. Evidence is also presented of the first observations of mass fractionation in cadmium in lunar soils.Changes have been observed in samples from the Apollo 14, 16 and 17 missions. In 114 [superscript 114]Cd/[superscript 113]Cd changes of 0.3% to 0.5% have been observed in lunar samples 60501,105, 65701,23 and 72161,73, of 0.4% and 0.8% in [superscript 158]Gd/[superscript 157]Gd in samples 14163,848 and 60501,105 and of 0.8%, 1.2% and 0.06% in [superscript 150]Sm/[superscript 149]Sm in samples 14163,848 and 60501,105 and 74220,125 respectively. This is the first time that neutron capture has been detected in cadmium. Mass fractionation effects of 0.30%, 0.53% and 0.54% per mass unit have been observed in lunar samples 60501,105, 65701,23 and 72161,73, the first cadmium mass fractionation observed in lunar soils. The cause of elemental mass fractionation on the Moon is not yet understood. These new data from cadmium, a volatile element with a large mass range from [superscript 106]Cd to [superscript 116]Cd is a valuable contribution to the debate.Thermal neutrons are captured preferentially at resonance energies of 0.03 eV by [superscript 155]Gd and [superscript 157]Gd, at 0.09 ++ / eV by [superscript 149]Sm and at 0.178 eV by [superscript 113]Cd. A comparison of the changes in [superscript 114]Cd/[superscript 113]Cd, [superscript 156]Gd/[superscript 155]Gd, [superscript 158]Gd/[superscript 157] and [superscript 15O]Sm/[superscript149]Sm due to neutron capture can therefore indicate the relative energies of the neutrons.Previous work has compared changes in [superscript 158]Gd/[superscript 157]Gd and [superscript 150]Sm/[superscript 149]Sm, this work extends the comparison with the new measurements of the changes in 114 [superscript 114]Cd/[superscript 113]Cd, This thesis shows that the intensity of the thermal neutrons peaks at a higher energy than the <0.1 eV assumed by Lingenfelter et al. (1972). The capture rate for gadolinium calculated by Lingenfelter, has been shown to be too high, this thesis shows that if a modified energy spectrum is considered, biased towards higher energies, it will bring the calculated neutron capture rate by gadolinium closer to the measured rates.The concentrations of cadmium, gadolinium and samarium in nine lunar samples have been measured for the first time by high precision isotope dilution mass spectrometry. Cadmium in 10017,341, 14310,615, 15041,188, 15059,240, 60501,105, 65701,23, 72161,73 and 74220,125 is 10.0_+0.2, 1.51_+0.02, 32.8+_0.6, 34.9+_0.3, 112+_2, 68.3_+0.8, 57.0+_0.6 and 300+_7 ppb respectively. Gadolinium concentrations of 21+_7 ppm in 14163,848, 3.26+_0.05 and 5.8_+0.3 ppm in 60501,105, and 8.1+_0.2 and 8.6+_0.1 ppm in 74220,125 are presented. Samarium concentrations of 24.3_+0.4 and 29.8_+0.5 ppm were found in 14163,848, 2.68+_0.04 and 14+2 ppm in 60501,105, and 6.3+_0.1 and 6.8+_0.1 ppm in 74220,125.The concentrations of cadmium, gadolinium and samarium in the seven geochemical reference materials BCR-1, BHVO-1, BIR-1, DNC1, MAG-1, PCC-1 and W-2 are also presented, some measured for the first ++ / time by isotope dilution mass spectrometry.

At present several groups are analysing quasar absorption spectra to search for variation of the fine structure constant, alpha, across space and time. These studies compare the wavelengths of several transitions observed in the absorption clouds with those seen in the laboratory, and interpret anomalies as variation in alpha. One group has already presented evidence that alpha may have been smaller at an early epoch. Other groups using different telescopes see no variation. These studies use the ???many-multiplet??? method, which relies on the utilisation of many transitions in many ions to enhance the size of the effects and remove sources of systematic error. While this method offers an order-of-magnitude improvement in sensitivity over the previously used alkali-doublet method, the alpha-dependence (relativistic shift) of every transition used in the analysis must be calculated ab initio. In this thesis we present a method for the precise calculation of relativistic shifts, based on an energy calculation involving combination of the configuration interaction method and many-body perturbation theory. The many-multiplet method also introduces a potential systematic error: if the relative isotope abundances of the absorbers differ from terrestrial abundances then there can be spurious shifts in the measured wavelengths, which may be incorrectly interpreted as variation of alpha. A ???conspiracy??? of several isotopic abundances may provide an alternative explanation for the observed spectral anomalies. To account for these systematic errors we need accurate values of the isotope shift. We calculate these shifts using the finite-field method to reduce the problem to that of an energy calculation, which in turn is done using the same method used for the relativistic shift. We present the results of our calculations for a variety of atoms and ions seen in quasar absorption spectra. The results of this research should allow astrophysicists to measure isotope abundances in the absorbers directly. This can provide a test for models of nuclear reactions in stars and supernovae, and of the chemical evolution of the Universe. Our calculations can also be used in conjunction with measurements to extract changes in nuclear charge radii between isotopes.

The present study aimed to determine whether stable isotope techniques can be universally applied to detect landfill leachate contamination in aquatic systems. Results of analysis of ��C in dissolved inorganic carbon ([delta]��C-DIC), deuterium and �⁸O in water ([delta]D-H₂O and [delta]�⁸O-H₂O), and �⁵N of dissolved inorganic nitrogen components ([delta]�⁵N-NH₄⁺ and [delta]�⁵N-NO₃⁻) were presented for leachate, surface, and ground water samples collected from seven landfills located throughout New Zealand between 2003 and 2006. The unique conditions within a landfill lead to measurable fractionations in the isotopic ratios of the products of degradation. Results of isotope and ancillary parameter analyses enabled the discernment of different types of leachate, resulting from different microbial processes within the landfill environment. The isotopic characterisation of leachate enabled improved interpretation of geochemical data from potentially impacted surface and ground waters, and provides useful insight to landfill development for landfill operators. A general isotopic fingerprint delineated by [delta]��C-DIC and [delta]D-H₂O values showed leachate to be isotopically distinct from uncontaminated surface and ground water for samples analysed in the present study. However, not all water samples identified as leachate-impacted via site-specific assessments exhibited isotopic values that overlapped with the general leachate fingerprint. This highlights the need to investigate each site individually, within the context of a possibly global leachate isotope signature. Site-specific investigations revealed the effectiveness of applying [delta]�⁸O-H₂O and [delta]�⁵N-NH₄⁺ or [delta]�⁵N-NO₃⁻, in addition to [delta]��C-DIC and [delta]D-H₂O analyses, to the detection of leachate impact on aquatic systems. Furthermore, ancillary parameters such as alkalinity and ammonium concentration enabled the construction of simple isotope mixing models for an estimate of the quantity of leachate contribution. Results of isotopic investigations of stream biota suggested potential for the development of bio-indicators to monitor leachate influence on aquatic ecosystems in landfill-associated streams. The present study demonstrated the probative power of stable isotope techniques applied to investigations of leachate impact on landfill-associated aquatic systems.

This thesis has developed a suite of compound specific stable isotope tools to monitor landfill leachate and identify the infiltration of leachate to ground water and surface water. These tools have the power to indicate the fractional contribution multiple discrete sources of pollution are making to a single location. This journey began by developing two solid phase extraction (SPE) methods to extract non-polar and polar organic compounds from leachate with minimal fractionation of hydrogen or carbon isotopes. Non-polar compounds were successfully extracted using ENV+ SPE cartridges and polar compounds were successfully extracted using Strata-X SPE cartridges. The isotopic fractionation of non-polar compounds during ENV+ extraction varied significantly (up to 245⁰/₀₀ and 1.8⁰/₀₀ for D and ��C respectively, when eluted with acetonitrile and ethyl acetate, as recommended by manufacturers) but the fractionation of compounds eluted with dichloromethane was negligible (less than instrumental precision). Polar compounds were eluted from Strata-X cartridges with negligible isotopic fractionation using methanol. The direct comparison of SPE and liquid-liquid extraction found SPE to extract slightly more compound from leachate then liquid-liquid extraction (especially for polar compounds) and the isotopic compositions of compounds did not change with extraction methods. These new analytical methods subsequently were used to determine the isotopic compositions of organic compounds dissolved in leachates from three New Zealand landfills. The molecular and isotopic signature of leachate varied significantly between landfills, indicating the isotopic fingerprint of organic compounds in leachate is unsuitable as a universal tracer of leachate. However, compounds such as terpien-4-ol, methylethylbenzene and juvabione maintained their isotopic composition over short geographical distance-indicating their potential as site-specific tracers of leachate. Organic compounds analysed on a transect across the landfill boundary indicated polar compounds were more mobile than semi-volatile compounds and possessed a more conservative isotopic composition. However, hexadecanoic acid extracted from leachate and ground water was highly depleted in ��C (-72 ⁰/₀₀ to -40⁰/₀₀), indicative of methanogenic and sulfate reducing bacteria. These bacteria only live in highly reducing environments such as leachate; therefore their presence in the pristine environment can potentially indicate the release of leachate from the landfill. The final experiments traced the uptake and utilisation of leachate by periphyton. The isotopic composition of bulk periphyton, fatty acids and phytol indicated that microbial assimilation and utilisation of nutrients is a complex process. Fatty acid biomarkers for green algae and diatoms showed signs of leachate derived nutrients, however the availability of nutrients (carbon, nitrogen, water and light) caused significant changes in metabolic processes and isotopic compositions. Under slow growing conditions, the [delta]��C composition of periphyton became enriched in ��C as solar irradiation levels decreased (including shading by detritus and periphyton), while the [delta]D composition of fatty acid was controlled by the internal recycling of hydrogen. This study indicated the power of compound specific isotope analysis as a tool to detect the release of landfill leachate from a landfill, especially at locations with multiple potential sources of contaminants, and provides a sound platform for future research.