published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Throughout North America, many species of aerial insectivorous birds – birds that capture flying insects – have exhibited sharp declines, with tree swallows (Tachycineta bicolor) experiencing a 2.8% annual decline in Canada since 1989. The timing of these declines coincides with major changes in agriculture, perhaps signaling a potential causal link. Declines may be influenced indirectly by agriculturally-driven reductions in the abundance of the swallow’s main diet of aerial insects through increased agrochemical use, wetland drainage and cropping intensity. My objectives were to determine how aerial insect abundance and biomass vary across an agricultural landscape in the Canadian Prairies, and determine how breeding tree swallows respond in terms of (i) foraging behaviours and return rates, (ii) body condition, and (iii) selected physiological responses (i.e. oxidative stress and feather corticosterone). Artificial nest-boxes were erected at each of 5 sites with varying levels of agricultural intensification in south-central Saskatchewan, 2012-2014. Insect abundance and biomass were monitored daily using passive aerial samplers at each of the sites throughout the breeding season. Tree swallows adults (n = 596) and nestlings (n = 1107) from each nest were captured, banded, measured and blood and feather sampled to determine condition and physiological status. In 2014, radio frequency identification (RFID) technology was employed at a subset of nest-boxes to determine adult foraging rates and durations. Despite strong differences in land use characteristics among sites, insect abundance and biomass varied by year but were similar between heavily cropped and grassland references sites. Regardless, birds responded to temporal changes in prey abundance and differences were found in measurements of foraging behavior and physiological measures of oxidative stress. Adult foraging rate (number of nest visits/hour) was primarily influenced by wind and nestling age, but was also positively related to daily insect biomass. Foraging intervals (i.e., length of time spent on each trip) differed between agricultural intensities; birds on agricultural sites spent more time away from the nest box, presumably foraging, suggesting reduced nest attentiveness. Return rates were primarily predicted by body condition and brood size from the previous breeding season, but were also higher on grassland sites, suggesting that agricultural sites may be less favorable. Older adults (based on banding records) and females displayed a higher body condition than did younger individuals and males, respectively. Measures of physiology (i.e. total antioxidant counts and oxidative damage) in both adult and nestling swallows were primarily driven by temporal changes in insect biomass and abundance. Similarly, adult body mass and physiology were related to local insect availability, but not land use (agriculture/grassland). Measures of nestling feather corticosterone were unrelated to any of the explanatory variables. These results provide valuable information pertaining to components of individual quality, foraging behaviour, and return status in response to prey availability under differing agricultural land use regimes, which may help to inform conservation strategies for a variety of insectivorous bird species of conservation concern.

Even though the role of oxidative stress in a variety of disease states is known, strategies to alleviate this oxidative stress by antioxidants have not been able to achieve clinical success. Particularly, treatment of oxidative stress by small molecule antioxidants has not received due attention because of the challenges associated with its delivery. Antioxidant polymers, where small molecule antioxidants are incorporated into the polymer backbone, are an emerging class of materials that can address some of these challenges. In this work, biodegradable polymers incorporating phenolic antioxidants in the polymer backbone were synthesized. Antioxidant polymers were then characterized for their in vitro degradation, antioxidant release and their effect on oxidative stress levels (redox state) in the cells. Trolox, a water-soluble analogue of vitamin E, was polymerized to synthesize poly(trolox ester) with 100% antioxidant content which undergoes biodegradation to release trolox. Nanoparticles of poly(trolox ester) were able to suppress oxidative stress injury induced by metal nanoparticles in an in vitro cell injury model. In another study, we polymerized polyphenolic antioxidants (e.g. curcumin, quercetin) using a modified non-free-radical polymerization poly(β-amino ester) chemistry. This synthesis scheme can be extended to all polyphenolic antioxidants and allows tuning of polymer degradation rate by choosing appropriate co-monomers from a large library of monomers available for β-amino ester chemistry. Poly(antioxidant β-amino esters) (PABAE) were synthesized and characterized for their degradation, cytotoxicity and antioxidant activity. PABAE degradation products suppressed oxidative stress levels in the cells confirming antioxidant activity of degradation products.

Olfactory sensory decline has been associated with normal aging as well as neurodegenerative disorders, yet the underlying mechanisms are unclear. The overall aim of this dissertation was to investigate the fundamental molecular and cellular mechanisms associated with olfactory neurodegeneration. This investigation uses an integrative approach, combining proteomics and gene expression analyses with cellular and tissuelevel characterization. Using these approaches, two model systems were investigated: 1) normally aging C57BL/6 mice of ages 1.5-, 6- and 20-months; and 2) a mouse model of elevated endogenous oxidative stress-associated neurodegeneration, namely, the Harlequin mutant mouse. The first specific aim was to test the hypothesis that oxidative stress is associated with aging of the olfactory system. Using proteomics, I demonstrated that olfactory aging was accompanied primarily by increased oxidative stress-, mitochondrial metabolism- and synaptic/transport-associated changes. The second specific aim was to test the hypothesis that the olfactory system accumulates oxidative stress-mediated macromolecular damage over time, predisposing it to neurodegeneration. Two types of protein oxidation, namely, carbonylation and nitration, accumulated with aging in the olfactory system. Protein and cellular targets of oxidative stress-associated damage were identified using redox proteomics coupled with immunohistochemical localization. The third specific aim was to test the hypothesis that elevated oxidative stress in the olfactory system results in apoptosis/neurodegeneration. The Harlequin mutant mouse was critically selected and validated as a model for studies of oxidative stress-associated olfactory neurodegeneration at both the cellular and molecular levels. The Harlequin mouse had decreased levels and altered distribution of apoptosis inducing factor protein in mature olfactory sensory neurons, increased oxidative DNA damage and apoptosis in the olfactory epithelium, and pronounced cytoskeletal disorganization. The molecular studies confirmed and extended our cellular data and identified several significantly regulated genes associated with elevated oxidative stress and apoptosis. This novel study, by combining contemporary proteomics and genomics with cellular and tissue-level analyses, has provided a road map for understanding fundamental molecular mechanisms of olfactory degeneration.

Advancing applications of engineered nanomaterials (ENM) in various fields create the opportunity for intended (e.g. drug and gene delivery) or unintended (e.g. occupational and environmental) exposure to ENM. However, the knowledge of ENM-toxicity is lagging behind their application development. Understanding the ENM hazard can help us to avoid potential human health problems associated with ENM applications as well as to increase their public acceptance. Ceria (cerium [Ce] oxide) ENM have many current and potential commercial applications. Beyond the traditional use of ceria as an abrasive, the scope of ceria ENM applications now extends into fuel cell manufacturing, diesel fuel additives and for therapeutic intervention as a putative antioxidant. However, the biological effects of ceria ENM exposure have yet to be fully defined. Both pro-and anti-oxidative effects of ceria ENM exposure are repeatedly reported in literature. EPA, NIEHS and OECD organizations have nominated ceria for its toxicological evaluation. All these together gave us the impetus to examine the oxidative stress effects of ceria ENM after systemic administration. Induction of oxidative stress is one of the primary mechanisms of ENM toxicity. Oxidative stress plays an important role in maintaining the redox homeostasis in the biological system. Increased oxidative stress, due to depletion of antioxidant enzymes or molecules and / or due to increased production of reactive oxygen (ROS) or nitrogen (RNS) species may lead to protein oxidation, lipid peroxidation and/or DNA damage. Increased protein oxidation or lipid peroxidation together with antioxidant protein levels and activity can serve as markers of oxidative stress. To investigate the oxidative stress effects and the mechanisms of ceria-ENM toxicity, fully characterized ceria ENM of different sizes (~ 5nm, 15nm, 30nm, 55nm and nanorods) were systematically injected into rats intravenously in separate experiments. Three brain regions (hippocampus, cortex and cerebellum) were harvested from control and ceria treated rats after various exposure periods for oxidative stress assessment. The levels of oxidative stress markers viz. protein carbonyl (PC), 3-nitrotyrosine (3NT), and protein bound 4-hydroxy-2-trans-nonenal (HNE) were evaluated for each treatment in each control and treated rat organ. Further, the levels and activities of antioxidant proteins, such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), were measured together with levels of heat shock proteins heme oxygenase -1 and 70 (HO-1 and Hsp-70). In addition, the levels of pro-inflammatory cytokines IL-1β, TNF-α, pro-caspase-3, and autophagy marker LC-3A/B were measured by Western blot technique. In agreement with the literature-proposed model of oxidative stress hierarchy mechanism of ENM-toxicity, the statistical analysis of all the results revealed that the ceria ENM-induced oxidative stress mediated biological response strongly depends on the exposure period and to some extent on the size of ceria ENM. More specifically, a single intravenous injection of ceria ENM induced tier-1 (phase-II antioxidant) response after shorter exposure periods (1 h and 20 h) in rat brain. Upon failure of tier-1 response after longer exposure periods (1 d to 30 d), escalated oxidative stress consequently induced tier-2 and tier-3 oxidative stress responses. Based on our observations made at chronic exposure period (90 d) after the single i.v. injection of ceria ENM, we could extend the model of oxidative stress hierarchy mechanisms for ceria-ENM-induced toxicity. Considering the evaluation of all the oxidative stress indices measured in 3-brain regions, oxidative stress effects were more prominent in hippocampus and the least in cerebellum, but no specific pattern or any significant difference was deduced.

Selenium (Se) has received a lot of attention for its antioxidant and immune modulating properties. Yet, comparably few studies have focused on the horse. Therefore the objectives of this research were to evaluate the influences of Se status on immune function and antioxidant defense in horses. Twenty eight horses were allocated to one of 4 dietary Se treatments: low (LS), adequate (AS), high organic (SP) and high inorganic (SS). First, horses assigned to LS, SP and SS were depleted of Se and received a low Se diet (0.07 ppm Se) for 35 wk, while AS received an adequate Se diet (0.14 ppm Se). During week 28 to 35 immune function was evaluated using a vaccine challenge with keyhole limpet hemocyanin (KLH) and equine influenza as antigens. Then, a 29 wk repletion phase followed. The LS and AS received the same diets described above while SP received an organic Se supplemented diet (0.3 ppm; Sel-Plex, Alltech, Nicholasville, KY) and SS an inorganic Se supplemented diet (0.3 ppm; sodium selenite). Immune function was assessed using a vaccine challenge with ovalbumin (OVA) and equine influenza as antigens during week 22 to 29. Samples collected throughout the depletion and repletion phases were used to assess change in Se status, antioxidant status and oxidative stress. Finally, a mild exercise test served to assess exercise induced oxidative stress. The experimental model responded as hypothesized, evaluated by blood Se and glutathione peroxidase (GPx) activity. Upon vaccination with KLH, antibody response was faster in AS than LS. Antigen specific mRNA expression of T-bet was also higher for AS than LS. Following OVA vaccination humoral and cell-mediated vaccination responses were similar across treatments. However, non-specific stimulation of peripheral blood mononuclear cells indicated suppressed mRNA expression of selected cytokines for LS compared to AS, SP and SS. Antioxidant capacity and oxidative stress were unaffected by change in Se status. A difference in GPx response post exercise was also noted between SP and SS. Low Se status impaired some measures of immune function. Supplementation at 0.3 ppm may benefit horses as indicated by higher GPx activity in idle and exercised horses.

There is an undeniable link between oxidative stress, inflammation, and disease. Currently, approaches using antioxidant therapies have been largely unsuccessful due to poor delivery and bioavailability. Responding to these limitations, we have developed classes of polymer and delivery systems that can overcome the challenges of antioxidant and anti-inflammatory therapy. In our initial studies, nanoparticles of poly(trolox), a polymeric form of trolox, were surface-modified with antibodies. This modification allows for specific targeting to endothelial cells, affording controllable and localized protection against oxidative stress. We have shown these targeted nanoparticles bind, internalize, and provide protection against oxidative stress generation and cytotoxicity from iron oxide nanoparticles. In a similar fashion, we have tested the ability of poly(trolox) to prevent rheumatoid arthritis in vivo. Poly(trolox) nanoparticles were encapsulated in a PEGylated polymer to enhance circulation and biocompatibility. These particles were shown to accumulate in inflamed joint tissue, recover natural antioxidant function, suppress protein oxidation, and inhibit inflammatory markers. Lastly, we developed a class of polyphenolic compounds utilizing a non-free radical based reaction chemistry of poly(β-amino esters). The polyphenol apigenin was investigated for its anti-inflammatory properties to inhibit inflammation-mediated tumor cell metastasis. PEGylated nanoparticles that incorporated apigenin poly(β-amino ester) were developed and found to retain their anti-inflammatory efficacy while providing a long term release profile. These inhibited the ability of tumor cells to adhere to inflamed vascular cells. We also have shown that these polymers can suppress markers of inflammation responsible in enhancing tumor cell adhesion.

Oxidative stress is characterized by the cellular accumulation of reactive oxygen species (ROS). Increased production of ROS, such as the superoxide anion (O2.-), or a deficiency in their clearance by antioxidant defenses, mediates the cellular pathology. Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder commonly manifesting in milieu of the metabolic syndrome. Oxidative stress is an important pathogenic mediator in NAFLD, and in its associated morbidities like atherosclerosis. The objective of my research was to investigate the regulation of oxidative stress and the antioxidant actions of natural health products (NHPs) in the context of NAFLD and its associated disorders. The O2.- generating NADPH oxidase contributes to atherogenesis by facilitating macrophage induced vascular injury. In manuscript I, the plant alkaloid berberine effectively abolished NADPH oxidase mediated O2.- production in lipopolysaccharide stimulated macrophages. Real-time PCR analysis and siRNA transfection studies revealed that berberine mediated its effects through down-regulation of the oxidase’s catalytic subunit gp91phox. Berberine also restored the activity of the O2.- clearing enzyme superoxide dismutase (SOD). High fat diet (HFD) fed rodents are a popular model for investigating NAFLD pathogenesis. In manuscript II, folic acid supplementation significantly reduced HFD-induced hepatic oxidative stress and liver injury in mice. Folic acid decreased NF-kB/DNA binding, down-regulated NADPH oxidase gene expression, and inhibited the oxidase. The antioxidant activities of SOD and catalase were restored and the reduced to oxidized glutathione ratio (GSH:GSSG) was re-established with folic acid supplementation. Folic acid’s hepatoprotective antioxidant effects were associated with a marked improvement in liver histology. Homocysteine (Hcy) levels are perturbed in NAFLD, but the etiology is unclear. In manuscript III, HFD fed mice exhibited decreased Hcy levels. Real-time PCR and Western Immunoblotting analysis revealed that Hcy catabolising enzymes cystathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE) were increased in the liver of these animals. The transsulfuration activities of these enzymes were elevated and coincided with enhanced hepatic hydrogen sulfide biosynthesis. Glutathione was maintained despite increased hepatic oxidative stress. Taken together, NHPs such as berberine and folate, and Hcy catabolising enzymes CBS and CSE, might have therapeutic potential for managing oxidative stress in NAFLD and its associated co-morbidities. / October 2015