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Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases. / Master of Science

M.Sc. (Chemistry) / An aqueous, simple, environmentally friendly synthetic method for the preparation of water-soluble CuS and CdS semiconductor nanoparticles has been developed. Different capping agents with active functional groups (-COOH, -NH and -OH) namely, alanine, glucuronic acid and thioglycolic acid, have been employed in the preparation copper sulfide (CuS) and cadmium sulfide (CdS) semiconductor nanoparticles as capping molecules. These capping agents were explained in Chapter 3 and 4. The reported nanoparticles display good optical and structural properties. The semiconductor nanoparticles were easily synthesized at low temperatures employing low cost materials. In Chapter 3 colloidal methods were used to develop CuS and CdS nanoparticles from metal acetate as a stabilizing agent at different low temperatures. The effect of temperature on the growth and solubility of nanoparticles was investigated. The absorption spectra of all samples prepared were blue shifted as compared to their bulk materials which signify small particles size. Water-soluble alanine-capped CuS and CdS semiconductor nanoparticles were obtained. The alanine-capped CuS in the form of rods, triangular and spherical like shaped with single crystal phase were successfully attained. Chapter 4 describes the uses of the acids, glucuronic and thioglycolic acids, as capping ligands. The glucuronic acid molecules were found to be the effective ligands to render solubility of the nanoparticles. Solubility tests revealed that both glucuronic acid-capped CuS and CdS nanoparticles were more soluble in water as compared to thioglycolic acid-capped CuS and CdS nanoparticles. In Chapter 5 sulphonated porphyrins were prepared and explored in combining them with nanoparticles. The results obtained from 1H NMR suggest that the porphyrins were sulphonated. The sulphonated porphyrins were conjugated with the water-soluble nanoparticles and this is described in Chapter 6. The results presented in Chapter 6 are in favour of the formation of nanoparticle-porphyrin complexes.

Nitric oxide (NO), a reactive diatomic gas and a potent signaling molecule, is required for proper cardiovascular functioning. Soluble guanylate cyclase (sGC), a heterodimeric heme protein, is the key intracellular NO receptor protein which, upon NO binding, undergoes conformational changes leading to catalysis and the cGMP signaling cascade. Several small molecules that allosterically stimulate sGC have been developed for treatment of pulmonary hypertension, but little is known about their binding site or how they stimulate activity. This dissertation describes experiments designed to uncover the molecular basis for signal transduction in sGC by NO and small molecule stimulators. The crystal structure of the α-subunit PAS domain from Manduca sexta (Ms) sGC was solved at 1.8 Å resolution revealing the expected PAS fold but with an additional β strand and a shorter Fα helix. CO binding measurements on different Ms sGC N-terminal constructs and the β₁ (1-380) construct revealed that the α-subunit keeps the β₁ H-NOX domain in an inhibited conformation and this inhibition is relieved by removal of the α-subunit or by addition of stimulatory compounds such as compound YC-1. Linked-equilibria measurements on the N-terminal constructs show that YC-1 binding affinity is increased in the presence of CO. Surface plasmon resonance (SPR) studies on the in-vitro biotinylated constructs showed that YC-1 binds near or directly to the β₁ H-NOX domain. Computational and mutational analysis of the β₁ H-NOX domain revealed a pocket important in allostery and drug action. Finally, we show that the coiled coil domain plays an important role in allosteric regulation of the β₁ H-NOX domain and possibly in signal transduction. Our data are consistent with a model of allosteric activation in which the α-subunit and the coiled coil domains function to keep heme in a low affinity conformation while YC-1 binding to the β₁ H-NOX domain switches heme to a high affinity conformation, and sGC to its high activity form.

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No description available.

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acase@tulane.edu / In an effort to construct and study aqueous supramolecular assemblies that mimick those of nature (proteins and viral capsids), a novel, water-soluble deep-cavity cavitand has been synthesized – tetra-exo-methyl octa-acid (TEXMOA). In addition, water-soluble cavitand tetra-exo-carboxy tetra-acid (TECTA) was synthesized as an anti-dimerization host to further explore the binding properties of a monomeric host-guest system. The synthetic processes are investigated and discussed in this dissertation. Driven by hydrophobic effect, long-chain alkane guests bind strongly to the pocket of TEMOA in aqueous media. Initial NMR studies suggest that the steric hindrance induced from the exo-methyl groups of TEXMOA is responsible for a non-monotonic assembly profile toward the guests (from monomeric to dimeric and mixture of both in some cases). The synthesis of TECTA required direct ortho metalation and careful purification steps. However, the host still displayed the binding properties as intended, since the carboxylate groups of TECTA in the exo position prevented capsule formation with various aliphatic guests in water. Based on previous findings, modified 1-adamantanoic acids were expected to be potential strong-binding guests for these hosts. Therefore, chloro-, bromo, and iodo-substituted adamantanoic acids were synthesized in order to expand the library of guest molecules. The Z-isomer of these halogenated adamantanoic acids were synthesized from the Z-2-hydroxy-5-adamantanoic acids with the corresponding mineral acids, and these reactions were found to be stereoselective. The amine-footed meta-basket was also synthesized as a potential detection ligand in biosensors via SPR (surface plasmon resonance). The synthesis of the host utilized a modified Staudinger reaction from the azido- precursor. / 1 / Thong Nguyen