The antibacterial efficacy of hydrogen peroxide treatments in four fruit juices was determined. Preservative free apple cider, white grape, and purple grape juice were inoculated with ~ 6.4 log CFU/ml of a five strain, acid adapted, nalidixic acid resistant E. coli O157:H7 cocktail. Orange juice was inoculated with a comparable Salmonella spp. cocktail. In the first study, 0.017% and 0.012% H₂O₂ was added in combination with 0.1% and 0.3% of the dominant organic acid (OA) to 4°C and 25°C juices, with samples taken each day for 21 days. H₂O₂ was a significant factor in all juices (p < 0.05) except white grape (lack of data), and both 0.017% H₂O₂ treatments reduced counts in apple cider, orange juice, and white grape to undetectable numbers within 48 hrs as cultured on tryptone soy agar + 0.05% nalidixic acid (TSAN). Treatments in purple grape juice were less effective overall, and more dependent on OA concentration (p < 0.001) than H₂O₂. There were instances where bacterial survival in apple cider, purple grape, and orange juice continued for 21 days after treatment, and sometimes outlasted the control. These occurrences were dependent on temperature (25°C) and H₂O₂ (0.012%), but not on OA. However, OA concentration was a significant factor (p < 0.05) overall in apple cider and purple grape juice, but not in orange juice. In the second study, 0.015% and 0.03% H₂O₂ was added to 10, 25, and 40°C apple cider and orange juice inoculated with 6.4 log CFU/ml E. coli O157:H7 and Salmonella spp. respectively. Only 0.03% H₂O₂ was effective in reducing counts to undetectable numbers in both juices. However, both temperature and H₂O₂ were significant factors (p < 0.0001) in bacterial destruction, with 0.03% H₂O₂ at 40°C giving undetectable numbers at ≤ 3 and ≤ 6 hours in orange juice and apple cider respectively. It has been demonstrated that at ~ ≥ 0.017%, H₂O₂ can provide a 5 log reduction of these pathogens in fruit juice. Increasing temperature and organic acid concentration can improve its rate of effectiveness in certain juices. However, sensory concerns may negate its use in some products. / Master of Science

Demyelinating disorders affect many people around the globe and are characterized by loss of myelin sheaths and oligodendrocyte death, ultimately compromising neuronal signal transmission across the Central Nervous System (CNS). Adult Neural Stem Cells (NSC) are multipotent stem cells with the ability to differentiate into the three types of CNS cells: oligodendrocytes, neurons and astrocytes. Hydrogen peroxide (H2O2) is an inflammatory mediator, often present in demyelinating events, commonly associated with oxidative stress and cell death. However, H2O2 also plays a major role as an intracellular signaling molecule. It has been seen that NSC exposed to H2O2 revealed an increase in proliferation and oligodendrogenesis. In this project, we tried to understand how oligodendrogenesis is modulated at a transcriptional level by H2O2. We have identified the genes Hes1, Foxo1, Nrf2 and Prdx6 as being downregulated in the presence of H2O2 when compared to the non-exposed controls. In order to understand if the differential gene expression is involved in the H2O2-induced oligodendrogenesis, we silenced the genes through siRNA transfection (mimicking the downregulation observed after H2O2 exposure) and analyzed the effects on the transcriptome of NSCs and the impact on cell proliferation and differentiation. Our findings indicate that Foxo1 silencing induced the greatest increase in cell proliferation and that Nrf2 silencing revealed the greatest impact on oligodendrogenesis. While not very significant, Foxo1 silencing seems to induce oligodendrogenesis, and Prdx6 silencing seems to inhibit it. The results obtained give important hints on the role that these genes play in NSCs differentiation and fate determination when exposed to oxidative stress and might allow a better understanding of this complex system.

Trälim utgör en stor del av alla lim globalt och är avgörande för skapandet av träkompositmaterial, som många industrier och produkter är beroende av. Bland de vanligaste trälimmen använder flera formaldehyd som en väsentlig komponent i deras polymera struktur. Medan formaldehyd är ett allmänt använt, billigt och pålitligt material, är det också giftigt och har generellt ursprung från petroleumkällor. Ett ersättningsmaterial med di- eller polyaldehydisk funktionalitet av grönt ursprung är därför önskvärt för framtida trälimkemi. En potentiell lösning involverar att oxidera polysackarider såsom stärkelse och dextrin för att funktionalisera de upprepande glukosenheterna med aldehyder, ketoner och karboxylsyror. Här visas att Fenton-liknande förhållanden, med väteperoxid som oxidationsmedel och järn som katalysator, kan användas för att utföra reaktionen effektivt under atmosfäriska förhållanden, vilket producerar minimala biprodukter såsom vätgas, vatten och järnkomplex. Betydande funktionalisering observerades i en lågmolekylviktsfraktion som frigörs från polysackariden vid oxidation, en fraktion som traditionellt förloras under upparbetning via centrifugering. Detta arbete antyder att retention av denna lågmolekylviktsfraktion i produktblandningen kan förbättra framtida limegenskaper vid låg grad av oxidation i material med hög polymerisationsgrad, även om kvarhållande av depolymeriserade fraktioner från material med lägre polymeriseringsgrad, såsom dextrin, kanske inte ger de önskade polyaldehydiska produkterna i tillräckliga koncentrationer. Kvarhållandet av lågmolekylviktsfraktionen antyder också att stora mängder oxidationsmedel och katalysator bör undvikas, eftersom högre oxidationsgrad kan resultera i oönskade monofunktionella molekyler på grund av hög depolymerisation. / Wood adhesives comprise a large part of all adhesives globally and are essential in creation of wood composite materials, which many industries and products are entirely reliant on. Amongst the most common wood adhesives, several utilize formaldehyde as an essential component of their polymeric structure. While formaldehyde is a widely used, cheap and reliable material, it is also toxic, and generally derived from petroleum sources. A replacement with di- or poly-aldehydic functionality of green origin is therefore desirable for future wood adhesive chemistry. One potential solution involves oxidizing polysaccharides such as starch and dextrin, to functionalize the repeating glucose units with aldehydes, ketones, and carboxylic acids. Herein, it is demonstrated that Fenton-type conditions, using hydrogen peroxide as oxidant and iron as catalyst, can be used to perform the reaction efficiently in atmospheric conditions, producing minimal byproducts such as hydrogen gas, water, and iron complexes. Significant functionalization was observed in a small molecular weight fraction released from the polysaccharide upon oxidation, a fraction which is traditionally lost during workup via centrifugation. This work suggests that retention of this small molecular weight fraction in the product mixture may enhance future adhesive properties at low degrees of oxidation in materials with high degrees of polymerization, though retention of depolymerized fractions of lower chain length materials such as dextrin may not provide the targeted poly-aldehydic products in sufficient concentrations. The retention of the small molecular weight fraction also suggests that large amounts of oxidant and catalyst should be avoided, as higher degree of oxidation may result in undesired mono-functional molecules due to higher degrees depolymerization.

Hydrogen peroxide (H2O2) is extensively used in a broad range of industrial and medical applications, such as aseptic processing of food and pharmaceuticals, disinfection, water treatment plants, and decontamination of industrial effluents. H2O2 is believed to be responsible for chemical degradation of polymer membranes in Polymer-Electrolyte-Membrane (PEM) fuel cells. Therefore, a versatile H2O2 sensor that functions in different environments with different conditions is of practical importance in various fields. This dissertation presents the fabrication of a fiber-optic H2O2 sensing probe (optrode) and its H2O2 sensing behavior in different conditions. An H2O2 optrode is fabricated using chemical deposition of Prussian blue (PB) onto the tip of a multimode optical fiber. Sensing tests are performed in aqueous solutions at a constant pH and different concentrations of H2O2. Sensing features of the optrode (i.e. repeatability, durability, and reproducibility) are assessed by performing multiple sensing tests with several optrodes. The results show the prepared optrode is able to detect concentrations of H2O2 in aqueous solutions at a constant pH of 4 and the optrode features a repeatable and durable response at this condition. The functionality of optrodes at different pH values is further investigated by performing additional sensing experiments. These experiments are carried out in aqueous solutions with different concentrations of H2O2 at different pH values (i.e. pH 2-7). The sensor detects the presence of H2O2 at a range of pH values. Sensing behavior of optrodes toward detection and measurement of H2O2 concentrations is studied at the pH value corresponding to an operating PEM fuel cell (i.e. pH 2). The optrode is able to detect concentrations of H2O2 at this condition with a repeatable and durable response. The stability of PB films, prepared through different conditions, is investigated to address the stability of optrodes at elevated temperatures. PB films are first deposited onto the glass slides through three different chemical processes, and then at different synthesis temperatures. The PB films are left in Phosphate-Buffer-Solutions (PBS) with pH 2 and at elevated temperatures for a day. Finally, PB films are characterized using Fourier transform infrared spectroscopy (FTIR) to analyze their stability following PBS processing at operating temperatures and pH value corresponding to an operating PEM fuel cell (i.e. 80 °C and pH 2). The results of these experiments illustrate the PB films prepared through the single-source precursor (SSP) technique and at synthesis temperatures above 60 °C remain stable after the PBS processing. The proposed optrode shows reliable sensing behavior toward detection and measurement of H2O2 concentrations in aqueous solutions at different conditions. The prepared optrode has the potential for being developed and used in different industrial and medical fields, as well as an operating PEM fuel cell, to detect and measure H2O2 concentrations. / Graduate / 0794 / 0548 / 0485 / hakbarik@uvic.ca

>Magister Scientiae - MSc / There is an influx in heavy metals into soils and ground water due to activities such as increased mineral mining, improper watering and the use of heavy metal contaminated fertilizers. These heavy metals are able to increase the ROS species within plants which may result in plant metabolism deterioration and tissue damage. Heavy metals may also directly damage plants by rendering important enzymes non-functional through binding in metal binding sites of enzymes. The heavy metal focused on in this study was vanadium due to South Africa being one of the primary produces of this metal. Two related Brassica napus L cultivars namely Agamax and Garnet which are economically and environmentally important to South Africa were exposed to vanadium. Physiological experiments such as cell death, chlorophyll and biomass determination were conducted to understand how these cultivars were affected by vanadium toxicity. A low cost, sensitive and robust vanadium assay was developed to estimate the amount of vanadium in samples such as water, soils and plant material. The oxidative state as well as the antioxidant profile of the two cultivars were also observed under vanadium stress. A chlorophyll assay which was conducted on the two cultivars exposed to vanadium showed a marked decrease in chlorophyll A in the suspected sensitive cultivar which was Garnet. However, the suspected tolerant cultivar Agamax fared better and the decrease in chlorophyll A was much less. A similar trend was observed for the two cultivars when the cell death assay was conducted. The vanadium assay showed that Garnet had higher concentrations of vanadium within its leaves and lower concentrations in its roots when compared to Agamax. This observation displayed that Agamax had inherent mechanisms which it used to localize vanadium in its roots and which assisted in its tolerance to the vanadium stress. The oxidative state was determined by doing assays for the specific reactive oxygen species namely hydrogen peroxide and superoxide. It was observed that vanadium treated Garnet leaves had higher reactive oxygen species (ROS) production when compared to the Agamax treated leaves. In-gel native PAGE activity gels were conducted to determine the antioxidant profile for the two cultivars which were exposed to vanadium. The antioxidant enzymes which were under investigation were ascorbate peroxide (APX), superoxide dismutase (SOD) and glutathione-dependent peroxidases (GPX-like) as these enzymes are known to be responsible for controlling the ROS produced in the plants. The GPX-like profile consisted of three isoforms. No isoforms were inhibited by vanadium treatments but one isoform had increased activity in both the Garnet and Agamax treated samples. The SOD profile for Garnet consisted of six isoforms and Agamax had seven isoforms. One isoform which was visualized in both Agamax as well as Garnet was inhibited by vanadium treatments. Agamax also had two isoforms which were up-regulated however the corresponding isoforms in Garnet showed no change. The Ascorbate peroxidase profile consisted of seven isoforms for both Garnet and Agamax. No isoforms were inhibited by vanadium treatment. Three isoforms were up-regulated in Garnet and Agamax under vanadium treatments. Here, it is illustrated that Garnet lacked certain mechanisms found in Agamax (and thus experienced more cell death, yield and chlorophyll loss) and performed worst under high vanadium concentrations. Although Garnet increased the activity of some of its antioxidant isoforms in response to increasing ROS levels it was not adequate to maintain a normal oxidative homeostasis. This disruption in oxidative homeostasis lead to plant damage. Agamax was observed to produce less ROS than Garnet and was able to control the ROS produced more effectively than Garnet and thus less damage was observed in Agamax.

This work is focused on study of electrical discharges in liquids, especially in water solutions. Generation of the discharge in water solutions leads to simultaneous effect of UV radiation, shock waves, electrical field and most importantly, chemically reactive species. This can be utilized in many applications such as sterilization, degradation of organic waste products, lithotripsy or other medical applications. The experimental part is concentrated on a diaphragm arrangement of the reaction system. This means that the reactor is divided into two electrode reservoirs connected only through a small orifice in a dielectric barrier. This barrier is made of Macor® non-porous ceramics with thickness of 1 mm, with the diameter of the orifice 0.6 mm, in the first part of work. In the second part, ShapalTM-M ceramics of thickness 1.0 mm and orifice diameter 0.6 mm was used. The experimental part is divided into two sections. For both, NaCl is chosen as an electrolyte to set the initial conductivity of the tested solutions to the value of 400 S/cm. Supplied direct voltage is regulated to attain power of 100 W in the system. In the first part, effect of addition of chosen alcohols (ethanol, isopropylalcohol and glycerol) on the efficiency of the discharge in their water solutions is studied. For this purpose, a special glass reactor was designed and constructed. The efficiency of the discharge is measured by a spectroscopic determination of concentration of complex formed by a titanium reagent and hydrogen peroxide, which is generated during the discharge. The results show no positive effect of addition of extra OH group to the reaction through the alcohols. The use of isopropylalcohol causes even a significant decrease in the amount of hydrogen peroxide generated. The subject of the second part is a comparison of effect of different electrode materials on the discharge. The efficiency is measured by the same method as in the first part. Materials chosen were stainless steel, platinum, aluminium, copper and carbon. Each material shows different hydrogen peroxide production rate under the same parameters. The most perspective material seems to be carbon, as an inert material, that can be expected not to initiate any decomposition of hydrogen peroxide. The least favourable appears to be copper. When used, no production of hydrogen peroxide was observed in one of the electrode parts of the reactor.

This thesis presents an experimental study of non-thermal atmospheric pressure gas plasmas in presence of liquid as an efficient source of transient and reactive species to initiate chemical reactions necessary for many important applications. Two types of liquid-containing plasmas are considered: discharges formed between a needle electrode and a liquid electrode, and plasma jets formed in a water vapour flow mixed in helium or argon gas. Two plasma modes (the pulsed and the continuous mode) are observed in the needle-to-liquid plasma. A comparative study of the needle-to-liquid plasma in the continuous mode with DC and AC excitations reveals that the plasmas are glow discharges, and AC excited plasmas have the highest energy efficiency. A study of helium/water vapour plasma jet shows that “plasma bullets” are formed even with water vapour in the gas mixture, but become quenched when the moist helium flow rate is above 300sccm (~1800ppm water concentration). Moderate amount of water vapour (~250ppm water concentration) is beneficial for active species production mainly due to the high electron density. Hydrogen peroxide production in saline solution with three different plasma sources is investigated due to the importance of H2O2 in several important applications. Long lifetime of H2O2 in the liquid after plasma treatment indicates an exciting possibility of plasma pharmacy.

L’apurinic/apyrimidic endonuclease 1 (APE1) est une protéine multifonctionnelle qui joue un rôle important dans la voie de réparation de l’ADN par excision de base. Elle sert également de coactivateur de transcription et est aussi impliquée dans le métabolisme de l’ARN et la régulation redox. APE1 peut cliver les sites AP ainsi que retirer des groupements, sur des extrémités 3’ créées suite à des bris simple brin, qui bloquent les autres enzymes de réparation, permettant de poursuivre la réparation de l’ADN, puisqu’elle possède plusieurs activités de réparation de l’ADN comme une activité phosphodiestérase 3’ et une activité exonucléase 3’→5’. Les cellules de mammifères ayant subi un knockdown d’APE1 présentent une grande sensibilité face à de nombreux agents génotoxiques. APE1 ne possède qu’une seule cystéine située au 65e acide aminé. Celle-ci est nécessaire pour maintenir l’état de réduction de nombreux activateurs de transcription tels que p53, NF-κB, AP-1, c-Jun at c-Fos. Ainsi, elle se retrouve impliquée dans la régulation de l’expression génique. APE1 passe également à travers au moins 4 types de modifications post-traductionnelles : l’acétylation, la désacétylation, la phosphorylation et l’ubiquitylation. La façon dont APE1 est recrutée pour accomplir ses différentes fonctions biologiques demeure un mystère, bien que cela puisse être relié à sa capacité d’interaction avec de multiples partenaires différents. Sous des conditions de croissance normales, il a été démontré qu’APE1 interagit avec de nombreux partenaires impliqués dans de multiples fonctions. Nous émettons l’hypothèse que l’état d’oxydation d’APE1 est ce qui contrôle les partenaires avec lesquels la protéine interagira, lui permettant d’accomplir des fonctions précises. Dans cette étude nous démontrons que le peroxyde d’hydrogène altère le réseau d’interactions d’APE1. Un nouveau partenaire d’interaction d’APE1, Prdx1, un membre de la famille des peroxirédoxines responsable de récupérer le peroxyde d’hydrogène, est caractérisé. Nous démontrons qu’un knockdown de Prdx1 n’affecte pas l’activité de réparation de l’ADN d’APE1, mais altère sa détection et sa distribution cellulaire à l’intérieur des cellules HepG2 conduisant à une induction accrue de l’interleukine 8 (IL-8). L’IL8 est une chimiokine impliquée dans le stress cellulaire en conditions physiologiques et en cas de stress oxydatif. Il a été démontré que l’induction de l’IL-8 est dépendante d’APE1 indiquant que Prdx1 pourrait réguler l’activité transcriptionnelle d’APE1. Il a été découvert que Prdx1 est impliquée dans la régulation redox suite à une réponse initiée par le peroxyde d’hydrogène. Ce dernier possède un rôle important comme molécule de signalisation dans de nombreux processus biologiques. Nous montrons que Prdx1 est nécessaire pour réduire APE1 dans le cytoplasme en réponse à la présence de H2O2. En présence de Prdx1, la fraction d’APE1 présent dans le cytoplasme est réduite suite à une exposition au peroxyde d’hydrogène, et Prdx1 est hyperoxydé suite à l’interaction entre les deux molécules. Cela suggère que le signal, que produit le peroxyde d’hydrogène, sur APE1 passe par Prdx1. Un knockdown d’APE1 diminue la conversion de la forme dimérique de Prdx1 vers la forme monomérique. Cette observation implique qu’APE1 pourrait être impliquée dans la régulation de l’activité catalytique de Prdx1 en accélérant son hyperoxydation. / Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein, which play important roles in base excision repair (BER) pathway and serve as transcriptional co-activator. APE1 is also involved in RNA metabolism and redox regulation. APE1 can cleave abasic sites and process 3’-blocking termini into 3’-OH for DNA repair replication as it posseses several DNA repair activities including AP endonuclease, 3’-phosphodiesterase and 3’ to 5’-exonuclease. Mammalian cells knockdown for APE1 are very sensitive to various DNA damaging agents. APE1 has a unique cysteine C65, which is required to maintain the reduced state of several transcriptional activators such as p53, NF-кB, AP-1, c-Jun, and c-Fos and therefore is involved in the regulation of gene expression. APE1 also undergoes at least four types of post-translational modifications that include acetylation, deacetylation, phosphorylation and ubiquitylation. How APE1 is being recruited to execute the various biological functions remains a challenge, although this could be directly related to its ability to interact with multiple different partners. Under normal growth conditions, APE1 has been shown to interact with a number of proteins that are involved in various functions. We propose that the oxidative state of APE1 governs its interacting partners thereby allowing the protein to perform specific functions. In this study we find that APE1 interactome alters in response to hydrogen peroxide. One novel APE1 interacting partner Prdx1, a member of the peroxiredoxin family that can scavenge hydrogen peroxide is characterized. We demonstrate that knockdown of Prdx1 did not impair APE1 DNA repair activity, but alters APE1 detection, and subcellular distribution in HepG2 cells leading to the induction of interleukin 8 (IL-8). IL-8 is a pro-inflammatory chemokine involved in cellular stress, under physiological and iv oxidative stress conditions. It has been shown that the induction of IL-8 is dependent on APE1 indicating Prdx1 may regulate APE1 transcriptional activity. Prdx1 has been discovered to be involved in the redox regulation of cell signaling initiated by hydrogen peroxide, which has important roles as a signaling molecule in the regulation of a variety of biological processes. Prdx1 exists as a dimer in the cells and we show that Prdx1 is required to reduce APE1 in the cytoplasm in response to H2O2. During this process, the dimeric form of Prdx1 is converted to the oxidized monomeric form. Interestingly, the H2O2-induced conversion of Prdx1 to the monomeric form is dependent upon the presence of APE1. These observations imply that there is a tight regulatory network existing between APE1 and Prdx1.

Nous avons récemment démontré que les espèces réactives oxygénées induisent une augmentation de l’expression des protéines Giα dans les cellules du muscle lisse vasculaire (CMLV) provenant d’aortes de rats spontanément hypertendus (SHR, de l’anglais spontaneously hypertensive rats). La présente étude a pour but d’étudier les effets du peroxyde d’hydrogène (H2O2), un oxydant qui induit le stress oxydatif, sur l’expression de Giα et sur l’activité de l’adénylate cyclase, et d’explorer les voies de signalisation sous-jacentes responsables de cette réponse. Nos résultats montrent que H2O2 induit une augmentation de l’expression des protéines Giα-2 et Giα-3 de manière dose- et temps-dépendante avec une augmentation maximale de 40-50% à 100 µM après 1 heure, sans affecter l’expression de Gsα. L’expression des protéines Giα a été maintenue au niveau normal en presence de AG 1478, AG1295, PD98059 et la wortmannine, des inhibiteurs d’EGF-R (de l’anglais epidermal growth factor receptor), PDGFR-β (de l’anglais platelet-derived growth factor receptor β), de la voie de signalisation ras-ERK1/2 (de l’anglais extracellular regulated kinase1/2), et de la voie de la PI3Kinase-AKT (de l’anglais phosphatidyl inositol-3 kinase), respectivement. En outre, le traitement des CMLV avec H2O2 a induit une augmentation du degré de phosphorylation d’EGF-R, PDGF-R, ERK1/2 et AKT; et cette expression a été maintenue au niveau témoin par leurs inhibiteurs respectifs. Les inhibiteurs d’EGF-R et PDGF-R ont aussi induit une diminution du degré de phosphorylation de ERK1/2, et AKT/PKB. En outre, la transfection des cellules avec le siRNA (de l’anglais, small interfering ribonucleic acid) de EGF-R et PDGFR-β a atténué la surexpression des protéines Giα-2 et Giα-3 induite par le traitement au H2O2. La surexpression des protéines Giα induite par H2O2 a été corrélée avec une augmentation de la fonction de la protéine Giα. L’inhibition de l’activité de l’adénylate cyclase par de faibles concentrations de GTPγS après stimulation par la forskoline a augmenté de 20% dans les cellules traitées au H2O2. En outre, le traitement des CMLV au H2O2 a aussi accru l’inhibition de l’activité de l’adénylate cyclase par les hormones inhibitrices telles que l’angiotensine II, oxotrémorine et C-ANP4-23. D’autre part, la stimulation de l’adénylate cyclase induite par GTPγS, glucagon, isoprotérénol, forskoline, et le fluorure de sodium (NaF) a été atténuée de façon significative dans les cellules traitées au H2O2. Ces résultats suggèrent que H2O2 induit la surexpression des protéines Giα-2 and Giα-3 via la transactivation des récepteurs des facteurs de croissance EGF-R, PDGFR-β et l’activation des voies de signalisation ras-ERK1/2 et PI3K-AKT Mot-cles: Protéines Giα, peroxyde d’hydrogène, stress oxydant, récepteurs des facteurs de croissance, MAP kinases, adénylate cyclase, hypertension / We recently have shown that reactive oxygen species contribute to the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). The present study was undertaken to examine if H2O2, an oxidant that induces oxidative stress could also enhance the expression of Giα proteins and associated adenylyl cyclase signalling in aortic VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of cells with H2O2 increased the expression of Giα-2 and Giα-3 proteins but not that of Gsα proteins in a concentration- and time-dependent manner. A maximal increase of 40-50% was observed at 100µM and 1h. The enhanced expression of Giα proteins was restored to control levels by AG 1478, AG1295, PD98059 and wortmannin, inhibitors of epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGFR-β), the mitogen-activated protein kinase (MEK1/2), and PI3 kinase respectively. In addition, treatment of VSMC with H2O2 also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2 and AKT and this increased phosphorylation was attenuated to control levels by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also attenuated the enhanced phosphorylation of ERK1/2 and AKT to control levels. Transfection of cells with EGF-R and PDGFR-β siRNA followed by H2O2 treatment restored the H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins to control levels. The increased expression of Giα proteins by H2O2 was reflected in the increased Gi functions. The inhibition of forskolin (FSK)-stimulated AC activity by low concentration of GTPγS (receptor- independent Gi functions) was increased by about 20% by H2O2 treatment. Moreover, treatment of cells with H2O2 also resulted in an increased Ang II-, C-ANP4-23, and oxotremorine-mediated inhibition of AC (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulation of AC by GTPγS, glucagon, isoproterenol, FSK, and NaF was significantly decreased in H2O2-treated cells. These results suggest that H2O2 increases the expression of Giα-2 and Giα-3 proteins in VSMC through the transactivation of EGF-R, PDGFR-β and associated ERK1/2 and PI3K signalling pathways. Keywords: Giα proteins, hydrogen peroxide, adenylyl cyclase, oxidative stress, MAP kinase, growth factor receptors, hypertension.

This thesis describes the catalytic asymmetric epoxidation of olefins mediated by chiral iminum salts. The first chapter introduces some of the most novel and effective catalytic asymmetric methods for preparing chiral oxiranes. The second chapter is divided into three sections. The first section of chapter two is dedicated to our efforts to develop new aqueous oxidative conditions using both hydrogen peroxide and sodium hypochlorite as efficient, green oxidants that remove the temperature boundaries observed with the use of Oxone® as the stoichiometric oxidant. A wider range of available temperatures was examined allowing optimization of both oxidative systems. Ethereal hydrogen peroxide was observed to mediate asymmetric epoxidation within an acetonitrile monophasic co-solvent system giving enantioselectivities of up to 56%. When sodium hypochlorite was used in a biphasic solvent system in conjunction with dichloromethane; it was observed to mediate oxidation of the substrate alkenes in up to 71% ee. The second and third sections of chapter two are dedicated to our efforts to synthesize chiral iminium salts as catalysts for asymmetric epoxidation based on a biphenyl azepinium salt catalyst structure. From previous work within the Page group, the asymmetric synthesis and subsequent defined stereochemistry of a chiral carbon atom α to the iminium nitrogen atom was shown to have significant effect on the enantiocontrol of epoxidation using the iminium salt catalyst. Work was completed on biphenyl azepinium salt catalysts, inserting an alkyl or aryl Grignard reagent into the iminium bond using a pre-defined dioxane unit as a chiral auxiliary. Oxidation of the subsequent azepine gave a single diastereoisomerically pure azepinium salt. The methyl analogue of this sub-family of azepinium catalysts has been shown to give up to 81% ee for epoxidation of 1-phenylcyclohexene, furthermore, the binaphthalene azepinium salt with an additional methyl group was also synthesized and was shown to give up to 93% for epoxidation of 1-phenylcyclohexene. Continuation of the substitution α to the nitrogen atom gave rise to an interesting tetracyclic (biphenyl) azepinum salt catalyst. Construction of an asymmetric oxazolidine ring unit encapsulating the azepinium nitrogen and one of the methylene carbon atoms was achieved. In doing so two chiral centres α to the nitrogen atom were generated. The azepinium chiral carbon atom was populated by an addition methyl group with variation in the substitution on the oxazolidine chiral carbon atom. The benzyl analogue of this sub-family of tetracyclic azepinium catalysts has shown to give up to 79% ee for epoxidation 1-phenylcyclohexene. The third chapter is the experimental section and is dedicated to the methods of synthesis and characterization of the compounds mentioned in the previous chapter. X-ray reports regarding the crystallographic analysis of the structures presented in chapter two are provided in appendix A. Appendix B contains the analytical spectra for the determination of enantiomeric excess of the epoxides.