The emergence of antibiotic resistance to current treatments of bacterial infection represents a major challenge that needs to be addressed with the development of new generations of inhibitors. The enzyme 5-enolpyruvylshikimate 3- phosphate synthase (EPSPS) catalyses the sixth step in the shikimate biosynthetic pathway, which is essential for the synthesis of aromatic compounds such as the aromatic amino acids phenylalanine, tryptophan and tyrosine. It occurs in plants, bacteria and some parasites. Since the pathway is absent in mammals but essential for the pathogenicity of a number of organisms, EPSPS is considered a prospective target for new inhibiter design. A number of EPSPS inhibitors have been reported in the literature. What we are lacking is an understanding of the features that are important for binding EPSPS. We have synthesized compounds to probe the active site of the enzyme based on the knowledge of an enzyme-catalyzed intermediate with a high cationic character. This will include assembling bipartite/tripartite inhibitors to discover what interactions or structural motifs are important for binding. Once the features important for binding to EPSPS are understood, the possibility of elaborating them to create potent inhibitors of EPSPS will be investigated. In addition, the synthesis of two shikimate analogs [5-^(18)O] shikimic acid and 4-deoxyshikimic acid were completed for further experiments to probe the enzyme mechanism in detail, and for transition state structure by transition state analysis. Transition state analysis using kinetic isotopic effects (KIE) will elucidate the transition state structure of the enzyme-catalyzed EPSP reaction, and provide a detailed starting point for designing EPSPS inhibitors. / Thesis / Master of Science (MSc)

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Organoboron compounds are ubiquitous in organic chemistry. Fundamental transformations utilizing organoboron compounds are a necessary addition to any organic chemist's synthetic toolbox. In addition to their extensive use as synthetic intermediates, organoboron compounds are being increasingly studied for their material and medicinal properties. Excitingly, significant advances have been made over the years towards the synthesis of a wide variety of organoboron substrates. In the case of vinylboronic acids, synthesis primarily occurs through cis addition of boron reagents across triple bonds. However, methods affording trans addition products are scarce. Furthermore, many current methods rely on the use of expensive and toxic transition-metal catalysts. Herein, we describe the development of trans addition of boron reagents across polarized triple bonds to afford novel vinylboronic acids. Emphasis is placed on the transition metal-free nature of the reactions as well as the regio- and stereoselectivity observed in the products. In addition, the synthetic utility of the resulting trans addition products is demonstrated in the synthesis of biologically relevant molecules. We first describe the Brønsted base-mediated trans silaboration of propiolamides in which two functional groups with orthogonal reactivity are simultaneously installed. We then go on to describe an organocatalyzed trans hydroboration of propiolate esters as well as a complementary Brønsted base-mediated trans hydroboration reaction of propiolamides. To conclude this portion, we demonstrate how the products from the previous methods can be used to synthesize difluoroborylacrylamides which possess unique and versatile reactivity. Herein we disclose the first small-molecule inhibitors of the sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2). While little is known in regard to the structure and function of SPNS2, previous studies have demonstrated the vital role SPNS2 plays in S1P mediated processes and have identified SPNS2 as a potential clinical target. For example, SPNS2 is critical to S1P-mediated lymphocyte egress from primary lymphoid tissues. Thus, small molecule inhibition of SPNS2 represents a novel therapeutic strategy for the treatment of autoimmune disorders such as multiple sclerosis. In this study, we report the discovery of small molecule inhibitors that display low micromolar activity using a novel yeast-based SPNS2 assay. Inhibitor structure-activity-relationship studies led to the discovery of the imidazole-based amine inhibitor 7.54. Furthermore, administration of 7.54 to mice recapitulates the lymphopenic phenotype observed in previous SPNS2 knockout studies. / Doctor of Philosophy / Boron-containing compounds are important in organic chemistry and are involved in the synthesis of a variety of materials and medicines used in everyday life. As such, the ability to efficiently and sustainably prepare boron-containing compounds has far reaching consequences. Access to an important class of boron-containing compounds known as vinylboronic acids has previously been established; however, product selectivity is often limited to what is referred to as cis addition products. Furthermore, access to the corresponding trans addition products is often limited to processes involving expensive transition metal catalysts that produce environmentally toxic waste. Herein, novel transition metal-free trans addition processes are described for preparing vinylboronic acids. In addition, the application of the resulting products is demonstrated through the synthesis of biologically relevant compounds. Sphingosine-1-phosphate (S1P) is an important signaling lipid that is involved in a variety of physiological processes. Improper balance in the amount of S1P in the body is associated with a variety of disease states such as autoimmunity and cancer. Two drugs that inhibit S1P-mediated processes have been approved by the FDA, fingolimod (Gilenya®) and siponimod (Mayzent®). However, there are drawbacks to targeting the S1P receptor directly, including dose-limiting side effects that are associated with these drugs. Consequently, recent efforts have focused on developing new ways to control the effects of S1P. Herein, we describe the discovery and development of the first reported inhibitors of the S1P transporter, spinster homolog 2 (SPNS2). A library of compounds was synthesized and tested for SPNS2 inhibition. The resulting structure-activity-relationship studies led to the discovery of the imidazole-based propanamine derivative 7.54. Furthermore, we demonstrate the potential of SPNS2 inhibition to control the effects of S1P in mice. These studies provide a foundation for future SPNS2-based drug discovery that will hopefully lead to the development of improved therapies for the treatment of autoimmune disease and cancer.

Ediacaran phosphorites provide a principal record of the paleoenvironmental and paleoecological conditions of the oceans during Earth's second major oxygenation and the evolution of complex life. Although the fidelity of this record is high, diagenesis and metamorphism frequently alter or overprint primary minerals and structures, necessitating validation of results from mineralogical and geochemical analyses and determinations of fossil affinities. Therefore, it is imperative to unravel the complications of post-depositional alteration, and thus provide a strong foundation for environmental and biological interpretations, via an integrated sedimentological, stratigraphic, petrographic, and geochemical approach. Transmitted light, cathodoluminescence, and scanning electron microscope petrography in conjunction with Raman spectroscopic and X-ray diffraction analyses were employed to determine the origin of phosphorites from the early Ediacaran (632 – 614 Ma) Doushantuo Formation at the Wanjiagou section, near Zhangcunping, Hubei Province in South China. Results suggest granular phosphorites were deposited during one to two episodes of reworking of pristine phosphorite hardgrounds, which originated during redox-controlled and/or microbially-mediated phosphogenesis. Granular laminae were then cemented by a ferric iron-phosphate mineral, phosphosiderite. As a product of oxidative weathering and/or thermal stabilization of ferrous iron-phosphates (e.g., vivianite), this cement is suggestive of precipitation from ferruginous porewaters. This is the first direct evidence for iron-phosphate minerals in Ediacaran phosphorites and substantiates previous hypotheses of P burial beyond primary calcium phosphate. If accumulation and burial of phosphorites during this interval was rapid enough to have limited P availability and thus primary productivity, their formation may have governed oxygen production prior to the Neoproterozoic Oxygenation Event (NOE). Oxygenation of the oceans during the NOE and the appearance of complex, multicellular life are suspected to be causally linked within the Ediacaran. However, a fragmented fossil record with insufficient analogues and varied taphonomic modes leaves much of the Ediacaran fauna with uncertain taxonomic and phylogenetic affinities, leading to ambiguity regarding their life modes and environmental associations. Furthermore, demonstrating biogenicity is an often overlooked, yet fundamental component of Ediacaran fossil identification and interpretation, something that has particularly affected the morphologically simple discoidal group of fossils known colloquially as Aspidella. Petrographic observations supported by Raman and energy dispersive spectroscopy provide evidence that discoidal concretions from the Ediacaran Miaohe Member near Maxi, Hubei Province in South China are diagenetic in origin but superficially resemble Aspidella's morphology. Erosion of these syn-compactional concretions produced concentric rings on bedding planes caused by internally deformed laminae resulting in Aspidella pseudofossils. These results highlight the necessity for critical evaluation of the origin of discoidal structures observed in Ediacaran sedimentary successions. / Master of Science / Contemporary and ancient phosphorus-rich sedimentary deposits, known as phosphorites, precipitate within the oceans as a result of intricate chemical and biological interactions. The Ediacaran Period (635 – 539 million years ago) contains the first truly extensive occurrences of phosphorites in addition to a fossil record of the earliest animal organisms. Deposited after the end of Earth's last global glaciation, the origins of Ediacaran phosphorites are affiliated with these dramatic climatic and evolutionary transitions as well as the rise of atmospheric and oceanic oxygen concentrations to near modern levels. Deposition of phosphorites often occurs in low-oxygen oceanic environments and their formation constitutes the dominant mechanism by which phosphorus is removed from the phosphorus cycle on time scales greater than 1000 years. Therefore, phosphorite occurrences provide a record of phosphorus cycling, oxygen availability, and biological productivity. However, microscopic and chemical analyses of phosphorites from the Ediacaran Doushantuo Formation in South China demonstrate they are partially composed of phosphorus minerals that likely formed in non-oxygenated environments. The presence of these atypical phosphorus minerals has been previously hypothesized, with the implication that they further limited the availability of phosphorus for use by photosynthetic organisms. Such a limitation on photosynthesis would have resulted in decreased oxygen production and thus the formation of these phosphorites may explain the rate and trend of the change in oxygen concentrations observed during the Ediacaran. Ediacaran fossils also act as a proxy for environmental conditions of the ancient oceans through inferences about the preserved organisms' requirements for life. Although most fossils of this age are the first of their kind in terms of biological complexity, they are typically simple in terms of their morphology, making identification difficult. Furthermore, providing evidence that such simple structures actually represent a fossilized organism is often problematic due to the inability to compare them with modern organisms. Microscopic and chemical analyses of disc-shaped structures from the Ediacaran Miaohe Member in South China reveal that they are concretions that were not created by an organism, even though their morphology very closely resembles the Ediacaran fossil Aspidella. Identification of these concretions as pseudofossils suggests that close examination of fossils from Ediacaran rocks is necessary.

The genus Phytophthora contains a large number of species that are known plant pathogens of a variety of important crops. Phytophthora sojae, a hemibiotroph, causes approximately 1-2 billion dollars (US) of lost soybean world-wide each year. P. infestans, the causative agent of the Irish potato famine, is responsible for over 5 billion dollars (US) worth of lost potato each year. These destructive plant pathogens facilitate pathogenesis through the use of small secreted proteins known as effector proteins. A large subset of effector proteins is able to translocate into host cells and target plant defense pathways. P. sojae Avr1b is able to suppress cell death triggered by BAX and hydrogen peroxide. The W-domain of Avr1b is responsible for this functionality, and is recognized by the Rps1b gene product to induce effector triggered immunity. These oomycete effector proteins translocate into host cells via a highly conserved N-terminal motif known as RXLR-dEER without the use of any pathogen encoded machinery. In fungi an RXLR-like motif exists, [R,K,H] X [L,F,Y,M,~I] X, that is able to facilitate translocation without pathogen encoded machinery. Both functional RXLR and RXLR-like motifs are able to bind phosphatidylinositol-3-phosphate (PtdIns- 3-P) to mediate entry into host cells. The use of novel inhibitory mechanisms has shown effector entry can be blocked either by sequestering PtdIns-3-P on the outer leaflet of plant and animal cells or by competitive inhibition of the binding pocket of the RXLR or RXLR-like motifs. / Ph. D.

Skeletal loss and bone deficiencies are major worldwide problem that is only expected to increase due to increase in aging population. As current standards in treatment autografts and allografts are not without drawbacks, there is a need for alternative bone grafts substitutes. The goal of this project was to utilize electrospinning and heat sintering techniques to create biodegradable full thickness three dimensional biomimetic polymeric scaffolds with macro and nano architecture similar to natural bone for bone tissue engineering. First we have investigated pretreatment with 0.1M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds when incubated in concentrated simulated body fluid (SBF)10XSBF. Mixture of 10% gelatin and PLLA resulted in the significantly higher degree of mineralization, increased mechanical properties, and scaffolds that supported cellular adhesion and proliferation. In the next step we applied heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. Fiber orientation can make a slight difference in nanofibrous scaffold compressive mechanical properties, but this difference is not as profound as the difference seen with increased mineralization. We also developed a technique to fabricate scaffolds that mimic the organization of an osteon, the structural unit of cortical bone. Resulting scaffolds consisted of concentric layers of electrospun gelatin/PLLA nanofibers wrapped around microfiber core with diameters that ranged from 200-600µm. Individual osteon-like scaffolds were heat sintered to fabricate three dimensional scaffolds contained a system of channels running parallel to the length of the scaffolds, as found naturally in bone tissue. Finally we combined two previously fabricated structures, sintered electrospun sheets and individual osteon-like scaffolds, to create novel scaffolds that mimic dual structural organization of natural bone with cortical and trabecular regions. Mineralization for 24 hr significantly increased mechanical properties of the scaffolds, both yield stress and compressive modulus under physiological conditions. Both nonminerlized and mineralized scaffolds were found to support cellular attachment and proliferation over 28 days in culture, but scaffolds mineralized for 24hr were found to better support osteoblastic differentiation and mineral deposition. / Ph. D.

Polymer – clay nanocomposites have improved thermal, mechanical, and barrier properties when compared with the pure polymer. The objective of this study was to examine if gas separation performance could be improved by introducing a layered nanopourous aluminum phosphate with a large aspect ratio into a polymeric matrix. The aluminum phosphate has eight membered rings, which could potentially serve as a size selective medium. A hexafluorinated polyimide, 6FDA-6FpDA-8%-DABA, was used as the polymeric matrix. The polyimide and the aluminum phosphate were synthesized separately according to well documented procedures. The two materials were blended and fabricated into nanocomposite membranes. The effect of mixing temperature and percentage of layered aluminum phosphate added to the polymer on the permeation properties were examined. These factors had a direct effect on the degree of intercalation and exfoliation of the nanocomposite structure. Transmission FTIR, TEM, DMTA, and X-ray diffraction were used to characterize the morphology, structure, and composition of these nanocomposite films. The permeation properties of the nanocomposite membranes were evaluated using pure gases (He, O₂, N₂, CH₄, CO₂) at 35°C and a feed pressure of 4 atm. In general, the permeability decreased and the selectivity coefficients increased when adding 10 wt% aluminum phosphate to the polyimide. Furthermore, the membranes showed size selectivity, consistent with the pore size in the layered aluminum phosphate. / Master of Science

The purpose of this investigation was to determine the extent of phosphorus readsorption on Occoquan sediments and the amount of subsequent removal because of settling of the particles with time. Occoquan sediments were placed in six polyurethane cylinders and submerged. In two separate experiments, the cylinders were driven anaerobic, which allowed phosphorus, previously bound in the sediments, to release. The sedimentâ water system was then reaerated and mixed with a porous air diffuser. Analyses were conducted before and after the mixing period. From the results obtained, the following conclusions were drawn: phosphate readsorption occurred upon mixing the system, and as the turbidity decreased, the phosphate concentration decreased; phosphate readsorption occurred in a two-step process; each suspension tended toward equilibrium after mixing; interstitial waters contributed significant amounts of phosphorus upon mixing of the sediment-water systems; Occoquan sediments released variable amounts of phosphorus even within samples from similar sites; sediments from the Occoquan Creek arm contained less phosphorus than Bull Run sediments; and sediments low in phosphorus concentrations removed greater amounts of phosphorus in a shorter period of time. / Master of Science

Master of Science

Organic ligands, such as EDTA, accelerate the dissolution of silicate and oxide minerals. In natural systems oxyanions can compete with organic ligands for mineral surface sites thereby affecting ligand-promoted dissolution rates. Oxyanions can either enhance or inhibit dissolution depending upon pH. It has been suggested that the type of surface complex formed by the oxyanion is responsible for differences in ligand-promoted dissolution rates with mononuclear being dissolution enhancing and bi- or multinuclear being dissolution inhibiting. However, recent research has demonstrated that the type of surface complex formed by the oxyanion is not responsible for differences in ligand-promoted dissolution rates of oxide minerals. Accordingly, this manuscript examines the influence of selenite, molybdate, and phosphate on the EDTA-promoted dissolution of goethite and proposes a mechanism for the observed differences in dissolution rates over a pH range of 4 - 8. We propose that the surface complex formed by EDTA is the controlling factor for the observed dissolution rate, with mononuclear EDTA surface complexes accelerating goethite dissolution compared to bi- or multinuclear complexes. These experimental results suggest that EDTA forms multinuclear complexes with goethite surfaces at pH values greater than or equal to 6 and mononuclear complexes with goethite surfaces at pH values < 6. Dissolution results show that when the oxyanion and the EDTA are present in the system at concentrations nearly equaling the surface sites available for adsorption, the oxyanion reduces the adsorption of EDTA and inhibits dissolution over the pH range of 4 - 8. However, a different mechanism occurs at pH values greater than or equal to 6 when the oxyanion is present at 0.1 mM. EDTA adsorbs but the number of carboxylic groups that can sorb to the surface is reduced causing the formation of mononuclear complexes. This shift from multi- to mononuclear surface complexes enhances the EDTA-promoted dissolution of goethite in the presence of the oxyanions compared to EDTA-promoted dissolution in their absence. / Master of Science