http://www.pharmacol.usyd.edu.au/thesis This thesis is primarily concerned with a class of chemical compounds known as pyridazinediones, being 6-membered aromatic rings containing two adjacent nitrogen atoms (pyridazine), doubly substituted with oxygen. In particular, the work focuses on pyridazine-3,6-diones, derivatives of maleic hydrazide (1). Understanding of the chemistry of these compounds is extended, using theoretical and synthetic techniques. This thesis is also concerned with two very important classes of receptors which bind amino acids in the brain: firstly, the inhibitory GABA receptor, which binds g-aminobutyric acid (GABA) (2) in vivo, and for which muscimol (3) is an agonist of the GABAA subclass; secondly, Excitatory Amino Acid (EAA) receptors, which bind glutamate (4) in vivo, and in particular the AMPA subclass, for which (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) (5) is an agonist. The connection between pyridazinediones and amino acid receptors is the design, synthesis, and evaluation of structures based on pyridazinediones as potential GABA and EAA receptor ligands. Techniques of theoretical chemistry, molecular modelling, synthetic chemistry, and in vitro pharmacology are used to explore pyridazine-3,6-dione derivatives as ligands.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Seizures have important consequences in terms of mortality and quality of life of the affected population, being a risk factor for the development of cognitive and behavioral abnormalities. Considering the promising pharmacological properties of molecules containing selenium, in article 1, we evaluated the anticonvulsant action of 1-(2,5-diphenylselenophen-3-yl)-3-methylpent-1-yn-3-ol, generically called 3-alkynyl selenophene (3-ASP) against seizures induced by pilocarpine (PC), pentylenetetrazole (PTZ) and kainate (KA) in 21-days-old rats. Animals were pre-treated with 3-ASP (10, 25 or 50 mg/kg; per oral, p.o.) or vehicle, 30 minutes before of intraperitoneally administration of PC (400 mg/kg), PTZ (80 mg/kg) or KA (45 mg/kg). 3-ASP pre-treatment (50 mg/kg) abolished seizures and the death induced by PC administration. 3-ASP (50 mg/kg) increased the latency to the first convulsive episode, as well as decreased the mortality and incidence of seizures caused by PTZ and KA. In article 1, the antioxidant activity of 3-ASP (10, 25 or 50 mg/kg; p.o.) against the oxidative stress induced by PC (400 mg/kg, i.p.) in 21-days-old rats was evaluated. Our results demonstrated that 3-ASP pre-treatment was effective in protecting against the inhibition of cerebral activity of superoxide dismutase, decreased ascorbic acid levels, stimulation of catalase activity and increase of reactive species levels caused by PC. Additionally, 3-ASP protected against the inhibition of acetylcholinesterase and Na+, K+-ATPase activities resulting from convulsions induced by PC. The involvement of glutamatergic and GABAergic systems in the anticonvulsant action of 3-ASP was investigated (Articles 1 and 2). The combination of sub-effective doses of 3-ASP (10 mg/kg, p.o.) and diazepam (GABA agonist; 0,5 mg/kg, i.p.), 5S,10R-(+)-5-methyl-10,11-dihydro- 5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist; 0.1mg/kg, i.p.) or 6,7-dinitroquinoxaline-2,3-dione (DNQX, a non-NMDA receptor antagonist; 5 mg/kg, i.p.) was effective in increasing the latency to the first convulsive episode, as well as, in decreasing the incidence of convulsions induced by PC. On the other hand, the combination of 3-ASP and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP, an antagonist of metabotropic glutamate receptor mGluR5; 0,5 mg/kg, i.p.) did not protect against convulsions. The oral administration of 3-ASP (50 mg/kg) caused an inhibition of 64% and 58% of GABA uptake in the cortex and hippocampus, respectively. However, no change in glutamate uptake after 3-ASP administration (50 mg/kg) was found. Additionally, in article 2, we investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors against PC-induced seizures in 21-days-old rats. To this, sub-effective doses of 3-ASP (10 mg/kg; p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake; 2 mg/kg; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor; 10 mg/kg, i.p.) were co-administrated in 21-days-old rats before of PC administration (400 mg/kg). The treatment with 3-ASP and DABA abolished the PC-induced seizures. Similar results were found when sub-effective doses of 3-ASP and AOAA were administrated in 21-days-old rats. Finally, in the article 3 we investigated the effect of 3-ASP or diazepam against convulsions, the increased susceptibility to the development of seizures and long term memory impairment resulting from febrile seizures induced by hyperthermia. 21-Days-old rats were pre-treated with 3-ASP (25, 50 or 100 mg/kg; p.o), diazepam (1 or 5 mg/kg; i.p.) or vehicle. After the treatment, animals were exposed to a stream of heated air to approximately 41°C. Thirty days after the exposure to hyperthermia, the susceptibility to the development of seizures and long term memory impairment were evaluated. We verified that the pre-treatment with 3-ASP or diazepam did not protect against stereotyped behavior, facial automatisms and body flexion induced by hyperthermia. The protective effect of 3-ASP (100 mg/kg) against the increased susceptibility to the development of seizures and long term memory impairment resulting from febrile seizures induced by hyperthermia was demonstrated. Diazepam (1 or 5 mg/kg) did not protect against long term memory impairment caused by febrile seizures. In addition, diazepam (1 mg/kg) treatment caused a significant cognitive impairment in animals kept at room temperature. These results suggest that 3-ASP had anticonvulsant action in 21-days-old rats and that this action appears to be mediated by glutamatergic and GABAergic systems. In addition, the anticonvulsant action of 3-ASP seems to be associated with its antioxidant activity. Finally, 3-ASP can represent an important tool to protect against increased susceptibility to seizures and cognitive impairment resulting from febrile seizures. / As convulsões têm conseqüências importantes em termos de mortalidade e qualidade de vida da população afetada, sendo um fator de risco para o desenvolvimento de alterações cognitivas e anormalidades comportamentais. Tendo em vista as promissoras propriedades farmacológicas das moléculas contendo selênio, no artigo 1 avaliamos a ação anticonvulsivante do (1-(2,5-difenilselenofeno-3-il)-3-metilpent-1-in-3-ol, que foi genericamente denominado de 3-alquinil selenofeno (3-ASP) frente as convulsões induzidas por pilocarpina (PC), pentilenotetrazole (PTZ) e cainato (KA) em ratos de 21 dias de vida. Os animais foram pré-tratados com 3-ASP (10, 25 ou 50 mg/kg; per oral, p.o.) ou veículo, 30 minutos antes da administração intraperitoneal (i.p.) de PC (400 mg/kg), PTZ (80 mg/kg) ou KA (45 mg/kg). Verificamos que o pré-tratamento com 3-ASP (50 mg/kg) aboliu as convulsões e a morte induzidas pela administração de PC. O 3-ASP (50 mg/kg) aumentou a latência para o primeiro episódio convulsivo, bem como, diminuiu a mortalidade e a incidência das convulsões causadas por PTZ e KA. Ainda no artigo 1, consideramos importante o estudo da ação antioxidante do 3-ASP (10, 25 e 50 mg/kg; p.o.) frente ao estresse oxidativo induzido pela PC (400 mg/kg, i.p.) em ratos de 21 dias de vida. Os resultados demonstraram que o pré-tratamento com 3-ASP mostrou-se eficaz na proteção contra a inibição da atividade cerebral da superóxido dismutase, diminuição dos níveis de ácido ascórbico, estimulação da atividade da catalase e aumento dos níveis de espécies reativas causadas pela PC. Adicionalmente, o 3-ASP protegeu contra a inibição da atividade da acetilcolinesterase e da Na+,K+-ATPase resultantes das convulsões induzidas pela PC. Em um segundo momento, o envolvimento dos sistemas glutamatérgico e GABAérgico na ação anticonvulsivante do 3-ASP foi verificado (Artigos 1 e 2). A combinação de doses sub-efetivas de 3-ASP (10 mg/kg, p.o.) e diazepam (agonista GABAérgico; 0,5 mg/kg, i.p.), 5S,10R (+)-5-metil-10,11-dihidro-5H-dibenzo [a,d] ciclohepteno -5,10- imina maleato (MK-801; antagonista não-competitivo do receptor NMDA; 0.1mg/kg, i.p.) ou 6,7-dinitroquinoxalina-2,3-diona (DNQX; antagonista de receptores não-NMDA; 5 mg/kg, i.p.) aumentou a latência para o primeiro episódio convulsivo, bem como diminuiu a incidência de convulsões induzidas pela PC. Por outro lado, a combinação de 3-ASP e 2-metil-6-feniletinil piridina hidroclorada (MPEP; antagonista do receptor glutamatérgico metabotrópico do tipo 5; 0,5 mg/kg, i.p.) não apresentou efeito protetor contra os episódios convulsivos. A administração oral de 3-ASP (50 mg/kg) causou uma inibição de 64% e 58% da captação de GABA no córtex e no hipocampo, respectivamente. Entretanto, nenhuma alteração na captação de glutamato após a administração de 3-ASP (50 mg/kg) foi observada. Adicionalmente, no artigo 2 investigamos a possível interação entre doses sub-efetivas de 3-ASP e inibidores da captação de GABA ou da GABA transaminase (GABA-T) frente às convulsões induzidas por PC em ratos de 21 dias de vida. Para isto, doses sub-efetivas de 3-ASP (10 mg/kg; p.o.) e ácido DL-2,4-diamino-n-butírico hidroclorado (DABA - um inibidor da captação de GABA; 2 mg/kg; i.p.) ou ácido aminooxiacético hemihidroclorado (AOAA um inibidor da GABA-T; 10 mg/kg, i.p.) foram co-administrados em ratos de 21 dias de vida antes da administração de PC (400 mg/kg; i.p.). A presença de episódios convulsivos foi avaliada. Verificamos que o tratamento com o 3-ASP e DABA aboliu as convulsões induzidas por PC, corroborando com nossos resultados neuroquímicos. O mesmo foi observado quando foram administradas doses sub-efetivas de 3-ASP e AOAA. Por fim, no artigo 3 investigamos o efeito do 3-ASP ou diazepam frente as convulsões, aumento da susceptibilidade ao desenvolvimento de convulsões e prejuízo na memória a longo prazo resultantes da convulsão febril induzida pela hipertermia. Os ratos de 21 dias de vida foram pré-tratados com 3-ASP (25, 50 ou 100 mg/kg; p.o), diazepam (1 ou 5 mg/kg; i.p.) ou veículo. Após o pré-tratamento, os animais foram expostos a uma temperatura de 41°C. Trinta dias após a exposição à hipertermia, avaliamos o aumento da susceptibilidade ao desenvolvimento de convulsões e prejuízo na memória a longo prazo. Verificamos que o pré-tratamento com 3-ASP ou diazepam não foi capaz de proteger contra o comportamento estereotipado, automatismos faciais e flexão corporal induzidos pela hipertermia. O efeito protetor do 3-ASP (100 mg/kg) contra o aumento da susceptibilidade ao desenvolvimento de convulsões e prejuízo na memória a longo prazo resultantes da convulsão febril induzida pela hipertermia foi verificado. O diazepam (1 ou 5 mg/kg) não protegeu contra o prejuízo na memória a longo prazo causado pela convulsão febril. Além disso, o tratamento com diazepam (1 mg/kg) nos animais mantidos a temperatura ambiente causou um significativo prejuízo cognitivo. Estes resultados sugerem que o 3-ASP apresenta ação anticonvulsivante em ratos de 21 dias de vida e que essa ação parece ser mediada pelos sistemas glutamatérgico e GABAérgico. Além disso, a ação anticonvulsivante do 3-ASP parece estar associada à sua atividade antioxidante. Por fim, o 3-ASP pode representar uma importante ferramenta para a proteção contra o aumento à susceptibilidade às convulsões e o prejuízo cognitivo resultantes das convulsões febris.

A moxidectina é uma droga antiparasitária, do grupo das milbemicinas, utilizada em animais domésticos. Em mamíferos seu mecanismo de ação envolve o GABA, neurotansmissor que tem um papel relevante na regulação do comportamento sexual. Assim, o presente trabalho estudou os efeitos da moxidectina no comportamento sexual de ratos machos. Uma vez que alterações na função motora podem interferir na manifestação deste comportamento, avaliou-se, inicialmente, a atividade geral no campo aberto e a coordenação motora na trave elevada, e posteriormente, o comportamento sexual de ratos inexperientes e experientes. A avaliação da atividade geral dos ratos observados no campo aberto mostrou que, mesmo em altas doses (2,0 e 20,0mg/kg), a moxidectina não altera o comportamento de ratos no campo aberto. Esta droga, porém, prejudicou a coordenação motora dos animais avaliados na trave elevada, sendo este efeito atribuído, ao menos em parte, à ação da moxidectina em receptores GABAérgicos. Os resultados obtidos na avaliação do comportamento sexual de ratos inexperientes mostraram redução da motivação sexual dos animais que receberam 0,2mg/kg de moxidectina e foram observados 24 ou 72horas depois. Nenhuma alteração significante foi observada nos diferentes parâmetros do comportamento sexual dos ratos experientes, indicando que a experiência sexual pode reverter os efeitos desta droga. Estes resultados mostraram que a moxidectina prejudicou o comportamento sexual de ratos machos inexperientes e a coordenação motora, efeitos estes atribuídos a uma ação central desta droga em receptores GABAA. / The moxidectin is an antiparasitic drug that is used in domestic animals. It is a semi-synthetic milbemycin. Its mechanism of action, in mammals, involves GABA, neurotransmiter that has an important role in the regulation of the sexual behavior. Thus, the present work studied the effects of the moxidectin in male rats sexual behavior. Due to the fact that alterations in the motor function can interfere in the expression of this behavior, it was evaluated, initially, the general activity in the open field and the motor coordination at wooden dowel, and later, the sexual behavior of naïve and experienced rats. The evaluation of the general activity of the rat observed in the open field showed that, even in high doses (2.0 and 20.0 mg/kg), the moxidectin do not alter the behavior of the rats in open field. However, this drug was able to impair the motor coordination of the animals at the wooden dowel. As a matter of fact, this effect is due, at least part of it, to the action of the moxidectin in GABAergics receptors. The results achieved in the evaluation of naïve male rats’ sexual behavior showed reduction of the sexual motivation of the animals that received 0.2 mg/kg of moxidectin and were observed after 24 or 72 hours later. No significant alteration was detected in the various parameters of the experienced rats’ sexual behavior, indicating that the sexual experience could revert the effects of this drug. The moxidectin impair the sexual behavior of naive male rats and the motor coordination, and the reasons that caused these effects were attributed to a central action of the moxidectin at GABAA receptors.

科学研究費補助金 研究種目:一般研究(C) 課題番号:01570633 研究代表者:大磯 ユタカ 研究期間:1989-1990年度

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

Made available in DSpace on 2015-07-22T20:50:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-26. Added 1 bitstream(s) on 2015-08-11T03:25:44Z : No. of bitstreams: 1 Publico-10777.pdf: 584483 bytes, checksum: e11ab9839fe48602b080e48c338291cc (MD5) / Introdução: A cefaléia crônica diária é constituída por um grupo de cefaléias, dentre elas a enxaqueca crônica, comórbida com patologias como a depressão, o abuso de medicamentos, a obesidade e, mais recentemente, associada a casos de hipertensão intracraniana idiopática sem edema de papila. Objetivos: Determinar a prevalência de hipertensão intracraniana idiopática sem edema de papila e os níveis liquóricos de glutamato e ácido gama-aminobutírico em pacientes com enxaqueca crônica comparado a outros grupos de pacientes. Métodos: Foram estudados pacientes com enxaqueca crônica, mediante a realização do exame do líquido cefalorraquidiano com medida da pressão de abertura e dosagens dos níveis liquóricos dos aminoácidos glutamato e ácido gama-aminobutírico pela técnica de cromatografia líquida de alta resolução. Resultados: Dos pacientes submetidos a punção lombar, seis pacientes, em grupo de sessenta, tiveram elevação na pressão liquórica maior que 200 mm H20 sem acusar edema de papila à fundoscopia. Os pacientes que abusavam de triptanos mostraram níveis liquóricos de glutamato menores que aqueles com uso abusivo de outros tipos de medicações analgésicas e pacientes que não abusavam de nenhum tipo de medicação. Quanto aos níveis de ácido gamaaminobutírico no líquido cefalorraquidiano, esses foram menores nos pacientes com enxaqueca crônica e depressão quando comparados aos pacientes que tinham apenas enxaqueca crônica. Conclusões: A realização do estudo do líquido cefalorraquidiano foi importante em pacientes com enxaqueca crônica para a exclusão da hipertensão intracraniana idiopática sem papiledema, possibilitando perspectivas futuras para o entendimento da fisiopatogênese e desenvolvimento de novas terapias medicamentosas para a enxaqueca e suas comorbidades. / Introduction: Chronic daily headaches consist of a group of headaches, among them chronic migraine, that is comorbid with depression, overuse of medication, obesity and recently, cases of idiopathic intracranial hypertension without papilloedema. Objectives: To establish idiopathic intracranial hypertension without papilloedema prevalence and glutamate and gamma-aminobutyric acid levels in cerebrospinal fluid from patients with chronic migraine compared to other groups of patients. Methods: We studied patients with chronic migraine, who underwent lumbar puncture to rule out idiopathic intracranial hypertension without papilloedema. Amino acids glutamate and gamma-aminobutyric acid levels were measured by high performance liquid chromatography in cerebrospinal fluid. Results: Six patients, among sixty, had CSF open pressure higher than 200 mm H20 without papilloedema on fundoscopy. Patients who overused triptans had glutamate levels lower than those with abuse of other analgesic types and nonoverusers. The gamma-aminobutyric acid levels in cerebrospinal fluid were lower in depressed patients when compared to patients without depression and controls. Conclusions: The study of the cerebrospinal fluid was important in patients with chronic migraine for the exclusion of idiopathic intracranial hypertension without papilloedema, opening perspectives for the understanding of the physiopathology and development of new drug therapies for migraine and its comorbidities. / TEDE / BV UNIFESP: Teses e dissertações

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

La compréhension des mécanismes de défense mis en place lors de la résistance des plantes vis-à-vis d'agents pathogènes a pour objectif de proposer des alternatives à l'utilisation de produits phytosanitaires utilisés en agriculture. Dans une première partie, nous avons étudié les mécanismes moléculaires impliqués dans la résistance induite aux pathogènes par l'acide β-aminobutyrique (BABA) chez la vigne. En effet, cet acide aminé non protéique favorise un état physiologique particulier, appelé potentialisation, dans lequel la plante est capable de mobiliser plus rapidement et/ou plus intensément ses réactions de défense en réponse à un stress. Contrairement aux éliciteurs comme les oligogalacturonates (OG), nous avons montré que le BABA seul n’induisait pas les événements précoces de signalisation sur suspensions cellulaires de vigne, tels que les variations de la concentration en calcium cytosolique libre ([Ca2+]cyt), la production de monoxyde d’azote (NO), la production d’H2O2, la phosphorylation de MAPkinases, ni l’expression de gènes de défense. Seules la production d’H2O2 et l’expression plus intense du gène RbohD codant une NADPH oxydase sont potentialisées par le BABA dans les suspensions cellulaires élicitées par les OG. In planta, le BABA potentialise également une production d’H2O2 en réponse à l’infection par l’oomycète Plasmopara viticola. L’utilisation d’un inhibiteur de NADPH oxydase abolit complètement cette production d’H2O2 et bloque partiellement la résistance induite par le BABA. Nous montrons donc que la potentialisation de la production d’H2O2 dépendante d’une NADPH oxydase contribue à l’établissement de la résistance induite par le BABA chez la vigne. Une deuxième partie a permis d’appréhender les événements cellulaires impliqués dans la résistance des plantes en se focalisant sur le mutant pad2 (phytoalexin deficient) d’Arabidopsis thaliana. Ce mutant présente une sensibilité accrue à différents pathogènes et contient un taux de glutathion de l’ordre de 20 % par rapport à l’écotype sauvage. Nous avons tout d’abord montré que le faible taux de glutathion dépendait d’une quantité réduite de la première enzyme de sa biosynthèse, la glutamate-cystéine ligase. Le glutathion étant impliqué dans la mise en place des réactions de défense, nous avons tenté de définir le lien entre la déficience en glutathion et la sensibilité de pad2 aux pathogènes. Nous avons tout d’abord montré que pad2 possédait un état redox du glutathion plus oxydé que le sauvage. Une analyse transcriptomique à l’état basal a révélé que la plupart des gènes différentiellement exprimés étaient réprimés chez pad2. Parmi ces gènes, certains codent des protéines impliquées dans les flux d’ions qui pourraient déréguler la dépolarisation membranaire. Nous avons ainsi confirmé que la dépolarisation de la membrane plasmique est amoindrie chez pad2 en réponse aux OG. De plus, des événements en aval tels que la production d’H2O2 et la production de NO sont également plus faibles chez le mutant par rapport au sauvage. Cette absence de la production d’H2O2 a également été spécifiquement observée sur plantes pad2 infectées par l’oomycète Phytophthora brassicae. Il en résulte un développement accru du pathogène corrélé à une absence de réponse hypersensible, une mort cellulaire localisée normalement observée dans le cas du sauvage résistant. En réponse aux OG ou à l’infection par P. brassicae, les analyses transcriptomiques font ressortir un fort enrichissement de gènes relatifs à la dégradation des protéines chez pad2. De manière globale, nos résultats suggèrent que la déficience en glutathion chez pad2 pourrait profondément modifier le turn-over des protéines, perturbant ainsi la signalisation cellulaire et les réponses biologiques associées. / Alternative strategies are required to reduce pesticide input into the environment for effective and sustainable plant protection. One solution is the activation of plant basal resistance that relies on the application of resistance inducer molecules. In the first part of this study, we analyzed the mode of action of β-aminobutyric acid (BABA), a non-protein amino acid, in the grapevine induced resistance. BABA confers a physiological state, called priming, during which plants are able to mobilize better and/or more rapidly defense responses to biotic or abiotic stress. Unlike oligogalacturonides (OG), we showed that BABA did not induce early signaling events in grapevine cells such as variations of cytosolic free calcium concentration, H2O2 and nitric oxide production, MAPkinase phosphorylation, nor the expression of defense-related genes. Among them, only H2O2 production and the expression of RbohD gene, which encodes a NADPH oxidase, are primed by BABA in OG-treated cells. Moreover, BABA-treated plants display a stronger accumulation of H2O2 in response to the oomycete Plasmopara viticola. Application of an NADPH oxidase inhibitor completely abolishes this H2O2 production and leads to a reduction of BABA-induced resistance against P. viticola. These data suggest that the priming of an NADPH oxidase-dependent H2O2 production contributes to BABA-induced resistance in grapevine. The second part consisted to analyze molecular events involved in plant resistance by using the pad2 (phytoalexin deficient) mutant of Arabidopsis thaliana which is susceptible to a broad range of pathogens. We showed that the glutathione depletion depends on the low amount of glutamate-cysteine ligase protein, the first enzyme involved in its biosynthesis. We studied molecular events, which are involved in defense mechanisms, to understand the impact of the glutathione content on pad2 susceptibility. Our results show that the redox state of glutathione is more oxidized in pad2 than in wild type Col-0. Since cellular redox state change is known to regulate gene expression, a basal transcriptome analysis has been performed in pad2 and wild type plants. Interestingly, most of the identified genes in pad2 are down-regulated, some of them encoding proteins involved in ion fluxes. As expected, the plasma membrane depolarization and events downstream like H2O2 and NO production are impaired in pad2 in response to OG. During infection with Phytophthora brassicae, the lack of H2O2 production is concomitant with an absence of the hypersensitive response, a localize cell death observed in the resistant wild type. After OG treatment or P. brassicae infection, microarray analysis brings out genes related to protein machinery including degradation in pad2. Taken together, these data suggest that the depletion of glutathione has an impact on protein turn-over which disturbs cell signaling events and related biological responses.

Gammaaminosmörsyrareceptorer typ A (GABAAR) är pentameriska ligandstyrda kloridkanaler som uppvisar neurohämmande egenskaper. Därmed är de primära läkemedelsmål för flera ångestdämpande och lugnande läkemedel som används för att minska förekomsten av aktionspotential i neuroner. Trots vikten av dessa receptorer har strukturen av öppen receptor för GABAAR inte lösts hittills, på grund av deras snabba desensibiliseringskinetik. Diazepambindande hämmare (DBI) är en neuropeptid som tidigare rapporterats vara en positiv modulerare för α5β3 GABAAR. I denna studie framställdes DBI genom rekombinant proteinexpression, och den positiva moduleringen undersöktes och karakteriserades med hjälp av voltage-clamp med två elektroder på Xenopus laevis oocyter. För att kunna studera DBI moduleringen skapades GABA dos-responskurvan, och dess karakteristik undersöktes. Baserat på resultaten verkar den positiva moduleringen av DBI vara koncentrationsberoende. Vidare orsakar moduleringen en 2,16-faldig ökning av GABA-framkallad ström vid dess maximala modulationskoncentration. Trots att ström signaler från voltage-clamp uppvisar en viss grad av variabilitet stämmer resultaten överens med tidigare rapporterade observationer som utredde DBI moduleringen respektive GABA dos-responskurvan för α5β3 GABAAR. Dessa resultat kan utnyttjas för att stödja framtida strukturella studier av GABAAR genom att använda denna kunskap om DBI för att potentiellt kunna stabilisera den öppna receptorn, såväl som för att förstå mekanismen för interaktionen mellan DBI och GABAAR. / γ-Aminobutyric acid type-A receptors (GABAARs) are pentameric ligand-gated chloride channels which exhibit neuro inhibitory effects. Hence, they are the primary drug-targets of multiple anxiolytic and sedative drugs used to inhibit the firing rate of neurons. Despite the importance of these receptors, the open structure of GABAAR has not been resolved, owing to their rapid desensitization kinetics. Diazepam binding inhibitor (DBI) is a neuropeptide previously reported to positively modulate the α5β3 GABAARs. In this study, DBI was recombinantly expressed, and this positive modulation was further investigated and characterized by using two-electrode voltage clamp of Xenopus oocytes. For the purpose of studying DBI modulation, GABA dose-response curve was generated, and its characteristics were assessed. Based on the results, the positive modulation of DBI appears to be concentration dependent. Furthermore, the modulation causes a 2.16-fold increase in GABA-elicited current at its maximum modulatory concentration. Although the current traces present some degree of variability, the results are supported by being consistent with previously reported findings investigating DBI modulation and the dose-response curve for α5β3 GABAARs, respectively. These findings can be used to support future structural studies of GABAARs by utilizing this knowledge of DBI to potentially stabilize the open structure of the receptor, as well as in understanding the mechanism of interaction between DBI and GABAARs.