Atrial fibrillation (AF) is the most sustained arrhythmia in the population and is associated with increased morbidity and mortality. A variety of cardiac disease entities, including valve disease, cardiomyopathies, hypertension and diabetes increase the risk of AF. However, present drug therapies are ineffective and can even increase the risk of dangerous ventricular tachyarrhythmias. Studies in animal models show that AF is associated with atrial remodeling that favors AF induction and maintenance. The mechanisms of atrial remodeling depend on the underlying pathology. Atrial remodeling caused by atrial tachycardia or AF itself is primarily electrical, associated with changes in the expression and function of ion channels. These changes are reflected in decreased atrial refractory periods, impaired refractoriness adaptation to rate and conduction slowing. Together, these electrical changes increase the incidence of AF. Heart failure (HF), an important cause of AF, induces another type of atrial remodeling. HF-remodeling is characterized by conduction heterogeneity and altered atrial structural properties. In particular, interstitial fibrosis plays a major role in the stabilization of reentry circuits and prolongation of AF duration.Although many studies have described a large number of the changes associated with each form of remodeling, the underlying mechanisms remain poorly understood. The goal of this thesis was to clarify the molecular mechanisms underlying the atrial remodeling associated with AF. In my first study, I put forward the hypothesis that atrial remodeling caused by atrial tachycardia and that resulting from HF differ in terms of the time course and nature of underlying changes in cardiac gene expression. I indeed discovered that the changes in gene expression induced by atrial tachycardia and HF were qualitatively different and evolved differently over time. The changes by atrial tachycardia were limited and showed time-dependent adaptation, whereas the changes induced by HF were qualitatively greater and more varied, and showed qualitative evolution with the development of the pathology. In my second study, I put forward the hypothesis that HF caused by ventricular "tachycardiomyopathy" causes differential gene-expression changes in atria versus ventricles and that these changes evolve over time. This study highlighted the signaling pathways implicated in atrial remodeling like those of MAP kinases, ubiquitin/proteasome systems and apoptosis, along with specific metabolic pathways like the electron-chain transport system and Krebs cycle at the ventricular level.Even though the large-scale study of gene-expression changes allowed us to identify certain signaling systems, they do not detect post-translational alterations. In our third study, I put forward the hypothesis that tachycardia-induced HF causes progressive changes in the proteins involved in different functional groups that play an important role in atrial pathophysiology. My results highlighted proteins linked to oxidative stress, metabolism and contractile proteins.In my final study, I decided to explore a new therapeutic avenue for AF prevention based on the molecular pathophysiology of AF. I had identified the MAP kinase ERK as a particularly important molecule in atrial remodeling caused by myocardial infarction in the rat and I had obtained results implicating a microRNA (miR21) in signaling leading to pathological ERK hyperphosphorylation. I therefore suggested that atrial interstitial fibrosis could be prevented by targeting underlying microRNA-related mechanisms and thereby reduce the inducibility and maintenance of AF caused by HF. My study is not yet completed but the preliminary results suggest beneficial effects of anti-miR21 treatment in preventing AF. In conclusion, my studies indicate the molecular pathophysiology underlying AF and suggest that it can be exploited to develop new therapeutic approaches. / La fibrillation auriculaire est l'arythmie supra-ventriculaire soutenue la plus commune et est associée à un taux élevé de morbidité et de mortalité. Les traitements pharmacologique actuels demeurent inefficaces et entraînent parfois même une augmentation du risque d'arythmies ventriculaires. Les études effectuées sur différents modèles animaux ont démontré que la fibrillation auriculaire était associée à un remodelage auriculaire qui favorisait son induction et son incidence. Les mécanismes de remodelage auriculaire diffèrent selon la pathologie sous-jascente. Le remodelage auriculaire induit par une tachycardie auriculaire ou une fibrillation auriculaire est principalement électrique, associé à l'altération de l'expression et de la fonction de canaux ioniques. Ces changements se reflète par un raccourcissement des périodes réfractaires, une diminution d'adaptation au rythme et un ralentissement de la vélocité de conduction, augmentant ainsi l'incidence de la fibrillation auriculaire. L'insuffisance cardiaque, induit un remodelage auriculaire principalement caractérisé par une hétérogénéité de conduction associée à une altération des propriétés structurelles de l'oreillette. Notamment, la fibrose interstitielle joue un rôle majeur dans la stabilisation de circuits de réentrée et la prolongation de la durée de la fibrillation auriculaire. Bien que plusieurs études aient décrit un grand nombre de changements associés à chacun de ces types de remodelage, les mécanismes qui sous-tendent ces changements demeurent mal connus. Dans notre première étude, nous avons émis l'hypothèse selon laquelle le remodelage auriculaire induit par la tachycardie auriculaire et celui induit par une défaillance cardiaque diffèreraient au point de vue de l'évolution temporelle et de la nature des changements au niveau génomique. Nous avons constaté que les changements d'expression génique induits par la tachycardie auriculaire et par la tachycardie ventriculaire étaient qualitativement différents et évoluaient de façon différente dans le temps. Comparativement aux changements survenant au niveau auriculaire, les changements ventriculaires observés au point de vue biochimique et histopathologique différaient en terme d'intensité et de progression temporelle. Dans notre deuxième étude, nous avons émis l'hypothèse que l'insuffisance cardiaque induite par une tachycardie soutenue entraînait des changements d'expression génique qui diffèreraient entre les oreillettes et les ventricules et ces changements seraient évolutifs. Cette étude nous a permis de mettre en évidence l'implication des voies de signalisations telles que la voie des MAP kinases, l'apoptose et le système ubiquitine/protéosome au niveau auriculaire et certaines voies métaboliques au niveau ventriculaire. Bien que l'étude des changements d'expression génique nous permette de mettre en évidence certaines voies de signalisation, les changements survenant au niveau post-transcriptionel ne sont pas toujours détectables par une approche génomique. Dans notre troisième étude, nous avons donc émis l'hypothèse que l'insuffisance cardiaque induite par une tachycardie ventriculaire entraînait un remodelage auriculaire qui impliquerait des changements évolutifs importants d'expression de protéines de différents groupes fonctionnels. Nos résultats ont montré un changement au niveau de protéines liées au stress oxydatif, au métabolisme et aux protéines contractiles. Dans notre dernière étude, nous avons exploré une nouvelle avenue thérapeutique dans le traitement préventif de la fibrillation auriculaire. Nous avons suggéré que la fibrose interstitielle pourrait être prévenue par la modulation de mécanismes de régulation de l'expression de gènes par des microARN, qui réduirait l'inductibilité et le maintien de FA en contexte d'insuffisance cardiaque. Nos résultats préliminaires suggèrent un effet bénéfique du traitement anti-miR21 pour réduire la fibrillation auriculaire.

Cystic fibrosis (CF) is the most common fatal genetic disease affecting Canadian Caucasians. The majority of CF patients suffered from perpetual inflammation caused by Pseudomonas aeruginosa (P. aeruginosa). Previous, we found cells lacking functionalCFTR exhibit IL6 hypersecretion phenotype and a concomitant hyperactivity of p38 MAPK in response to P. aeruginosa. Therefore, I dedicated my master's thesis to investigate the link between increased IL6 production and p38 MAPK. ZC3H12A, an IL6-specific ribonuclease, was found to be a novel substrate of p38 MAPK and negatively regulated IL6 production from P. aeruginosa-stimulated airway epithelial cells. Together, these discoveries demonstrated a direct link between a specific signal transduction pathway and am RNA stability modulator, both of which contribute to the regulation of IL6 gene expression at a post-transcriptional level. / La fibrose kystique (FK) est la maladie génétique mortelle la plus fréquente parmi les canadiens d'origine caucasienne. La majorité des personnes atteintes souffrent d'une inflammation chronique causée par la bactérie Pseudomonas aeruginosa (P. aeruginosa). Précédemment, nous avons découvert que des cellules dépourvues de protéine CFTR fonctionnelle présentent un phénotype d'hypersécrétion d'interleukin-6 et d'hyperactivité concomitante de la MAPK p38 en réponse à la bactérie. J'ai donc consacré ma maîtrise à étudier le lien potentiel entre la MAPK p38 et la production élevée d'IL6. La ribonuclease ZC3H12A, spécifique envers IL6, fut découverte comme un nouveau substrat de la MAPK p38. Ces découvertes ont démontré un lien direct entre une voie spécifique de transduction du signal et un modulateur de stabilité de l'ARNm, les deux contribuant à la régulation post-transcriptionnelle de l'expression d'IL6.

Background: Multiple sclerosis (MS) is an autoimmune disease characterized by the destruction of oligodendrocytes (OLGs) and myelin, as well as neuronal axonal loss. OLGs are the myelinating cells of the central nervous system (CNS). The function of myelin is to ensure rapid saltatory conduction along axons. People with MS often suffer from many symptoms including muscle spasms and weakness, pain, fatigue, and blurred vision. The prevalence of MS in North America ranges from one in 500 to one in 1000 people depending on the region. Current therapies include anti-inflammatory drugs with little pharmacological selectivity. Recently, the δ and γ isoforms of the phosphoinositide 3-kinase (PI3K) family have been suggested as potential targets to treat inflammatory diseases, such as MS. However, it is not known whether these PI3K subtypes are expressed in OLGs. Our laboratory has previously shown that the PI3K/Akt pathway is essential for OLG growth and differentiation. PI3K, a key regulator of cell survival and metabolic control, catalyzes phosphorylation of the inositol ring of PI at the 3' position. PI3Ks are divided into three classes based on their size, structure, function and substrate. Class I PI3Ks consist of four isoforms (α, β, δ, and γ) and their differential activation may control downstream cellular processes, depending on cell type and context. One key downstream effector of PI3K is the Ser/Thr kinase Akt. Upon its activation by PDK1, Akt can phosphorylate and, consequently, inactivate pro-apoptotic factors leading to cell growth and proliferation. Objective: The goal of my project is to functionally characterize the specific isoforms of PI3K in terms of their involvement in OLG development and microglial growth. Methods: Primary cultures of OLG progenitors (OLPs) and microglia were prepared from the brains of newborn Sprague-Dawley rats. The specific PI3K isoforms expressed in these glial cells were determined by Western blotting using antibodies against the α, β and γ isoforms. In order to determine whether specific PI3K isoforms affect microglial and OLG growth and development, OLGs and microglia were stimulated with IGF-1 and GM-CSF respectively, and then treated with PI3K isoform-specific inhibitors. These glial cultures were then tested for survival and proliferation using MTT and 3H-thymidine assays, respectively. As an index of PI3K activity, Akt phosphorylation was also assessed under similar conditions. Results: My results demonstrate that OLGs express PI3Kα throughout all stages of development, whereas PI3Kβ is only expressed in OLPs, while PI3Kγ was not detected. Furthermore, PI3Kα was expressed in all CNS glial cells, whereas PI3Kγ expression was restricted to microglia. Using selective inhibitors, the data also suggest that PI3Kα, but not β or γ, is required for IGF-1 stimulated OLG survival. PI3Kα and surprisingly, PI3Kγ were required for OLG proliferation. GM-CSF proved to be an effective stimulant for both microglia survival and proliferation. PI3Kα but, surprisingly, not PI3Kγ was required for GM-CSF stimulated microglial survival and proliferation. Conclusion: Our results indicate that PI3Kγ might be a good potential target to treat the inflammatory component in MS since this isoform is not expressed in OLGs but expressed at high levels in microglia. / N/A

Kainate-type ionotropic glutamate receptors (iGluRs) fulfill important neuromodulatory roles in the mammalian CNS, and as such have been subject to much structural and functional analysis. However, proposed models of partial agonism at these receptors still suffer from several issues. For example, contrary to conventional understanding, agonist efficacy at KARs does not always correlate with the cleft closure of its agonist binding domain (ABD). And it is not yet determined how activation of these receptors leads to responses containing a large transient component, followed by a considerably smaller sustained component. Recent work from the Bowie lab showed the importance of the KAR ABD closed-cleft stability for the activation of the transient component response. My research work identifies the role of another region of kainate receptor, the ABD dimer interface, for the steady state component of KAR response. In support of this, my work reveals that stabilizing the dimer interface by covalent crosslinking locks the KAR into low conductance states with macroscopic and single-channel properties similar to that of wild-type KAR steady-state response. Surprisingly, restriction of movement at the dimer interface renders L-glutamate a weak partial agonist to such an extent that the receptor spends much of its time cycling through long-lived, closed states between activations. This finding diverges from the established understanding that stabilizing the dimer interface prevents onset of KAR desensitization. From our data, we also suggest that protein interactions formed at the dimer interface during transient and steady state activation of the receptor are agonist-specific. Thus, my work demonstrates how the dimer interface stability and the ABD cleft closure stability work together to generate specific components of the KAR response. / Les récepteurs ionotropiques de glutamate (iGluRs) de type kaïnate (KAR) remplissent des rôles neuromodulatoires importantes dans le système nerveux central (SNC) mammalien, et ainsi plusieurs études structurelles et fonctionnelles y sont consacrées. Cependant, les modèles d'efficacité d'agonistes chez ces récepteurs souffrent encore de divers problèmes. Par exemple, contrairement à l'entente conventionnelle, l'efficacité d'agoniste chez les récepteurs de kaïnate ne reste pas avec le degré de fermeture du domaine de liaison des ligands (ABD). De plus, ce n'est pas encore déterminé comment l'activation de ces récepteurs résulte dans des réponses contenant une portion large et transitoire, et une autre portion beaucoup plus petite et soutenue. Des résultats obtenus récemment dans le laboratoire Bowie démontrent l'importance de la stabilité de la fermeture du ABD suivant liaison de l'agoniste pour l'activation de la portion de réponse transitoire. Data obtenue de ma recherche identifie une autre région du récepteur, l'interface dimère de l'ABD, étant responsable pour la portion de KAR réponse constante. À l'appui de ceci, mon travail de recherche démontre pour la première fois qu'en stabilisant l'interface dimère avec réticulations covalentes, le KAR est bloqué dans des niveaux de conductance bas avec des propriétés macroscopiques et microscopiques similaires à celles du KAR (type sauvage) portion constante. Curieusement, ce restriction du mouvement à l'interface dimère rend aussi le neurotransmitteur glutamate comme agoniste partiel aussi faible que le récepteur reste la majorité de son temps dans des niveaux fermés et longs entre activations. Cette découverte est divergente de l'entente établie que la stabilisation de l'interface dimère prévient la désensibilisation du KAR. Nous suggérons aussi que les interactions formées à l'interface dimère durant l'activation transitoire et constante du récepteur sont spécifiques à l'agoniste. Ainsi, les résultats de ma recherche démontrent comment l'interface dimère et la stabilité de la fermeture du ABD travaillent ensemble pour générer des composants spécifiques de la réponse agoniste chez le KAR.

Abnormal embryonic development can arise from maternal or paternal exposure to therapeutic agents, environmental toxicants or social habits. Such exposures prior to conception may damage the gametes and have detrimental effects on the developing embryo. When male rats are exposed to the chemotherapeutic agent, cyclophosphamide, the genomic integrity of the male germ cells is altered. The goals of these studies were to determine the impact of paternal preconceptional exposure to cyclophosphamide on embryonic development and to elucidate how cleavage stage embryos respond to DNA damage in the male genome. Paternal exposure to cyclophosphamide induces sperm DNA damage and leads to the alteration of chromatin compaction during spermiogenesis. Exposure to cyclophosphamide alters the rate of sperm decondensation, as manifested by the difference in the number of zygotes within each sperm decondensation stage compared to controls. DNA double strand breaks, detected by gamma H2AX small and large foci, are enhanced during sperm decondensation, indicative of chromatin remodelling and DNA damage recognition, respectively. The damaged male genome leads to the formation of micronuclei during the first zygotic division and to a gradual developmental delay in cleavage stage embryos. The capacity of cleavage stage embryos to mount an efficient DNA damage response against the damaged male genome prevents the propagation of DNA damage to all blastomeres in subsequent cellular divisions. The activation of DNA damage responses was inappropriate, as indicated by a decrease in PARylation, in the presence of an accumulation of DNA damage in the form of large gamma H2AX foci in eight-cell embryos sired by cyclophosphamide exposed males. Thus, DNA damage induced by paternal cyclophosphamide exposure is transmitted to the early embryo, altering the progression of developmental events and activating DNA damage responses that are likely to determine embryonic fate. Furthermore, the assessment of the quality of cleavage stage embryos and developmental competence with biomarkers of the DNA damage response, such as gamma H2AX foci and PAR polymers, may be useful in developmental medicine and infertility clinics. / Un développement embryonnaire anormal peut être induit par l'exposition maternelle ou paternelle à des produits thérapeutiques, à des produits toxiques présents dans l'environnement ou à des habitudes sociales néfastes. De telles expositions peuvent endommager les gamètes mâtures et avoir des conséquences néfastes sur le développement embryonnaire. Lorsque des rats mâles sont exposés à un agent anticancéreux, la cyclophosphamide, l'intégrité des cellules germinales mâles est modifiée. Le but de ces études était de déterminer les conséquences d'un génome mâle endommagé par la cyclophosphamide sur le développement et les mécanismes de reconnaissance de l'ADN modifié dans des embryons au stade de division cellulaire rapide. L'exposition paternelle à la cyclophosphamide altère l'ADN et la condensation de la chromatine du spermatozoïde durant la spermatogénèse. Par conséquent la progression du spermatozoïde suivant la fertilisation durant les différents stades de la décondensation de la chromatine est affectée puisque les nombres de zygotes observés à chaque stade de décondensation étaient différents entre le groupe contrôle et celui soumis au traitement. Le nombre de cassures de l'ADN double-brin détectées par la méthode de comptage des petits et grands focis gamma H2AX est augmenté dans le groupe traité aussitôt que la chromatine commence à être décondensée dans les spermatozoïdes, démontrant ainsi un remodelage chromatinien et une altération de l'ADN, respectivement. Lors de la première division du zygote, nous avons observé la formation de micro-noyaux provoquant un retard du développement embryonnaire au stade de division cellulaire rapide. La capacité des embryons à induire une réponse appropriée aux dommages causés à l'ADN des spermatozoïdes durant ces stades de divisions rapides prévient la propagation des dommages à l'ADN d'un blastomère à un autre durant les subséquentes divisions. Les mécanismes de défense normalement activés dans les embryons sont inefficaces puisque le niveau de PARylation ne refléte pas la quantité des dommages causés à l'ADN des spermatozoïdes, tels que démontré par l'accumulation de grands focis gamma H2AX, dans les embryons à huit cellules fertilisés par des mâles exposés à la cyclophosphamide. Ces résultats démontrent que les dommages à l'ADN causés par l'exposition paternelle à la cyclophosphamide sont transmis aux embryons ayant des conséquences néfastes sur la progression du développement embryonnaire et l'activation des mécanismes de défenses affectant ainsi leur survie. L'analyse d'embryons au stade de division rapide ainsi que l'utilisation des marqueurs gamma H2AX et PAR polymère comme marqueurs de qualité embryonnaire et compétence du développement s'avèrent potentiellement utiles dans le domaine de la médecine du développement et en cliniques de fertilité.

The objectives of this thesis were to investigate the intracellular signaling pathways involved in insulin-like growth factor-I (IGF-I)-mediated oligodendrocyte progenitor survival and proliferation; as well as to assess whether activation of muscarinic acetylcholine receptors (mAChR) could support the survival of these cells and to characterize the underlying mechanisms. The main signaling pathways studied were the PI3K (phosphatidylinositol 3-kinase)-PDK1 (3-phosphoinositide-dependent kinase-1)-Akt, the MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and Src tyrosine kinases. / IGF-1 was found to protect oligodendrocyte progenitors from apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by PI3K inhibition but not by inhibition of MEK1. Interestingly, PP2, a specific Src tyrosine kinase inhibitor, blocked tyrosine phosphorylation of Fyn and Lyn and activation of Akt stimulated by IGF-I, yet had no significant effects on caspase-3 activation or progenitor survival. Furthermore, while treatment with dominant negative Akt-mutants or a pharmacological inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. / IGF-I promoted oligodendrocyte progenitor proliferation through PI3K/Akt, MEK/ERK and Src tyrosine kinase pathways. Thus, IGF-I stimulated a transient phosphorylation of PDK1 and ERKI/2 and a rapid and sustained activation of Akt. Furthermore, inhibitors of PI3K, MEK1, and Src tyrosine kinases blocked ERK1/2 activation. / Similar to IGF-I, activation of mAChR significantly protected oligodendrocyte progenitors from apoptosis following growth factor withdrawal. This action was reversed by inhibitors of PI3K, Akt and Src tyrosine kinases, but not by MEK inhibitors. Furthermore, the acetylcholine analog carbachol blocked caspase-3 cleavage and stimulated tyrosine-phosphorylation of Fyn, a member of the Src tyrosine kinase family. Both Akt and ERK1/2 activation were dependent on the upstream action of the Src tyrosine kinases. In conclusion, we showed that PI3K/Akt promotes both survival and proliferation effects of IGF-I on oligodendrocyte progenitors, whereas MEK/ERK1/2 mediates only the mitogenic effect. Src tyrosine kinases act upstream of Akt and ERK1/2. Akt independent signaling downstream of PI3K is also implicated in IGF-I-stimulated cell survival. mAChRs play a role in the activation of Src tyrosine kinases and PI3K/Akt as well as in oligodendrocyte progenitor survival.

Chronic bladder pain is becoming more and more prevalent and yet there is a lack of effective treatment because there is so little known about the mechanisms causing this type of pain. A possible etiology of chronic bladder pain is persistent inflammation. It has been proposed that inflammation initiates neurological changes in the bladder and CNS which could lead to sensitization of the bladder to cause chronic pain and frequency. Both autonomic and sensory innervation changes in the periphery can lead to hyperalgesia and allodynia, thus we have chosen to look for autonomic and sensory innervation changes that occur in the bladder in a model of chronic cystitis. / In this study, we used a well established animal model to investigate changes in the peptidergic and parasympathetic innervation of the bladder following chronic bladder inflammation. Adult female Sprague-Dawley rats were injected with either 70mg/kg cyclophosphamide diluted in saline, i.p., once every 3 days or saline. After 10 days, all animals were tested for urinary frequency and number of low volume voids, as well as symptoms of spontaneous pain. At the end of 12 days, all animals were perfused with histological fixatives and the urinary bladders processed for immunofluorescence using antibodies against calcitonin gene-related peptide (CGRP) and the vesicular acetylcholine transporter (VAChT) as markers, respectively, of peptidergic primary afferent fibres and parasympathetic efferent fibres. We show that animals treated with cyclophosphamide had inflamed bladders and displayed high urinary frequency as well as some indicators of spontaneous pain, such as piloerection and a rounded-back posture. Furthermore, they had a significant increase in the density of both parasympathetic and peptidergic sensory fibres in the bladder mucosa and an increase in peptidergic sensory fibres in the detrusor muscle. / Thus from our results, we propose that parasympathetic and sensory peptidergic innervation changes can occur in the bladder following chronic cystitis. Since we observed that the parasympathetic and peptidergic fibres often wrapped around one another or were in close proximity, these two fibre populations may be interacting with each other to lead to and maintain sensitization. These innervation changes may be responsible for prolonged and exacerbated urinary symptoms and may play a role in diseases where there is chronic urinary pain, such as in interstitial cystitis.

The sympathetic division of the autonomic nervous system is known to play a role in the genesis of neuropathic pain. In the skin of the rat lower lip (hairy skin), sympathetic and parasympathetic fibres normally innervate the same blood vessels in the lower dermis but do not occur in the upper dermis. However, we have shown that sympathetic fibre migration into the upper dermis occurs following mental nerve lesions (1. Compo Neurol. 422:287-296, 2000). As sensory denervation has a dramatic effect on sympathetic fibre innervation patterns in the rat lower lip skin, we decided to investigate the possible changes in the other autonomic fibre type in the skin - the parasympathetic fibre. Sensory denervation of the rat lower lip was achieved by bilateral transection of the mental nerve, and animals were allowed to recover for one to eight weeks. Lower lip tissue was processed for double labelling light microscopic immunocytochemistry (ICC), using antibodies against substance P (SP), which labels a subpopulation of peptidergic sensory fibres, and against the vesicular acetycholine transporter (VAChT), used as a marker for parasympathetic fibres. In sham-operated rat, SP-immunoreactive (IR) sensory fibres were found in the epidermis and upper and lower dermal regions, whereas VAChT-IR fibres were confined to the lower dermis. Mental nerve lesions induced the gradual disappearance of SP-IR fibres from all skin layers accompanied by the progressive migration of VAChT-IR fibres into the upper dermis. Cholinergic fibre migration was evident by the second week post-surgery and the ectopic innervation of the upper dermis by these fibres persisted even at the last time point studied (8 weeks). VAChT-IR fibres were observed in the upper dermis, well above the opening of the sebaceous glands into the hair follicles. These results show that considerable changes occur in the innervation patterns of parasympathetic fibres following mental nerve lesions. The unique cutaneous innervation of the trigeminal region - sensory, sympathetic, and parasympathetic - presents novel opportunities for close interactions between these three fibre types and may have implications for improving the existing understanding and treatment of neuropathic pain in this region.

In drug-tolerant rats, acute administration of systemic nicotine stimulates locomotor activity. Several lines of evidence suggest that this effect is mediated by nicotinic acetylcholine receptors located on the neurons of the mesolimbic dopaminergic (MLDA) pathway. However, a report published recently has challenged this idea, finding that destruction of the MLDA with 6-hydroxydopamine (6-OHDA), a catecholamine specific neurotoxin, had no effect on nicotine-induced hyperlocomotion. The objective of this thesis was therefore to re-examine the role of the MLDA in mediating the locomotor stimulant effect of nicotine in rats. Vehicle or 6-OHDA was bilaterally infused into the ventral tegmental area, the somatodendritic region of the MLDA pathway, of rats that had been pre-treated with nicotine. Drug-induced locomotor activity was then assessed. The locomotor activity of lesioned animals in response to nicotine (0.4 mg/kg base sc) and amphetamine (0.5 mg/kg salt sc) decreased by 75% and 87% respectively in comparison to non-lesioned rats, while the saline and scopolamine-induced (0.5 mg/kg salt sc) locomotor activity was not significantly altered. Autoradiographic examination of rat brains using 125[I]-RTI 55, a dopamine transporter ligand, revealed that binding within the cell body and terminal region of the MLDA pathway was decreased by 70% and 85% respectively in lesioned animals. A significant correlation (r = 0.68) was found between 125[I]-RTI 55 binding in the nucleus accumbens, the main projection of the MLDA system, and nicotine-induced locomotor activity. These findings strongly implicate the MLDA pathway in mediating the locomotor stimulant effect of nicotine in rats.

Studies of long-acting benzodiazepines (LABZ) use in elderly patients found an increased risk of deterioration in cognitive function, falls, hip fractures and car accidents. The purpose of this study was to identify the characteristics of physicians who prescribe LABZ to elderly patients. French language, increased number of years since medical school graduation, training as a general practitioner, physician gender and practice in a long-term care setting were risk factors for excessive prescribing of LABZ. Differences were also noted between the various provincial medical schools. University affiliation was associated with a decreased risk of being an excessive prescriber of LABZ. / Reduction of the amount of LABZ prescribed and drug-related illnesses will depend on the identification of potentially modifiable physician risk factors.