Este estudo visou observar os efeitos plásticos induzidos pelo exercício a curto prazo em regiões motoras do encéfalo de ratos. Observou-se a expressão das subunidades GluR1 e GluR2/3. Os animais foram divididos em grupos de: 3 dias(COR3), 7 dias(COR7) e 15 dias(COR15); e um grupo controle(CONT). Empregaram-se as técnicas de imuno-histoquímica e immunoblotting. A expressão de GluR1 no cerebelo, demonstrou um decréscimo em COR3. No hipocampo houve uma queda na expressão em COR3(40%), retornando aos níveis basais em COR7. No córtex cerebral observou-se uma queda da expressão com máxima queda em COR7(52%), retornando à expressão basal em COR15. O estriado não sofreu alterações na expressão de GluR1 ao longo dos primeiros 7 dias, tendo um aumento em COR15(90%). A expressão de GluR2/3 não foi alterada, exceto no cerebelo, onde houve um decréscimo em dois momentos distintos, COR3(55%) e COR15(25%), retornando à expressão basal em COR7. Os nossos dados revelam que o exercício físico a curto prazo foi capaz de promover alterações plásticas ao longo do treinamento. / This study aimed at analyzing the plastic effects of the short-term exercise upon the rat motor area. We check the expression of GluR1 and GluR2/3. We divided into 3 experimental groups based on duration of exercise: 3 days(COR3), 7 days(COR7), and 15 days(COR15); and a control group(CONT). The experimental animals were subjected to a treadmill exercise protocol. The brains were subjected to the techniques of immunohistochemistry and immunoblotting. In the cerebellum, there was a decrease for COR3(17%). In the hippocampus, there was a decrease of the GluR1 expression for COR3 (40%). In the cerebral cortex there was a drop of GluR1 expression for COR3 and COR7(52%). In the striatum, there was no change of GluR1 expression during the first seven days, with a increase for COR15(90%). The GluR2/3 expression did not change in any brain structure analyzed, except in the cerebellum, where there was a significant decrease for two distinct groups, COR3(55%) and COR15(25%). Our data show that short-term physical exercise was able to promote plastic changes during training.

No description available.

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.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.

El glifosat, N-(fosfonometil) glicina, és un dels herbicides més utilitzats arreu del món a causa de la seva baixa toxicitat i al seu ampli espectre d'aplicació. A conseqüència del gran ús que se'n fa, és necessari monitoritzar aquest compost i el seu principal metabòlit, l'àcid aminometilfosfònic (AMPA), en el medi ambient. S'han descrit diversos mètodes instrumentals basats en cromatografia de gasos (GC) i de líquids (HPLC), sent aquesta darrera l'opció més favorable a causa del caràcter polar dels anàlits. Per assolir nivells de concentració baixos cal, però, la preconcentració dels anàlits.En aquest treball s'estudien diferents alternatives amb aquest objectiu. S'ha avaluat la tècnica de membrana líquida suportada (SLM) on la membrana consisteix en una dissolució orgànica, que conté un transportador (en el nostre cas, un bescanviador d'anions comercial, Aliquat 336), que impregna un suport polimèric microporós que se situa entre dues solucions aquoses: la de càrrega, que conté els anàlits inicialment, i la receptora, on es retenen els anàlits després del seu transport a través de la membrana. Les condicions d'extracció més adequades s'obtenen treballant en medi bàsic amb NaOH on els anàlits estan en forma aniònica i les majors recuperacions s'obtenen amb HCl 0,1 M o NaCl 0,5 M, la qual cosa indica que l'ió clorur és la força impulsora del transport.Un cop dissenyat el sistema, es duen a terme experiments de preconcentració amb dues geometries diferents: un sistema de membrana laminar (LSLM) on recircula la fase receptora i un sistema de fibra buida (HFSLM). Els millors resultats s'obtenen amb el mòdul de fibra buida, amb factors de concentració de 25 i 3 per a glifosat i AMPA, respectivament, fent recircular durant 24 hores 100 ml de solució de càrrega i 4 ml de solució receptora. També s'aplica una tècnica més selectiva, la cromatografia d'afinitat amb ió metàl·lic immobilitzat (IMAC), basada en la interacció entre els anàlits i un metall immobilitzat en una resina a través d'un grup funcional d'aquesta. En aquest estudi s'immobilitza pal·ladi al grup funcional 8-hidroxiquinoleïna de la resina amb matriu acrílica Spheron Oxine 1000 i s'avalua per a l'extracció i preconcentració de glifosat i AMPA. Per a ambdós anàlits l'adsorció és del 100 % i les recuperacions són superiors al 80 % i al 60 % per a glifosat i AMPA, respectivament, utilitzant HCl 0,1 M + NaCl 1 M com a eluent. Aquests resultats es comparen amb els obtinguts amb dues resines més, també carregades amb pal·ladi: Iontosorb Oxin 100, que té el mateix grup funcional però matriu de cel·lulosa, i Spheron Thiol 1000, on el grup funcional és un tiol i la matriu també és acrílica. Per al glifosat els resultats són similars amb totes les resines, però per a l'AMPA la resina Spheron Thiol és la única que proporciona recuperacions superiors al 93 %.Finalment, una altra opció estudiada és l'acoblament de dues columnes de cromatografia líquida (LC-LC). En l'estudi l'objectiu és millorar el mètode existent per a glifosat i AMPA en aigües naturals on el LOD era de 0,25 ug/l. El mètode consisteix en la derivatització precolumna amb el reactiu fluorescent FMOC i l'anàlisi amb l'acoblament LC-LC-fluorescència. Variant lleugerament les condicions de derivatització s'aconsegueix quantificar 0,1 ug/l de glifosat i AMPA. Es fortifiquen aigües naturals amb 0,1, 1 i 10 ug/l dels anàlits per validar el mètode. S'obtenen recuperacions d'entre el 85 % i el 100 %, amb desviacions estàndard relatives inferiors al 8 %. Aplicant una tècnica de preconcentració prèvia a la derivatització i anàlisi utilitzant una resina de bescanvi aniònic, Amberlite IRA-900, es millora la sensibilitat del mètode i s'assoleix un LOD per al glifosat de 0,02 ug/l. / Glyphosate, N-(phosphonomethyl)glycine, is one of the most widely used herbicides in the world due to its low mammal toxicity. Therefore, there is a great interest in the monitoring of this compound and its major metabolite aminomethylphosphonic acid (AMPA), in environmental matrices. A great variety of methods based on gas chromatography (GC) and liquid chromatography (HPLC) have been applied for their determination. HPLC is the most popular option due to the polar nature of both compounds. However, the analysis of these compounds at residue levels requires the use of preconcentration techniques.In this work we have studied different alternatives to this aim. We have evaluated the supported liquid membrane (SLM) technique where the membrane consists of an organic solution, which contains the carrier (in this case a commercial anion exchanger extractant, Aliquat 336), that impregnates a microporous polymeric support placed between two aqueous solutions: the feed solution, which initially contains the analytes, and the stripping solution, where the analytes are retained after their transport through the membrane. The best conditions were achieved with NaOH as feed solution, where the analytes are present in their anionic form and 0.1 M HCl or 0.5 M NaCl as stripping solutions indicating that the chloride ion is the responsible for the transport.Under these chemical conditions some preconcentration experiments were run using two different geometries of the membrane: a laminar membrane system (LSLM) where the stripping solution can be recirculated and a hollow fiber liquid membrane (HFSLM). The best results were obtained for the liquid membrane system in HF configuration, with concentration factors of 25 and 3 for glyphosate and AMPA, respectively, after recirculation during 24 hours of 100 ml of feed solution and 4 ml of stripping solution.A more selective technique is also studied, immobilised metal ion affinity chromatography (IMAC), which is based on the interaction between the analytes and a metal immobilised in a resin through a functional group. In this work we have immobilised palladium through the chelating group 8-hydroxyquinoline of the acrylic resin Spheron Oxine and it has been evaluated for the preconcentration of glyphosate and AMPA. 100 % adsorption has been obtained for both analytes and the recoveries are higher than 80 % and 60 % for glyphosate and AMPA, respectively, using 0.1 M HCl + 1 M NaCl as eluent. These results are compared with the results obtained working with two different resins also charged with palladium: Iontosorb Oxin, which has the same functional group but cellulose matrix, and Spheron Thiol, which a thiol functional group and acrylic matrix. The results for glyphosate are similar with all the resins tested, but for AMPA the best results are obtained with Spheron Thiol with recoveries higher than 93 %.Finally, another option studied is the coupling of two liquid chromatographic columns (LC-LC). In this work the objective is to improve the existing method for the analysis of glyphosate and AMPA in natural waters which had a LOD of 0.25 mg/l. The method consists of the pre-column derivatization with the fluorescent reagent FMOC and the analysis by LC-LC-fluorescence detection. The derivatization step has been slightly modified with the aim of decreasing the limits of quantification of glyphosate and AMPA down to 0.1 mg/l. Different water samples spiked at three concentration levels (0.1, 1, 10 mg/l) were analysed by the improved method yielding recoveries between 85-100 % with relative standard deviations lower than 8 %.Additionally, the potential of an anion-exchange resin, Amberlite IRA-900, for preconcentration of glyphosate, previously to the derivatization step, has been investigated. In this way the LOD of the parent compound has been lowered to 0.02 mg/l.

Major depression is a devastating disease that affects up to 20% of world population and is classified today as a leading cause of disability-adjusted life years. Since late 50s with the serendipitous discovery of the first antidepressant agents, pathophysiology and therapeutics of depression are governed by the monoaminergic hypothesis. Monoaminergic-based treatment, although still in use today, was proven inefficient to treat a significant proportion of cases and presents a delayed onset of action. Recent research has unveiled an array of new mechanisms through which antidepressant medication helps restore neuronal plasticity and neurotransmission that is disrupted in mood disorders and in animal models of depression. Glutamatergic transmission, in particular AMPA receptor, and signal transduction cascades have been implicated both in antidepressant action and the pathophysiology of depression, as here and now regulators that mediate persistent changes. In this study, AMPA receptors positive modulators demonstrated antidepressant-like effects in a chronic model of depression and preliminary data suggest a faster onset of action than conventional antidepressants. The ERK/MAPK signaling pathway was also studied as a major integrator of synaptic plasticity modifications that links extracellular signals to gene expression regulation via its downstream molecular partners. We used a new class of inhibitors of the ERK pathway, whose design was based on the particular property of ERK to bind to its downstream targets via specific docking domains. A considerable amount of data provided evidence for an antidepressant action of selective inhibition of the ERK/Elk1 signaling complex in multiple animal models of depression. Overall, the findings of this work reveal novel, promising targets for the treatment of depression.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

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.

Le syndrome de Coffin-Lowry (CLS), une déficience intellectuelle liée à l'X, est causée par des mutations du gène RPS6KA3 codant pour la kinase RSK2 régulée par les facteurs de croissance.Pour comprendre les conséquences du déficit en RSK2 dans l'hippocampe nous avons effectué une comparaison des profils d'expression génique d'hippocampes de souris Rsk2-KO et WT. Elle a révélé l'expression différentielle de 100 gènes, codant pour des protéines agissant dans divers processus biologiques. Nous avons analysé les conséquences de la dérégulation de l'un de ces gènes Gria2 codant pour GluR2, une sous-unité du récepteur glutamate AMPA. Un niveau d'expression doublé de GluR2 a été relevé dans l'hippocampe des souris Rsk2-KO et les études électrophysiologiques y ont révélé une réduction des transmissions AMPAR et NMDAR. L’activité de ERK1/2 était aussi anormalement augmentée dans l'hippocampe des souris Rsk2-KO, ainsi que le niveau de P-Sp1. Ensemble, mes résultats ont suggéré que la surexpression de GluR2 dans les neurones déficients en RSK2, était causée par une augmentation de l'activité transcriptionnelle de Sp1 sur le gène Gria2, qui, elle-même, est le résultat de l’augmentation anormale de l’activité de ERK1 / 2. / Coffin–Lowry Syndrome (CLS), an X-linked form of intellectual disability, is caused by mutations of the RPS6KA3 gene encoding the growth factor regulated kinase RSK2. To understand the consequences of RSK2 deficiency in the hippocampus we performed a comparison of the hippocampal gene expression profiles from Rsk2-KO and WT mice. It revealed differential expression of 100 genes, encoding proteins acting in various biological pathways. We further analyzed the consequences of deregulation of one of these genes, Gria2 encoding GluR2, a subunit of the glutamate AMPAR. An abnormal two-fold increased expression of GluR2 was found in the hippocampus of Rsk2-KO mice. Electrophysiology studies showed a reduction of basal AMPAR and NMDAR mediated transmission, in the hippocampus of Rsk2-KO mice. Activity of ERK1/2 was also abnormally increased in the adult hippocampus of Rsk2-KO mice. P-Sp1 level was also significantly higher in RSK2 deficient cells. Together, my results suggested that over expression of GluR2 in RSK2 deficient cells, is caused by increased Sp1 transcriptional activity on the Gria2 gene, which, itself, is the result of ERK1/2 increased signaling.