Domoic acid (DA) is a naturally occurring cyanotoxin, which upon ingestion, is responsible for amnesic shellfish poisoning (ASP) in both humans and animals. Produced by the marine diatom, Pseudonitzschia, DA is accumulated by a number of marine organisms including shellfish, clams and mussels which upon consumption can lead to headaches, nausea and seizures. Possessing a variety of functional groups the structure of DA contains three carboxyl groups, a pyrrole ring and a potent conjugated diene region allowing for binding to glutamate receptors in the dorsal hippocampus of the brain causing the described detrimental effects. Although limitations have been placed regarding the amount of DA that may be contained in seafood no limitations have been placed on the amount present in drinking water. Natural degradation of the toxin may occur through reactive oxygen species such as the hydroxyl radical and singlet oxygen at the conjugated diene region. In this work the photooxidation of DA via singlet oxygen has been studied using sorbic acid as a model compound. The three major reaction pathways observed during the photooxdiation process for both acids include 2 + 4 cycloaddition to produce endoperoxides , 2 + 2 reaction to afford aldehydes and ketones or an ene reaction to generate hydroperoxides. Under similar reaction conditions for SA and DA, the endoperoxide has been seen to be the major product for photoxidation of SA while the hydroperoxide has been seen to be the dominant product during photooxidation of DA.

Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), themineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), influences hippocampalneuron vulnerability to injury. Excessive activation of GR may compromise hippocampalneuron survival after several types of challenge including ischemic, metabolic, and excitotoxicinsults. In contrast, MR prevents adrenalectomy-induced loss of granule neurons in the dentategyrus. The present thesis addresses the respective roles of MR and GR in modulating neuronalsurvival following two forms of neuronal injury, excitotoxicity and traumatic brain injury. MaleSprague-Dawley rats were pretreated with MR antagonist spironolactone or GR antagonistmifepristone (RU486) and subsequently injected with kainic acid, an excitotoxic glutamateanalog, or injured with a controlled cortical impact. Twenty-four hours following injury,hippocampal neuron survival was measured to test the hypotheses that MR blockade wouldendanger and GR blockade would protect hippocampal neurons following injury. MessengerRNA levels of viability-related genes including bcl-2, bax, p53, BDNF, and NT-3 were alsomeasured to test the hypothesis that ACR regulation of these genes wouldcorrelate with neuronal survival. In addition, ACR mRNA levels were measured followingreceptor blockade and injury to test the hypothesis that glucocorticoid signaling is alteredfollowing neuronal injury via regulation of ACR expression.Mineralocorticoid receptor blockade with spironolactone increased neuronal vulnerability toexcitotoxic insult in hippocampal field CA3, and GR blockade with RU486 prevented neuronalloss after traumatic brain injury in field CA1. These results are consistent with the hypothesesthat MR protects and GR endangers hippocampal neurons. Adrenocorticosteroid receptorblockade decreased mRNA levels of the anti-apoptotic gene bcl-2 in select regions of uninjuredhippocampus, yet ACR regulation of bcl-2 did not consistently correspond with measures ofneuronal survival after injury. Kainic acid decreased MR mRNA levels in CA1 and CA3, whileboth kainic acid and controlled cortical impact dramatically decreased GR mRNA levels indentate gyrus. These data suggest that injury modulation of glucocorticoid signaling throughregulation of ACR expression may influence hippocampal neuron viability following injury.

A epilepsia é uma das desordens mais frequente do sistema nervoso central com aproximidamente 2% de prevalência mundial e 30% de refratariedade. O uso das plantas medicinais é uma possibilidade de identificar novas moléculas com potencial terapêutico. Nesse estudo, utilizamos o modelo de convulsão induzida pelo ácido caínico em zebrafish adulto, para explorar o potencial terapêutico da samambaia Blechnum brasiliense Desv. Primeiramente, utilizando cromatografia liquida de ultra eficiência, foi validado método analítico para quantificação do ácido rosmarínio na fração acetato de etila, obtida por fracionamento do extrato bruto da planta. A fração apresentou teor de 0,0764 g% de ácido rosmarínico. A partir desta fração foi realizado isolamento em coluna preparativa para obtenção do composto para testes em peixe-zebra. Nos ensaios toxicológicos em embriões de zebrafish foram avaliados o extrato bruto e a fração acetato de etila de B. brasiliense, bem como ácido rosmarínico isolado, possibilitando o cálculo das doses letais médias (DL50). Os valores foram 363,3 μg/mL para o extrato, 196,3 μg/mL para a fração e 250,3 μM para o ácido rosmarínico. Os resultados demonstraram que as amostras apresentaram toxicidade aos embriões apenas nas maiores concentrações testadas. Nos ensaios realizados com zebrafish adultos, todas as amostras foram capazes de modular o perfil e a intensidade da convulsão causada pelo ácido caínico, bem como a latência para a primeira crise clônica. As doses ativas foram: 20 mg/kg para o extrato bruto, 5 e 10 mg/kg para a fração acetato de etila e 1 mg/kg para o ácido rosmarínico, sendo capazes de reduzir as crises e aumentar o tempo de latência no modelo de epilepsia em zebrafish. Assim, nosso estudo indica a espécie Blechnum brasiliense como fonte de substância com potencial terapêutico a ser explorada para o tratamento da epilepsia. / Epilepsy is one of the most frequent disorders of the central nervous system with approximately 2% of world prevalence and 30% of refractoriness. The use of medicinal plants is a possibility to identify new molecules with therapeutic potential. In this study, we used the caynic acid-induced convulsion model in adult zebrafish to explore the therapeutic potential of the Blechnum brasiliense Desv fern. Firstly, using ultra-efficient liquid chromatography, we validated an analytical method for the quantification of rosmarinic acid in the ethyl acetate fraction, obtained by plant crude extract fractionation. The fraction presented a content of 0.0764 g% of rosmarinic acid. From this fraction was carried out the preparative column isolation of the compound for tests in zebrafish. In the toxicological tests using zebrafish embryos, were evaluated the crude extract and the ethyl acetate fraction of B. brasiliense, as well as, the isolated rosmarinic acid, allowing the calculation of median lethal doses (LD50). The values were 363.3 μg/mL for the extract, 196.3 μg/mL for the fraction and 250.3 μM for the rosmarinic acid. The results showed that samples presented toxicity to the embryos only in the highest concentrations tested. In the trials with adult zebrafish, all samples were able to modulate the profile and intensity of the convulsion caused by kainic acid, as well as, the latency for the first clonic crisis. The active doses were 20 mg/kg for the crude extract, 5 and 10 mg/kg for the ethyl acetate fraction, and 1 mg/kg for rosmarinic acid, being able to reduce the crises and increase the latency time in the zebrafish model of epilepsy. Thus, our study indicates the species Blechnum brasiliense as a source of substance with therapeutic potential to be explored for the treatment of epilepsy.

Nrf2 project: The protein nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that provides protection against oxidative stress and the dysfunction of this pathway has been suggested to be implicated in many neurodegenerative diseases. The aim of this thesis was to identify novel Nrf2 activators that disrupt the protein-protein interaction between Nrf2 and Keap1 and thereby induce increased expression of antioxidant enzymes and protective genes. The crystal structure of the Keap1-Nrf2 interface was used to perform a virtual screen and compounds from the screen were assayed using a cellular nuclear complementation assay that measures the nuclear translocation of Nrf2 from the cytosol. Although two novel compounds were found to increase the Nrf2 nuclear translocation, they had low activity and further characterisation did not provide sufficient evidence of a Nrf2-Keap1 robust interaction. iGluRs project: AMPA and kainate receptors are ionotropic glutamate receptors (iGluRs) that are important for excitatory transmission and synaptic plasticity and are linked to several neurological disorders such as epilepsy, schizophrenia and autism. This project aimed to find novel allosteric modulators binding in the ligand-binding domain (LBD) of the GluA2 and GluK1 and GluK2 subtypes of AMPA and kainate receptors, respectively, using protein purification and X-ray crystallography methodologies. Fragment screening for GluA2 identified eight novel fragments, five of which were located at the dimer interface and three located in a novel site near the glycine-threonine dipeptide linker. As regards kainate receptors, structural information on the Gluk1 and GluK2 LBD was obtained, both proteins were soaked with in-house fragments with one compound displaying 20% occupancy in the GluK2 dimer interface. These data form the basis of future studies in the search for novel drugs for the treatment of epilepsy and schizophrenia.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

A epilepsia é uma das desordens mais frequente do sistema nervoso central com aproximidamente 2% de prevalência mundial e 30% de refratariedade. O uso das plantas medicinais é uma possibilidade de identificar novas moléculas com potencial terapêutico. Nesse estudo, utilizamos o modelo de convulsão induzida pelo ácido caínico em zebrafish adulto, para explorar o potencial terapêutico da samambaia Blechnum brasiliense Desv. Primeiramente, utilizando cromatografia liquida de ultra eficiência, foi validado método analítico para quantificação do ácido rosmarínio na fração acetato de etila, obtida por fracionamento do extrato bruto da planta. A fração apresentou teor de 0,0764 g% de ácido rosmarínico. A partir desta fração foi realizado isolamento em coluna preparativa para obtenção do composto para testes em peixe-zebra. Nos ensaios toxicológicos em embriões de zebrafish foram avaliados o extrato bruto e a fração acetato de etila de B. brasiliense, bem como ácido rosmarínico isolado, possibilitando o cálculo das doses letais médias (DL50). Os valores foram 363,3 μg/mL para o extrato, 196,3 μg/mL para a fração e 250,3 μM para o ácido rosmarínico. Os resultados demonstraram que as amostras apresentaram toxicidade aos embriões apenas nas maiores concentrações testadas. Nos ensaios realizados com zebrafish adultos, todas as amostras foram capazes de modular o perfil e a intensidade da convulsão causada pelo ácido caínico, bem como a latência para a primeira crise clônica. As doses ativas foram: 20 mg/kg para o extrato bruto, 5 e 10 mg/kg para a fração acetato de etila e 1 mg/kg para o ácido rosmarínico, sendo capazes de reduzir as crises e aumentar o tempo de latência no modelo de epilepsia em zebrafish. Assim, nosso estudo indica a espécie Blechnum brasiliense como fonte de substância com potencial terapêutico a ser explorada para o tratamento da epilepsia. / Epilepsy is one of the most frequent disorders of the central nervous system with approximately 2% of world prevalence and 30% of refractoriness. The use of medicinal plants is a possibility to identify new molecules with therapeutic potential. In this study, we used the caynic acid-induced convulsion model in adult zebrafish to explore the therapeutic potential of the Blechnum brasiliense Desv fern. Firstly, using ultra-efficient liquid chromatography, we validated an analytical method for the quantification of rosmarinic acid in the ethyl acetate fraction, obtained by plant crude extract fractionation. The fraction presented a content of 0.0764 g% of rosmarinic acid. From this fraction was carried out the preparative column isolation of the compound for tests in zebrafish. In the toxicological tests using zebrafish embryos, were evaluated the crude extract and the ethyl acetate fraction of B. brasiliense, as well as, the isolated rosmarinic acid, allowing the calculation of median lethal doses (LD50). The values were 363.3 μg/mL for the extract, 196.3 μg/mL for the fraction and 250.3 μM for the rosmarinic acid. The results showed that samples presented toxicity to the embryos only in the highest concentrations tested. In the trials with adult zebrafish, all samples were able to modulate the profile and intensity of the convulsion caused by kainic acid, as well as, the latency for the first clonic crisis. The active doses were 20 mg/kg for the crude extract, 5 and 10 mg/kg for the ethyl acetate fraction, and 1 mg/kg for rosmarinic acid, being able to reduce the crises and increase the latency time in the zebrafish model of epilepsy. Thus, our study indicates the species Blechnum brasiliense as a source of substance with therapeutic potential to be explored for the treatment of epilepsy.

A epilepsia é uma das desordens mais frequente do sistema nervoso central com aproximidamente 2% de prevalência mundial e 30% de refratariedade. O uso das plantas medicinais é uma possibilidade de identificar novas moléculas com potencial terapêutico. Nesse estudo, utilizamos o modelo de convulsão induzida pelo ácido caínico em zebrafish adulto, para explorar o potencial terapêutico da samambaia Blechnum brasiliense Desv. Primeiramente, utilizando cromatografia liquida de ultra eficiência, foi validado método analítico para quantificação do ácido rosmarínio na fração acetato de etila, obtida por fracionamento do extrato bruto da planta. A fração apresentou teor de 0,0764 g% de ácido rosmarínico. A partir desta fração foi realizado isolamento em coluna preparativa para obtenção do composto para testes em peixe-zebra. Nos ensaios toxicológicos em embriões de zebrafish foram avaliados o extrato bruto e a fração acetato de etila de B. brasiliense, bem como ácido rosmarínico isolado, possibilitando o cálculo das doses letais médias (DL50). Os valores foram 363,3 μg/mL para o extrato, 196,3 μg/mL para a fração e 250,3 μM para o ácido rosmarínico. Os resultados demonstraram que as amostras apresentaram toxicidade aos embriões apenas nas maiores concentrações testadas. Nos ensaios realizados com zebrafish adultos, todas as amostras foram capazes de modular o perfil e a intensidade da convulsão causada pelo ácido caínico, bem como a latência para a primeira crise clônica. As doses ativas foram: 20 mg/kg para o extrato bruto, 5 e 10 mg/kg para a fração acetato de etila e 1 mg/kg para o ácido rosmarínico, sendo capazes de reduzir as crises e aumentar o tempo de latência no modelo de epilepsia em zebrafish. Assim, nosso estudo indica a espécie Blechnum brasiliense como fonte de substância com potencial terapêutico a ser explorada para o tratamento da epilepsia. / Epilepsy is one of the most frequent disorders of the central nervous system with approximately 2% of world prevalence and 30% of refractoriness. The use of medicinal plants is a possibility to identify new molecules with therapeutic potential. In this study, we used the caynic acid-induced convulsion model in adult zebrafish to explore the therapeutic potential of the Blechnum brasiliense Desv fern. Firstly, using ultra-efficient liquid chromatography, we validated an analytical method for the quantification of rosmarinic acid in the ethyl acetate fraction, obtained by plant crude extract fractionation. The fraction presented a content of 0.0764 g% of rosmarinic acid. From this fraction was carried out the preparative column isolation of the compound for tests in zebrafish. In the toxicological tests using zebrafish embryos, were evaluated the crude extract and the ethyl acetate fraction of B. brasiliense, as well as, the isolated rosmarinic acid, allowing the calculation of median lethal doses (LD50). The values were 363.3 μg/mL for the extract, 196.3 μg/mL for the fraction and 250.3 μM for the rosmarinic acid. The results showed that samples presented toxicity to the embryos only in the highest concentrations tested. In the trials with adult zebrafish, all samples were able to modulate the profile and intensity of the convulsion caused by kainic acid, as well as, the latency for the first clonic crisis. The active doses were 20 mg/kg for the crude extract, 5 and 10 mg/kg for the ethyl acetate fraction, and 1 mg/kg for rosmarinic acid, being able to reduce the crises and increase the latency time in the zebrafish model of epilepsy. Thus, our study indicates the species Blechnum brasiliense as a source of substance with therapeutic potential to be explored for the treatment of epilepsy.

Kainic acid has been used for nearly 50 years as a tool in neuroscience due to its pronounced neuroexcitatory properties. However, the significant price increase of kainic acid resulting from the disruption in the supply from its natural source, the alga Digenea Simplex, as well as inefficient synthesis of kainic acid, call for the exploration of functional mimics of kainic acid that can be synthesized in a simpler way. Aza kainoids analog could be one of them. The unsubstituted aza analog of kainoids has demonstrates its ability as an ionotropic glutamate receptor agonist and showed affinity in the chloride dependent glutamate (GluCl) binding site. This opened a question of the importance of the presence of one nitrogen or both nitrogens in the aza kainoid analogs for binding to glutamate receptors. Therefore, two different pyrrolidine analogs of kainic acid, trans-4-(carboxymethyl)pyrrolidine-3-carboxylic acid and trans-2-carboxy-3-pyrrolidineacetic acid, were synthesized through multi-step sequences. The lack of the affinity of both pyrrolidine analogs in GluCl binding site indicated that both nitrogens in aza kainoid analogs are involved in hydrogen bonding with receptors, significantly enhancing their affinity in GluCl binding site. Another potential functional mimic of kainic acid is isoxazolidine analogs of kainoids whose skeleton can be constituted directly via a 1, 3 dipolar cycloaddition as the key step. The difficulty in synthesizing N-unsubstituted isoxazolidines when applying such common protecting groups as alkyl, phenyl and benzyl groups, and the requirement of a desired enantioselectivity due to the three chiral ceneters in kainic acid, pose great challenges. Hence, several different protected nitrones were studied to establish that diphenylmethine nitrone may be a good candidate as the dipole in that the generated isoxazolidines can be deprotected in mild conditions with high yields. Our investigations also indicated that the exo/endo selectivity of the 1, 3 dipolar cycloaddition can be controlled by Lewis acids, and that the application of a directing group in dipolarophiles can accomplish a satisfied enantioselectivity. Those results demonstrated the synthesis of isoxazoldines analogs of kainic acid is very promising.

For glutamate and the developing optic pathway, glutamate and its ionotropic receptor subunits are expressed widely in retina and ventral diencephalon, and in cells that are related to the chiasm formation. These studies indicate that glutamate may act as a communicator or attractor to coordinate with other factors to affect the retinal axon pathfinding in the prenatal optic pathway. / Furthermore, for the function of glutamate, AMPARs and KARs in the optic chiasm formation, we did retinal explant culture experiment at E14 in vitro, with application of different concentration of L-glutamate (500muM -1mM), AMPAR antagonists: CP465022 hydrochloride (2-20muM) and GYK15466 dihydrochloride (25-150muM), and KAR antagonists: CNQX (50-500muM) and UBP301 (5-25muM). The results show that L-glutamate promotes retinal axon outgrowth; AMPA receptor antagonists inhibit that; and KAR antagonists have no effect on that. In the presence of different combinations of ionotropic receptor antagonists (including NMDAR antagonist), they suggest that the blockage of glutamate iontroptic receptors displays an obvious effect of inhibiting neurite outgrowth in E14 retinal explants. However, inhibiting kainate receptors show little effect on retinal neurite outgrowth which is different from that of blocking AMPARs. We also did E13 and E15 brain slice culture experiments, and found that blocking of glutamate ionotropic receptors affects crossed axon projection in the midline at early stage, but has no effect to the uncrossed one. / Glutamate is the dominant amino acid neurotransmitter in the central nervous system naturally occurring in the L-form. Glutamate ionotropic receptors can be further a-amino-3-hydroxy-5-methy1-4-isoxazole-propionate divided into three types by their ligand (AMPA, specificities: GluR1-4), N-methyl-D-aspartate (NMDA, NR1-3) and kainate (KA, GluR5-7 and KA1-2) receptors, which function as ligand-gated ion channels. In this study, we focus on the AMPARs and KARs which are expressed in the developing brain. / Here, we used semi-quantitative RT-PCR to analyze mRNA expression levels of AMPAR and KAR subunits in the mouse retina and ventral diencephalons at different developmental stages, and in adult retina. The results show that both AMPAR and KAR subunits can be detected in retina and ventral diencephalon at as early as E13. We also used specific antibodies to investigate glutamate, AMPAR and KAR subunit expression in the mouse retinofugal pathway. We found that: 1) Glutamate is expressed at as early as E13. In retina, it tends to localize in retinal ganglion cells (RGCs) and their axons; in ventral diencephalon, it is most intense in optic stalk, optic chiasm and optic tract. It is also localized with chiasmatic neurons, which are related to the formation of optic chiasm. 2) For the individual AMPAR and KAR subunits, all of them are expressed at as early as E13. The immunoreactive GluRl and GluR5/6/7 are distributed preferentially in the RGCs and their axons; the staining of GluR2/3 and GluR4 are largely found in RGCs and the supporting cells around the pathway, but for GluR4, its staining is weakly detected in optic fibers and strongly in the midline of chiasm. Although the staining patterns of these specific subunits are different, they are all localized in chiasmatic neurons in diencephalon. / Cheng, Xiaojing. / "November 2010." / Adviser: Sun On Chan. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.