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Localização das diferentes formas de GnRH no encefálo de Astyanax altiparanae (Garutti e Britski, 2000) e Danio rerio (Hamilton, 1822). / Localization of different forms of GnRH in the brain of Astyanax altiparanae (Garutti and Britski, 2000) and Danio rerio (Hamilton, 1822).Chayrra Chehade Gomes 22 October 2010 (has links)
O GnRH é um decapeptídeo que está envolvido na reprodução, estimulando a hipófise a liberar gonadotropinas (LH e FSH), as quais regulam a esteroidogênese e gametogênese. Ainda, o GnRH age como neuromodulador atuando no comportamento sexual. A distribuição de suas isoformas pode ajudar a revelar a função específica de cada GnRH. A principal ênfase deste estudo foi detectar a presença e distribuição por imuno-histoquímica, e a expressão gênica de diferentes formas de GnRH no encéfalo de Astyanax altiparanae e Danio rerio, os quais têm importância comercial, ecológica e acadêmica. Em Astyanax altiparanae foi encontrado GnRH3 em diversos corpos celulares, inclusive em corpos celulares ligados à função reprodutiva, juntamente com fibras que inervam a neuro-hipófise. O GnRH1 foi encontrado apenas em fibras. Em Danio rerio foi encontrado GnRH3 nos núcleos da região hipotalâmica e em um grande número de fibras, incluindo as que inervam a neuro-hipófise. A expressão gênica de GnRH2 e 3 foi observada em Danio rerio. / GnRH is a decapeptide involved in reproduction, stimulating the pituitary to release gonadotropins, which, in turn, regulate the steroidogenesis and gametogenesis. In addition to the reproductive function, the GnRH displays neuromodulatory roles with implications in the modulation of sexual behavior. Therefore, the main emphasis of this study is to detect the presence, distribution, and gene expression of different forms of GnRH in the brain of the freshwater teleosts Astyanax altiparanae and Danio rerio, which have commercial, ecological and academic importance. The immunohistochemical method of peroxidase was used to detect GnRHs in the brain and pituitary. In A. altiparanae immunoreactivity to anti-GnRH3 was found in various cell bodies, including those related to reproductive functions, and fibers which innervate the neurohypophysis. Immunoreactivity for GnRH1 was found only in fibers. In D. rerio immunoreactivity to anti-GnRH3 was found in hypothalamic nuclei and in a large number of fibers, including the ones which innervate the neurohypophysis.
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Studies on the interaction of chemicals with cellular efflux transporter proteins Danio rerio Abcb4 and Homo sapiens ABCB1Burkhardt-Medicke, Kathleen 27 February 2018 (has links)
ABCB1, a member of the ATP binding cassette (ABC) transporter family, hydrolyses ATP as energy source for the translocation of substrate chemicals across the cell membrane. ABCB1-like transporters are found in all studied species. Typically, these transporters are abundant in tissues that separate compartments of the body such as the blood-brain barrier. Among the ABC transporters the ABCB1-like transporter proteins are of particular interest because they accept a broad variety of substrates and are therefore able to confer multidrug resistance (MDR) and multixenobiotic resistance (MXR) in wildlife, respectively. Inhibitors of the ABCB1-like transporter function can cause chemosensitisation, i.e. accumulation and increased sensitivity of organisms towards potentially harmful (natural/man-made) ABCB1-like substrate chemicals. In zebrafish (Danio rerio) Abcb4 was identified as functionally homologous to ABCB1.
The aim of this study was to further characterise Danio rerio Abcb4 and to provide a database to approach the question to what extent ABCB1-like transporter related functions/effects are of ecotoxicological relevance. Main objectives are whether and how known ABCB1 ATPase stimulators and inhibitors interact with Abcb4 ATPase activity; to what extent ABCB1 ATPase assay data are transferable to Abcb4 ATPase assay data; and whether and how environmental chemicals interact with Danio rerio Abcb4 ATPase activity.
In this study we established a test system – the ATPase assay with recombinant Danio rerio Abcb4 – to study the interaction of chemicals with the ATPase activity of the transporter protein. To relate obtained data to data for the well-known Homo sapiens ABCB1 and because available data for Homo sapiens ABCB1 were not in all cases suitable for a comparison, the ATPase assay with recombinant ABCB1 was adapted accordingly. Chemicals were tested up to concentrations in the range of their water solubilities to modulate basal and stimulator co-treated Abcb4 and/or ABCB1 ATPase activities. ATPase stimulators are often transported substrates. However, lipophilic compounds stimulating the transporter ATPase activity are not or little transported by transporter action. Therefore, experiments revealing whether compounds are translocated by transporters chemical interference with the transporter protein will not be indicated. Chemicals inhibiting the stimulator (here verapamil) co-treated ATPase activity compete with the verapamil to stimulate ATPase activity or are non-competitive inhibitors. When tested individually, these chemicals can be stimulators or inhibitors of basal ATPase activity, or do not interact with basal ATPase activity. ATPase inhibitors mitigate ATPase activity and ABCB1-like transporter mediated translocation of substrate chemicals. Obtained ATPase assay data were analysed with regard to concentrations at half-maximal effects (EC50s) and effect strengths (percent modulation).
ATPase assays with recombinant Abcb4 (at 27 °C) are comparable to ABCB1 ATPase assay data obtained at 37 °C. Danio rerio Abcb4 seems less temperature-sensitive than ABCB1. Calculated activation energies for Abcb4 ATPase activities (40.75 kJ/mol for basal ATPase activity) were up to half as high as those for ABCB1 ATPase activities (81.61 kJ/mol for basal ATPase activity). Larger activation energies were previously proposed to be indicative for larger conformational rearrangements and hence possibly smaller rearrangements take place in Abcb4 compared to ABCB1. Known standard modulators of Homo sapiens ABCB1 ATPase activity interacted specifically with Danio rerio Abcb4 ATPase actitiy. The EC50s of the tested chemicals – 16 of 17 tested chemiacals interacted with the ABCB1 and the Abcb4 ATPase activity – ranged from 0.09 to 296 µM for ABCB1 and from 0.14 to 171 µM for Abcb4. Qualitative ATPase assay data for ABCB1, as interaction or not, seems transferable to Danio rerio Abcb4. Furthermore, when aligning amino acid sequences of mammalian ABCB1 transporter proteins and Danio rerio Abcb4 and comparing ABCB1 residues known to bind to (lipophilic) chemicals no obvious hints were found that chemical binding to Abcb4 is certainly different from ABCB1. Twenty-five of 33 studied environmental chemicals modulated the Abcb4 ATPase activity as stimulators and/or inhibitors. Stimulation of basal Abcb4 ATPase activity was lower for environmental chemicals than for known standard modulators. EC50s of environmental chemicals ranged from below 10 to 357 µM. Effects by environmental chemicals on Abcb4 ATPase activity with EC50s close to their water solubilities may be rather unspecific.
The results of this work underline that Abcb4 function is of ecotoxicological importance as on the one hand several environmental chemicals were identified to inhibit Abcb4 ATPase activity – likely acting as chemosensitisers, while on the other hand chemicals stimulating basal ATPase activity suggest that these chemicals are possibly transported. A number of environmental chemicals also inhibited the basal Abcb4 ATPase activity. Especially non-transported inhibitors of the basal Abcb4 ATPase activity would be of ecotoxicological relevance as organisms (here Danio rerio) exposed to these chemicals would not be protected by Abcb4 mediated multixenobiotic resistance and were moreover threatened by chemosensitisation.
Future studies should systematically elucidate under which circumstances chemicals are apparently net transported by ABCB1-like transporters and relate these findings to concentrations of environmental chemicals and ABCB1-like transporter protein abundance in wildlife.
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Die Wirkung von Pharmaka und Pestiziden einzeln und in Kombination auf die Embryonalentwicklung des Zebrabärblings (Danio rerio)Kehrer, Anja 19 December 2008 (has links)
Pharmaka werden nach ihrer Einnahme bzw. Verabreichung über verschiedene Pfade in die Umwelt eingetragen. Obwohl Arzneimittel zu den toxikologisch best-untersuchten und -charakterisierten Stoffen gehören, ist ihre Wirkung auf die Umwelt und die darin lebenden Organismen weit weniger gut untersucht. Wenn in der Literatur Daten zur Ökotoxizität vorhanden sind, so beziehen sich diese meist nur auf die Wirkung von Einzelstoffen. In der Umwelt sind die Organismen jedoch gegenüber Mischungen exponiert. Aufgrund der geschilderten Problematik wurden eine Reihe von Arzneimitteln unterschiedlicher Indikationsgruppen einzeln und in Kombination mit dem Embryotest mit dem Zebrabärbling (Danio rerio, DarT) untersucht. Dieses Testsystem wurde durch Schulte & Nagel (1994) als Alternativmethode zum akuten Fischtest nach OECD 203 entwickelt und bietet den Vorteil neben letalen auch eine Reihe von subletalen Endpunkten erfassen zu können. Es handelt sich zudem nach dem deutschen Tierschutzgesetz nicht um einen Tierversuch. Die generelle Vergleichbarkeit der ermittelten Werte mit Daten aus akuten Fischtests nach OECD 203 sowie die Anwendbarkeit für verschiedenste Fragestellungen konnten in einer Reihe von Studien gezeigt werden (Nagel, 2002). Für die hier vorgestellten Untersuchungen wurden zunächst 32 Pharmaka und drei Pflanzenschutzmittel als Einzelstoffe mit dem DarT untersucht. Basierend auf den Ergebnissen der Einzelstofftests wurden Mischungen sowohl aus Substanzen mit ähnlichen als auch unähnlichen Wirkmechanismen getestet. Es zeigte sich, dass unabhängig vom Wirkmechanismus die Mischungstoxizität durch das Konzept der Konzentrationsadditivität gut vorhergesagt wurde, während das Konzept der Unabhängigen Wirkung die Mischungstoxizität unterschätzte. Ebenfalls konnte gezeigt werden, dass die Kombination der Stoffe auf Basis der NOEC, die im DarT anhand der Herzschlagfrequenz nach 48 Stunden ermittelt wird, zu deutlichen Mischungseffekten führt.
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Relating genotoxicity to DNA repair and reproductive success in zebrafish (Danio rerio) exposed to environmental toxicantsReinardy, Helena C. January 2012 (has links)
The potential for environmental toxicants to cause genetic damage (genotoxicity) in organisms is a prominent concern because effects on DNA can compromise reproductive success and survival in organisms. Genotoxicity in male germ cells is of particular concern because damage to DNA in sperm may not be repaired and the consequences of damaged genetic material may be transgenerational (from parent to offspring). An integrated approach across multiple levels of biological organization is necessary to establish linkages between exposure to genotoxicants and subsequent effects at molecular and higher levels of biological organization. This thesis addresses the relation between toxicant-induced genotoxicity and reproductive success in zebrafish, and focuses on a model genotoxicant (hydrogen peroxide) and dissolved metals (radionuclide or non-radioactive forms) under controlled laboratory conditions. Uptake and depuration kinetics of a mixture of radionuclides (54Mn, 60Co, 65Zn, 75Se, 109Cd, 110mAg, 134Cs, and 241Am) were investigated, and radiation dose estimations were computed to link exposure and bioaccumulation with radiation dose. Cobalt (Co, non-radioactive) was selected as an environmentally relevant toxicant for investigation of genotoxicity and effects on reproductive success with a focus on male fish. Chronic exposure (12-d) to 0 – 25 mg l-1 Co resulted in reduced numbers of spawned eggs, lower fertilization success, and reduced survival of larvae to hatching. In male fish, DNA damage was detected in sperm and genes involved in DNA repair (xrcc5, xrcc6, and rad51) were induced in testes from some Co treatments, generally consistent with reduced reproductive success. No change in expression of repair genes in larvae spawned from parents exposed to Co was observed. Overall, results indicate that DNA damage and induction of DNA repair genes can occur rapidly after exposure to genotoxicants and that, if exposure levels are elevated, negative effects on reproduction can occur. Results are considered with particular focus on implications of male genotoxicity on reproductive success and the potential for transgenerational effects of toxicants.
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Efeitos da dominância sobre as dimensões de personalidade em Danio rerio / Effects of dominance on the personality dimensions in zebrafishPINHEIRO, Marcelo de Sena 13 May 2016 (has links)
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Previous issue date: 2016-05-13 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Danio rerio é amplamente utilizado em diversas áreas da pesquisa como modelo experimental, o fenômeno dominância e submissão estão presentes nessa espécie, e relacionasse diretamente com o comportamento de agressão, estes podem estar ligados a dimensões de personalidade que podem ser usados para prever e explicar a conduta de um indivíduo, gerando diferentes perfis de comportamentos sobre determinados aspectos, essas variações de comportamento podem ser reflexo de vários fatores dentre eles diferenças corporais, a disponibilidade de alimento, a pressão de predadores, a competição intra-especifica, entre outros. Para tentar responder se sujeitos dominantes respondem de maneira diferente em situações envolvendo estímulos apetitivos e aversivos, foi realizado uma bateria de testes envolvendo ambos previamente testados para determinar seu estado de dominancia, em seguida foram submetidos aos seguintes testes como de encardumeamento, inspeção de predador e campo aberto. Os resultados desta pesquisa, mostraram que o perfil de dominância em danio rerio, parece estar relacionada a diferenças de padrões comportamentais, quando associados a possíveis parâmetros de aproximação de estímulos aversivos (predador) e estímulos não aversivos (cardume), que podem ser relacionadas a duas grandes dimensões de personalidade, extroversão e neuroticismo. / The zebrafish is widely used in various areas of research as experimental model, dominance and submission phenomenon are present in this species, and relate directly with the aggressive behavior, these can be linked to personality dimensions that can be used to predict and explain the conduct of an individual, creating different profiles of behaviors on certain aspects, these behavioral changes may reflect several factors, among them bodily differences, the availability of food, the pressure of predators, the intraspecific competition, among others. To try to answer whether dominant subjects respond differently in situations involving appetitive and aversive stimuli, was performed a battery of tests involving both previously tested to determine its state of dominance, then were subjected to the following tests as scooling, predator inspection and open field. These results showed that the dominance profile in zebrafish, appears to be related to differences in the behavioral patterns when associated with possible parameter approximation of aversive stimuli (predator) and not aversive stimuli (shoals), which may be related to two large personality, extroversion and neuroticism.
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A intensificação do comportamento tipo ansiedade induzido por cafeína em Daniorerio(zebrafish) é prevenida pelo tratamento com α-Tocoferol e L-NAMECARVALHO, Tayana Silva de 23 January 2015 (has links)
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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.
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Sensibilização dependente de tempo em paulistinhas adultos como modelo de transtorno de estresse pós-traumático: papel do óxido nítricoLIMA, Monica Gomes 11 August 2015 (has links)
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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.
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From DNA to Protein: a study of genomic instability candidate genes during zebrafish developmentGriffett, Kristine 01 January 2011 (has links)
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.
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Avalia??o da toxicidade aguda do inseticida metomil e o seu efeito sobre a atividade da acetilcolinesterase do peixe Danio rerioSantos, Paula Ivani Medeiros dos 05 October 2009 (has links)
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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
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Desenvolvimento de ferramentas biotecnológicas a partir de recursos naturais para o tratamento da doença de AlzheimerSantos, Rosiane dos 14 February 2013 (has links)
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.
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