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

No / Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose¿response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

Yes / There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1 and 10 μM induced increases of over ∼10-fold in the percentage of apoptotic cells (p ≤ 0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.

Yes / We investigated the protective role of myricetin bulk and nanoforms, against reactive oxygen species (ROS)-induced oxidative stress caused by hydrogen peroxide and tertiary-butyl hydro peroxide in lymphocytes in vitro from healthy individuals and those from pre-cancerous patients suffering with monoclonal gammopathy of undetermined significance (MGUS). The change in intracellular reactive oxygen species was measured once cells were treated with myricetin bulk forms and nanoforms with and without either hydrogen peroxide or tertiary-butyl hydro peroxide co-supplementation. The direct and indirect antioxidant activity of myricetin was spectrofluometrically measured using the fluorescent dye 2',7'-dichlorofluorescin diacetate and using the Comet assay, respectively. Hydrogen peroxide (50 µM) and tertiary-butyl hydro peroxide (300 µM) induced a higher level of reactive oxygen species-related DNA damage and strand breaks. Addition of myricetin nanoform (20 µM) and bulk (10 µM) form could, however, significantly prevent hydrogen peroxide- and tertiary-butyl hydro peroxide-induced oxidative imbalances and the nanoform was more effective. Glutathione levels were also quantified using a non-fluorescent dye. Results suggest that myricetin treatment had no significant effect on the cellular antioxidant enzyme, glutathione. The current study also investigates the effect of myricetin on the induction of double-strand breaks by staining the gamma-H2AX foci immunocytochemically. It was observed that myricetin does not induce double-strand breaks at basal levels rather demonstrated a protective effect.

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.