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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Information is transmitted from the cell surface to various specific targets in the cell via several cellular signaling pathways. Cytosolic free calcium (Ca2+)is one of the most versatile and ubiquitous intracellular messengers since it is able to regulate diverse number of functions such as proliferation, secretion, fertilization, metabolism, learning and memory. In the last couple of years, evidence has been accumulating that Ca2+ ion is able to integrate information from multiple signaling pathways and convert this information into a code which regulates events ranging from contraction to modification of gene expression (Berridge et al. 1998). It was shown that Ca2+ concentration displays oscillatory behavior in response to agonist stimulation in a variety of cells(Goldbeter 1996) and the frequency of these oscillations increases with the concentration of agonist, a behavior called frequency encoding which has led to the concept that many Ca2+-regulated processes are controlled by these codes(Berridge 1998). Although the presence of Ca2+ oscillations and the sources of Ca2+ pools involved is known in many cell types, it is yet not known how the various frequencies of Ca2+ oscillations are converted into codes that regulate the numerous cellular events. Recently a number of cellular targets that decode Ca2+ signals and are tuned to the frequency of Ca2+ oscillations have been identified. Prominent among them are calcium-calmodulin kinase II (CAM II) and protein kinase C (PKC). The objective of this work is to study and mathematically model the oxytocin and vasopressin-induced Ca2+ oscillations in cells of normal rat liver (Clone 9) and cells of pregnant human myometrium. The proposed model accounts for the receptor-controlled Ca2+ oscillations involving positive feedback leading to activation of phospholipase C (PLC) and negative feedback from PKC onto G-proteins which simulates many of the features of observed intracellular Ca2+. The model also incorporates the concept that coordinated Ca2+ signals in a group of hepatocytes require both effective gap junctions and the presence of agonist at each cell surface. Another objective of this research is to understand the relevance of frequency-encoded signals by performing an analysis of frequencies of Ca2+ oscillations using the Fast Fourier Transform and the Wavelet Transform. The validity of the model was confirmed by using statistical tests to check if the frequencies and amplitudes of the experimental Ca2+ oscillations match with those of the modelled oscillations.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les variations de la concentration cytosolique de Ca[indice supérieur 2+] contrôlent divers processus biologiques tel la contraction, la division cellulaire et la transactivation de gènes. Chez les cellules non excitables, la stimulation de certains GPCRs (G protein coupled receptor) ou récepteurs tyrosine kinase mène à l'activation d'une phospholipase C qui produit de l'inositol 1,4,5-trisphosphate (IP[indice inférieur 3]). L'IP[indice inférieur 3] active un récepteur-canal au niveau du réticulum endoplasmique (RE) et permet une relâche de Ca[indice supérieur 2+] du RE. Il existe trois isoformes distinctes d'IP[indice inférieur 3]R (IP[indice inférieur 3]R-1 à -3) différemment exprimées dans les tissus. Dans ces travaux, nous avons étudié un mode de régulation de l'IP[indice inférieur 3]R-2 par la PKC, une kinase elle-même activée lors de la réponse calcique. Nous avons aussi vérifié l'implication de chaque isoforme d'IP[indice inférieur 3]R dans l'activité transcriptionnelle des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB.Les cellules AR4-2J expriment principalement l'IP[indice inférieur 3]R-2 (86%), ce qui en fait un bon modèle d'étude pour les mécanismes de régulation de l'IP[indice inférieur 3]R-2. Dans la première étude, nous avons vérifié si la PKC influençait l'activité calcique de l'IP[indice inférieur 3]R-2. D'abord, nous avons montré que l'IP[indice inférieur 3]R-2 est majoritairement exprimé dans les cellules AR4-2J. Nous avons montré par phosphorylation in vitro et in cellulo que l'IP[indice inférieur 3]R-2 est phosphorylé par la PKC. Nous avons montré que le traitement par la PKC réduit la réponse calcique induite par l'IP[indice inférieur 3] sur des cellules perméabilisées. Finalement, nous avons démontré que la réponse calcique induite par le CCh ou l'EGF sur des cellules entières était réduite avec l'activation de la PKC. Ces résultats indiquent que l'IP[indice inférieur 3]R-2 est une autre cible de la PKC permettant de contrôler l'intensité de la réponse calcique. Dans la deuxième étude l'objectif était de déterminer l'implication des isoformes d'IP[indice inférieur 3]R sur l'activité des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB. Par l'approche des gènes rapporteurs de l'activité de NFAT ou CREB, nous avons montré que NFAT était activé par la voie de la calcineurine et que CREB par les voies de CamKII et de la calcineurine dans les cellules HEK 293A. Nous avons démontré que l'invalidation de l'IP[indice inférieur 3]R-2 diminuait la réponse calcique induite par le CCh et que la costimulation avec le VIP pouvait la restaurer. Nous avons montré que l'activité transcriptionnelle de NFAT était affectée, contrairement à celle de CREB, par l'invalidation de l'IP[indice inférieur 3]R-2. Finalement, nous avons montré que l'IP[indice inférieur 3]R-2, et aussi l'IP[indice inférieur 3]R-1, semblent jouer un rôle secondaire, différent de celui de transporteur de Ca[indice supérieur 2+], dans le mécanisme d'activation de NFAT. Ces résultats laissent envisager que l'IP[indice inférieur 3]R serait impliqué dans la formation d'un complexe protéique facilitant l'activation de NFAT.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Der Glutamattransporter GLT1v, eine Spleißvariante von GLT1, kommt hauptsächlich im Zytoplasma von Neuronen vor. Es wurde gezeigt, dass GLT1v ein putatives PDZDomänen- Bindungsmotiv am C-Terminus enthält und mit PICK1, ein mit PKC interagierendes Protein, interagiert. Es ist daher denkbar, dass durch Interaktion zwischen GLT1v und PICK1 die GLT1v-Translokation über eine PKC-abhängigen Phosphorylierung reguliert wird. In der vorliegenden Untersuchung wurden kultivierte zerebelläre Körnerzellen aus der Maus benutzt, um mittels Immunzytochemie und Biotinilierung/Westernblot zu zeigen, ob eine GLT1v-Translokation über einen PKC-abhängigen Signalweg reguliert wird und sollte dies der Fall sein, ob diese Regulation vom elektrophysiologischen Status der zerebellären Körnerzellen abhängt. Vergleichstudien wurden mit EAAC1 durchgeführt. Die Körnerzellen wurden in einem Medium mit 27 mM KCl (chronisch depolarisierte Körnerzellen) und mit 5 mM KCl (ruhende Körnerzellen) kultiviert. Eine 30 minütige PKC-Aktivierung durch Phorbol-Myristat-Acetat (PMA) ergab in ruhenden Körnerzellen eine 41 % bzw. 31 % (signifikante) Zunahme in der Zelloberflächenexposition von GLT1v bzw. EAAC1 im Vergleich zur Kontrolle. Vergleicht man Körnerzellen nach PMA- mit solchen nach 30 minütiger Staurosporinbehandlung (PKC-Inhibitor), so beträgt die Oberflächenzunahme nach der PMA-Behandlung bei GLT1v bzw. EAAC1 115% bzw. 69%. Zerebelläre Körnerzellen, die mit 27 mM KCl kultiviert wurden (chronische Depolarisation), ergaben demgegenüber keine signifikanten Änderungen in der Oberflächenexpression von GLT1v und EAAC1, beim Vergleich der verschiedenen experimentellen Bedingungen (PMA, Staurosporin). Die immunzytochemischen Untersuchungen ergaben, dass bei ruhenden Körnerzellen (5mM KCl) nach PKC-Aktivierung mittels PMA zahlreiche, große Varikositäten (präsynaptische Elemente der Neuriten) auftreten, die eine intensive Immunreaktivität für GLT1v und EAAC1 zeigen. Wir konnten auch nachweisen, dass beide Transporter in getrennten Vesikelpopulationen vorkommen. Die Immunelektronenmikroskopie am Kleinhirn der adulten Maus hat ergeben, dass GLT1v und EAAC1 in Varikositäten der Parallelfasern von Körnerzellen lokalisiert sind. Dieses in situ Ergebnis stimmt somit mit den kultivierten Körnerzellen überein. Insgesamt lassen die Untersuchungen den Schluss zu, dass die Oberflächenexpression von GLT1v und EAAC1 (1) ähnlich reguliert zu werden scheint, (2) in Varikositäten von glutamatergen Körnerzellen stattfindet, aus denen Glutamat freigesetzt wird, und (3) vom elektrophysiologischen Status der zerebellären Körnerzellen abhängt. / The glutamate transporter GLT1v, a splice variant of GLT1, is present mainly in the cytoplasm of neurons. It is shown that GLT1v contains a putative PDZ domain binding motif and interacts with PICK1, a protein kinase C (PKC) interacting protein. The interaction between GLT1v and PICK1 could regulate trafficking of GLT1v via PKC dependent phosphorylation. In the present study we used cultured cerebellar granule cells (CGCs) from mice to demonstrate, applying immunocytochemistry and biotinylation/ Western blotting, whether GLT1v trafficking is regulated by PKC and if so, whether this depends on the electrophysiologic state of CGCs. Comparative studies were performed with EAAC1. The CGCs were cultured in high-potassium medium (chronic depolarization of CGCs) and in low-potassium medium (resting CGCs). Stimulation of PKC by phorbolester resulted in resting CGCs in a 41% and 31% (significant) increase of cell surface localization of GLT1v and EAAC1, respectively, compared to controls, and a 115% and 69% increase, respectively, compared to staurosporine inhibition. No significant changes were observed in chronically depolarizing CGCs. In resting CGCs stimulation of PKC enhanced the formation of large varicosities in neurites showing intense immunoreactivity for GLT1v and EAAC1. We showed also that both transporters were contained in different vesicle populations, and were localized in situ in varicosities of CGC parallel fibres. These findings provide evidence that the surface exposition of GLT1v and EAAC1 (1) seems to be similarly regulated, (2) takes place in varicosities of glutamatergic CGCs, where glutamate is thought to be released, and (3) depends on the electrophysiologic state of CGCs.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.