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<p>Uranium oxides are the most common form of uranium in both nuclear waste and in the environment. As such, the uranyl <i>di</i>oxo framework (UO₂)²⁺, and its strong U&equiv;O triple bond interactions, dominate the research. It is well-established the oxo ligands are thermodynamically robust and kinetically inert, thus making the uranyl fragment very unreactive. Interestingly, microbial activation of uranyl is a vital component in the sequestration of uranium in uranium-contaminated environments, likely requiring functionalization of the normally unreactive oxo ligands. However, there is little evidence available for the mechanism of this process and model systems describing this chemistry are scarce. </p><p> In contrast to uranium oxide chemistry, the chemistry of uranium and the softer Group 16 elements (e.g., S, Se, Te) is substantially less developed even though it has been proposed that separation of radiotoxic fission products in spent nuclear fuel would be greatly enhanced by employment of soft-donor extractants. This postulation has been rationalized on the basis of increased covalency within actinide-chalcogenide bonds. As such, developing soft-donor, heteroatom-substituted analogues of the uranyl moiety will allow for greater insight into the extent of <i>f</i>-orbital participation, and thus, covalency, within uranium-ligand multiple-bonding frameworks and facilitate the design of novel waste extractants. </p><p> This first part of this dissertation describes the activation of uranyl via reductive silylation. By employing strongly donating equatorial co-ligands, the nucleophilic nature of the oxo-ligands is exploited by addition of Me₃SiI. This substrate promotes reduction to U(V) and silylation of <i> both</i> oxo ligands. Subsequent addition of Lewis bases results in facile reduction to U(IV) and clearly displays the complete transformation of the uranyl fragment to better described 'alkoxide-like' complexes. </p><p> The second part of the dissertation attempts to expand the library of uranium-chalcogenide complexes, targeting molecules which exhibit multiple-bonding ligation modes reminiscent of uranyl. In this regard, a highly reducing U(III) starting material was employed and found to readily activate chalcogen sources to yield several complexes which exhibit <i>terminally</i>-bound chalcogenide ligands. These complexes were fully characterized to better understand the electronic nature of the uranium chalcogenide bonds.</p>

<p> Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), is one of the deadliest infectious diseases. Emergence of drug resistant strains of <i>Mtb</i> and co-infection with HIV has made TB both difficult and expensive to treat. New TB therapies are needed to shorten treatment and be effective against all strains and metabolic states of the organism. Development of inhibitors of 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (Dxr), an essential enzyme for <i>Mtb,</i> is a novel approach toward the development of a new TB chemotherapy. Natural product fosmidomycin inhibits Dxr and kills other organisms (<i>Plasmodium falciparum,</i> <i>Escherichia coli</i>) reliant on this enzyme. Interestingly, fosmidomycin is not effective against <i>Mtb.</i> The goals of this work are to rationally design inhibitors that will specifically inhibit <i>Mtb</i> Dxr and enhance cellular uptake. Two series of compounds were designed and synthesized. Compounds from both series inhibit <i> Mtb</i> Dxr and demonstrate enhanced whole cell activity. The synthetic and biological results of this work will be presented.</p>

<p> The pygmy sperm whale (<i>Kogia breviceps</i>) is currently the second-most commonly stranded cetacean in the Southeastern United States (SEUS), but information concerning their population structure is severely limited. This study utilized stable isotope analysis to investigate the possible migratory patterns and population structure of <i>K. breviceps </i> among six different regions in the SEUS. Combined growth layers from different regions of the teeth were subsampled via dental drill and analyzed representing four different age classes: calf, juvenile, sub-adult, and adult, as well as four yearlings that had stranded with their mothers. Stable isotope ratios of carbon and nitrogen were measured in the organic component of 46 teeth, and oxygen isotope ratios were measured in the inorganic (hydroxyapatite) component of 21 teeth obtained from stranded individuals. There was a high degree of individual variability in &delta;¹³C, &delta;¹⁵N, and &delta;¹⁸O resulting in no significant differences between the six different regions: South Carolina, Georgia, Northern, Central, and Southern Florida, and the Gulf of Mexico. Differences between the age classes were significant for &delta;¹³C and &delta;¹⁵N. Adults exhibited significantly more negative &delta;¹³C than subadults. These results support a previously hypothesized inshore-offshore migration for <i>Kogia breviceps.</i> Yearlings displayed significantly higher &delta;¹⁵N values than all other age classes due to nursing. A slight increase in &delta;¹⁵N from juvenile to adult supports a possible ontogenetic shift in the trophic level of prey. Results from this study provide the first carbon and nitrogen isotope values from different age classes of pygmy sperm whales as well as the first reported oxygen isotopes values for this species.</p>

<p> Surface plasmon resonance (SPR) active nanohole array substrates offer a diverse biosensing platform with high sensitivity and unique characteristics. This dissertation investigates the sensitivity and fundamental SP features of various nanohole array substrates and demonstrates higher sensitivity than conventional continuous gold platforms, tunability to specific analytes, and great enhancement of the local field intensity. Novel instrumentation and analytical techniques are developed and utilized to assess the nanohole array SPR sensing substrates in the near infrared as well as with interaction of other nanostructures.</p><p> The nanohole array substrates are evaluated throughout the near-infrared (NIR) region by novel SPR instrumentation and methodology that extends the working SPR wavelength range and measurement reliability. Development of a robust NIR-SPR instrument allows access to higher wavelength ranges where sensitivity is improved and novel SP modes and plasmonic materials may be investigated. Different aspects of the NIR-SPR instrument, including temporal stability, mechanical resilience and sensitivity, are evaluated and presented. Furthermore, a method is developed for improving precision and accuracy of empirically determined SP penetration depth, a merit of SPR spectroscopy sensitivity. The technique incorporates an adsorbate-metal bonding effect which improves the consistency in the penetration depth value calculated at different adsorbate thicknesses from 41-1089% relative deviation (without bonding effect) to 2-11% relative deviation (with bonding effect). It also improves the experimental agreement with theory, increases the accuracy of assessing novel plasmonic materials and nanostructures, and increases the precision in adsorbate parameters calculated from the penetration depth value, such as thickness, binding affinity, and surface coverage.</p><p> Utilizing this NIR-SPR instrument and improved technique for calculation of penetration depth, the sensitivity and various SP modes of the nanohole arrays throughout the NIR range are evaluated, and an improvement in sensitivity compared to conventional continuous gold is observed. Both the Bragg SPs arising from diffraction by the periodic holes and the traditional propagating SPs are characterized with emphasis on sensing capability of the propagating SPs. There are numerous studies on the transmission spectroscopy of nanohole arrays; however this dissertation presents one of the few studies in Kretschmann mode, and the first in the near infrared, where greater surface sensitivity is observed. The sensitivity profile of various nanohole array parameters (periodicity, diameter, excitation wavelength) and SP modes is also presented. </p><p> Further control and enhancement of the SP field is pursued by interaction between nanohole array substrate and nanoparticles to exploit field intensification between plasmonic structures, i.e. gap mode enhancement. Under specific conditions, the SPs couple together and the electric field between the structures is amplified and localized, which may be exploited for sensing purposes and surface enhanced techniques, including tip enhanced Raman spectroscopy (TERS) or surface enhanced Raman spectroscopy (SERS). A technique for observing nanohole array-nanoparticle distance dependent SP interaction is developed and utilized to demonstrate SP interaction. Scanning probe microscopy controls the position of a single nanoparticle (SNP) affixed to an atomic force microscope probe, and the location specific interaction of the SNP-nanohole array surface plasmons is measured by darkfield surface plasmon resonance spectroscopy. Coupling of the nanoparticle to the nanohole array exhibits a maximum when the SNP resides within a nanohole, which resulted in a maximum SPR wavelength shift of 17 nm and an increase in scatter intensity. This dissertation presents the first empirical observations of SPM controlled gap mode enhancement of more complex nanostructures and allows for optimization of positioning prior to use in sensing.</p>

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3822. Adviser: Zaida Luthey-Schulten. Includes bibliographical references (leaves 92-106). Available on microfilm from Pro Quest Information and Learning.

<p> Cancer cells are characterized by elevated ROS levels, which provide these cells with a distinct survival advantage, promoting proliferation, invasion, and resistance to apoptotic stimuli. In order to maintain ROS below a critical threshold that would otherwise result in death, cancer cells have appropriately adapted their anti-oxidative machinery. Here, I studied superoxide dismutase 1 (SOD1) as a potential contributor to cancer cell survival under conditions of high oxidative stress. I determined that SOD1 is up-regulated in a majority of cancer cells in which the activity of another dismutase, MnSOD, is reduced, and found evidence to suggest that SOD1 is essential for maintaining mitochondrial integrity in these cells. Additionally, I explored a possible mechanism by which mitochondrial SOD1 increases in cancer cells, and found evidence indicating that this is due to decreased MULAN levels. Last, I tested whether blocking SOD1 activity could sensitize cancer cells to chemotherapeutic treatment, and determined that additional mechanisms must exist to compensate for SOD1 activity loss.</p>

<p> This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data. Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and several substrates. These experiments were combined with lead optimization techniques to create a potent irreversible inhibitor which protects 95% of cells in vitro. Dynamics studies on a TcdB cysteine protease domain were performed to an allosteric communication pathway. Comparative analysis of the static and dynamic properties of the TcdA and TcdB glucosyltransferase domains were carried out to determine the basis for the differential lethality of these toxins. Large scale biological data is readily available in the post-genomic era, but it can be difficult to effectively use that data. Two bioinformatics methods were developed to process whole-genome data. Software was developed to return all genes containing a motif in single genome. This provides a list of genes which may be within the same regulatory network or targeted by a specific DNA binding factor. A second bioinformatic method was created to link the data from genome-wide association studies (GWAS) to specific genes. GWAS studies are frequently subjected to statistical analysis, but mutations are rarely investigated structurally. HyDn-SNP-S allows a researcher to find mutations in a gene that correlate to a GWAS studied phenotype. Across human DNA polymerases, this resulted in strongly predictive haplotypes for breast and prostate cancer. Molecular dynamics applied to DNA Polymerase Lambda suggested a structural explanation for the decrease in polymerase fidelity with that mutant. When applied to Histone Deacetylases, mutations were found that alter substrate binding, and post-translational modification.</p>

<p> Calmodulin (CaM) plays an important role in calcium (Ca²⁺)-dependent signal transduction. Ca²⁺ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca²⁺-dependent inactivation (CDI) process in store-operated Ca²⁺ entry (SOCE), by interacting with the N-terminus of the hexameric plasma membrane Ca²⁺ channel Orai1. To understand the relationship between Ca²⁺-induced hydrophobicity of CaM and the CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) were used as an informative probe to better understand the functionality of each EF-hand. ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca²⁺ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM, as depicted by ANS fluorescence and binding affinity. Such a conclusion is consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai1. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (&Delta;H = &minus; 5.02 &plusmn; 0.13 kcal/mol) and binding affinity (K_a = 8.92 &plusmn; 1.03 x 105 M^&minus;1). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ?H than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. Circular Dichroism studies suggested differences in binding most likely resulted from changes in chimera three-dimensional structure rather than secondary structure, as the extent of ?-helical content from apo-, Ca²⁺-, and Orai-CMBD-bound proteins remained similar. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 &plusmn; 0.08 s^&minus;1 by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76, indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the unique, open binding mode suggested by already published structural studies.</p>

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