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<p>Breast cancers are the most common source of metastases to bone of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with a multiplicity of determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can depress the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that has been found to be released <em>via</em> the system x_C^-cystine/glutamate antiporter on cancer cells of types that frequently metastasize to bone, including breast cancers. This project examines the hypothesis that limiting glutamate release from cancer cells metastasized to bone will reduce bone tissue disruption and cancer-induced bone pain. A mouse model of cancer-induced bone pain was established with intrafemoral human breast cancer cells (MDA-MB-231), and behavioural measurements were taken for weight bearing and induced paw withdrawal thresholds. The system x_C^- inhibitors sulfasalazine and (S)-4-carboxyphenylglycine both attenuated glutamate release from cancer cells in a dose-dependent manner <em>in vitro</em>. Treatment with sulfasalazine induced a moderate delay in the onset of behavioural indicators of pain in mouse models, and treatment with (S)-4-carboxyphenylglycine had no apparent results. This data suggests that the limitation of extracellular glutamate released from cancers in bone with sulfasalazine may provide some alleviation of the often severe and intractable pain associated with bone metastases.</p> / Master of Science (MSc)

Cocaine is a highly abused psychostimulant with repeated use potential culminating in addiction, a disease associated with compulsive drug seeking, use and high rates of relapse despite adverse consequences. It is well established that cocaine acts by binding to and blocking monoamine transporters therefore increasing synaptic extracellular monoamine concentrations. Cocaine also increases extracellular levels of the excitatory amino acid glutamate within the neural circuitry comprising the ascending dopamine system. Cocaine induces a number of behavioral and neurochemical manifestations following acute and repeated administration. As such, elucidating the molecular mechanisms involved in the behavioral and neuromodulatory effects of cocaine are critical to the development of effective pharmacotherapies for cocaine addiction. The overall aim of this research was to identify a novel kinase that may be involved in the behavioral effects of cocaine. Thus, we chose to investigate glycogen synthase kinase-3 (GSK3), which has recently gained attention as being critical in dopaminergic and glutamatergic signal transduction. GSK3 is a critical mediator of many intracellular signaling systems. The activity of GSK3 is regulated by several kinases including Akt, with inactivation occurring via phosphorylation of the inhibitory serine-21(α-isoform) and serine-9 (β-isoform) residues. It is well established that acute cocaine administration causes hyper-locomotion in animal models and that repeated cocaine administration elicits a sensitized or increased response to the locomotor-stimulating properties of the drug. The studies outlined herein sought to determine whether non-selective and selective inhibition of GSK3 would regulate acute cocaine-induced hyper-locomotion. Further, we investigated the role of GSK3 in the development of cocaine-induced locomotor sensitization. Results of the research outlined herein demonstrate that pharmacological inhibition of GSK3 reduced both the acute behavioral responses to cocaine and the long-term neuroadaptations produced by repeated cocaine, therefore suggesting a role for GSK3 in the behavioral manifestations associated with cocaine exposure. Previous studies have assessed the role of the dopamine D1 receptor in locomotor behaviors. As cocaine indirectly activates dopamine D1 receptors, we investigated whether activation of GSK3 was necessary for the expression of dopamine D1 receptor-mediated behaviors. To assess the role of GSK3 in dopamine D1 receptor-induced hyperactivity, GSK3 was inhibited prior to administration of the selective dopamine D1 receptor agonist SKF-82958. Selective inhibition of GSK3 reduced ambulatory and stereotypic activity produced by SKF-82958. These data implicate a role for GSK3 in the behavioral manifestations associated with dopamine D1 receptor activation. To further assess the importance of GSK3 in cocaine-induced behaviors we investigated the role of GSK3 in various facets of cocaine-conditioned reward. We show that selective inhibition of GSK3 prevented the development of cocaine-conditioned reward using a conditioned place preference paradigm, indicating a reduction in the rewarding properties of cocaine. Relapse to drug-seeking can be precipitated by certain stimuli including the drug itself, drug-paired contextual cues and stress. Memory of drug-paired cues is highly resistant to extinction and the molecular mechanisms underlying relapse have not been clearly defined. Our results demonstrate that inhibition of GSK3 interfered with the reconsolidation of cocaine-associated contextual memories by preventing the retrieval of cocaine conditioned place preference. Inhibition of GSK3 in a neutral environment 24 hours prior to the test for reinstatement, however, did not prevent reinstatement of cocaine place preference following a cocaine priming injection. Thus, our results indicate that GSK3 serves an important role in cocaine-conditioned reward and is a critical intracellular signaling protein for the development of cocaine place preference. GSK3 is also essential to the reconsolidation and subsequent retrieval of cocaine-associated contextual cues. In addition to studying the role of GSK3 in cocaine-induced behaviors, we assessed the neuromodulatory effects of cocaine on GSK3 activity. As stated previously, the activity of GSK3 is regulated by a number of kinases including Akt (protein kinase B). Recent evidence suggests that psychostimulants regulate the activity of Akt and subsequently GSK3 in various brain regions. Here, the ability of cocaine to regulate the activity of Akt and GSK3 was investigated. Enzymatic activity was assessed by determining protein phosphorylation in the brain. Mice administered acute injections of cocaine showed a significant decrease in phosphorylated Akt (Thr. 308) and GSK3β; in the caudate putamen as determined by Western blot analysis. Cocaine did not alter pAkt (Thr. 308) or pGSK3β; in the nucleus accumbens or frontal cortex. The role of dopaminergic and glutamatergic receptors on cocaine-induced attenuation of pAkt (Thr. 308) and pGSK3β; was also assessed. Blockade of the dopamine D1, D2 or glutamatergic NMDA receptor prevented cocaine-induced attenuation of pGSK3β; in the caudate putamen. Only blockade of the dopamine D2 receptor prevented the effect of cocaine on pAkt (Thr. 308) levels in the caudate putamen. The results of the present study indicate that the activity of Akt and GSK3 is selectively regulated in the brain following acute cocaine, an effect that is contingent upon both dopaminergic and glutamatergic receptor regulation. In summary, the experiments described in this dissertation tested the initial hypothesis that GSK3 mediates acute cocaine-induced hyperactivity and locomotor sensitization. Acute cocaine administration increased the phosphorylation of GSK3 in the caudate putamen, therefore enhancing kinase activity. Further, the increase in GSK3 activity following cocaine administration is contingent upon activation of the dopamine D1 and D2 receptors and the glutamatergic NMDA receptor. Results presented herein also demonstrate a role for GSK3 in cocaine-conditioned reward. Selective inhibition of GSK3 prevented the development of cocaine conditioned place preference. Inhibition of GSK3 also prevented the retrieval of cocaine contextual memories, therefore playing an important role in reconsolidation. Thus, the results presented in this dissertation indicate that GSK3 is a neuromodulator of cocaine-induced behaviors and may be an important factor underlying cocaine addiction. / Pharmacology

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Les récepteurs du glutamate sont impliqués dans les maladies neurodégénératives telles que le dommage cérébral caractéristique des maladies de Huntington, Alzheimer et Parkinson, en plus d'être associés à l'ischémie, aux traumatismes crâniens, à l'encéphalopathie liée au SIDA et à l'épilepsie. Ils sont donc importants dans les mécanismes du système nerveux central et son bon fonctionnement. Parmi les récepteurs du glutamate, on retrouve le récepteur kaïnate. Ce mémoire présente une nouvelle approche synthétique des kaïnoïdes et la synthèse de trois nouveaux analogues des kaïnoïdes à partir de la trans-4-hydroxyproline. Les étapes comprennent la protection du carbonyle et de l'amine, l'oxydation de l'alcool, l'alkylation régiospécifique de la cétone en position 3, la formation d'un énol triflique, le couplage à un acide arylboronique et enfin la déprotection pour obtenir les acides (2S)-A-arylkaïniques. Les trois kaïnoïdes générés varient par le groupement aryle qui est soit le phényle, le 2-méthoxyphényle ou le 2-naphtyle.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24668号 / 農博第2551号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5449(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 栗原 達夫, 教授 伊福 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The foundational studies of this dissertation concern the characterization of segmented polyurethanes and polyureas synthesized without the use of chain extenders'molecules that are typically used to promote a microphase separated morphology that gives these materials their useful characteristics. Polyurethanes in which a single asymmetric diisocyanate comprising the whole of the hard segment were found to display poor microphase separation. Conversely, polyurethanes in which a single symmetric diisocyanate composed the hard segment were found to display good microphase separation. The more efficient packing of the symmetric hard segments also led to an increase in hard segment connectivity and hence higher values of storage moduli in these systems. When hydroxyl-terminated diisocyanates were replaced with amine-terminated diisocyanates, polyureas were formed. Here too, diisocyanate symmetry was found to play a key role with symmetric diisocyanates leading to better microphase separation. In addition, the polyurea materials displayed broader service temperature windows than their polyurethane counterparts as the relatively stronger bidentate hydrogen bonding replaced monodentate hydrogen bonding in these materials. A thread-like, microphase separated morphology was visually confirmed using atomic force microscopy. Other techniques such as ambient temperature tensile testing, and wide and small angle x-ray scattering were employed to confirm the presence of the microphase separated structure. The investigation into the effects of diisocyanate chemistry and its symmetry was broadened to incorporate non-chain extended polyurethane materials with different soft segment molecular weights, as well as polyurethanes that did contain chain extenders. Once again the effect of using symmetric versus asymmetric diisocyanates was evident in the structure–property behavior of these systems, with symmetric diisocyanates forming materials that displayed better microphase separation and more connectivity of their hard domains. Lastly, in a departure from the segmented copolymer area, a study was conducted into the influence of casting variables on the solid-state structure of synthetic polypeptide films based on glutamate chemistry. The effect of solvent evaporation was determined to play a key role in the morphology of these polypeptide films. Measured small angle light scattering patterns were compared to computer calculated patterns to reveal information about the structure, shape, and length scale of the polypeptide structure. / Ph. D.

PTSD is a moderately heritable disorder that causes intense and chronic suffering in many afflicted individuals. The pathogenesis of PTSD is not well understood, and genetic mechanisms are particularly elusive. Neurotransmitter systems are thought to contribute to PTSD etiology and are the targets of most pharmacotherapies used to treat PTSD, including the only two FDA approved options and a wide array of off-label options. However, the degree to which variation in genes which encode for and regulate neurotransmitter receptors increase risk of developing PTSD is unclear. Recently, large collaborative groups of PTSD genetics researchers have completed genome-wide association studies (GWAS) using massive sample sizes and have made summary statistics available for public use. In 2018, a new technique for high-powered analysis of GWAS summary statistics called GSA-SNP2 was introduced. In order to explore the relationship between PTSD and genetic variants in widely theorized molecular targets, this study applied GSA-SNP2 to manually curated neurotransmitter receptor gene-sets. Curated gene-sets included nine total "neurotransmitter receptor group" gene-sets and 45 total "receptor subtype" gene-sets. Each "neurotransmitter receptor group" gene-sets was designed to capture concentration of genetic risk factors for PTSD within genes which encode for all receptor subtypes that are activated by a given neurotransmitter. In contrast, "receptor subtype" gene-sets focused on specific subtypes and also accounted for intracellular signaling; each was designed to capture concentration of genetic risk factors for PTSD within genes which encode for specific receptor subtypes and the intracellular signaling proteins through which they exert their effects. Due to practical considerations, this work used summary statistics derived from a GWAS with far fewer participants (2,424 cases; 7,113 controls) than initially planned (23,212 cases; 151,447 controls). Prior to controlling for multiple comparisons, 7 of the investigated gene-sets reached statistical significance at the p ≤ .05 level. However, after controlling for multiple comparisons, none of the investigated gene-sets reached statistical significance. Due to limited statistical power of the current work, these results should be interpreted very cautiously. The current study is best interpreted as a preliminary study and is most informative in relation to refining study design. Implications for next steps are emphasized in discussion and nominally significant results are synthesized with the literature to demonstrate the types of research questions that might be addressed by applying a refined version of this study design to a larger sample. / Doctor of Philosophy / Though nearly all individuals will be exposed to a potentially traumatic event in their lifetime, only a small percentage will experience PTSD, which is a severe psychological disorder. Though genetics are known contribute to an individual's level of risk for developing PTSD, relatively little is known about which particular genetic differences are key. Neurotransmitter receptors are thought to contribute to the risk for PTSD and are a key aspect of medications for PTSD. However, little is known about whether genetic differences in neurotransmitter receptors contribute to risk for developing PTSD. Recently, large collaborative groups of PTSD genetics researchers have completed studies which investigate genetic risk factors from across the genome using massive sample sizes and have made the statistical output of these studies available to the public. In 2018, a new technique called GSA-SNP2 was created to help assist with efforts to analyze aspects of that statistical output that have not been previously analyzed. This study used GSA-SNP2 to analyze the degree to which groups of neurotransmitter receptor genes contribute to the risk of developing PTSD. Due to the coronavirus pandemic, the researcher did not have access to the computing power needed to analyze the initially planned data which included 23,212 individuals with PTSD and 151,447 individuals without PTSD. As a substitute, the current work is an analysis using statistical output data from a study which included 2,424 individuals with PTSD and 7,113 individuals without PTSD. Based on a level of statistical significance that is typically used in most psychological studies, seven of the investigated gene-sets contribute highly to the risk for PTSD. However, it was necessary to use a different threshold for statistical significance due to the testing of many different groups of genes. After making that adjustment, none of the investigated gene-sets reached statistical significance. Due to limited statistical power of the current work, these results should be interpreted very cautiously. The current study is best interpreted as a preliminary study and is most informative in relation to refining study design. Implications for next steps are emphasized in discussion and nominally significant results are synthesized with the literature to demonstrate the types of research questions that might be addressed by applying a refined version of this study design to a larger sample.

Blast-induced traumatic brain injury (bTBI) remains a significant problem among military populations. When an explosion occurs, a high magnitude positive pressure rapidly propagates away from the detonation source. Upon contact, biological tissues throughout the body undergo deformation at high strain rates and then return to equilibrium following a brief negative pressure phase. This mechanical disruption of the tissue is known to cause oxidative stress and neuroinflammation in the brain, which can lead to neurodegeneration and consequently poor cognitive and behavioral outcomes. Further, these clinical outcomes, which can include chronic headaches, problems with balance, light and noise sensitivity, anxiety, and depression, may be sustained years following blast exposure and there are currently no effective treatments. Thus, there is a need to investigate the acute molecular responses following bTBI in order to motivate the development of effective therapeutic strategies and ultimately improve or prevent long-term patient outcomes. It is important to not only understand the acute molecular response, but how the brain tissue mechanics drive these metabolic changes. The objective of this work was to identify the interplay between the tissue-level biomechanics and the acute bTBI pathophysiology. In a rodent bTBI model, using adult rats, intracranial pressure was mapped throughout the brain during blast exposure where frequency contributions from skull flexure and wave dynamics were significantly altered between brain regions and were largely dependent on blast magnitude. These findings informed the subsequent spatial and temporal changes in neurometabolism. Amino acid molecular precursor concentrations decreased at four hours post-blast in the cortex and hippocampus regions. This motivates further investigation of amino acids as therapeutic targets aimed to reduce oxidative stress and prevent prolonged injury cascades. However, neurochemical changes were not consistent across blast magnitudes, which may be explained by the disparities in biomechanics at lower blast pressures. Lastly, we investigated the acute changes in metabolic regulators influencing excitotoxicity where it was found that astrocytes maintained normal clearance of excitatory and inhibitory neurotransmitters prior to astrocyte reactivity. Outcomes of this work provide improved understanding of blast mechanics and associated acute pathophysiology and inform future therapeutic and diagnostic approaches following bTBI. / Doctor of Philosophy / Blast-induced traumatic brain injury (bTBI) remains a significant problem among military populations. When an explosion occurs, a high magnitude positive pressure wave rapidly propagates away from the detonation source. Upon contact, biological tissues throughout the body undergo deformation that can cause injury. This mechanical disruption of the tissue is known to trigger negative biological processes that lead to persistent cognitive and behavioral deficits. Further, these clinical outcomes, which can include chronic headaches, problems with balance, light and noise sensitivity, anxiety, and depression, may be sustained years following blast exposure. There are currently no effective treatments that can help those afflicted, and biomarkers for injury diagnostics are limited. Thus, there is a great need to investigate the early biological responses following bTBI in order to motivate the development of effective therapeutic strategies and ultimately improve or prevent long-term patient outcomes. It is important to not only understand the immediate responses, but also how the brain tissue mechanics drive these metabolic changes. The objective of this work was to identify the interplay between the brain biomechanics and the acute bTBI pathophysiology. Using a translational animal model, pressure inside the brain was measured with pressure sensors during blast exposure. Subsequent spatial and temporal changes in neurochemical concentrations were quantified. The results showed (1) significant disparities in the pressure dynamics inside the brain and it varied across brain regions, (2) neurochemical precursors may have therapeutic potential post-injury, and (3) biomechanical and neurochemical responses were dependent on blast severity. Outcomes of this work provide improved understanding of blast mechanics and associated pathophysiology and inform future therapeutic and diagnostic approaches to prevent prolonged injury cascades.