Thesis (M.S.)--University of Missouri-Columbia, 2008. / 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. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.

Thesis (Ph. D.)--University of Canterbury, 2009. / Typescript (photocopy). Includes bibliographical references (leaves 191-230). Also available via the World Wide Web.

Chemical sensors capable of detecting a specific atom or molecule under various conditions have been utilized in biological and environmental analyses. Fluorescence based sensors are particularly advantageous in these studies because of their high sensitivity, relative ease in handling, and low technical costs. This dissertation focuses on the detection of two analytes, H+ and hypochlorous acid, which are of interest in biology because the presence of abnormal quantities of these analytes may be indicative of disease. We have established a new platform for which sensitive changes in various regions of pH can be detected using fluorescence. The aminomethylrhodamine (AMR) scaffold is highly versatile, i.e. the pH range in which the sensor is active can be tuned by introducing different substituents on the amine moiety. Overall this systematic approach to the design of pH sensitive fluorophores has allowed for a library of compounds that are responsive over a broad range of pH (pH 3 - 10) by simply changing the substituent on the amino group. We report the synthesis and characterization of a silicon analog of rhodamine for the fluorescence based detection of hypochlorous acid. This fluorophore exhibits a 90 nm bathochromic shift in its absorption and emission, relative to its oxygen counterpart. Hypochlorous acid is a biological agent linked to certain diseases. Therefore, the longer wavelength properties of the this far-red fluorescent sensor will be of significant benefit to imaging experiments of this analyte in biological media and tissue due to its spectral proximity of the so called NIR optical window. Furthermore, the novel synthetic methodology of this sensor possesses a key intermediate, which could potentially lead to future fluorescence based sensors. The characterization of a fluorescent probe designed for the detection of hypochlorous acid (HOCl) using a silicon analog of fluorescein (SiF) was also reported. Over a range of pHs, the probe reacts with a stoichiometric amount of HOCl resulting in a mixture of two pH dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorometric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore. Finally, an attempt was made at developing a pH responsive photodynamic therapy agent. This system was designed to target the relatively low extracellular pH found around tumors. A di-bromohydroxymethylrhodamine system was synthesized and the photophysical properties were characterized. This system absorbs weakly under acidic conditions (ca. pH 3), however was shown to be a moderate photosensitizer under acidic conditions.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

The synthesis and development of new materials and the improvement of the existing materials are a continuous quest for scientists. There is a constant need for new materials that have more advantages over the existing ones. The research to develop new materials can be appreciated in high magnitude, as the new materials improve the livelihood. In this dissertation, we are presenting our progress in developing new materials, such as fluorescence fluorophores as probes, and esterification catalysts. In the first part of the dissertation, the synthesis and characterization of a novel silicon analog of fluorescein (silyl fluorescein) was studied in detail. The photo-physical properties and toxicity studies of these fluorophores are also discussed. Compared to fluorescein, silyl fluorescein displayed a 90 nm longer wavelength in its absorption and emission. Silyl fluorescein showed good solubility in water and organic solvents, and has a considerable fluorescence quantum yield compared to fluorescein. Silyl fluorescein was found to be moderately toxic under light and non-toxic in dark, where as fluorescein toxicity was found to be greater than silyl fluorescein both in the dark and under light. Two probes from our novel silyl fluorescein fluorophore were developed and studied. SIF HySOx is probe for hypochlorous acid (HOCl), which is a reactive oxygen species (ROS) found in cells that are stressed due to various diseases. The HOCl probe was synthesized and its photo-physical properties were studied. It was found to be selective and sensitive only to HOCl. A fluoride probe (DTBDMS SIF P) was also developed from silyl fluorescein. Its synthesis and photo-physical properties were also studied. The probe displayed excellent selectivity and sensitivity to fluoride (TBAF) in the presence of other halides and bases. Future directions on novel fluorophore are briefly mentioned. Second part of the dissertation is focused on the screening of two organic esterification catalysts which were synthesized by Dr. Liu and Dr. Wong. The catalysts which are synthesized from pyridine and dinitrobenzene moieties were screened with several carboxylic acids and alcohols. Catalyst 8-4 was shown to be moderately active in the esterification of carboxylic acids and alcohols. It has no selectivity in the esterification of primary, secondary, tertiary alcohols, and the carboxylic acids that are attached to those carbons. Catalyst 9-1 however, was more efficient in the esterification of carboxylic acids and alcohols. Catalyst 9-1 displayed selectivity in the esterification of primary, over secondary, over tertiary alcohols, and the carboxylic acids that are attached to those carbons. Catalyst 9-1 gave up to 95% isolated yields.

No description available.

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

>Magister Scientiae - MSc / To address the urgent need for safe potable water in South Africa’s rural areas, sustainable systems for water disinfection at the village-scale of operation are required.In this thesis, the development of a small-scale water chlorination system that runs on salt and solar panels is described. The system combines a membrane-based hypochlorite generator, or “membrane electrolyser”, with an automated hypochlorite dosing system.The system was designed to (i) coordinate hypochlorite production and dosing automatically in a flow-through system, and (ii) fit inline with low pressure pipelines from overhead storage tanks or raised water sources. Low cost materials were used for construction, and water-powered mechanisms were devised to control both brine supply to the electrolyser and regulation of water flow. The capacity of the system was based on the maximum daily output of the electrolyser at ~20 g of sodium hypochlorite. This was sufficient chlorinate up to 10 kL of water per day using less than 80 g of salt and less than 0.1 kW.h of electricity. The cost of the system was estimated at ~R10 000 and therefore potentially affordable for communities up to 100 people, e.g. small farms and villages.Testing of the system was carried out at a farm site in Worcester (Western Cape) using remote monitoring of current levels in the electrolyser. Operation of the system over a two month test period, dosing at ~4 mg/L, produced consistent chlorination measured as(FAC). Community participation in maintenance of the brine supply was managed and chlorinated water was made available to the community after a brief social survey was conducted. Community awareness of chlorination was minimal. No significant history of diarrhoea was reported. However, the community regularly boiled their tap water in response to turbidity increase in summer.The system was affected by turbidity increase in the local water, which caused a drop in electrolyser current and chlorine production due to particle blockage of the membrane in the electrolyser. However, turbidity at acceptable levels for chlorination was found to have no detrimental effect on the system’s performance. The system showed promise for rural implementation providing low turbidity was maintained. Therefore,groundwater sites, and surface waters with appropriate clarification systems are recommended for the system’s installation. Further testing of the system will be required to establish its long term viability in the hands of a rural community.

Très répandues dans le monde vivant, les protéines à doigt de zinc constituent une super-famille dont les membres possèdent un site à zinc de formule générale [ZnII(Cys)4-X(His)X] (x=0, 1 or 2). Tandis que la majorité de ces sites joue un rôle purement structural, certains présentent une fonction réactive, comme la détection de stress oxydant par exemple. En effet, les sites doigt de zinc de Hsp33 et de RsrA ont été décrits comme des interrupteurs rédox[1,2] : transmettant l'information « stress oxydant » sous forme d'un signal structural, via l'oxydation/réduction des cystéines coordonnées au zinc, détruisant/reformant le domaine doigt de zinc. Cependant, certains aspects de l'étape d'oxydation restent mal compris.Étant donné le grand nombre des protéines à doigt de zinc et leurs rôles clés, il est de tout intérêt d'identifier les facteurs contrôlant leur réactivité afin de comprendre pourquoi certaines espèces réactives de l'oxygène (ERO) sont capable d'oxyder des doigts de zinc in vivo, contrairement à H2O2. Durant ce projet, nous avons décidé de nous focaliser sur deux ERO très puissantes et jouant un rôle important en biologie : l'acide hypochloreux (HOCl) et l'oxygène singulet (1O2). Nous étudierons la réactivité des doigts de zinc vis-à-vis de ces deux oxydant en utilisant des modèles peptidiques, reproduisant parfaitement la structure de doigts de zinc courants. / Widely spread in the living world, zinc finger proteins constitute a large superfamily, with a zinc site of general formula [ZnII(Cys)4-X(His)X] (x=0, 1 or 2) as a common feature. Whereas the majority of such sites plays a purely structural role, a few of them exhibit a reactive function (e.g. oxidative stress detection). Indeed, zinc finger sites of Hsp33 and RsrA have been shown to act as redox switches[1,2]: transmitting the information “oxidative stress” as a structural signal, by means of oxidation/reduction of its ZnII-coordinating cysteines. However, the precise mechanism of the oxidation step remains poorly understood.Given the occurrence of zinc finger proteins and their key roles, it is of high biological interest to identify factors controlling their reactivity and to understand why some ROS are able to oxidize them in vivo, on the contrary to H2O2. In this project, we decided to focus on two major ROS: hypochlorous acid (HOCl), a key player of the immune response, and singlet oxygen (1O2), produced in significant amount by photosynthetic organisms. By use of peptide model complexes, reproducing perfectly the structure of some archetypal zinc fingers, we investigated the reactivity of zinc fingers toward those ROS.