Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-17T19:14:35Z No. of bitstreams: 1 Ribeiro, Anézia Lima Chaves [Dissertação, 2011].pdf: 3144917 bytes, checksum: 028a1b168bde80a2bfe9b881b9c28b08 (MD5) / Made available in DSpace on 2017-03-17T19:14:35Z (GMT). No. of bitstreams: 1 Ribeiro, Anézia Lima Chaves [Dissertação, 2011].pdf: 3144917 bytes, checksum: 028a1b168bde80a2bfe9b881b9c28b08 (MD5) / O glifosato é um herbicida organofosforado amplamente utilizado que constitui um poluente potencial do meio ambiente; ácido aminometilfosfônico (AMPA) é o seu principal metabólito. O objetivo deste estudo foi desenvolver um método direto rápido e sensível para a quantificação dos herbicidas, a fim de monitorar seus resíduos na água de diferentes fontes. Além disso, este estudo teve como objetivo elaborar um procedimento para a determinação simultânea de glifosato, AMPA e F-, Cl-, Br-, NO3 -, SO4 2- e PO4 3- na água. Em ambos os casos, foram usados a cromatografia de troca iônica com detecção condutométrica. A determinação de glifosato pode ser realizada em modo isocrático (6,0 mmol L-1 Na2CO3 e 2,0 mmol L-1 NaHCO3), enquanto a separação simultânea de glifosato, AMPA e ânions foi realizada por gradiente com as seguintes fases móveis: A: 15,0 mmol L-1 NaOH + 1,0 mmol L-1 Na2CO3 e B : Na2CO3 - 15,0 mmol L-1. A confirmação de espécies AMPA (m / z = 110) e glifosato (m / z = 168) foi realizada pelo IC-MS. A determinação do isótopo 31P por ICPMS permitiu a determinação indireta destas espécies e um teste de recuperação resultou em cerca de 105%. O método foi validado utilizando matrizes de agua ultrapura, agua superficial, mineral e água subterrânea. Por eluição isocrática, os limites de detecção (LODs) encontrados para o glifosato foram: 10 μgL-1 em água ultrapura , 54 μgL-1 em água subterrânea. Para a determinação simultânea de glifosato e AMPA, os LODs foram: 9,6 μgL-1 , 9,8 μgL-1 (água ultrapura), 9,5 μgL-1 , 60 μgL-1 (água mineral), 12,4 , 29,7 μgL-1 (agua superficial) respectivamente para glifosato e AMPA. A determinação de glifosato por IC/Condutimetria mostrou-se adequada à aplicação em águas naturais, alcançando valores de LD muito abaixo dos permitidos pela legislação vigente (500 μgL-1; Portaria MS 518 de 25/03/2004 e Resolução CONAMA 396 de 03/04/2008) e, também outros órgão internacionais como EPA, sem necessidade de preparação prévia da amostra como extração, pré-concentração e derivatização / Glyphosate is a widely used organophosphorated herbicide, which constitutes a potential pollutant of the environment; aminomethylphosphonic acid (AMPA) is its main metabolite. The purpose of this study was to develop a direct, rapid and sensitive method for quantification of the herbicide in order to monitor its residues in water from different sources. Furthermore, this study aimed to draw up a procedure for the simultaneous determination of glyphosate, AMPA and F-, Cl-, Br-, NO3 -, SO4 2- and PO4 3- in water. In both cases, ion-exchange chromatography with conductimetric detection were used. The determination of glyphosate could be performed in isocratic mode (6.0 mmol L-1 Na2CO3 and 2.0 mmol L-1 NaHCO3), while the simultaneous separation of glyphosate, AMPA and anions was performed by gradient elution with the following mobile phases: A - 15.0 mmol L-1 NaOH + 1.0 mmol L-1 Na2CO3 and B - 15.0 mmol L-1 Na2CO3. Confirmation of species AMPA (m/z = 110) and glyphosate (m/z = 168) were carried out by IC-MS. The determination of the isotope 31P by ICPMS allowed the indirect determination of these species and a recovery test resulted in about 105%. The method was validated using ultrapure, river, mineral and groundwater. With isocratic elution, limits of detection (LODs) of 10 μg L-1 in ultrapure water and 54 μg L-1 in groundwater were found for glyphosate. For the simultaneous determination of glyphosate and AMPA, LODs were, respectively, 9.6 μg L-1, 9.8 μg L-1 (ultrapure water), 9.5 μg L-1, 60 μg L-1 (mineral water), 12.4 μg L-1, 29.7 μg L-1 (river water). The determination of glyphosate IC / Conductimetry proved to be suitable for application in natural waters, reaching LD values far below those allowed by legislation (500 μg L-1), MS portaria 518 of 25/03/2004 and CONAMA Resolution 396 of 03 / 04/2008), and also other international bodies such as EPA, without previous sample preparation and extraction, pre-concentration and derivatization

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex. / Medicine, Faculty of / Graduate

Ketamine is a non-competitive NMDA antagonist that has been shown to have antidepressant properties both clinically as well as in preclinical studies when administered at a subanesthetic dose. In vivo electrophysiological recordings were carried in male Sprague Dawley rats 30 minutes following ketamine administration (10 mg/kg) to first assess its effects on monoaminergic firing. Whilst no change in the firing activity of serotonin (5-HT) neurons was observed in the dorsal raphe nucleus (DRN), an increase in the firing activity was observed for dopamine (DA) and noradrenergic (NE) neurons in the ventral tegmental area (VTA) and locus coeruleus (LC), respectively. The effect of ketamine on these electrophysiological parameters was prevented by pre-administration of the AMPA receptor antagonist NBQX 10 minutes prior to ketamine administration. In a second series of experiments, an increase in AMPA-evoked response was observed within 30 minutes in the CA3 layer of the hippocampus (HPC) following acute ketamine administration. These findings suggest that acute ketamine administration produces a prompt enhancement of AMPA transmission in the forebrain and also results in increased catecholaminergic activity. These effects may play a crucial role in the rapid antidepressant effects of ketamine observed shortly following its infusion in the clinic.

AMPA receptors mediate excitatory synaptic transmission throughout the central nervous system via activation by their natural agonist glutamate. Several other molecules have been recognized as receptor agonist or antagonist, and recently allosteric modulators have been developed that potentiate the currents generated by these receptors. The goal of this thesis has been to address specific and as yet unresolved questions regarding the binding interactions between the AMPA receptors and these classes of molecules. For instance AMPA receptors are seemingly converted to have lower affinity for agonist as they move towards synapses and we evaluate two hypotheses put forward to explain the molecular mechanisms responsible for this. Additionally, guanine nucleotides competitively inhibit AMPA receptors and a second goal has been to further characterize guanine nucleotide binding, and to create mutations that selectively diminish this so that the function of the inhibition can be evaluated. A third goal has been to characterize the molecular factors that influence the effects of the allosteric modulators in order to explain why their efficacy differs greatly between brain regions. Experiments pertaining to these three goals were carried out sequentially and are described below as Projects 1 (guanine nucleotide inhibition), Project 2 (agonist affinity), and Project 3 (allosteric modulators). Project 1. Guanine nucleotides competitively inhibit AMPA-Rs (AMPA receptors) and because this inhibition is ubiquitous among virtually all types of glutamate receptors from fish to mammals, it likely serves a physiological function. Evaluation of this would be greatly facilitated if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. Therefore we mutated the homologous lysine (K445) in AMPA-R subunit GluR1 plus 12 additional residues around the glutamate binding pocket with the expectation that this would reduce nucleotide binding even further. Nucleotide affinity was determined by measuring the displacement of [3H]fluorowillardiine. As expected, the guanine nucleotide affinity was decreased about five-fold in R1-K445A mutants and the agonist affinity was seemingly unchanged. However, when tested by electrophysiology, characteristics of the mutant such as desensitization and the EC50 for glutamate were found to be altered. None of the other mutations were more successful at decreasing nucleotide affinity selectively. Nonetheless, these studies have given new insight into the docking mode of guanine nucleotides. The loss of binding in R1-K445A was much larger for GTP and GDP than for GMP, and guanosine binding, which is much lower, was unaffected by the mutation. These data suggest that the first phosphate of GMP determines the higher affinity of the phosphorylated nucleotides, and that K445 stabilizes the binding of the second and third phosphates of GDP and GTP. This along with various other observations suggest that the guanine base docks deep within the agonist binding pocket and that bulky additions, such as the phosphates, are accommodated by projecting out of the cleft in the vicinity of lysine 445. However, the exact docking mode of guanine nucleotides would have to be determined by crystallography. Project 2. Agonist binding to AMPA-R in brain consists of a high and low affinity components with KDs of 9-28 nM and 190-700 nM. Previous studies have suggested that newly synthesized receptors have high affinity and are converted to lower affinity by a secondary process. Two particular processes have been implicated, namely the conversion of receptor glycosylation from immature to complex, and modulation by receptor associated proteins. Both hypotheses were evaluated in this project using homomeric receptors GluR1-4 expressed in HEK 293 cells. The role of glycosylation was tested mostly with GluR4 receptors because they are expressed in distinct populations that exhibit either immature or complex glycans and their binding consists of high and low affinity components similar to those previously seen in brain receptors. Cells were treated with castanospermine or deoxymannojirimycin to decrease the proportion of receptors with complex glycosylation, or with cycloheximide plus chloroquine to increase the number of receptors with complex glycosylation. Although 70% of receptors from cells treated with cyloheximide/chloroquine exhibited complex glycans compared to <5% with other treatments, the affinity decreased at most 2-fold. Also, the low affinity component was nearly 80% of the total binding in receptors that exhibited virtually no complex glycans. Taken together these data indicate that complex glycosylation is not the key factor that confers low affinity. To test the second hypothesis GluR1i or GluR2i were co-expressed with stargazin which associates to receptors in neurons and affects their kinetics and trafficking. Considering the affinities of the two components seen in brain, we expected stargazin to cause a 20-fold or greater decrease in binding affinity. This was not the case, however our results did suggest that stargazin caused the appearance of a low affinity component but this was small and remained largely masked by the more abundant high affinity component. Recently, experiments with brain membranes have revealed preliminary evidence that an associated protein of ~85kDa may cause receptors to have low affinity. This hypothesis is currently under investigation. Project 3. Ampakines are cognitive enhancers that potentiate AMPA receptor currents at excitatory synapses. The efficacy of these drugs varies substantially among neurons in different brain regions, being for example about three times larger in the hippocampus than in the thalamus. Binding assays have shown that these compounds also increase the affinity of receptors for agonists. Importantly, the efficacy of these drugs to increase synaptic responses and agonist binding exhibit a positive correlation. Indeed, we have found that the increase in agonist binding (Emax) induced by the prototypical ampakine CX546 is highly variable across eight brain regions and that there is a 3-fold difference between the hippocampus and the thalamus which is similar to the difference reported for physiological efficacy. Therefore, binding assays or receptor autoradiography can potentially be used to predict the physiological efficacy of these drugs in a particular brain region. An important goal of this project has been to identify factors that may be responsible for the regionally different efficacies. Ampakines show some preference for receptor subunits but various considerations suggest that other factors must be involved. In this project we evaluated the role of a novel class of proteins called TARPs (transmembrane AMPA receptor regulatory proteins) that have recently been discovered to be tightly associated with AMPA receptors and to regulate their kinetics. Four of these proteins, named lambda;2(stargazin),λ3,λ4,and λ8 are abundant in the brain, but they exhibit highly selective regional distribution. We determined the maximum increase in agonist binding (Emax ) caused by saturating CX546 in three different AMPA receptor subunits, GluR1i, GluR2i, and GluR4i without and with co-expression of the four TARPs. Without TARPs, both Glu2i and GluR4i showed an Emax value of 100% over baseline binding. Co-expression of TARPs increased the Emax in GluR2i and this was largest for λ3 and λ8 (~130%). However, TARPs decreased the Emax of CX546 in GluR4i and this was most notable with λ2 and λ4 (~72%). Agonist binding in GluR1i was increased by only 15% and it was not significantly changed by TARPs. The expression patterns of TARPs and AMPA-R subunits in the brain have been partially characterized in the literature. Thus, it was previously reported that GluR4i transcripts are abundant in the thalamus but minor in the hippocampus. Using western blots we confirmed that this is also true for protein content; in the thalamus expression of GluR1, GluR2, GluR3, and GluR4 was 4%, 33%, 40%, and 147% respectively, of that in the hippocampus. When considering the known expression patterns of TARP variants, the hippocampus can be described as being enriched in GluR2, λ3 and λ8 while GluR4, λ2 and λ4 are prevalent in the thalamus. In comparison between these specific subunit/TARP combinations, the Emax values for those representative of the hippocampus (GluR2i/λ3 or λ8) were ~2-times larger than the Emax values of thalamic combinations (R4i/λ2 or λ4). Thus we can conclude that the differences in the expression of both TARP variants and AMPA-R subunits are critical factors for determining the variable efficacy of ampakines across brain regions.

Objective: The purpose of this literature study was to evaluate the effect of perampanel as an adjunct therapy in patients, 12 years of age or older suffering from pharmacoresistant epilepsy. Methods: This thesis is a literature work based on scientific articles sought and obtained from the medical database PubMed. The five articles were selected with the criteria that perampanel is used for therapy-resistant epilepsy, as an adjunct therapy in patients who do not respond to other anti-epileptic drugs, in patients using 1-3 antiepileptic drugs, participants 12 years or older and the drug is used in humans. The result measured was the percentage change in seizure rate per 28 days and ≥ 50% responder rate. Results: Study 1 and 5 compared the effect and safety of different doses of perampanel with placebo. A statistically significant change in seizure rates was shown in the perampanel group, 34.5% for 8 mg/day and 26.3% for 12 mg/day, compared to placebo 21.0%. The impact of perampanel in study 5 also showed a statistically significant difference in responder rate in perampanel group, 64.2% compared to placebo 39.5%, study 1 showed no statistically significant difference here. Study 2 evaluated the safety and tolerability of long-term treatment of perampanel up to 12 mg/day. In each 13-week interval of perampanel treatment, the overall median percentage reduction was 39.3% for 14-26 weeks (n=1,114), 46.5% for weeks 40-52 (n=731) and 58.1% for weeks 92104 (n=59). Similarly, in the case of the decrease in the responder rate of 41.4%, 46.9% and 62.7% for the respective 13-week intervals. Study 3 compared the efficacy and safety of perampanel with placebo. A statistically significant change in seizure rates was shown in perampanel group, -34.8% for 8 mg/day and -35.6% for 12 mg/day compared to placebo who had -18.0%. An increase of the 50% responder rate was also seen in the perampanel group, 40.9% for 8 mg/day and 45.0% for 12 mg/day. In Study 4, the effect and safety of perampanel were evaluated in patients who had received placebo in previous Phase III double-blinded core studies and switched to perampanel in extension study 307. The percentage decrease in the overall seizure rate in double blinded studies increased from 18.6% to 44.3% for placebo group and from 31.7% to 41.4% for perampanel group. Responder rate for placebo was 20.3% increased to 44.3% and perampanel was 34.0% increased to 43.4% in the double blinded studies. Conclusions: All included studies analyzed the effect on seizure frequency, tolerability and adverse reactions. Studies 1–5 showed a clear decrease in seizure rates. But it was also found that there were differences between the youth group and the study pool both as a whole as well as at different doses. The question in this literature study can thus be answered with yes, perampanel has a positive effect as an adjunct therapy against therapy-resistant epilepsy. However, it is of great importance that the dose and titration are adjusted individually.

The thesis deals with the determination of selected ion herbicides in surface waters and in the first part describes the herbicides. Given the broad spectrum herbicide further work deals mainly with glyphosate. The next section describes the electromigration methods and the methods by which the herbicides were determined. Finally, it describes how the development of an electrolyte system along with the use of graphical possibilities of computer technology. In conclusion are presented and commented on the results of measurements of glyphosate in surface waters, compared with foreign literature and assessing the suitability of methods for the analysis of contaminants in the environment.

yes / Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression. / Medical Research Council, the European Research Council and the Wellcome Trust

Yes / The activity-regulated cytoskeleton associated protein (Arc/Arg3.1) plays a key role in determining synaptic strength through facilitation of AMPA receptor (AMPAR) endocytosis. Although there is considerable data on the mechanism by which Arc induction controls synaptic plasticity and learning behaviours, several key mechanistic questions remain. Here we review data on the link between Arc expression and the clathrin-mediated endocytic pathway which internalises AMPARs and discuss the significance of Arc binding to the clathrin adaptor protein 2 (AP-2) and to endophilin/dynamin. We consider which AMPAR subunits are selected for Arc-mediated internalisation, implications for synaptic function and consider Arc as a therapeutic target. / The work in S.A.L.C. laboratory is supported by the BBSRC (BB/H018344/1 and BB/J02127X/1) and Wellcome Trust 200646/Z/16/Z. The work in M.J.W. Laboratory is supported by ERUK.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23923号 / 工博第5010号 / 新制||工||1782(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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