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

In vorausgegangenen Experimenten unseres Labors war bereits gezeigt worden, dass die Applikation von Glutamat zur transienten Erhöhung der cytosolischen Calciumkonzentration sowie zu einer Depolarisation der Plasmamembran von Mesophyllzellen führt. Pharmakologische Studien weisen auf mögliche Orthologe tierischer ionotroper Glutamatrezeptoren (iGLuRs) hin. Ziel dieser Arbeit war eine vertiefte molekulare und funktionelle Analyse der Glutamatrezeptoren (GLRs) aus Arabidopsis thaliana. Dabei konnten folgende Erkenntnisse gewonnen werden: i. Zugabe von extrazellulärem Glutamat in Kombination mit Glycin führt in Abhängigkeit der extrazellulären ATP-Konzentration (eATP) zu einer transienten Erhöhung der Calciumkonzentration in Mesophyllzellen. ii. Die Reaktion von Mesophyllprotoplasten auf die Applikation von Glutamat und Glycin ist im Vergleich zum intakten Blatt stark reduziert, kann jedoch in Gegenwart von eATP oder Glucose signifikant gesteigert werden. iii. Diese Responsibilität von Mesophyllprotoplasten ist zum Zeitpunkt des Einsetzens der Zellwandsynthese (48h) am höchsten. iv. In Patch-Clamp Experimenten führt die photolytische Freisetzung von extrazellulärem caged-Glutamat bei 34 % der gemessenen A. thaliana Mesophyllprotoplasten zu einem verstärkten Kationentransport über die Plasmamembran. Dieser Kationenstrom kann durch gleichzeitige Anwesenheit von extrazellulärem ATP noch verstärkt werden und ist durch einen Desensitivierungsprozess gekennzeichnet. Die Empfindlichkeit dieser Ströme gegenüber Antagonisten tierischer iGluRs stellt eine Verbindung zu der Genfamilie der AtGLRs dar. v. Coexpressionexperimente ausgewählter AtGLRs geben erste Hinweise auf eine Homo- bzw. eine Heteromerbildung von AtGLR1.1 und AtGLR1.4. Aufgrund fehlender Kanalaktivität konnten einzelne, in Oozyten exprimierte AtGLRs bislang nicht funktionell charakterisiert werden. vi. Studien zur transienten und stabilen Überexpression im homologen Expressionssystem zeigen einen cytotoxischen Effekt bei funktioneller Überexpression ausgewählter AtGLRs. vii. Die Analyse der Stellung des Glutamatrezeptors 3.4 in kälteregulierten Signalnetzwerken via Microarrays weist auf ein überlappendes Aufgabenspektrum der Familie der AtGLRs hin. viii. Im Rahmen von Microarray Transkriptionsanalysen an der glr3.4-1 Mutante konnte ein bisher nicht charakterisiertes, co-reguliertes Protein identifiziert werden. Dieses Protein ist durch den Besitz einer transmembranen Region sowie einer ATP-Bindedomäne charakterisiert und könnte einen möglichen Regulator des AtGLR3.4-Kanals darstellen. ix. Die Überprüfung einer möglichen Beteiligung der pflanzlichen Glutamatrezeptoren an der Generierung der glutamatabhängigen Ca2+-Signale sowie die Suche nach Regulatoren bzw. fehlenden Untereinheiten erfolgte mithilfe Mutanten-„Sreenings“. Die Analyse der bis dato identifizierten, Glutamat-insensitiven Mutanten konnte im Rahmen dieser Arbeit nicht abgeschlossen werden. / Preliminary work has shown that glutamate application results in a transient increase of cytosolic calcium and leads to a depolarisation of the plasma membrane potential in Arabidopsis mesophyll cells. Pharmacological studies indicate that orthologs of mammalian ionotropic glutamate receptors (iGLuRs), members of the AtGLR family, participate in this process. Aim of this work was to analyse the molecular and functional properties of Arabidopsis thaliana glutamate receptors (GLRs). During the course of this work the following results were obtained: i. The extracellular application of glutamate in combination with glycine leads to a transient rise in cytosolic calcium concentration in Arabidopsis mesophyll cells which was strongly enhanced in the presence of extracellular ATP (eATP). ii. The response of mesophyll protoplasts upon an application of glutamate and glycine was strongly reduced in comparison to intact leaves, but was increased significantly in the presence of eATP or glucose. iii. This response peaked out at the inset of cell wall synthesis i.e. after 48 hours. iv. After a photolytic release of caged-glutamate the probability of observing glutamate-activated cation currents across the plasmamembrane was about 34 % of all measured A. thaliana. mesophyll protoplasts. The observed cationic currents could be further stimulated in the presence of eATP and were characterized by desensitization. The responsiveness of these currents to antagonists of mammalian iGluRs constitutes a connection to the gene family of the AtGLRs. v. Coexpression-experiments of selected AtGLRs provide a first indication to a homo- and heteromerization of AtGLR1.1 and AtGLR1.4. Due to channel inactivity of individual AtGLRs expressed in oocytes, a functional characterization, however, was not possible so far. vi. Transient and stable overexpression in homologous expression systems indicated a cytotoxic effect, when individual AtGLR were functionally overexpressed. vii. Analysis of AtGLR3.4 function in cold-induced signal transduction networks via microarrays point to overlapping tasks among members of the glutamate-receptor-family. viii. The microarray-transcript-analysis of the glr3.4-1 mutant resulted in the identification of a not yet characterized, co-regulated gene. The deduced protein contains a putative signal peptide, a single transmembrane region, as well as an ATP-binding domain and could represent a putative regulator of the AtGLR3.4-channel. ix. Participation of plant glutamate receptors in generating glutamate-dependent calcium signals as well as the search for novel regulators was carried out by using an EMS-based mutant-screening procedure. The analysis of the so far identified, glutamate-insensitive mutants could not be completed within this thesis.

Hypoglutamatergic function is implicated in the pathogenesis of schizophrenia, and supersensitivity of platelet N-methyl-D-aspartate (NMDA) receptors has been reported in schizophrenia. The aim of this study was to examine the platelet glutamate receptor sensitivity in patients with schizophrenia as well as other psychotic conditions, and matched controls, in order to assess if this is a specific marker of schizophrenia or occurs in other psychotic conditions. Glutamate receptor sensitivity was assessed using the intracellular calcium response to glutamate measured with spectrofluorometry. The percentage responses to glutamate stimulation of the schizophrenic subjects and those with depression with psychotic features were significantly greater than control subjects (p<0.005). The mania with psychotic features group was not significantly different to controls. This data suggests that platelet glutamate receptors may be supersensitive in schizophrenia and depression with psychotic features. The platelet may be a possible peripheral marker of glutamate function in schizophrenia and depression with psychotic features.

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Ph.D. / Omslagstitel: Metabotropic glutamate receptor 4 activation and expression.

Prevention of the neurotoxic effect that excess glutamic acid has on its receptors can lead to the treatment of a number of neurodegenerative diseases and pathological conditions. Developments of new methods for the stereospecific synthesis of novel amino acids will allow identification of compounds which might be effective at these receptors to be prepared. (2S)-Pyroglutamic acid was used as a chiral template for the synthesis of a key aldehyde for use in a novel synthesis of glutamate antagonists. Subsequent ring transformation ('ring-switching') reactions using bisnucleophiles then afforded a number of homochiral pyridazines that, when deprotected and oxidized, would possess the structural requirements of glutamate antagonists. A number of other routes requiring protection of the aldehyde were studied with the aim of synthesising a library of substituted homochiral pyridazines. Ring transformations involving an a, ß-unsaturated lactam with methyl hydrazine were also briefly studied, furnishing chiral pyrazolidin-3-ones with potential for glutamate receptor activity. The second area of research involved investigation of a stereoselective route to 5- alkylated 6-oxopipecolates from a 5-substituted enaminone. Initial attempts to prepare the enaminone tert-butyl ester from tert-butyl (2S)-N-tert-butoxycarbonyl-6- oxopipecolate were unsuccessful, but the enaminone methyl ester was eventually successfully prepared from methyl (2S)-N-tert-butoxycarbonyl-6-oxopipecolate. This enaminone was reacted with a number of Grignard reagents to afford a range of 5- alkylidene derivatives which were all (E)-isomers. Catalytic reduction of these was stereoselective and so an effective route to methyl (2S, 5S)-5-alkyl-6-oxopipecolates has been developed. A route involving alkylation of methyl (2S)-N-tert-butoxycarbonyl-6- oxopipecolate followed by enhancement of stereoselectivity using a hindered proton source was also investigated.

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University, Diss., 2001--Konstanz. / Parallelt.: The role of dopamine and glutamate in associative learning by the pigeon (Columba livia). Text. engl.

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