Thesis (doctoral)--Uppsala Universitet. / Includes bibliography.

Thesis (doctoral)--Uppsala Universitet. / Includes bibliography.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1930. / Typescript. Includes bibliographical references.

Typescript. / Photocopy. [S.l. : s.n., 1976]. 29 cm. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1976. / Bibliography: leaves 231-243. / Microfilm / xiii, 243 leaves ill

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine 2015 / The dissertation aims to discuss the disulphide and thiol chemistry for use in drug delivery. In particular it focuses on the use of the modified native ovalbumin polymer as a vehicle for the thiol containing captopril. The binding capabilities of thiols expand the area in which peptides and proteins can be used as potential therapeutic drug carriers. It is important that drug delivery systems enhance drug storage stability and in vivo particle stability while delivering the drug efficiently. As part of the developing novel drug delivery systems, thiol-based chemical reactions are distinctive role players in stabilizing disulphide bioconjugated nanostructures for use as efficient drug carrier vehicles in vivo. A review of the current approaches for designing, optimizing and functionalizing nanostructures and conjugates by thiol chemistry modifications was explored. Captopril (Cp) is an Angiotensin-Converting Enzyme (ACE) inhibitor, which acts as an anti-hypertensive, structurally contains a free reactive thiol that binds variably via the thiol/disulphide reaction. A single dose of captopril can regulate hypertension for up to eight hours and the duration of the antihypertensive action of a single dose of 35-75 mg would be taken at 8 hour intervals for 24 hours. Hence the necessities in developing a sustained controlled release ovalbumin carrier system to maintain relatively constant blood pressure levels for 24 hours. The research focused on the construction, characterization and optimization of the thiol conjugated complex for sustained oral drug delivery. The thiol/disulphide-functionalized captopril-ovalbumin conjugate complex was assessed in terms of the structural characteristics and the thiol-disulphide covalent substitution reaction. For analysis of the conjugation complex, the Fourier Transmission IR-spectroscopy (FTIR), H+ NMR and Differential Scanning Calorimetry (DSC) was performed and used to confirm conjugation. Preliminary studies focused on a comparative study of sodium alginate, polyvinyl alcohol and hydroxypropylmethylcellulose hydrogel formulations for the release testing and drug entrapment of the ovalbumin-captopril conjugate complex. Utilizing this data, a series of process variables were used to achieve an optimized formulation through a Box- Behnken statistical design. Furthermore the drug release profiles of the optimised formulation were then analyzed in vitro and in vivo. The captopril released from the formulation was high with a cumulative release of 82%. In vivo analysis was the final testing to verify the validity of the ovalbumin-captopril conjugate complex encapsulated in sodium alginate and utilized a pig model. Ultra Performance Liquid Chromatography (UPLC) blood analysis revealed increased blood levels of captopril (Cmax Cp=33.2ng/mL) in relation to conventional dosage forms validating prolonged (24 hour) site-specific release and increased bioavailability. In conclusion, our validated method was successfully applied to the pharmacokinetic studies of captopril in the blood plasma samples.

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Master of Science / Division of Biology / Ana Lorena Passarelli / Baculoviruses are a large, diverse, and an ecologically-important group of entomopathogens. The ac78 gene of the prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is one of the 38 genes conserved among all baculoviruses sequenced to date. Previous studies show that Ac78 is essential for optimal production of occlusion-derived virions (ODVs) and budded virions (BVs), which are two virion types produced during baculovirus infection. However, the biochemical mechanism by which Ac78 is involved in these processes remains unknown. The AcMNPV sulfhydryl oxidase ac92 is a conserved gene, and its product, Ac92, is ODV and BV envelope-associated. Recently, the Ac78 and Ac92 homologs in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) were reported to interact and co-localize to the site of BV and ODV formation. To investigate the relationship between Ac78 and Ac92, we determined their localization in the presence and absence of AcMNPV infection, performed co-immunoprecipitations to assess interaction relationships, and provided an updated report of Ac78 and Ac92 homology with other proteins. We concluded that in the absence of viral infection, Ac78 and Ac92 localized perinuclearly in the cytoplasm and that localization of Ac92 was not affected by Ac78. During AcMNPV infection, Ac78 and Ac92 co-localized within the nucleus and surrounding virus replication and assembly sites (ring zone). Co-immunoprecipitation experiments showed that at least two differentially-tagged Ac78 proteins were part of a complex in the presence of other AcMNPV proteins. Ac78 did not associate with Ac92 during AcMNPV infection. Our characterization of the relationship between Ac78 and the AcMNPV sulfhydryl oxidase is a preliminary step in a broader effort to elucidate important biochemical pathways underlying the poorly described structural changes in capsid proteins and other proteins involved in virion stability, folding, and infectivity. In a separate project, the same approach was applied in a different virus system to determine the relationship between the small accessory protein C and the measles virus (MeV) replication complex. Co-immunoprecipitation experiments showed that during MeV infection, C associated with large protein (L) and phosphoprotein (P), which comprise the MeV replication complex, and nucleoprotein (N), which encapsidates the RNA genome. Expression constructs for full-length MeV L were generated, and L was successfully expressed following transfection. Subsequent co-immunoprecipitation experiments showed that C did not precipitate with L, P, nor N when transfected in isolation from MeV infection, indicating that another factor resulting from MeV infection is necessary for the association of C with the MeV replication complex. The results of this investigation are an important step in elucidating a biochemical mechanism underlying the function of C as a quality control factor in MeV replication. MeV has been attenuated and is a highly effective vaccine against pathogenic MeV and an active subject of clinical research as an oncolytic agent for treating a number of human cancers. Taken together, the investigations of Ac78 and C and their respective relationships with the AcMNPV sulfhydryl oxidase and the MeV replication complex adds knowledge of biochemical mechanisms underlying the important functions of small accessory proteins containing less than 200 amino acids as mediators in viral replication processes of two different viral systems.

<p>Nanomaterials often exhibit enhanced reactivity relative to their larger colloidal counterparts because of the high specific surface area and number of imperfections on the crystal lattice at the nanoscale. Management of ecosystems, remediation of contaminated waters, and assessment of the potential risks from the industrial use on nanomaterials requires an understanding of the environmental factors that control the reactivity and bioavailability of natural and manufactured nanomaterials. Dissolved organic matter (DOM) acts as a moderator of reactivity and bioavailability for dissolved and particulate moieties in natural waters. DOM consists of a range of low and high molecular weight species that are complex and heterogeneous. It has been historically categorized based on operational definitions, rather than physical properties. In order to understand the effect of DOM on nanomaterials, there is an urgent need for information regarding specific properties of DOM, such as ligand groups. </p><p>The goal of this research was to study how cysteine, a low molecular weight metal-binding ligand, affects the composition and reactivity of nanoparticulate zinc sulfide and metallic silver. Zinc sulfide was used as a representative of nanoparticulate metal sulfide which occurs naturally in sulfidic environments. Metallic silver nanoparticles were also studied because of its wide use in consumer products. Both types of nanomaterials contain metal constituents (zinc and silver) that are expected to strongly bind to sulfhydryl-containing ligands (such as cysteine) in the environment. Serine is structurally similar to cysteine, with the only difference of a hydroxyl group in the place of the sulfhydryl group of cysteine. Therefore, serine was used for comparison as a hydroxyl-containing analogue to cysteine. </p><p>The aggregation kinetics of zinc and other metal sulfide nanoparticles in the presence of cysteine and serine were investigated using dynamic light scattering. Cysteine decreased aggregation rates of the particles, while serine had no effect on their aggregation behavior. Further experiments revealed that the mechanism of stabilization occurred through the adsorption of cysteine on zinc sulfide, which induced electrostatic charge on the particles surface. A direct link was established between the amount of cysteine sorbed and attachment efficiency, an indicator of the tendency of particles to aggregate. These results shed light on discrepancies in the literature between metal sulfide precipitation experiments conducted in our lab and work on the formation and aggregation of zinc sulfide nanoparticles on biofilms of sulfate reducing bacteria. </p><p>The early-stage growth and aggregation kinetics of zinc sulfide nanoclusters in the presence of cysteine was studied in detail using a suite of complementary techniques. Growth and aggregation experiments have been traditionally difficult to conduct due to instrumental precision issues, but newly developed analytical tools and software products have made it possible to study the early-stage formation of nanoclusters. Experiments with small angle X-ray scattering, X-ray diffraction, dynamic light scattering, and X-ray absorption spectroscopy at the extended fine structure range showed that cysteine controlled the growth and aggregation of zinc sulfide nanoclusters. The molar ratio between zinc, sulfide, and cysteine was a determining factor in the precipitation process. When zinc and sulfide were in equimolar concentrations with cysteine, very small nanoclusters of about 2.5 nm formed within 12 hours and aggregated to structures with hydrodynamic diameter larger than 100 nm. When cysteine was in excess of zinc and sulfide, aggregation was held to a minimum, but monomer nanoclusters were able to grow to about 5 nm in 12 hours. Overall, these results indicate the importance of thiol ligands on the monomer size, extent of aggregation, and aggregate structure of zinc sulfides. </p><p>The effect of metal ligands on metal bearing particle surfaces is of particular interest for manufactured nanoparticles, because they are typically coated with an organic coating during the production process. These coatings are sorbed on the particles surface and are likely to interfere between the metallic surface and the ligand. Dissolution experiments using citrate and polyvinylpyrrolidone (PVP) coated zero valent silver nanoparticles in the presence of cysteine and serine showed that cysteine dissolved both types of particles, while serine did not. Dissolution rates depended on the aggregation state of the particles exposed to cysteine. As indicated by zeta potential and adsorption measurements, cysteine replaced the coating on the particles surface and altered their aggregation pattern. X-ray absorption spectroscopy near the absorption edge showed partial oxidation of silver and formation of Ag(+I)-sulfur bonds, indicating that the thiol group in cysteine formed chemical bonds with oxidized surface silver atoms. A comparison between the two coatings showed that citrate coated particles dissolved approximately three times faster than PVP coated particles. Overall, these results show that metal binding ligands can drastically change the fate of manufactured silver nanoparticles in the environment and that this effect is moderated by surface coatings. </p><p>The results of this study suggest that cysteine, a metal binding ligand was able to induce and control transformations, such as growth, aggregation, dissolution, and surface reactivity of zinc sulfide and metallic silver nanoparticles. Cysteine adsorbed on metal sites on both ZnS and Ag particles, inducing changes on their surface charge. Aggregation of ZnS particles was slowed because of a net decrease in zeta potential compared to the bare particles. On the contrary, cysteine enhanced the aggregation of Ag particles, by replacing the citrate and PVP coatings on the particles surface. Finally, the cysteine-Ag(+I) bonds caused strong polarization on the particles surface and lead to the oxidative dissolution of the particles. </p><p>Overall, this research provides a better understanding of the fate of natural and manufactured nanoparticles in anaerobic waters, where thiols are present in significant amounts. It may also be used for risk assessment of manufactured nanomaterials and the production of safer and environmentally responsible materials.</p> / Dissertation

Bethoxazin is a broad spectrum industrial biocide with commercial applications as a material preservative; however its mechanism of action has not been investigated. In this study, the chemical reactivity of bethoxazin towards biologically important nucleophiles was assessed with UV-Vis spectroscopy. Bethoxazin reacted with molecules containing free sulfhydryl groups such as glutathione and human serum albumin but not with amino, acetate or phenol containing compounds. Bethoxazin was shown to potently inhibit the growth of the K562 human cancer cell line with an IC50 value in the micromolar range. The sulfydryl fluorescent label ThioGlo-1 was used to investigate the biological effects of bethoxazin in K562 cells and explore its mechanism of action. Bethoxazin inhibited the formation of covalent adducts in K562 cells between the free sulfhydryl group of biomolecules and ThioGlo-1, implying that bethoxazin reacts with molecules containing free sulfhydryl groups. Likewise, when glutathione was depleted in K562 cells, by buthionine sulfoximine, high concentrations of bethoxazin were able to inhibit the formation of covalent adducts between sulfhydryl biomolecules and ThioGlo-1. The growth inhibition assay (MTS) was used to investigate the effect of continuous bethoxazin treatment versus wash out in K562 cells. The MTS assay revealed a reduction in the potency of bethoxazin due to the wash out effect, suggesting that the growth inhibition effects of bethoxazin are likely not due to glutathione depletion. A two-colour flow cytometry analysis of bethoxazin treated K562 cells for eight hours demonstrated that bethoxazin provokes necrosis induced cell death in K562 cells. Taken together, these experimental results demonstrate that the reaction of bethoxazin with proteins containing an accessible sulfhydryl group is more likely to be the mechanism of action of the cell growth inhibition effects rather than glutathione depletion.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references (p. 122-142).