Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. 1,4-Benzoquinone (BQ) is a reactive quinone that we have used to help predict target residue covalent binding by such compounds. Hydroquinone glutathione conjugates (HQ-GSH) cause renal cell necrosis by producing reactive oxygen species (ROS) and by adducting proteins preferentially localized in the S3 segment of the renal proximal tubules. In vitro experimentation using model peptides and proteins have identified cysteine, lysine, arginine, and glutamic acid as amino acids targeted for quinone-thioether adduction. By mimicking a standard protein digestion protocol (100 mM ammonium bicarbonate pH 7.5, or 50 mM Tris-HCl pH 7.5), we demonstrated that cysteine-BQ adducts are unstable. Taken together, these results indicate that BQ-adduct formation on cysteine residues may be a transient interaction, where physiological conditions may play a role in adduct stability. In vivo experimentation following administration of 2-(glutathion-S-yl)HQ (MGHQ, 400 μmol/kg, iv, 2 hr) to Long Evans rats identified the specific site of quinone-thioether protein adduction on a number of proteins. Urinary proteins were isolated, and either trypsin digested en masse and analyzed by multi-dimensional protein identification technology (MuDPIT) or, following SDS-PAGE, single immunopositive bands were excised, trypsin digested and analysed by LC-MSMS. Following site-specific identification of adducts, 3-dimensional protein modeling of adducts on the protein was performed as a way to reveal the potential structural consequence of the modification on 3D structure. The outer stripe of the outer medulla (OSOM) is the target site of protein adduction caused by quinone-thioethers. Using a 2DGE-Western blot approach, in combination with an extensive knowledge of quinol-thioether chemistry, LC-MSMS, and the latest MSMS analysis software, we identified the specific amino acid site of adduction on 17 unique peptides from 34 target proteins within the OSOM. Of the 22 bands analyzed, adducted peptides were identified in 11 of them. Many of the target proteins identified have previously been identified as a target of other electrophiles, producing additional evidence that such protein adduction is selective rather than random. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response.

Metastatic bone cancer is the most common cause of pain in patients with malignant tumors. Prolonged opioid treatment remains the primary method to treat pain in these patients. Sustained morphine exposure enhances both bone cancer-induced pain and bone loss in mice implanted with sarcoma cells. Sustained treatment of bone marrow cultures with morphine results in COX-2 dependent upregulation of RANKL and PGE2, and suppression of OPG. This results in increased osteoclastogenesis which was dependent on COX-2 and OPG/RANKL regulatory axis. Treatment with morphine does not induce any direct changes in osteoclasts or sarcoma cells. The in vitro data was validated in the animals where morphine induces an increase in the osteoclastogenesis and RANKL, and suppresses OPG. These data indicate that morphine enhances osteoclastogenesis by modulating the OPG/RANKL regulatory axis in osteoblasts through a COX-2 dependent mechanism.Prolonged opioid exposure induces an opioid-receptor dependent hyperalgesia in humans and in animals. Studying the direct effect of opioids on primary sensory neurons we demonstrate a modest increase in CGRP cellular content that was not opioid-receptor dependent. Although dynorphin A (2-13) and PGE2 enhanced the release of the neuropeptide, pretreatment with opioids does not influence the capsaicin or KCl evoked CGRP release. These date indicate that the neurochemical changes seen in vivo may be dependent on factors upregulated in the periphery and/or the CNS.It has been demonstrated that sensory neurons innervating the femur express markers of neuronal injury and the intramedullary region of the femur becomes devoid of nerve fibers as the tumor expands. In this study we demonstrate that the sarcoma cells generate high levels of ROS and release hydrogen peroxide into the surrounding space, which induces death and injury to both sensory neurons and glia. This death was prevented by the anti-oxidants BHA and catalase. The present study provides evidence that ROS released by cancer cells can directly lead to injury and death of sensory neurons. ROS induced injury may be one of the mechanism through which sensory neurons are injured in the murine bone cancer pain model.

Mechanisms of cell death extend beyond the simple apoptosis/necrosis relationship to include regulated modes of cell death that do not readily fit either of the classic descriptors. One such mechanism of cell death involves poly(ADP-ribose)polymerase-1 (PARP-1)-mediated cell death. 2,3,5-Tris(Glutathion-S-yl)-hydroquinone (TGHQ), a reactive oxygen species (ROS) generating nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes necrotic renal cell death, the basis for which is unclear. We therefore investigated TGHQ-mediated cell death in human renal proximal tubule epithelial HK-2 cells. TGHQ induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, loss of mitochondrial membrane potential, and subsequent necrotic cell death. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Inhibition of PARP-1 with PJ34 blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers, NAD consumption, and the consequent necrotic cell death. However, HK-2 cell death was only delayed by PJ34, and cell death remained necrotic in nature. In contrast, chelation of intracellular Ca2+ with BAPTA-AM completely abrogated TGHQ-induced necrotic cell death. Ca2+ chelation not only prevented the collapse in the mitochondrial potential but also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Moreover, TGHQ caused a sequential oxidation of peroxiredoxin III (PrxIII), a protein considered the primary antioxidant defense within mitochondria. Thus, TGHQ induced two acidic shifts in PrxIII, with both pI shifted spots representing oxidized forms of PrxIII. Transient expression of a dominant negative version of PrxIII resulted in a significant increase in TGHQ-induced cytotoxicity, whereas overexpression of wild-type PrxIII significantly attenuated cytotoxicity. Our studies provide new insights into PARP-1-mediated necrotic cell death. Changes in intracellular Ca2+ concentrations appear to couple PARP-1-hyperactivation to subsequent cell death, but in the absence of AIF release from mitochondria. NAD depletion, mitochondrial membrane depolarization, Ca2+-mediated calpain activation, and PrxIII oxidation, all contribute to TGHQ-driven ROS-mediated necrotic cell death.

Assessing hazards due to energetic or reactive chemicals is a challenging and complicated task and has received considerable attention from industry and regulatory bodies. Thermal analysis techniques, such as Differential Scanning Calorimeter (DSC), are commonly employed to evaluate reactivity hazards. A simple classification based on energy of reaction (-H), a thermodynamic parameter, and onset temperature (To), a kinetic parameter, is proposed with the aim of recognizing more hazardous compositions. The utility of other DSC parameters in predicting explosive properties is discussed. Calorimetric measurements to determine reactivity can be resource consuming, so computational methods to predict reactivity hazards present an attractive option. Molecular modeling techniques were employed to gain information at the molecular scale to predict calorimetric data. Molecular descriptors, calculated at density functional level of theory, were correlated with DSC data for mono nitro compounds applying Quantitative Structure Property Relationships (QSPR) and yielded reasonable predictions. Such correlations can be incorporated into a software program for apriori prediction of potential reactivity hazards. Estimations of potential hazards can greatly help to focus attention on more hazardous substances, such as hydroxylamine (HA), which was involved in two major industrial incidents in the past four years. A detailed discussion of HA investigation is presented.

Two projects were undertaken to study the non-energy conserving alternative pathway present in the plant mitochondrial ETC. In the first project, a tobacco AOX2 gene was cloned and characterized. AOX2 showed tissue specificity in expression and could not be induced by common stresses. In the second project I carried out a physiological characterization of transgenic tobacco plants with increased or decreased expression of AOX1 subjected to cold stress. Under non-stress condition, a strong inverse relationship between levels of AOX1 and levels of oxidative damage was observed, while after cold treatment AOX1 transgenic lines and WT showed more complicated and differential responses in aspects of oxidative damage and the capacity of antioxidant system. I also discovered that the pool sizes of monosaccharides after temperature shift were proportional to AOX1 levels. These results indicated that AOX1 might have crucial but complex impacts on ROS balance and carbon metabolism during cold stress.

Two projects were undertaken to study the non-energy conserving alternative pathway present in the plant mitochondrial ETC. In the first project, a tobacco AOX2 gene was cloned and characterized. AOX2 showed tissue specificity in expression and could not be induced by common stresses. In the second project I carried out a physiological characterization of transgenic tobacco plants with increased or decreased expression of AOX1 subjected to cold stress. Under non-stress condition, a strong inverse relationship between levels of AOX1 and levels of oxidative damage was observed, while after cold treatment AOX1 transgenic lines and WT showed more complicated and differential responses in aspects of oxidative damage and the capacity of antioxidant system. I also discovered that the pool sizes of monosaccharides after temperature shift were proportional to AOX1 levels. These results indicated that AOX1 might have crucial but complex impacts on ROS balance and carbon metabolism during cold stress.

Various commodity plastics used today are based on fossil fuels. Most of these plastics are non-biodegradable and will persist in the environment over a long time. The bioplastics from renewable resources have the potential to support a greener economy. The two of such renewably resourced bioplastics are poly (hydroxyl butyrate-co-valerate), PHBV and poly (butylene succinate), PBS. We have used petro-based PBS in our study, but renewable resource based PBS is expected to be available on a commercial scale in a very near future. These two bioplastics are both biodegradable. These two bioplastics are both biodegradable. In our study we have used PBS from petroleum resource but PHBV is a brittle bioplastic with a high modulus value and a low elongation at break while PBS is a low modulus bioplastic with a high elongation at break. Complementary properties can be obtained by blending PHBV and PBS. The direct melt blends showed poor mechanical properties due to limited interaction between PHBV and PBS phases. This research focuses on increasing the interaction between PHBV and PBS blends by using compatibilizers. The compatibilizers used in this thesis were dicumyl peroxide, DCP and trimethylolpropane triacrylate, TMPTA. Use of an in situ compatibilization method was done for the melt mixing of PHBV and PBS yielding blends with improved characteristics. The investigations were performed at three different ratios of PHBV and PBS blends. The increase in the compatibility between the two phases was demonstrated through various thermal, thermo-mechanical, rheological and morphological means. The increase in elongation at break was used as a primary marker for compatibilization. The optimization of DCP and TMPTA was carried out, which showed the enhanced interaction between PHBV and PBS phases, with the successful stress transfer from PHBV phase to the PBS phase resulting in increase in elongation at break. Inward shifts in tan delta peak on addition of DCP and TMPTA to the blends also showed increase in compatibility between the two phases. The interfacial adhesion between a brittle and ductile polymer, PHBV and PBS respectively was increased by using DCP and TMPTA. This opened gateways to various novel applications of PHBV and PBS blends via in situ reactive extrusion process. / The Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA) for their financial support via New directions research program (SR9235). We would also like to thank Canadian Foundation for Innovation (CFI) and Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA) for their equipment supports.

This thesis concerns the investigation of the neuroprotective effects of the peptides relaxin-2 and relaxin-3. Previous studies have shown that intracerebral relaxin-2 reduces brain lesion size in an in vivo model of stroke, thereby providing evidence of a neuroprotective action of relaxin-2. This thesis set out to extend this work to determine whether or not relaxin-2 and relaxin-3 protected neural tissues from stroke in vivo and to determine the mechanisms by which relaxin-2 and relaxin-3 may protect astrocytes from injury by affecting migration, resistance to hypoxia and prevention of apoptosis. The first set of experiments show that relaxin-2 and relaxin-3 pre- and post-treatments following stroke induction protect neural tissues from cerebral damage in vivo. The next experiments show that relaxin-2 and relaxin-3 increase astrocyte migration in vitro through nitric oxide, phosphoinositide 3-kinase and matrix metalloproteinase-mediated pathways. A third set of experiments show that relaxin-2 and relaxin-3 treated astrocytes exhibited a higher viability compared to untreated astrocytes when exposed to oxygen glucose deprivation for 24 hours. Astrocytes that were cultured with relaxin-2 or relaxin-3 also showed a lower production of reactive oxygen species compared to astrocytes that were exposed to oxygen glucose deprivation alone. Finally, relaxin-2 and relaxin-3 protected astrocytes from 24-hour apoptosis injury that was induced by tumor necrosis factor alpha and hydrogen peroxide. Taken together these experiments provide evidence that relaxin-2 and relaxin-3 peptides protect neural tissues from the deleterious effects of cerebral ischemia in vivo and help elucidate some of the cellular mechanisms by which relaxin peptides might protect the brain. Furthermore, these data show that relaxin-2 and relaxin-3 act directly on astrocytes, the most numerous cell type in the brain, to increase astrocyte migration and to protect these cells from some of the deleterious effects of stroke, namely hypoxia and apoptosis.

This thesis examines Kantian conceptions of freedom. Beginning with Kant himself, I show how Kant’s response to Hume concerning the rational justification of causal judgements results in his claim that the sensible world is governed a priori by causal principles. Kant’s moral philosophy, however, requires a robust conception of freedom for moral agency to be possible. These two features leave Kant in an apparent contradiction, for it is unclear how we, as members of the physical, causal world, can be truly free if all events are governed by causal laws. I show that Kant’s solution to this contradiction lies in an important aspect of his transcendental idealism: the noumenal/phenomenal distinction. I argue, further, that his solution is problematic due to the fundamentally unknowable quality of the noumenal realm, wherein freedom is located. John McDowell’s Mind and World is introduced as an alternative to the extreme Kantian dualism between noumena and phenomena, while remaining within a broadly Kantian framework. Like Kant, McDowell locates our freedom in our ability to operate through reason, though unlike his predecessor, he situates “the space of reasons” within nature. This becomes possible by extending our conception of nature to include a “second nature”, thus making our initiation into the space of reasons—into the realm of freedom—a natural process. Remaining Kantian in spirit, however, McDowell’s account inherits a problematic Kantian feature. He maintains the distinction between two modes of intelligibility—between naturalistic and rational modes of explanation—thus leaving room for a hard-nosed naturalist to question the autonomy of the latter. I argue that Peter Strawson’s proposal in “Freedom and Resentment” is able to assuage this worry in McDowell’s otherwise plausible model. In it, Strawson provides an account of why the autonomy of rational explanations can never be undermined by purely naturalistic explanations, even in the face of a theoretical conviction in determinism. Strawson argues that our “personal reactive attitudes” (like gratitude and resentment)—attitudes that express our commitment to a moral life and are representative of our functioning within the space of reasons—could never be undermined by the truth of determinism, and this reveals the extent to which our conception of ourselves as rational agents is immune from assault by the determinist. The result is a compelling form of compatibilism that persuasively retains the space of reasons without appeal to Kantian noumenalism. / Thesis (Master, Philosophy) -- Queen's University, 2007-09-14 14:36:23.511

Previous investigations have demonstrated that reactive oxygen species such as hydrogen peroxide (H2O2) have the ability to alter electrophysiological and mechanical properties of rat ventricular cardiac myocytes. However, despite the breadth of the literature, there is little definitive consensus on the cellular mechanisms. The purpose of this study, therefore, was to study the cellular mechanism of action of H2O2 and test whether H2O2-mediated affects were partially a result of reverse-mode Na+/Ca2+ exchanger (NCX) activity. Unloaded cell shortening, intracellular Ca2+ transients, caffeine-induced Ca2+ transients, L-type Ca2+ channel recordings, and action potential waveforms were recorded in the presence of combinations of different compounds including Cd2+, H2O2, and KB-R7943. H2O2 was found to cause significant positive inotropy by an increase in contractility of 80 ± 20 % (n=6) and an increased amplitude of Ca2+ transients by 24 ± 14 % (n=8), relative to pre-treatment values. Interestingly, H2O2 caused an increase in contractility even in the presence of Cd2+ block from 4 ± 1 % (n=9) to 15 ± 3 % (n=5) of resting cell length. Using caffeine pulse experiments to induce unloading of the sarcoplasmic reticulum (SR), we found that 100µM H2O2 did not significantly alter SR Ca2+ load. Under control conditions, H2O2 significantly increased L-type Ca2+ currents while this H2O2-induced increase was not observed in myocytes pretreated with Cd2+. Positive inotropy in the presence of H2O2 was blocked using 10µM KB-R7943, a selective reverse-mode inhibitor of the NCX. However, it was found that 10µM KB-R7943 alone altered action potential profile and suppressed normal contraction. Altogether, the major finding of this study is that H2O2 has the ability to enhance myocardial contractility, even under conditions of L-type Ca2+ channel inhibition, through a mechanism that likely involves reverse-mode of the NCX. / Thesis (Master, Physiology) -- Queen's University, 2009-03-31 14:00:34.21