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A dissertation submitted to the Faculty of Science, University of the Witwatersrand in fulfilment of the requirements for the degree of Master of Science. University of the Witwatersrand, Johannesburg, March 2016 / A liquid phase microextraction based on single hollow fibre followed by liquid chromatographic determination was developed for the extraction and quantification of the hallucinogenic muscimol and its two precursors, tryptophan and tryptamine from urine samples. A multivariate design of experiment was used in which a half fractional factorial approach was applied to screen six potential factors (donor phase pH, acceptor phase concentration, supported liquid membrane composition, stirring rate, extraction time and salt content) for their extent of vitality on the extraction of muscimol, tryptophan and tryptamine using the developed method. Four factors were identified as essential for an enhanced enrichment of each of the three research analytes from diluted urine samples. The paired vital factors were then optimized using central composite designs where empirical quadratic response models were used to visualize the response surface through contour plots, surface plots and optimization plots of response output. When the muscimol-based optimum factor levels were applied for the simultaneous extraction of the three research analytes, a composite desirability of 0.687 was obtained implying that the set conditions were ideal for a combined extraction of the analytes from the donor phase into the acceptor phase across a supported liquid membrane impregnated with a carrier molecule. This was an acceptable result considering that only the optimized muscimol factor levels were set as universal factor values. Muscimol was the analyte of interest in this research. The composite desirability value was predicted by setting the extraction conditions to 20% (w/w) di-(2-ethylhexyl) phosphoric acid (DEHPA) in dihexyl ether (DHE) supported on the walls of a hollow fibre into a 200 mM HCl acceptor phase inside the hollow fibre from a 20% (v/v) diluted urine donor phase spiked in the 0.1 – 10 μg mL-1 analyte concentration range maintained at pH 4 and stirred at 800 rpm for 60 mins. Experimentally, average enrichments of 4.1, 19.7 and 24.1 were obtained for muscimol, tryptophan and tryptamine, respectively. iv The complexity of urine and the anionic nature of the carrier molecule embedded on the supported liquid membrane resulted in interfering peaks that could not be completely resolved from the analyte peaks. Thus matrix-based calibration curves were used to address matrix effects. Various statistical approaches were used to validate suitability of the developed method for its potential use in quantifying muscimol and its precursors from urine samples. These validation measures were used as a way of determining the method’s ability to maintain the extraction process at equilibrium over a specific range of analyte concentrations over a period of analyte existence in a urine sample. The r² values of the matrix-based linear regression prediction models ranged from 0.9933 to 0.9986. The linearity of the regression line of the matrixbased calibration for each analyte was directly linked to the analyte enrichment repeatability. Simultaneous analyte enrichment repeatability over a 0.1 – 10 μg mL-1 analyte spiking concentration ranged from an RSD value of 8.3% to 13.1%. Limits of detection were 0.021 μg mLˉ¹, 0.061 μg mL-1 and 0.005 μg mL-1 for muscimol, tryptophan and tryptamine, respectively. Other validation parameters that were considered included specificity (and selectivity), accuracy, robustness, extraction range and system suitability. The accuracy of the developed method was reported as the reproducibility of enrichment factor values over six spiking concentrations used in constructing matrix-based calibration curves. System suitability was limited to an HPLC-UV approach. Method suitability was addressed through a comparative summary in which the LOD, LOQ and r² values for the developed method were compared to other methods that have been used to extract muscimol from urine samples. The relevance or acceptability of the enrichment factor values obtained for the extraction of the three analytes was achieved by comparison with enrichment factor values of several compounds with similar polarity that have been extracted from urine samples using carrier-mediated hollow fibre liquid phase microextraction. / GR2016

by Qi Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 192-199). / Acknowledgment --- p.i / Abstract --- p.ii / Table of Contents --- p.iii / Index of Componds --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Survey of Inclusion Chemistry --- p.1 / Chapter 1.2. --- The Importance of Hydrogen Bonds --- p.5 / Chapter 1.3. --- "Classical Inclusion Compounds of Urea, Thiourea and Selenourea" --- p.8 / Chapter 1.4. --- Research Strategy --- p.12 / Chapter 2. --- Description of Crystal Structures --- p.16 / Chapter 2.1. --- Urea-Anion Inclusion Compounds --- p.16 / Chapter 2.1.1. --- Halide complexes --- p.18 / Chapter 2.1.2. --- Bicarbonate complexes --- p.30 / Chapter 2.1.3. --- Allophanate complexes --- p.36 / Chapter 2.1.4. --- Borate and pentaborate complexes --- p.44 / Chapter 2.1.5. --- Complex featuring both host-host and host-guest hydrogen bonding --- p.60 / Chapter 2.1.6. --- Tetraethylammonium and phosphonium chloride complexes --- p.65 / Chapter 2.2. --- Thiourea-Anion Inclusion Compounds --- p.71 / Chapter 2.2.1. --- Halide Complexes --- p.73 / Chapter 2.2.2. --- Bicarbonate Complexes --- p.76 / Chapter 2.2.3. --- Nitrate Complexes --- p.87 / Chapter 2.2.4. --- Formate Complexes --- p.101 / Chapter 2.2.5. --- Acetate Complexes --- p.113 / Chapter 2.2.6. --- Oxalate and Fumarate Complexes --- p.127 / Chapter 2.2.7. --- Unsymmetrical quaternary ammonium ions as guests --- p.138 / Chapter 2.3. --- Selenourea-Anion Inclusion Compounds --- p.152 / Chapter 3. --- Summary and Discussion --- p.161 / Chapter 3.1. --- Structural Features and Relationships --- p.161 / Chapter 3.2. --- Hydrogen Bonding in Urea/Thiourea/Selenourea-Anion Inclusion Compounds --- p.164 / Chapter 3.3. --- Linkage Modes of Urea and Thiourea Molecules --- p.168 / Chapter 3.4. --- Comolecular Aggregates of Urea and Other Host Components --- p.173 / Chapter 3.5. --- Comolecular Aggregates of Thiourea and Other Host Components --- p.175 / Chapter 4. --- Experimental --- p.177 / Chapter 4.1. --- Preparation --- p.177 / Chapter 4.2. --- Crystallography --- p.182 / Chapter 5. --- References --- p.192 / Appendix A: Tables of Atomic coordinates and thermal parameters --- p.200 / Appendix B: Publication Based on Results Reported in This Thesis --- p.243

Investigation on a series of hydrogen-bonded networks constructed with N-heteroaryl acids is described in Section 3.4. In this section, we focused on the connection modes within the heteroaryl dimer. The study of co-crystals and inclusion compounds based on 2-thiobarbituric acid (TBA) or trithiocyanuric acid (TCA) indicated that the dimer of TBA is present in all three crystals in the forms of ribbon, tetramer or separated dimer. In the case of 5-nitrobarbiturate, its dimer occurs in two ammonium salts and in three of its four thiourea complexes, but is absent in all three urea complexes. / Self-assembly of two-dimensional hydrogen-bonded honeycomb grids exhibiting the rosette motif has been conducted with the guanidinium cation and various anions as the building blocks, tetraalkylammonium ions of suitable bulk being employed as interlayer templates. It is noteworthy that the rosette layer constructed from three different trigonal-planar molecular components has been achieved. In addition, deviating from conventional topological design, the generation of new rosette layers, albeit highly distorted, has also been accomplished with 1,2-dithiosquarate and the dianionic form of 1,1'-biphenyl-2,2',6,6'-tetracarboxylate that do not conform to C3-symmetry. Although threefold molecular symmetry is regarded as a sacrosanct requirement for molecular building blocks in the construction of hydrogen-bonded rosette motif, this study shows that rosette motifs can be generated even if one of the building blocks does not have inherent threefold symmetry. / Study of compounds containing the deprotonated forms of Kemp's triacid (H3KTA) has revealed the chair or twist-boat conformation in six crystal structures. X-ray structural analysis showed that [C(NH2) 3+] · [C6H6(CH3) 3(COOH)2(COO-)] (2.2.2) exhibits a corrugated layer structure which mimics the rosette motif constructed from the guanidinium ion and the hydrogen carbonate dimer. The tricarboxylate form of Kemp's triacid KTA3- in 3[C(NH2) 3+] · [C6H6(CH3) 3(COO-)3] (2.2.4) registers a record number of eighteen acceptor hydrogen bonds involving the convergent N--H donor sites from nine guanidinium ions. The crystal structure of 3[(C2H5)4N+] · 20[C(NH 2)3+] · 11[C6H6(CH 3)3(COOH) (COO-)2] · [C6H6(CH3)3(COOH)2(COO -)]·17H2O (2.2.3) features a hydrogen-bonded aggregate with a centrosymmetric pseudo-octahedral arrangement of H2KTA- anions surrounding an inner core composed of eight guanidinium ions. The unusual twist-boat conformation of KTA3- is found in [(CH3)4N +] · 2[C(NH2)3+] · [C6H6(CH3)3(COO- )3] · 2H2O (2.2.6), which is stabilized by the co-existence of guanidinium and tetramethylammonium cations. / Systematic investigation on hydrogen-bonded supramolecular assembly using aromatic carboxylic acids bearing linear or bent skeletons with urea/guanidinium resulted in the formation of mainly R228 and R126 synthon motifs. In addition, isostructures were also constructed by varying the length of the linker between two carboxylate groups, as in naphthalene-2,6-dicarboxylate (2.3.2) and biphenyl dicarboxylate (2.3.3). / This thesis reports a systematic investigation on the generation of new inclusion compounds by the combined use of urea/thiourea, guanidinium ion and various organic anions as building blocks of hydrogen-bonded host lattices and selected quaternary ammonium ion as the enclosed guests. / Various acids bearing specific functional groups have been explored as structure building components, including boric acid, Kemp's triacid, heterocyclic (thio)urea derivatives, aryl and N-heteroaryl carboxylic acids and (dithio)squaric acid. All the co-crystals and inclusion compounds built of molecular components in the afore-mentioned categories have been characterized by single-crystal X-ray analysis. As a result, the complexes exhibit a rich variety of inclusion topologies, such as networks containing isolated cages, open channels, intersecting tunnels, double-layer systems, and sandwich-like as well as wave-like layer structures. / Han, Jie. / Adviser: Thomas C. W. Mak. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0337. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 204-218). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Chi-Keung Lam. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 202-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Digitized by Kansas Correctional Industries

HpUreI is a pH-gated urea channel found in the pathogenic bacterium Helicobacter pylori. This protein is an essential component of the mechanism of acid acclimation, which allows Helicobacter pylori to survive in the acidic conditions of the stomach. HpUreI conducts urea into the cytoplasm, where it is hydrolyzed by urease into carbon dioxide and ammonia. These products then transit back into the periplasm, where they function as a buffer and proton consumer respectively. HpUreI is an attractive target for small molecule inhibitors for the treatment of H. pylori infections as mutant strains lacking this protein no longer survive under acidic conditions. Despite the importance of HpUreI, it remains biochemically uncharacterized and many questions remain as to how this channel performs its roles. We have solved many of the technical issues regarding the heterologous expression and purification of HpUreI, allowing us to investigate this protein in detail. A robust stopped-flow light-scattering assay was developed which was used to determine the permeability of urea (or other solute) through HpUreI reconstituted proteoliposomes. With slight modifications this assay was be used to measure a wide range of characteristics and variables. Our results show that HpUreI is a hexameric protein that has a relatively weak affinity for urea (˜160mM). Proteoliposome studies indicate that HpUreI is highly selective for urea and hydroxyurea, and is able to conduct water. Interestingly, water and urea conduction is pH-gated, suggesting that both solutes share a common conduction pathway. HpUreI displayed a pH-dependent activity profile with a pH of half maximum activity of ˜5.9. Based on these results an updated mechanism of acid acclimation was proposed. HpUreI is a pH-gated channel; only conducting urea under acidic conditions. The mechanism by which this occurs is not well understood, but can be localized to the periplasmic loops. Chimeric proteins were prepared by swapping the periplasmic loops of HpUreI and StUreI, a pH-independent UreI channel from Streptococcus thermophilus. Our results show that the pH-gating behavior of HpUreI was lost if either periplasmic loop was replaced with the corresponding loop from StUreI. Conversely, pH-gating was gained by StUreI when both periplasmic loops were swapped for those of HpUreI. A model of pH-gating was proposed which takes these findings into account. The mechanism of urea conduction was also examined. The recent crystal structure of HpUreI revealed a ladder of tryptophan residues lining one face of the conduction pathway. Mutation of these residues resulted in lower rates of urea conduction and reduced urea affinity. Our findings indicate that urea interacts directly with the tryptophan residues, via stacking and dipole-dipole interactions, to facilitate urea conduction. These studies have greatly increased our understanding of HpUreI and the role it plays in H. pylori. Further research is required to fully elucidate the mechanisms by which HpUreI operates. However, this is a starting point with which to pursue the ultimate goal of developing small molecule drugs to inhibit HpUreI, culminating in the eradication of H. pylori infections and prevention of gastric cancer.

Bibliography: leaves 132-147.

We present a statistical mechanical approach for quantifying thermodynamic properties of proteins in mixed solvents. This approach, based on molecular dynamics simulations which incorporate all atom models and the theory of preferential binding, allows us to compute transfer free energies with experimental accuracy and does not incorporate any adjustable parameters. Specifically, we applied our approach to the model proteins RNase A and T1, and the solvent components water, glycerol, and urea. We found that the observed differences in the binding of glycerol and urea to RNase T1 and A are predominantly a consequence of density differences in the first coordination shell of the protein with the cosolvents, but the second solvation shell also contributes to the overall binding coefficients. The success of this approach in modeling preferential binding indicates that it incorporates the important underlying physics of proteins in mixed solvent systems and that the difficulty in quantitative prediction to date can be surmounted by explicitly incorporating the complex protein-solvent and solvent-solvent interactions. / Singapore-MIT Alliance (SMA)

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (February 9, 2007) Includes bibliographical references.