Made available in DSpace on 2016-06-02T19:29:00Z (GMT). No. of bitstreams: 1 TeseRF.pdf: 4503749 bytes, checksum: 9ddaa154f7e50a29fcda9669257e2a6e (MD5) Previous issue date: 2006-06-05 / In order to evaluate the physical, chemical, biological and ecotoxicological parameters of Monjolinho River system (in laboratory and in situ), water and sediment samples (from the river and from eight of its tributaries) we obtained in july/03, October/03, January/04 and April/04. Juveniles and adult individuals of Danio rerio and Poecilia reticulata were used in the ecotoxicological studies. Mortality, juvenile biometry, and deletery effects observed on the gills tissue were considered in the tests. The limnological data revealed the higher concentrations of heavy metals and organochlorides in the dry season, being over the limits established for aquatic organisms protection. The concentrations of zinc and heptachlore found in the sediments of the last study sites resulted in deletery effects. The higher concentrations of the analyzed parameters were found in the study sites UFSCar, Confluência and Córrego Tijuco, probably due to the higher concentrations of clay and silt found in these areas. The partially chronic tests performed using samples obtained from the natural environment revealed significant toxicity for at least one fish species and one parameter. Tests in situ showed more drastic effects, especially in July/03. P. reticulata was more vulnerable when compared to D. rerio in all of the analyses, especially considering the juvenile stages. Two contaminants were used in the laboratory ecotoxicological analyzes, using D. rerio juveniles as a test organism. Cadmium showed a CL(I)50, 96h, of 1894,73µg/L. In the tests of static partial chronic toxicity, the chronic value obtained was 189,73µg/L, for both survival and growth, indicating that in the same concentration, the effects on the survival and biometry were similar. Gills morphological alterations increased with the increasing concentrations: 0.6 µg/L, 6 µg/L, 60 µg/L and 600 µg/L, being the fusion of the secondary lamellaes observed in the last concentration. Regarding to the endossulfan sulphate, the semi-static CL(I)50, 96h, was 7.24 µg/L and the chronic value, considering survival rates, was 0.948 µg/L. Histological effects were observed in all of the concentrations (0.03 µg/L, 0,3 µg/L and 3 µg/L), being the hyperplasia and secondary lamellae fusions more drastic in the last concentration. The toxicity tests, as well as the limnological analyses, showed that the Monjolinho system in highly disturbed, with inadequate conditions for the conservation of the aquatic organisms and may represent risks for the human health. / Com o objetivo de avaliar as características físicas, químicas, biológicas e ecotoxicologicas (laboratoriais e in situ) da água e sedimento, do sistema Monjolinho, foram realizadas coletas em julho/03, outubro/03, janeiro/04 e abril/04 (rio e 8 tributários). Na avaliação ecotoxicológica, utilizouse Danio rerio e P. reticulata, juvenis e adultos. A mortalidade, biometria final (dos juvenis) e efeitos deletérios nos tecidos branquiais foram as variáveis consideradas em testes. Os resultados limnológicos revelaram que, em geral, o período seco apresentou as maiores concentrações das diferentes variáveis analisadas, incluindo-se os metais e organoclorados, acima dos limites recomendados para proteção da vida aquática. Nos sedimentos, o zinco e o heptacloro apresentaram-se em concentrações capazes de provocar efeitos deletérios nos organismos biológicos, nos últimos pontos do sistema. O sedimento dos pontos UFSCar, Confluência e córrego Tijuco, apresentaram as maiores concentrações das variáveis analisadas, possivelmente devido à fração de silte e argila detectadas. Nos testes crônicos parciais com amostras ambientais, pelo menos uma das espécies e variáveis analisadas, indicaram a toxicidade do rio Monjolinho. Nos testes in situ, os efeitos foram mais pronunciados para ambas as espécies, especialmente em julho/03. P. reticulata demonstrou a maior sensibilidade comparando-se à espécie D. rerio, em todas as situações avaliadas, principalmente considerando-se a fase juvenil. Foram escolhidos dois poluentes para avaliação ecotoxicológica em laboratório, utilizando-se D. rerio juvenis como organismos-teste. O cádmio apresentou uma CL(I)50,96h de 1894,77µg/L. Nos testes de toxicidade crônica parcial estático, obteve-se um valor crônico de 189,73µg/L tanto para sobrevivência como para a biometria final, indicando que na mesma concentração os efeitos na sobrevivência e biometria foram semelhantes. As alterações branquiais foram progressivas nas concentrações de 0,6µg/L, 6µg/L, 60µg/L e 600µg/L, sendo detectado na última, a junção de várias lamelas secundárias. Considerando-se o endossulfan sulfato, a CL(I)50, 96h, semi-estático foi de 7,24µg/L e o valor crônico, considerando-se a sobrevivência foi de 0,948µg/L os efeitos histológicos foram obtidos em todas as concentrações (0,03µg/L, 0,3µg/L e 3µg/L), com o efeito mais pronunciado na última concentração, com hiperplasia e conseqüente junção de lamelas secundárias. Os testes de toxicidade refletiram o estado de degradação instalado em todo o sistema Monjolinho, confirmada pelas análises limnológicas, as quais em última instância comprometem também a saúde da população.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-01T13:47:03Z No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-07T13:37:56Z (GMT) No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) / Made available in DSpace on 2017-08-07T13:37:56Z (GMT). No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) Previous issue date: 2017-04-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Bebidas alco?licas s?o popularmente consumidas em diversas culturas humanas. Por?m, o consumo alco?lico excessivo e a longo prazo pode promover danos sociais, f?sicos e psicol?gicos de dif?cil revers?o. De fato, o ?lcool exerce diferentes efeitos no organismo. Baixas concentra??es promovem euforia, relaxamento, e al?vio do estresse/ansiedade (efeito ansiol?tico), e a abstin?ncia, ap?s doses m?dias cr?nicas, aumentam o estresse/ansiedade (efeito ansiog?nico), podendo interferir na aprendizagem. No entanto, h? poucas informa??es sobre como essa droga altera aspectos cognitivos e psicol?gicos em situa??es de estresse/ansiedade que envolvam aprendizagem. O que demanda investiga??es aprofundadas com a proposi??o de modelos animais para uso translacional. Diante disso, caracter?sticas como: f?cil manuten??o e reprodu??o em laborat?rio, homologia gen?tica superior a 70% ? do genoma humano, e facilidade na administra??o de f?rmacos, tornam o peixe paulistinha (Danio rerio) um modelo ideal em pesquisas translacionais que envolvam drogas de abuso. Nesse sentido, o presente trabalho buscou investigar o efeito de diferentes tratamentos alco?licos (cr?nico e agudo) no desdobramento da resposta de ansiedade e na aprendizagem associativa aversiva do peixe paulistinha. Para isso, foram testados os efeitos do ?lcool na aprendizagem associativa aversiva do paulistinha (artigo 1) e os efeitos do ?lcool e do enriquecimento ambiental (EE) na impossibilidade do peixe evitar um estressor (artigo 2). Os resultados indicaram que comportamentos tipo-ansiosos em paulistinha s?o alterados por mudan?as nas concentra??es alco?licas, no regime de exposi??o ou no ambiente. ?lcool agudo aumenta ansiedade e potencializa a percep??o em rela??o ao eletrochoque. J? o EE promove efeito ansiol?tico e diminui a percep??o do eletrochoque. Por fim, sugerimos que esse trabalho serve de base para pesquisas neurofisiol?gicas gen?ticas e comportamentais sobre os efeitos da intera??o entre drogas de abuso e ambiente. / Alcoholic beverages are popularly consumed in various human cultures. However, excessive and long-term alcohol consumption can promote social, physical and psychological damages that are difficult to reverse. In fact, alcohol has different effects on the body. Low concentrations promote euphoria, relaxation, and stress/anxiety relief (anxiolytic effect), and abstinence after chronic medial doses increases stress / anxiety (anxiety) and may interfere with learning. However, there is little information on how this drug alters cognitive and psychological aspects in stress/anxiety situations involving learning. Therefore, in-depth investigations are required with the proposition of translational animal models. As a result, characteristics such as: easy maintenance and reproduction in the laboratory, genetic homology up to 70% to that of the human genome, and ease of administration of drugs, make zebrafish (Danio rerio) an ideal model in translational research involving drugs of abuse. In this sense, the present study sought to investigate the effect of different alcoholic treatments (chronic and acute) in the unfolding of the anxiety response and in the aversive associative learning of zebrafish.For this, the effects of alcohol in the aversive associative learning of zebrafish (manuscript 1) and the effects of alcohol and environmental enrichment (EE) on the impossibility of the fish to avoid a stressor were tested (manuscript 2). The results indicated that anxiety-like behaviors in zebrafish are altered by changes in alcoholic concentrations, in the exposure regime or in the environment. Acute alcohol increases anxiety and potentiates the electroshock perception. While EE promotes anxiolytic effect and decreases the perception of electroshock. Finally, we suggest that this work serves as a basis for genetic and behavioral neurophysiological research regarding the effects of the interaction between drugs of abuse and environment.

Orientador: Cláudia Vianna Maurer-Morelli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T13:52:07Z (GMT). No. of bitstreams: 1 Barbalho_PatriciaGoncalves_M.pdf: 8177547 bytes, checksum: 9a3923a19cb47c5609f933774ee68213 (MD5) Previous issue date: 2013 / Resumo: O Danio rerio é um peixe teleósteo popularmente conhecido como zebrafish que têm se destacado como modelo animal favorável para investigações genéticas devido à facilidade de manipulação in vivo, por sua transparência nas fases embrionária e larval e por seu desenvolvimento externo. Recentemente, foi demonstrado que o zebrafish é capaz de exibir padrão comportamental e alteração da atividade eletrográfica durante crise epiléptica como visto em roedores, tornando-o um modelo promissor para as investigações moleculares das epilepsias. Os estudos sobre os diferentes eventos já conhecidos das epilepsias no zebrafish estão apenas começando e, portanto, há ainda muito que ser investigado para uma melhor caracterização deste modelo para estudos em epilepsia. Estudos clínicos e em modelos animais mostraram que a crise epiléptica eleva os níveis da interleucina-1 beta e induz morte neuronal. Nesse sentido, o presente trabalho se propôs a investigar (i) o perfil temporal de expressão do transcrito do gene da interleucina-1 beta (il1b) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol por transcriptase reversa-PCR quantitativa e também, sua relação da expressão com a idade em que é realizada a indução da crise no cérebro imaturo e (ii) a morte neuronal pela histoquímica do Fluoro-Jade B (FJB) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol. Neste trabalho conseguimos estabelecer com sucesso as condições ideais para o acasalamento desta espécie e obtenção de embriões e, para a criação de larvas. Além disso, padronizamos o processamento histológico para a criosecção do cérebro do zebrafish. A análise temporal do transcrito do gene il1b revelou um padrão de expressão similar ao observado em roedores. A marcação por FJB não identificou inequivocadamente a presença de morte neuronal após crise epiléptica / Abstract: Danio rerio is a teleost fish popular named as zebrafish that has emerged as a suitable animal model for genetic investigations due to its features such as: facility for in vivo manipulation, transparency during embryonic and larval stages and external development. Recently, It was demonstrated that larvae and adult zebrafish (Danio rerio) seizure model mimics the behavior, eletrographic and c-fos features that are well established in rodent models of epilepsy, making the zebrafish as a promising model for studying the molecular mechanisms underlying epilepsies. However, the potential of the zebrafish model for epilepsy studies has been partly described since the molecular changes and neuronal loss after seizure has not yet been investigated. Proinflammatory cytokines, such as interleukin-1beta (il1b), has shown to be upregulated in surgical specimens of pharmacoresistent patients and in experimental rodent models of seizure, however the profile of this cytokine in zebrafish model for epilepsy is unknown. For this reason, and in view of the importance of inflammatory response in the pathophysiology of epilepsy, we sought to investigate the temporal transcript profile of interleukin-1b (il1b), a proinflammatory cytokine, in adult and immature (larvae) zebrafish brain after Pentylenetetrazole-evoked seizure, as well its aged-related expression in the developing brain by reverse transcriptase-quantitative PCR and neuronal death by Fluoro-Jade B (FJB) staining in adult zebrafish brain. Zebrafish breeding was successfully established during this work as well as the protocol for histological procedures using the zebrafish brain. Our results showed a short up-regulation of il1b mRNA levels after seizure in immature and adult zebrafish brain similar as with those patterns observed in rodent models. FJB did not show a reliable neuronal death staining in the zebrafish brain after seizure / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

Indiana University-Purdue University Indianapolis (IUPUI) / Oxidative stress can disrupt cellular homeostasis, leading to cellular dysfunction and apoptosis. The Nrf2 transcription factor regulates the antioxidant response in cells by binding to antioxidant response elements (ARE) in DNA and activating genes of enzymes that combat oxidative stress. During the pathogenesis of diabetes mellitus (DM), β-cells are exposed to increased amounts of reactive oxygen species (ROS) that cause oxidative stress. Zebrafish (ZF) are excellent models for studying the dynamic mechanisms associated with DM pathogenesis, and we recently developed a ZF model of β-cell apoptosis caused by ROS. Two paralogs of Nrf2 have been identified in ZF, Nrf2a and Nrf2b, but their roles in pancreas development and/or β-cell survival are unknown. To investigate their roles, Nrf2a and Nrf2b antisense morpholinos (MO) were injected into Day 0 ZF embryos and analyzed over time. While Nrf2a MO showed no obvious phenotypes compared to WT, Nrf2b MO exhibited reduced pancreas size and islets with disrupted morphology. Ins:NTR Nrf2a MO showed reduced β-cell loss upon exposure to Metronidazole (MTZ) under generation of ROS compared to WT. Sequence analysis of ZF nrf2b in 3-day post-fertilization (dpf) embryos revealed a novel splice variant containing an additional exon that has not been described. Further investigation of Nrf2a and Nrf2b is likely to yield additional insights regarding the function and regulation of the NRF2-signaling pathway and their roles in β-cell protection under oxidative stress.

La fecundación es el proceso de unión entre el espermatozoide y el ovocito durante la reproducción sexual para dar lugar a un nuevo individuo. En vertebrados la fecundación puede ser interna o externa. La fecundación interna implica que el encuentro de los gametos tiene lugar dentro de la hembra y es característica de mamíferos, reptiles y aves. En la fecundación externa la unión de ambos gametos ocurre fuera del cuerpo de la hembra y se da principalmente en peces y anfibios. Los espermatozoides de mamíferos adquieren la capacidad fecundante en el tracto reproductor femenino en un proceso dinámico llamado capacitación, el cual puede requerir entre algunos minutos hasta varias horas en función de la especie. Sabemos que la capacitación espermática implica cambios moleculares como la eliminación de colesterol de membrana, la entrada de calcio o la fosforilación de proteínas implicadas en la hiperactivación. Entre las diversas modificaciones, se le ha otorgado un papel destacado al glucocáliz debido a su implicación en el proceso de fecundación en mamíferos. Los glucoconjugados que constituyen el glucocáliz espermático se reorganizan considerablemente durante la capacitación, preparándose así para interaccionar con la zona pelúcida y el oolema del ovocito. Esta reorganización de azúcares de membrana plasmática ha sido ampliamente estudiada en especies como el ratón, jabalí, cabra, toro y primates no-humanos. Sin embargo, los cambios estructurales y funcionales de los espermatozoides durante la capacitación en humanos actualmente permanecen poco definidos. Además, la elevada heterogeneidad de las muestras de semen humanas y la limitación de las técnicas convencionales para predecir la funcionalidad espermática, hace necesario profundizar en la fisiología espermática durante la capacitación. En la Fase I de este trabajo se utilizaron múltiples biomarcadores espermáticos con el fin de evaluar el impacto de diferentes tiempos de capacitación (una y cuatro horas) sobre la calidad del espermatozoide humano. Los resultados demostraron que los espermatozoides recuperados tras cuatro horas de capacitación presentaron mayor funcionalidad que aquellos que únicamente capacitaron una hora. En concreto, se observó un número significativamente mayor de células con morfología normal y tasas de fragmentación de ADN más bajas. Además, tras capacitar durante cuatro horas se observó un incremento significativo de espermatozoides con fosforilación en los residuos de tirosina de la pieza principal, así como una mayor redistribución de los azúcares presenten en la membrana plasmática. El análisis multivariante, considerando diferentes biomarcadores morfológicos y moleculares, mostró que la subpoblación de espermatozoides recuperada tras cuatro horas de capacitación fue más homogénea que la subpoblación recuperada tras una hora de capacitación. Estos hallazgos subrayan la importancia del tiempo de capacitación como variable a considerar en los métodos de selección espermática, tanto en investigación básica como en las técnicas de reproducción asistida. La finalidad de las técnicas de reproducción asistida es favorecer el embarazo en casos de infertilidad masculina, femenina o de ambos. En la mujer, una de las causas más comunes de infertilidad es la presencia de endometriosis. La endometriosis es una enfermedad crónica que consiste en el crecimiento de tejido endometrial fuera de la cavidad uterina y se asocia con disfunciones ovulatorias secundarias, alteraciones en la foliculogénesis, en el líquido peritoneal y/o en el sistema inmune. El líquido peritoneal de las mujeres con esta patología contiene una elevada concentración de sustancias inflamatorias y debido a la permeabilidad de las trompas es capaz de difundir al interior y afectar a la calidad de los gametos. Dado que es una enfermedad femenina, pocos estudios han relacionado el efecto del líquido peritoneal procedente de mujeres con endometriosis sobre la calidad espermática. Entre ellos, se ha visto que el líquido peritoneal de estas pacientes deteriora parámetros espermáticos como: la motilidad, la reacción acrosómica, la fragmentación del ADN y la capacidad de unión a la zona pelúcida del ovocito. Sin embargo, la implicación del líquido peritoneal procedente de pacientes con endometriosis en la redistribución de los glucoconjugados en la membrana del espermatozoide humano no ha sido estudiada hasta el momento. En la Fase II de este trabajo se evaluó el efecto del líquido peritoneal procedente de mujeres con y sin endometriosis sobre el espermatozoide humano mediante diversos biomarcadores. Para ello, se realizaron cultivos espermáticos durante 24 y 48 horas con líquido peritoneal al 20%. Los resultaron mostraron que la viabilidad espermática y la reacción acrosómica espontánea no se vieron afectadas en ninguna de las condiciones experimentales. No obstante, tras 48 horas, los espermatozoides cultivados con líquido peritoneal procedente de mujeres con endometriosis presentaron significativamente menor motilidad, menor fosforilación en los residuos de tirosinas y diferente reorganización de azúres de superficie en comparación con las células control. Estos datos aportan información sobre la fisiología de los espermatozoides humanos al interaccionar con el líquido peritoneal de mujeres con endometriosis, destacando el deterioro de la calidad espermática conforme aumenta el tiempo de exposición. Los modelos biomédicos son una fuente de gran valor en todos los campos de investigación. El pez cebra (Danio rerio) se ha convertido en un modelo animal con un elevado potencial en la biología del desarrollo, la genética, la inmunología, el comportamiento, la fisiología y la nutrición, debido a las numerosas ventajas que ofrece frente a otras especies. Además, presenta una elevada homología genética con la especie humana y sus costes de mantenimiento son relativamente bajos. En la fisiología reproductiva y del desarrollo, el modelo biológico del pez cebra destaca por presentar una tasa de fecundación elevada, un ciclo reproductivo corto y un desarrollo embrionario rápido. La fecundación y el desarrollo son externos, condición que facilita el estudio directo de las etapas tempranas de la ontogenia. Además, una de las características más relevantes es que tanto los embriones como las larvas tempranas son transparentes lo que permite el seguimiento visual durante el desarrollo de los distintos tejidos y órganos. Este hecho ha conllevado que la gran mayoría de los estudios realizados se hayan concentrado en diferentes aspectos de las etapas del desarrollo, siendo muy pocos los que han analizado la fisiología del espermatozoide. Se sabe que los espermatozoides de Danio rerio al igual que la gran mayoría de peces no presentan acrosoma. Además, tras la activación de la motilidad, debido al deterioro que les produce el choque hipoosmótico, tienen apenas un minuto para llegar al ovocito y fecundarlo. Por otra parte, al tratarse de un pez con fecundación externa, los espermatozoides están sometidos a una elevada competencia espermática, ya que tienen que competir con los espermatozoides de los otros machos por fecundar los ovocitos. Un estudio previo caracterizó la influencia negativa de la elevada competencia espermática en la descendencia de Danio rerio. Sin embargo, nada se sabe acerca de los cambios fisiológicos antes y tras la activación de la motilidad al someter al espermatozoide de Danio rerio a diferentes niveles de competencia. En la Fase III de este trabajo se analizó el efecto de la competencia espermática a distintos tiempos de activación (30 segundos, 1, 5 y 10 minutos) sobre diferentes biomarcadores en el espermatozoide de Danio rerio. Para ello, se partió de espermatozoides procedentes de machos expuestos a diferentes niveles de competencia espermática, alta competencia (dos machos y una hembra) y baja competencia (un macho y dos hembras). Los resultados mostraron un incremento de espermatozoides con flagelos enrollados y fragmentación de ADN a medida que incrementaba el tiempo de activación. No obstante, los espermatozoides procedentes de machos sometidos a elevada competencia, presentaron significativamente menor porcentaje de espermatozoides con flagelos enrollados tras la activación pero mayor porcentaje de células con fragmentación en el ADN antes de la activación en comparación con las células procedentes de baja competencia. Adicionalmente, mediante morfología y morfometría geométrica (aislando forma y tamaño como variables independientes) se analizaron las distintas partes del espermatozoide (cabeza, pieza intermedia y flagelo) tras 30 segundos de activación en ambos tratamientos de competencia. Los gametos procedentes de machos expuestos a elevada competencia mostraron cabezas de menor tamaño, piezas intermedias más grandes y flagelos más largos que los espermatozoides procedentes de baja competencia. Por lo tanto, los datos sugieren que la presencia de un macho rival podría tener un efecto positivo en el fenotipo del espermatozoide pero un efecto negativo sobre la integridad del ADN. Estos hallazgos proporcionan una perspectiva nueva de como el entorno social puede afectar a los parámetros espermáticos y su influencia en la biología de la reproducción.

Ce travail de thèse s'est intéressé aux effets de l'uranium appauvri (U) sur le poisson zèbre, Danio rerio. L'hypothèse de travail majeure est que les effets de l'U peuvent se traduire par des modifications du métabolisme. Par conséquent nous avons caractérisé la performance physiologique par le biais de la théorie des bilans d'énergie dynamique (DEB) car c'est la seule théorie qui quantifie simultanément l'ingestion, l'assimilation, la croissance, la reproduction, la maturation, la maintenance et le vieillisse¬ment au cours du cycle de vie entier à des niveaux de nourriture variable. Un modèle DEB a ainsi été construit et a permis de quantifier et de prédire la manière dont la performance physiologique du poisson zèbre dépend de son niveau de nutrition (et de la température). Nous avons montré que le développement s'accélère après la naissance jusqu'à la métamorphose où l'accélération cesse. De plus les coûts de maintenance somatique sont très élevés.Un module spécifiant la toxico-cinétique de l'U, chez un individu qui se nourrit, croit et se reproduit, a été incorporé dans le modèle DEB. Le modèle a été appliqué aux données de toxicité (publiés et acquis pendant la thèse) afin de découvrir quel processus est affecté par l'U. Les résultats montrent qu'à partir de 0 nM, l'U augmenterait les coûts de croissance et diminuerait l'assimilation et/ou augmenterait le coût de la maintenance somatique. Nous n'avons pas pu détecter d'effets notables sur la maturation. Une étude histologique révèle que l'U altère l'intégrité de la paroi intestinale et pourrait perturber l'homéostasie des interactions hôte-bactéries. / The aim of this dissertation is to characterize the toxicity of depleted uranium (U) on the metabolism of zebrafish, Danio rerio. The underlying hypothesis of this work is that effects of U show up as effects on the metabolism of the individual. Consequently, we characterized physiological performance using Dynamic Energy Budget (DEB) theory since it is the only theory which simultaneously specifies ingestion, assimilation, growth, reproduction, maturation, maintenance and ageing over the whole life-cycle at varying food availability. Thus a DEB model was built which quantifies and predicts how the physiological performance of zebrafish relates to food level (and temperature). We showed that development accelerates after birth until metamorphosis after which acceleration ceases. Furthermore, somatic maintenance costs are very high.A module specifying toxico-kinetics of U in a feeding, growing and reproducing individual was incorporated into the DEB model. The model was then applied to toxicity data (from the literature or acquired during this thesis) in order to determine which processes are affected by U. Our results show that, from 0 nM onwards, U increases costs for growth and either increases somatic maintenance or decreases assimilation. We were unable to detect effects on maturation. A histological study showed that U alters histology of the gut wall and may perturb host-microbe homeostasis. By accounting for differences in initial conditions between individuals we were able to explain a number of seemingly contradictory results. The take home message is: observations on individuals should not be averaged for groups of individuals.

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.

No description available.

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.