Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-02-09T17:43:43Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_IntensificacaoComportamentoAnsiedade.pdf: 965220 bytes, checksum: a80564a45b42482d8611ce52d7e7f125 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-02-10T12:19:50Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_IntensificacaoComportamentoAnsiedade.pdf: 965220 bytes, checksum: a80564a45b42482d8611ce52d7e7f125 (MD5) / Made available in DSpace on 2015-02-10T12:19:50Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_IntensificacaoComportamentoAnsiedade.pdf: 965220 bytes, checksum: a80564a45b42482d8611ce52d7e7f125 (MD5) Previous issue date: 2014 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O crescente consumo de bebidas com elevado teor de cafeína pode resultar no aparecimento de sintomas provenientes do transtorno de ansiedade induzida por essa droga. Atualmente, tem-se utilizado a cafeína como um indutor farmacológico do comportamento tipo ansiedade e essa indução pode facilitar a melhor compreensão da relação entre alterações comportamentais e os mecanismos de ação envolvidos nesse efeito, portanto o presente trabalho propôs que a via nitrérgica poderia ser um mecanismo chave para explicar os efeitos comportamentais produzidos pela cafeína e que esses efeitos poderiam ser revertidos por um antioxidante, logo, no presente trabalho nós tivemos como objetivo avaliar o possível efeito do L-NAME e do α-tocoferol no comportamento tipo ansiedade ampliado pela cafeína nos testes de preferência claro/escuro (PCE) e distribuição vertical eliciada pela novidade (DVN) em Daniorerio. Foram utilizados peixes da espécie Daniorerio(n=178) subdivididos nos seguintes grupos experimentais: SAL – salina 0,9%; CAF – cafeína 100 mg/kg; DMSO – dimetilsulfóxido 0,1%; L-NAME - (N -Nitro-L-arginina-metil éster hidrocloreto) 10 mg/kg; TF – α-tocoferol 1 mg/kg (receberam apenas uma injeção por i.p); SAL + SAL; DMSO + SAL; SAL + CAF; L-NAME + SAL; L-NAME +CAF; TF + CAF (receberam duas injeções seguidas, uma injeção de cada substância na forma de cotratamento, por i.p). Os animais foram submetidos ao teste de preferência claro/escuro e de distribuição vertical eliciada pela novidade. Todos os testes foram filmados e os vídeos foram avaliados utilizando o X-PLO-RAT. Os dados foram expressos em média ± erro padrão. Foi aplicado o teste de normalidade utilizando o teste Shapiro-Wilk e o teste paramétrico ANOVA de uma via com pós-teste Tukey, considerando significativos valores com p<0,05. Nós demonstramos que o α-tocoferol na dose de 1 mg/kg reverteu todos os parâmetros do comportamento tipo ansiedade ampliado pela cafeína nos testes de PCE e do DVN e esse efeito foi semelhante ao observado quando administrado um inibidor da enzima óxido nítrico sintase (NOS), L-NAME. Portanto, o presente trabalho demonstrou pela primeira vez que o efeito comportamental ampliado pela cafeína no teste escotáxico e no DVN pode ser modulado pelo sistema nitrérgico e que o α-tocoferol reverte esse efeito comportamental induzido pela cafeína de forma total. / The growing consumption of beverages with high caffeine content can result in the appearance of symptoms from anxiety disorder induced by this drug. Currently, it has been used as a pharmacological caffeine inductor anxiety-like behavior and this induction may help facilitate better understanding of the relationship between behavioral changes and the mechanisms involved in this effect. Therefore, this study proposed that the nitrergic pathway could be a key mechanism to explain the behavioral effects produced by caffeine and that these effects could be reversed by an antioxidant, hence, the present work we had to evaluate the possible effect of L –NAME and - tocopherol expanded in anxiety-like behavior by caffeine in light/dark preference (LDP) test and novel tank dividing (NTD) test in Daniorerio. Zebrafish fish species (N=178) used were divided into the following experimental groups: SAL - 0.9% saline; CAF - Caffeine 100 mg/kg; DMSO - dimethylsulfoxide 0.1% ; L-NAME - (N -nitro -L - arginine methyl ester hydrochloride) 10 mg/kg; TF - -tocopherol 1 mg/kg (received only one injection by i.p.); SAL + SAL; DMSO + SAL; SAL + CAF , L-NAME + SALT , L-NAME + CAF ; TF + CAF, received two followed injections, one injection of each substance in the form of co-treatment, by i.p. The animals were submitted to the light/dark preference test and novel tank dividing test. All tests were filmed and the videos were evaluated using X-PLO-RAT. Data were expressed as mean ± SEM. The normality test was applied using the Shapiro-Wilk test and the ANOVA parametric test one-way with Tukey post-hoc, with the significance level set at p<0.05. The - tocopherol at a dose of 1 mg/kg, reversed all anxiety-like behavior parameters expanded by caffeine in LDP and NTD tests and this effect was similar to that observed when given one inhibitor of the enzyme nitric oxide synthase (NOS), L- NAME. Therefore, this study first demonstrated that the behavioral effect magnified by caffeine in scotaxis test and DVN test can be modulated by the nitrergic pathway and that the -tocopherol reverses completely this anxiety-like behavioral effect induced by caffeine.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-03-22T12:39:55Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_SensibilizacaoDependenteTempo.pdf: 3802322 bytes, checksum: 4d551ae62b6172f1523a4f782dd2f542 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-03-24T16:45:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_SensibilizacaoDependenteTempo.pdf: 3802322 bytes, checksum: 4d551ae62b6172f1523a4f782dd2f542 (MD5) / Made available in DSpace on 2017-03-24T16:45:16Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_SensibilizacaoDependenteTempo.pdf: 3802322 bytes, checksum: 4d551ae62b6172f1523a4f782dd2f542 (MD5) Previous issue date: 2015-08-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Transtorno de Estresse Pós-Traumático (TEPT) é classificado como um transtorno relacionado ao trauma e a estressores, um conjunto de doenças neuropsiquiátricas severamente debilitantes que se caracterizam por uma desregulação de respostas de estresse após um evento traumático. O paulistinha (Danio rerio Hamilton 1822) tem emergido como um modelo importante para o estudo de funções genéticas, neurofarmacológicas e comportamentais, como no estudo sobre ansiedade e estresse. O óxido nítrico (NO) é um gasotransmissor que parece ter um papel importante na regulação de respostas neurocomportamentais ao estresse, inclusive no paulistinha. É diante deste cenário que propomos um modelo comportamental para TEPT, com a avaliação da sensibilização dependente de tempo do comportamento do paulistinha em decorrência da exposição à substância de alarme co-específica (SA) – um potente estressor. Com esse modelo, verificaremos o papel do sistema nitrérgico nesse processo de sensibilização. Os animais serão expostos à SA e mantidos livres de estresse por 24 h; após esse período, o comportamento dos animais será analisado. Realizaremos 5 experimentos que visam investigar: i) o efeito atrasado da substância de alarme sobre diferentes tarefas comportamentais em paulistinhas, ii) a comparação da sensibilização dependente de tempo nos fenótipos shortfin e longfin, iii) a aplicação de Critérios Comportamentais de Corte na sensibilização dependente de tempo, iv) a quantificação de glutamato extracelular e nitrito tecidual no telencéfalo após exposição à substância de alarme, e v) Participação do NO na iniciação e consolidação da sensibilização dependente de tempo. Nossos resultados revelaram que: i) a substância de alarme produz sensibilização atrasada da ansiedade (aumento da geotaxia, diminuição da habituação, aumento do nado errático, aumento da frequência de thrashing no teste de distribuição vertical eliciada pela novidade; diminuição do tempo no branco, aumento do nado errático, avaliação de risco e tigmotaxia, no teste de preferência por escuridão) e hiperexcitação (aumento da distância percorrida na primeira tentativa e a inclinação da habituação no teste de reatividade de sobressalto). ii) em relação aos animais shortfin, a exposição de animais longfin produziu maior sensibilização do tempo no compartimento branco, da avaliação V de risco e da tigmotaxia, enquanto os animais shortfin apresentaram frequência de nado errático maior. iii) 25,74% dos animais que foram expostos à SA alcançaram o critério de Resposta Comportamental Extrema (RCE) e 20% atingiram o critério para Resposta Comportamental Mínima (RCM); em animais não-expostos, apenas 4% alcançaram o critério de RCE e 96% alcançaram o critério de RCM. Animais classificados como RCE dispenderam menos tempo no compartimento branco, com entradas de menor duração, maior tigmotaxia e mais nado errático em relação a animais classificados como RCM e controles não-expostos; iv) o tratamento com L-NAME 30 minutos antes da exposição à SA não bloqueou a sensibilização comportamental no teste de preferência por escuridão; v) o tratamento com L-NAME 30 minutos após a exposição à SA bloqueou a sensibilização da escototaxia e da avaliação de risco; vi) o tratamento com L-NAME 90 minutos após a exposição à SA bloqueou a sensibilização da avaliação de risco, nado errático e tigmotaxia. Esses resultados sugerem que a sensibilização dependente de tempo no paulistinha pode ser um bom modelo para estudo do TEPT e apontam o NO com um importante mediador nesse processo. / Post-traumatic stress disorder (PTSD) is classified as a trauma- and stressor-related disorder, a set of severely debilitating neuropsychiatric disorders characterized by the disregulation of stress responses after a traumatic event. Zebrafish (Danio rerio Hamilton 1822) emerged as an important model organism for the study of genetic, neuropharmacological and behavioral functions, such as the study of anxiety and stress. Nitric oxide (NO) is a gaseous transmitter that appears to have an important role in the regulation of neurobehavioral responses to stresss, including in zebrafish. In this scenario, we propose a behavioral model for PTSD in the evaluation of the time-dependent sensitization of behavior in zebrafish as a consequence of the exposure to conspecific alarm substance (AS) – a potent stressor. Using this model, we will verify the role of the nitrergic system in this process of sensitization. Animals will be exposed to AS and kept stress-free for 24 h; after this interval, animals' behavior will be analyzed. 5 experiments will be made to investigate: i) the delayed effect of alarm substance on different behavioral tasks in zebrafish, ii) a comparison of time-dependent sensitization on shortfin and longfin phenotypes; a comparação da sensibilização dependente de tempo nas linhagens shortfin e longfin, iii) the application of Behavioral Cutoff Criteria on timedependent sensitization, iv) the quantification of extracellular glutamate and tissue nitrite in the telencephalon after exposure to alarm substance, and v) the participation of NO on the initiation and consolidation of time-dependent sensitization. Our results revealed that: i) alarm substancce produces a delayed sensitization of anxiety (increased geotaxis, decreased habituation, increased erratic swimming and thrashing in the novel tank test; decreased time on white, increased erratic swimming, risk assessment and thigmotaxis on the ligh/dark test) and arousal (increased swim distance on the first trial and increased habituation slope in the startle reactivity test). ii) In relation to shortfin animals, exposure of longfin zebrafish to AS sensitized time on white, risk assessment and thigmotaxis more, while shortfin animals had more erratic swimming. Iii) 25.74% of AS-exposed animals reached criteria for Extreme Behavioral Response (EBR), and 20% reached criteria for Minimal Behavioral Response VII (MBR); in non-exposed animals, only 4% reached criteria for EBR and 96% reached criteria for MBR. Animals classified as EBR spent less time in the white compartment, with shorter entries, more thigmotaxis and more erratic swimming than animals classified as MBR and non-exposed controls. iv) treatment with L-NAME 30 minutes before AS exposure did not block the behavioral sensitization in the light/dark test; v) treatment with L-NAME 30 minutes after AS exposure blocked the sensitization of scototaxis and risk assessment; vi) treatment with L-NAME 90 minutes after AS exposure blocked the sensitization of risk assessment, erratic swimming and thigmotaxis. Theses results suggest that time dependent sensitization is a good model to study PTSD and point to NO as a important mediator in this process.

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation during the early stages of embryogenesis, that can be detected with the mosaic eye assay at 48-72 hpf. Using published genome and mapping data, several candidate genes for two of the gin mutations were identified and studied during early zebrafish development. The gin mutations are heritable, ENU-induced, and have both maternal and zygotic effects during zebrafish development. The first aim of this project was to study the normal gene characteristics of the gin-10 candidate genes, synbl, rfx4, and sir2 that are located on chromosome 18. Semi-quantitative RT-PCR, whole-mount in situ hybridization, and gene knockdown (using morpholino oligonucleotides) techniques were utilized in both wildtype and transgenic (Tg-synbl) zebrafish lines to gain an understanding of the function of each of these genes during zebrafish embryogenesis. Additionally, the synbl paralog, ric8a, was also explored, as it has been implicated in the control of asymmetric cell division in C. elegans. Single gene knockdowns were performed for each candidate in the golden heterozygous (pigment mutant) zebrafish background to test for genomic instability activity. Genomic instability activity was not observed, however the results showed that these genes are expressed throughout zebrafish embryogenesis, and are necessary for the proper development of the central nervous system, notochord and tail, as well as metabolic functions in the early embryo. Moreover, the transgenic line used for the paralog studies of synbl and ric8a was incorrectly genotyped. Using PCR analysis and sequencing, it was found that the viral insert for the Tg-synbl fish was disrupting the cry1b gene on an adjacent contig. The second aim focused on the gin-12 region on chromosome 4, where the mdm1 gene is located. Originally cloned from a transformed mouse cell line with mdm2, the function of the mdm1 gene in these cells or during development had not yet been identified. To allow the Mdm1 protein to be evaluated, custom antibodies targeting Mdm1 were produced and the detection of Mdm1 optimized in zebrafish embryos. This would allow us to then determine whether Mdm1 was a possible regulator of the p53-Mdm2/Mdm4 pathway. Additionally, the mdm1 gene was studied in situ and in vivo to determine the normal gene expression patterns and developmental role in the embryonic zebrafish. Moreover, this gene was also studied in the golden heterozygous zebrafish line to assess whether it had a role in modulating genomic instability activity using the mosaic eye assay. Collectively, morpholino oligonucleotides, RNA rescue, whole-mount antibody staining, and overexpression studies suggest that the mdm1 gene is involved in the development of the eye and portions of the central nervous system, but did not appear to be the gin-12 mutant. While the genes in this study did not appear to have genomic instability activity in the embryonic zebrafish based on the mosaic eye assay in the golden heterozygotes, normal developmental gene expression patterns were identified for synbl, ric8a, rfx4, sir2, and mdm1 in wildtype zebrafish embryos. Additional information was gained by the reverse genetic studies using gene knockdowns, which identified the functional roles of these genes at various stages of embryogenesis. Notably, it was determined that the mdm1 gene may be involved in retinal degenerative diseases based on our studies and recently published data. Future research of the Mdm1 protein should identify protein interactions and the specific role during eye development and retinal diseases.

Made available in DSpace on 2014-12-17T14:02:00Z (GMT). No. of bitstreams: 1 PaulaIM.pdf: 2175384 bytes, checksum: 7697a3aa18d03cba26e4a0d38dbd3b26 (MD5) Previous issue date: 2009-10-05 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Methomyl (Lannate?) is an insecticide from the carbamate group, frequently used in pest control in various types of crops. This compound works inhibiting the activity of the enzyme acetylcholinesterase. The use of physicochemical and ecotoxicological analysis is the most efficient strategy for the correct characterization and control of residues of metomil. The main objectives of this study were to evaluate the acute toxicity of methomyl in 96 hours of exposure and, through a sublethal assay of 5 hours, to assess its effect on the activity of acetylcholinesterase present in brain and squeletic dorsal muscle of the Danio rerio fish. The results showed that the LC50-96 found to D. rerio was 3.4 mg/L and it was found through the average of four definitive tests. In vitro assays were used to test the inhibitory action of methomyl directly over soluble AChE, extracted from the squeletic dorsal muscle, with maximum inhibition of 68.57% to the insecticide concentrations of 0.2 mg/L. In sublethal tests with D. rerio, inhibitory effect of methomyl was found over the soluble form of AChE in the squeletic dorsal muscle, both in one and five hours of fish exposure to the insecticide. In both period, the average values of inhibition were around 61%. In the same condition, no significant inhibitory effect of methomyl soluble and membrane AChE of the D. rerio was observed in the 0.42, 0.85, 1.70 and 2.50 mg/L concentrations and in both times of fish exposure / O Metomil (Lannate?) ? um inseticida do grupo dos carbamatos utilizado no combate a pragas em diversos tipos de lavouras, sua forma de a??o ? atrav?s da inibi??o da enzima acetilcolinesterase. Para uma caracteriza??o adequada e controle de seus despejos, a estrat?gia mais eficiente ? o uso integrado de an?lises qu?micas, f?sicas e ecotoxicol?gicas. O presente trabalho teve por objetivo avaliar a toxicidade aguda em 96h do metomil, e atrav?s de um ensaio subletal de cinco (5) horas, estimar o seu efeito sobre a atividade da acetilcolinesterase no c?rebro e m?sculo esquel?tico dorsal da esp?cie Danio rerio. Os resultados demonstraram que no Danio rerio o valor da CL-50-96h encontrada foi de 3,40 mg/L, obtida atrav?s da m?dia de quatro testes realizados. Nos ensaios in vitro foi atestada a a??o inibit?ria da metomil diretamente sobre a AChE sol?vel extra?da do m?sculo esquel?tico dorsal do D. rerio com uma inibi??o m?xima de 68,57 % para a concentra??o de 0,2 mg/L do inseticida. Nos testes subletais com D. rerio foi constatado o efeito inibit?rio do metomil sobre a forma sol?vel da AChE de m?sculo esquel?tico dorsal, tanto em uma como em cinco horas de exposi??o do peixe ao inseticida. Em ambos os hor?rios os valores m?dios de inibi??o foram em torno de 61%. Nas mesmas condi??es n?o foi observada inibi??o significativa da atividade da AChE de membrana do m?sculos. N?o foi observado efeito inibit?rio do metomil sobre as AChE sol?vel e de membrana de c?rebro de D. rerio, nas concentra??es 0,42, 0,85, 1,70, 2,50 mg/L e em ambos os hor?rios de exposi??o dos peixes

Alzheimer´s disease (AD) is a neurodegenerative disorder responsible for the largest number of cases of dementia in the elderly. The brain regions associated with the frontal cortex and the hippocampus are the most affected by the changes arising from the AD, resulting in loss of neuronal function, and the loss of memory and cognitive ability. In the cellular level, AD is associated with reduced levels of acetylcholine (ACh) in the synaptic process, decreasing cortical cholinergic neurotransmission. ACh is a neurotransmitter found in the brain and at(in the) neuromuscular junctions, forming part of the parasympathetic nervous system, your activity and staying in the synaptic cleft is regulated by hydrolysis catalyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes that modulate the levels of Ach in the nervous system. The outlook for new targets cholinesterase inhibitors has stimulated research using bioactive compounds of natural products and also the use of an animal model that provides better understanding of the mechanisms of cognitive development. Because they are microorganisms that produce secondary given this, metabolites of great importance for bioprospecting, a study of the anticholinesterase activity of compounds isolated from the endophytic fungus Phomopsis sp. (PE) associated with mangabeira (Hancornia speciosa). Furthermore, it was investigated the effect of menthol on the adult zebrafish memory in the passive avoidance test. The menthol was selected taking to account the screening conducted by our research group in previous work, where this compound showed potent anticholinesterase activity. In the search for promising compounds to anticholinesterase drugs, the present study aimed to evaluate the in vitro activity of the crude extract of the fungus, as well as their isolated compounds 5-methyl mellein (1), nectriapyrone (2), tyrosol (3) and tryptophol (4) in AChE and BuChE in vivo evaluation the effect of menthol on the acquisition of learning and memory consolidation. In all assays were obtained encouraging results. The crude extract of PE and its isolated compounds were effective to inhibit cholinesterase, and the tryptophol (4) was the most promising because it showed dual action on the enzymes being selective to BuChE. The study of exposure to menthol zebrafish showed that this compound produces no effect on memory. On the other hand, at all doses tested menthol reverses amnesia induced by scopolamine. This study, therefore, demonstrated the potencial use of these compounds isolated from PE as source of novel agents for the treatment of AD and that menthol may be indicated as therapeutic alternative for the treatment of memory deficits. / A doença de Alzheimer (DA) é uma desordem neurodegenerativa responsável pelo maior número de casos de demência em idosos. As regiões cerebrais associadas ao córtex frontal e ao hipocampo são as mais comprometidas pelas alterações decorrentes da DA, resultando em perda da função neuronal, além de perda progressiva da memória e declínio cognitivo. Em nível celular, a DA está associada à redução das taxas de acetilcolina (ACh) no processo sináptico, diminuindo aneurotransmissão colinérgica cortical. A ACh é um neurotransmissor encontrado no cérebro e nas junções neuromusculares, compondo parte do sistema nervoso parassimpático, sua atividade e permanência na fenda sináptica são reguladas por hidrólise catalisadapela acetilcolinesterase (AChE) e pela butirilcolinesterase (BuChE) enzimas que modulam os níveis de ACh no sistema nervoso. A perspectiva por novos alvos inibidores das colinesterases tem estimulado pesquisas utilizando compostos bioativos de origem natural e também a utilização de modelo animal que proporcione melhor compreensão dos mecanismos do desenvolvimento cognitivo. Diante disso, pelo fato deserem microrganismos produtores de metabólitos secundários de grande importância para bioprospecção, foi realizado um estudo da atividade anticolinesterásica de compostos isolados do fungo endofítico Phomopsis sp. (PE) associado à mangabeira (Hancornia speciosa Gomes). Além disso, foi investigado o efeito do mentol sobre a memória de zebrafishadulto através do teste da esquiva passiva. O mentol foi selecionado tomando-se como base o screening realizado por nosso grupo de pesquisa, onde esse composto apresentou potente atividade anticolinesterásica. Na busca por compostos promissores a fármacos anticolinesterásicos, opresente trabalho teve como objetivo a avaliação in vitroda atividade do extrato bruto do fungo, e de seus compostos isolados 5-metil meleína (1), nectriapirona (2), tirosol (3) e triptofol (4) frente às enzimas AChE e BuChE, assim como a avaliação in vivodo efeito do mentol sobre a aprendizagem e consolidação da memória. O extrato bruto do PE e os seus compostos isolados apresentaram efeito na inibição das colinesterases, sendo o triptofol (4) o mais promissor, pois apresentou ação dual sobre as enzimas sendo seletivo para BuChE. O estudo da exposição de zebrafishao mentol revelou que este composto não produz efeito sobre a memória. Por outro lado, em todas as concentrações testadas o mentol foi capaz de reverter a amnésia induzida por escopolamina. Diante dos resultados, conclui-se que os compostos isolados do PE podem ser considerados como alternativas para o desenvolvimento de novos fármacos anticolinesterásicos e que o mentol pode ser indicado como alternativa terapêutica para tratamento de déficits de memória.

The zebrafish (Danio rerio) promises to meet the growing needs of gerontological and neurobehavioral research by possessing highly conserved anatomy and physiology with all other vertebrates, while having low maintenance costs and requiring only simple care. The neurological and physiological bases of learning, memory formation, and memory retention have been studied in a variety of model organisms, such as the rat, mouse, sheep, and several teleost fishes, the notable example being the zebrafish. Unfortunately, most of these animals are poorly suited to senescence research due to costs, care requirements, or long life spans. My research expands upon our rapidly growing understanding of zebrafish neurobiology, learning processes, sensory modalities, and memory retention. Two pairs of distinct aversive conditioning experiments using classic shuttlebox designs compared the effects of sensory modality and conditioned-unconditioned stimulus (CS-US) intervals, the time delay between application of conditioned sensory stimulus and delivery of the stressful unconditioned stimulus in the event of failure to avoid it, upon memory formation and retention. These studies yielded a general spectrum of results against which future conditioning studies may be compared. Both visual and olfactory stimuli were tested, as were 10 second and 15 second CS-US intervals. Successes were scored when the fish crossed the shuttlebox hurtle within the CS-US interval, thereby avoiding the negative unconditioned stimulus. After a three-month delay, ten additional trials were conducted to compare the long-term memory retention resulting from each protocol. When testing a 15s CS-US interval, olfactory conditioning was significantly more likely (39%) to produce a successful outcome (memory formation) than visual conditioning. Grouped results reveal that the second pair of experiments, each with a 10s CS-US interval, yielded significantly more successful memory formation than a 15s CS-US interval. A significant difference was found when comparing the results of any two experiments, except between the results of the visual and olfactory 10s interval experiments). Only the olfactory experiment using a 15s CS-US interval yielded memory retention results significantly higher than the mean of memory retention results from the four experiments. These findings offer inconclusive evidence supporting olfaction’s strong role in memory formation and retention in zebrafish. The results expand our understanding of the relationship between the olfactory and visual senses and memory in the zebrafish and indicate the olfactory sense’s key role in vertebrate neurobiology, warranting further research into the effects of aging on the olfactory-memory modality.

In fish, maternal stress prior to spawn has been reported to have effects on offspring phenotype. Cortisol, the main glucocorticoid (GC) stress hormone, has been proposed as a potential mediator of such effects because of its organizational role in early teleost development. The present thesis tested whether maternal social stress or treatment with cortisol (as a proxy for maternal stress) prior to spawn affects the cortisol response to stress and anxiety-related behaviours in offspring. In zebrafish (Danio rerio), offspring of dominant females exhibited greater boldness at 6 days post-fertilization (DPF). Interestingly, offspring of females that engaged in social interactions, regardless of the resulting social status of the two females, exhibited greater survival at 1 DPF, a greater fear-related decrease in activity in response to bright light at 6 DPF, and decreased baseline whole-body cortisol content at 0 and 30 DPF. A field experiment with wild largemouth bass (Micropterus salmoides) revealed that maternal cortisol treatment prior to spawn also affected offspring phenotype; offspring of cortisol-treated females had higher masses right after hatch, had greater fear responses, were less bold and less anxious, and exhibited an attenuated cortisol response to an acute stressor. Together, the results of the present thesis suggest that effects of maternal stress prior to spawn on offspring survival, growth, responses to stress, and anxiety-related behaviours are mediated, at least in part, by elevated maternal cortisol but not likely via increased deposition of maternal cortisol into eggs. The effects of maternal stress and cortisol treatment on offspring reported in the present thesis also suggest that maternal stress may prime offspring with adaptive traits to better survive in a stressful environment.

Meis transcription factors belong to the group of TALE (three amino acids loop extension) homeodomain proteins. Meis2 proteins have a potential role in regulation of neural crest cells development and in differentiation of their derivates. Zebrafish genome has two paralogues of meis2 gene, meis2a and meis2b. CRISPR/Cas9 technology was used to prepare mutant lines of both paralogues, meis2a and meis2b, for the purpose of study of function of Meis2 transcription factors. Specific morpholinos that reduce the expression of meis2a and meis2b were used as controls. Craniofacial and cardiac development in mutant fish was analyzed in the meis2a line by RNA in situ hybridization, histological cartilage staining, and computed tomography. While we observed impaired craniofacial and cardiac development after injection of specific Morpholinos, we did not detect similar changes in the meis2a KO line. Our genetic approach has not clearly shown that the meis2a paralogue itself plays an important role in craniofacial development and cardiac development. For more detailed analysis, further experiments on fish lines with combined meis2a and meis2b knock-outs are needed. Key words Mutagenesis CRISPR, Danio rerio, neural crest cells, Meis2, transcription factor

Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...

Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...