Francisella tularensis is a highly virulent bacterial pathogen with an extremely low infectious dose (~10 CFU) and high rates of mortality if left untreated (30-60%). F. tularensis has an extensive history as a bioweapon, and there is no vaccine currently licensed. For these reasons the CDC considers F. tularensis a Tier 1 Select Agent. The unlicensed F. tularensis subsp. holarctica Live Vaccine Strain (LVS) provides moderate protection against virulent strains; however, we have discovered that various “wild type” lab stocks differ in their virulence and ability to confer immunity. Genome sequencing of high virulence (RML, LD50 ~200 CFU) and low virulence (ATCC, LD50 ~9,000 CFU) strains has identified nine differences, of which four are non-synonymous substitutions. One such mutation occurs in the ferrous iron uptake gene feoB in RML. While iron is required for cellular function, ferrous iron (Fe2+) can participate in the Fenton reaction with H2O2, leading to inactivation of essential iron-sulfur cluster enzymes. Part of the innate immune response involves mitochondria-derived reactive oxygen species in the cytosol. Fully virulent strains of F. tularensis are known to be highly resistant to such host defenses, and have low levels of intracellular iron. Accordingly, the RML strain was highly resistant to exogenous H2O2in vitro relative to the ATCC strain. An iron-responsive lacZ reporter had ~2-fold higher induction in the RML strain relative to ATCC during iron limitation. Overexpression of the functional feoB allele, but not the RML allele, leads to significantly increased sensitivity to H2O2 and increased killing by primary macrophages stimulated with the cytokine interferon-gamma. Given the connection of iron and H2O2 toxicity, I revisited a previously published transposon screen to determine if any of the mutants identified had a role in iron homeostasis and oxidative stress resistance. One such gene was annotated as bacterioferritin (bfr), which in other bacteria forms a hollow, spherical multimer that oxidizes Fe2+ to Fe3+ and stores the oxidized form in the interior of the sphere. The Δbfr mutant was ~10-fold more sensitive to H2O2 and was attenuated nearly 8-fold in murine intranasal infection in terms of LD50 relative to the parental RML strain. Importantly, the Δbfr mutant allowed us to test the hypothesis that H2O2 resistance is critical for the RML LVS to stimulate productive immunity. At six weeks post-infection, mice previously infected with either RML or the Δbfr mutant were challenged with an infection of 25 CFU of the fully virulent F. tularensis Schu S4 strain. All mice immunized with RML survived this challenge, while all mice immunized with Δbfr succumbed; only displaying a slight increase in time to death. These results are consistent with the hypothesis that the H2O2 resistance of RML LVS mediates increased fitness in a host.

The stereoselective formation of 1,2-cis-2-amino glycosides remains a challenging obstacle for researchers seeking to study glycan function in nature. A variety of techniques to form α-linked C(2)-aminoglycosides are examined herein. The most prominent of these techniques is the nickel catalyzed stereoselective coupling of C(2)-N-benzylidine protected trichloroacetimidates to form 1,2-cis-2-amino sugars. This protocol demonstrates excellent α-selectivity and is applicable to a large structural variety of C(2)-aminoglycosyl donors and acceptors. The application of the nickel catalyzed stereoselective coupling of C(2)-N-benzylidine protected trichloroacetimidates toward the synthesis of pseudosaccharides of glycosylphosphatidyl inositol (GPI) anchors and mycothiol (MSH) in good yield and with excellent α-selectivity was also examined. In stark contrast, employing conventional Lewis acids to activate trichloroacetimidate donors provided the desired pseudodisaccharides with poor α-selectivity. Additionally, the facile synthesis of both C(1)- and C(6)-hydroxyl myo-inositols bearing differentiated protecting groups from a common and easily attainable intermediate allows access to a wide variety of GPI anchor and MSH pseudosaccharides. The highly α-selective and scalable synthesis of the Fmoc-protected GalNAc-threonine amino acid and TN antigen in large quantities is also described. The challenging 1,2-cis-2-amino glycosidic bond is addressed through a coupling of threonine residues with C(2)-N-ortho-(trifluoromethyl)benzylidenamino trihaloacetimidates mediated by Ni(4-F-PhCN)4(OTf)2. The desired 1,2-cis-2-amino glycoside was obtained in large quantities with α-only selectivity and subsequently transformed into the Fmoc-protected GalNAc-threonine and TN antigen. With the establishment of 1,2-cis-selective synthesis of heparan disaccharides, we sought to develop multivalent inhibitors of heparanase. A model study of protein/glycan interactions, in which various macromolecular architectures were examined, was developed using Concanavalin A as the model protein. Preparations of the highly-ordered monoantennary, homofunctional diantennary, and heterofunctional diantennary glycopolymers of α-mannose and beta-glucose were achieved via ring opening metathesis polymerization. Isothermal titration calorimetry measurements of these synthetic glycopolymers with Concanavalin A, which has been reported to bind strongly to α-mannose unit, revealed that heterofunctional diantennary architectures bearing both α-mannose and non-binding beta-glucose residues, glucose units, enhanced binding affinity.

Central sensitization refers to abnormal pain modulation present which is characterized by non-aversive or mildly aversive stimuli promoting feelings of pain. Many conditions referred to as Functional Somatic Syndromes (FSS)s are characterized by abnormal pain modulation, including pain in areas of the body not thought to be related to the specific FSS with which the patient has been diagnosed. Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a diagnosis of exclusion characterized by pelvic pain and urologic symptoms that shares many environmental and psychosocial correlates with FSSs. Treatment is generally non-satisfactory for patients despite substantial healthcare expenditures. Preliminary evidence suggests abnormal pain modulation in IC/BPS. Inflammatory dysregulation is an underexplored mechanism in the pain experience in IC/BPS and FSSs. The purpose of the current project is to explore the role of dysregulated inflammatory processes in IC/BPS with an emphasis on painful symptoms in three distinct papers. Paper one examines the role of inflammation in IC/BPS patients with particular emphasis on the association of Toll-Like Receptor (TLR) - 4 mediated inflammation with symptoms of pelvic pain. Paper two expands on the findings of paper one by exploring the association of TLR-4 mediated inflammation with the presence of comorbid FSSs and widespread pain. Paper three evaluates the predictive ability of these previously explored baseline inflammatory measures by testing the association between TLR-2 and 4-mediated inflammation and diurnal cortisol rhythms with symptom trajectories and symptom flares over one year of observation. Finally, the significance of these novel findings is explored.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in the success of head and neck cancer therapy. Despite efforts by several groups to understand the mechanisms of resistance to tyrosine kinase inhibitors such as erlotinib, there has been little success in improving the patient survival. Given that there are a number of ongoing clinical trials testing the efficacy of erlotinib in head and neck cancer, it is essential to investigate the novel mechanisms of erlotinib resistance to improve its efficacy and patient survival. This dissertation addresses this issue of erlotinib resistance in head and neck cancer, underscoring the role of inflammatory cytokine signaling. Chapter 1 introduces the problem of erlotinib resistance and discusses the potential link between inflammatory signaling and cancer progression and erlotinib resistance in head and neck squamous cell carcinoma. Chapter 2 discusses the role of the cytokine interleukin-6 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 3 describes the role of IL-1 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 4 discusses the specific role of IL-1α (an agonistic ligand for IL-1 signaling) in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 5 discusses ideas to test for future work in this field. Altogether, this dissertation endeavors to emphasize the contributory role of inflammatory cytokine signaling in erlotinib resistance in head and neck squamous cell carcinoma so that it helps in the development of effective anti-cancer therapies and biomarkers of resistance and/or response in HNSCC.

The goal of this research was to learn more about the flow experiences of novice art learners in the art room context in an effort to inform lesson design and the creation of an effective learning environment. The research questions in this study sought to answer whether novice art learners were entering into flow and what conditions of flow related to classroom activities contributed or hindered this process. Results revealed that novice learners do enter into flow and are more likely to do so when they find projects interesting and enjoyable. Students who entered into flow were also more likely to feel challenged by the art activities than their counterparts who did not enter into flow.

The nitrogenous bisphosphonates pamidronate, alendronate, risedronate, and zoledronate are used clinically in the treatment of bone disease. All of these drugs inhibit the enzyme farnesyl diphosphate synthase (FDPS), which mediates production of farnesyl diphosphate (FPP). However, because it is a branch point in isoprenoid biosynthesis, FPP is involved in the biosynthesis of several different substrates at the same time. One key enzyme downstream of FDPS in isoprenoid biosynthesis is geranylgeranyl diphosphate synthase (GGDPS) which affords the geranylgeranyl diphosphate (GGPP) necessary for prenylation of the small GTPases such as Ras, Rab, Rho and Rac, that are important signaling proteins. Non-nitrogenous analogues of the clinical drugs, including mono- and bisisoprenoid bisphosphonates, have been developed more recently. These new analogues have been found to inhibit GGDPS selectively. Because it is important to inhibit the generation of GGPP, selective inhibition of GGDPS is highly desirable. In previous research, digeranyl bisphosphonate (DGBP) was discovered to show good inhibition of GGDPS. In order to obtain more potent analogues of the compound DGBP, and to study the biological effect of an á-alkoxy group on bisphosphonate compounds, a series of ether bisphosphonates has been prepared and studied. A second important enzyme in isoprenoid biosynthesis is geranylgeranyl transferase II (GGTase II). This enzyme transfers GGPP to Rab proteins, and thus converts the parent proteins to lipoproteins which are essential for their proper cellular localization. One known inhibitor of this enzyme is the chemical 3-PEHPC, but a high concentration of this compound is necessary to generate any cellular effects. In an effort to study the cellular effects that result from inhibition of this enzyme, and to develop more potent inhibitors, my research has focused on modification of 3-PEHPC to obtain derivatives that may have improved biological activity. Both the known compounds 3-PEHPC and 3-PEPC, and new structures, including the first generation PEHPC N-oxides and the second-generation compounds prepared through click chemistry, have been prepared and tested for activity in this system.

The main objective of deformation theory is to study the behavior of mathematical objects, such as modules or group representations, under perturbations. This theory is useful in both pure and applied mathematics and has led to the solution of many long-standing problems. For example, in number theory, universal deformation rings of Galois representations played an important role in the proof of Fermat’s Last Theorem by Wiles and Taylor. In this thesis, we consider the case when SDn is a semidihedral 2-group of order 2n+1 for n ≥ 3 and k is an algebraically closed field of characteristic 2. The indecomposable kSDn-modules have been completely described by Bondarenko and Drozd, and Crawley-Boevey. We concentrate on so-called endo-trivial kSDn-modules, which possess a well-defined universal deformation ring by work of Bleher and Chinburg. Using the classification of Carlson and Thevenaz of all endo-trivial kSDn-modules, we show that the universal deformation ring of every endo-trivial kSDn-module is isomorphic to the group ring W [ℤ/2 x ℤ/2], where W = W (k) is the ring of infinite Witt vectors over k.

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, reduced adiposity and diet-induced obesity, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.

Nanocrystalline ZSM-5 zeolite, which is a well-known catalyst used in a variety of applications in industry, environment, and medicine, can be synthesized using different methods. However, a big challenge in synthesizing nanocrsytalline ZSM-5 is the use of an organic template such as TPAOH, which is very expensive. The template is required to facilitate the growth of the nanocrsytalline ZSM-5 during the synthesis. However, to use the nanocrsytalline ZSM-5, the template has to be removed by a calcination process to open the pores and reveal the active surface of the nanocrystalline ZSM-5. The calcination process requires a high temperature for a long time to remove the organic template. Consequently, synthesizing nanocrystalline ZSM-5 by using a templated method is considered to be time, energy, and materials inefficient. In addition, the production of CO2 from the calcination process is a negative impact on the environment. Therefore, finding another method to synthesize nanocrystalline ZSM-5 without using an organic template would be beneficial. Here, nanocrystalline ZSM-5 was synthesized successfully in high yield and quality by using a seed-assisted method and without using the organic template. In addition, the effect of synthesis temperature, synthesis time, basic environment, amount of seeds, size of seeds, aging time, and use of calcined and uncalcined seeds are investigated in this study. The synthesized nanocrystalline ZSM-5 materials were characterized by using X-ray diffraction (XRD), gas adsorption isotherm (BET/BJH), and transmission electron microscopy (TEM).

Regulation of the Pseudomonas aeruginosa type III secretion system (T3SS) is controlled by the transcriptional activator ExsA, a member of the AraC family of regulators. Members of this family are characterized by a conserved DNA-binding domain, which contains two helix-turn-helix DNA-binding motifs and is generally located in the carboxy-terminal domain (CTD). Previous work from our lab has characterized the interaction between promoter DNA and the ExsA CTD. Two monomers of ExsA bind promoter DNA at binding sites 1 and 2 (centered at -41 and -65 relative to the transcriptional start site, respectively) to recruit RNA polymerase and activate transcription of all T3SS promoters. This interaction is required for T3SS gene expression, making it an attractive target for inhibitors designed to disrupt T3SS activity. In this study, I have characterized a group of N-hydroxybenzimidazole compounds that disrupt the ExsA-DNA interaction, leading to decreased gene expression and T3SS-mediated cytotoxicity. Furthermore, N-hydroxybenzimidazoles interact with the ExsA DNA-binding domain, and due to the conserved nature of this domain, these compounds have broad-spectrum activity against ExsA homologs. The amino-terminal domain (NTD) of AraC family proteins is poorly conserved at the primary amino acid sequence level; however, oligomerization and/or ligand binding is commonly mediated by the NTD of AraC family proteins. The ExsA NTD is required for both self-association and interaction with the anti-activator protein ExsD. In addition to DNA-binding by the ExsA CTD, I have shown that ExsA self-association is required for maximal activation of T3SS promoters, providing another target for ExsA inhibitors. In the current model for ExsA interactions with promoter DNA, ordered occupation of binding site 1 followed by occupation of binding site 2 is facilitated by an interaction between ExsA monomers. In this study, I identified an α-helix required for ExsA self-association and showed that ExsA self-association serves two distinct roles to promote occupation of binding site 2 by a second ExsA monomer: (1) self-association relieves NTD-mediated inhibition of site 2 occupation, and (2) self-association facilitates occupation of low-affinity binding sites. Lastly, the interaction between ExsD and ExsA prevents both ExsA self-association and DNA-binding, resulting in loss of activation at T3SS promoters. An understanding of the molecular basis for ExsA inhibition by ExsD is unknown but could provide valuable insight for the development of ExsA inhibitors. I performed site-directed and random mutagenesis to identify ExsA residues involved in the ExsA-ExsD interaction. Residues required for this interaction were identified within the ExsA self-association helix and elsewhere in the NTD; therefore, ExsD appears to prevent ExsA self-association by contacting multiple regions of ExsA, including the self-association helix. As a whole, these studies were performed to further our understanding of the protein-protein and protein-DNA interactions that are integral to the regulatory activity of ExsA and to provide direction for the development of therapeutic strategies that prevent expression of the P. aeruginosa T3SS.