Canonical Wnt signaling is involved in many aspects of development including axis specification and anterior-posterior neuroectoderm formation during vertebrate embryogenesis. Axin2, a homologue of Axin1, is thought to have a similar regulatory role within the cell, but differences in their expression and binding partners suggest Axin2 is not completely redundant with Axin1. To better understand Axin2 in canonical Wnt signaling, I utilized several approaches to explore its expression and function. In the zebrafish embryo, I found Axin2 is expressed in known active domains of Wnt signaling, suggesting an inducible regulatory role. Additionally, canonical Wnt signaling was sufficient and necessary to induce Axin2 expression and Axin2 was sufficient and necessary to inhibit Wnt signaling. As Wnt signaling is important in development and its dysregulation has been implicated in diseases such as colorectal cancer, this study helps advance our understanding of how Wnt signaling regulates itself through the use of negative feedback inhibitors, such as Axin2.

The diploma thesis summarizes the experimental method for the rearing of zebrafish Danio rerio (Hamilton, 1822) screed. The work of figuring out which of the methods of rearing fry in the transitional period of the ontogenetic development is best for the survival rate and growth of the standard length of the surveyed individuals. The test was carried out on two basic lines of zebrafish Wild Type and Casper. At the beginning of the test were created four groups divided by feeding mode. The control group was fed once a day with pellet feed GEMMA Micro 75 and one day from the 10th day after fertilization the fed with nauplii stages of brine shrimps Artemia salina (Linnaeus, 1758). The second group was fed only once a day with pellet feed GEMMA Micro 75. The last two groups were fed five times a day saltwater rotifer species Brachionus plicatilis (Müller, 1786), and only one of them and the other was still three times daily nourished with nauplii stages of brine shrimps. For each group of four representative have been created initially, thirty tanks of fish at the age of five days after fertilization. The results of this method have shown that the methods they use to feed the rotifers, did not have a statistically significant impact on the survival rate of individuals. In the growth of this method has proven to be statistically significant result in the group where the fed in combination with brine shrimps, where to achieve the average standard size of juvenile fish 16.02 +/- 0.80 mm for the Wild type and 17.39 +/- 0.81 mm for Casper, compared to the control group, that has been the standard length 11.63 +/- 0.64 mm for the Wild Type and 9.54 +/- 0.56 mm for Casper. This method has great potential and breeding is therefore necessary to further develop this method and to adapt it to individual zebrafish facilities.

Memoria para optar al título Profesional de Ingeniero Agrónomo Mención Producción Animal / No disponible a texto completo / El pez cebra (Danio rerio) es uno de los vertebrados más utilizados como organismo modelo en biología molecular y del desarrollo, fisiología y toxicología. Recientemente se ha propuesto como organismo modelo en estudios de nutrición y crecimiento donde los resultados podrían aplicarse a peces de importancia acuícola. Un requisito indispensable para utilizar el pez cebra como modelo en acuicultura es generar un gran número de peces adultos, sin embargo, no se han establecido los protocolos adecuados para la crianza larval lo que dificulta la tarea de generar abundante población adulta. La sobrevivencia de las larvas depende tanto de la alimentación como de las condiciones fisicoquímicas del medio donde se encuentran, sin embargo, la mayoría de estos parámetros sólo están relativamente establecidos para el mantenimiento de peces adultos. Por ejemplo, el nivel de oxígeno disuelto en el agua en la etapa larval no ha sido descrito. El objetivo de este trabajo fue determinar las condiciones óptimas de crianza larval del pez cebra desde los 8 a los 28 días post fertilización (dpf), una vez que ocurre la transición de larva a juvenil, y conocer el crecimiento de las larvas hasta los 98 dpf, previo a la madurez sexual. Se evaluó la sobrevivencia de las larvas sometidas a condición de nula aireación y aireación constante en 24 familias de peces durante los primeros 28 dpf. Se comparó la tasa de crecimiento de los peces sometidos a aireación constante desde los 28 dpf hasta los 98 dpf, alimentados con dos dietas experimentales que difieren en el origen de la proteína (animal v/s vegetal). Adicionalmente se obtuvieron curvas de crecimiento de los peces, alimentados con la dieta experimental a base de proteína animal y alimento comercial. Los resultados indican que la mayor mortalidad de las larvas se registra en peces sometidos a condición de nula aireación con un 52,81% de mortalidad versus un 4,95% en aquella población sometida a aireación constante. En ambos ensayos la mayor mortalidad se registra entre los 12 y 14 dpf, adicionalmente en el tratamiento sin aireación se observó un aumento de la mortalidad entre los 22 y 25 dpf, probablemente debido a las condiciones de hipoxia del medio. Por otra parte, las poblaciones alimentadas con dieta que difiere en el origen de la proteína presentaron significativamente mayores valores en peso y longitud en los peces alimentados con proteína de origen animal versus los alimentados con proteína de origen vegetal (181,964 ± 62,952 mg y 15,963 ±1,862 mm versus 135,204 ± 55,701 mg y 14,227 ± 2,340 mm respectivamente). Al comparar las curvas de crecimiento de los peces alimentados con dieta en base a proteína animal y dieta comercial se observa en ambos casos su modelación se ajustó al crecimiento potencial de la forma , siendo aquella población alimentada con dieta comercial la que presenta los mayores incrementos en peso y longitud (P > 0,05). / The zebrafish (Danio rerio) is one of the most common vertebrates as a model organism in molecular and developmental biology, physiology and toxicology. Recently it has been proposed as a model organism in studies of nutrition and growth. However, have not been established protocols for larval rearing which makes it difficult to generate large numbers of adult fish, a prerequisite for using zebrafish as a model for aquaculture. Recently it has been proposed as a model organism in studies of nutrition and growth where the results could be applied to important fish in aquaculture. A prerequisite for using zebrafish as a model for aquaculture is to generate a large numbers of adult fish, however, have not established appropriate protocols for larval rearing which makes the task of generating plenty of adults. The survival of the larvae depends on both the supply and physicochemical conditions of the environment where they are, however, most of these parameters are relatively established only for the maintenance of adult fish. For example, the level of dissolved oxygen in the water in the larval stage has not been described. The aim of this study was to determine the optimal conditions for rearing larval zebrafish from 8 to 28 days post fertilization (dpf), once the transition occurs from larva to juvenile, and know the growth of the larvae until 98 dpf, prior to sexual maturity. We evaluated the survival of larvae exposed to a condition of no aeration and constant aeration in 24 families of fish during the first 28 dpf. We compared the growth rate of fish under constant aeration from 28 dpf to 98 dpf, fed with two experimental diets differing in protein source (animal v/s plant). Additionally, growth curves were obtained for fish fed with experimental diet based on animal protein and commercial food. The results indicate that increased mortality of fish larvae register under no aeration condition with a mortality 52.81% versus 4.95% in that population under constant aeration. In both trials, the highest mortality was recorded between 12 and 14 dpf, in addition to the treatment without aeration showed an increase in mortality between 22 and 25 dpf, probably due to environmental hypoxic conditions. Moreover, the populations fed diets differing in protein source had significantly higher values in weight and length in those fish fed with animal protein versus those fed plant protein (181.964 ± 62.952 mg and 15.963 ±1.862 mm versus 135.204 ± 55.701 mg versus 14.227 ± 2.340 mm). By comparing the growth curves for fish fed diet based on animal protein and commercial diet were fitted to potential model ( for growth, where that population fed commercial diet that showed greatest increases in weight and length (P > 0.05).

Improved therapeutic strategies for patients with leukemia remain in great demand and beckon better understanding of the mechanisms underlying leukemic treatment resistance and relapse. Accordingly, discoveries in leukemic pathophysiology have been achieved in various animal models. Danio rerio—commonly known as the zebrafish—is a vertebrate organism well suited for the investigation of human leukemia. Zebrafish have a conserved hematopoietic program and unique experimental strengths. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of numerous zebrafish leukemia models. Additionally, improved imaging techniques, combined with the transparency of zebrafish, have revealed exquisite details of leukemic initiation, progression, and regression. Finally, advances in high-throughput drug screening in zebrafish are likely to hasten the discovery of novel anti-leukemic agents. Zebrafish provide a reliable experimental system for leukemic disease research and one in which investigators have accumulated knowledge concerning the genetic underpinnings of leukemic transformation and treatment resistance. Without doubt, zebrafish are rapidly expanding our understanding of disease mechanism and are helping to shape therapeutic strategy for improved patient outcomes.

Die Entwicklung des Schädeldachs beginnt beim Menschen bereits in der frühen Embryogenese und ist erst im Erwachsenenalter abgeschlossen. Das Wachstum der Schädelknochen muss sich während der Entwicklung fortwährend dem Gehirnwachstum anpassen. An den Stellen, wo zwei Schädelknochen aufeinandertreffen, formen sich Schädelnähte, die aus mesenchymalem Bindegewebe bestehen und als Wachstumsfugen des Schädels dienen. Tritt eine frühzeitige Verknöcherung innerhalb einer oder mehrerer Schädelnähte auf, spricht man von einer Kraniosynostose. Als Konsequenz wird ein weiteres Knochenwachstum verhindert, sodass sich das Neurokranium in dieser Region nicht dem expansiven Wachstum des Gehirns anpassen kann. Dies geht in der Regel mit einem kompensatorischen Wachstum des Schädels und infolgedessen mit kraniofazialen Dysmorphien und einem erhöhten intrakraniellen Druck einher. Klinische Studien und Forschungen an Modellorganismen konnten bereits eine Vielzahl an Genen mit der Entstehung von Kraniosynostosen assoziieren, darunter die Transkriptionsfaktoren TCF12 und TWIST1. Beim Menschen sind heterozygote Mutationen in TCF12 und TWIST1 mit Kraniosynostosen der Koronarnaht assoziiert. Bei Mäusen hingegen führt eine heterozygote Tcf12 Mutation nur in Kombination mit einer heterozygoten Twist1 Mutation zu Fusionen der Koronarnaht. Der Zebrabärbling (Danio rerio, überwiegend auch Zebrafisch genannt) weist eine bemerkenswerte Ähnlichkeit bezüglich der Anatomie und Morphologie des Schädeldachs zum Menschen auf. Um die genaue Funktion von TCF12 bei der Ausbildung der Schädelnähte zu untersuchen, wurde im Rahmen dieser Arbeit der Zebrafisch als in vivo Modell für die Entstehung tcf12-induzierter Kraniosynostosen etabliert. Zu Beginn der Arbeit wurde das Expressionsmuster von tcf12 über die Entwicklung hinweg analysiert. Ein besonderer Fokus lag dabei auf einem Expressionsnachweis während der Entwicklung der Schädelplatten und der Schädelnähte. Ein erster Expressionsnachweis von tcf12 mittels PCR-Analysen und Whole-mount RNA in-situ Hybridisierungen zeigte eine breite Expression von tcf12 ab dem 1-3 Somiten Stadium an. Für tiefergehende in vivo Analysen wurden im Zuge dieser Arbeit tcf12:EGFP Reportergenlinien generiert. Mit diesen gelang ein Nachweis der tcf12 Expression entlang der Wachstumsfronten der Schädelplatten, innerhalb der Schädelnähte sowie im Periost und der Dura mater. Mit den tcf12:EGFP Fischen als Referenz wurde in weiterführenden Experimenten die Aktivität drei hochkonservierter CNEs (engl. conserved non-coding elements) in vivo im Zebrafisch untersucht. Zwei der CNEs konnten als tcf12 Enhancer verifiziert werden, die eine Genexpression während der Neurogenese des zentralen Nervensystems (ZNS) steuern. Die beiden Enhancer-Elemente zeichnen sich durch eine hohe Konservierung vom Menschen bis hin zum Zebrafisch aus. Aufgrund der unterschiedlichen Sensitivität gegenüber einem Funktionsverlust von TCF12 und TWIST1 in Mensch und Maus sollte die Auswirkung eines Knockouts der orthologen Gene auf die Entwicklung der Schädelnähte des Zebrafisches untersucht werden. Mittels CRISPR/Cas9 wurden verschiedene Knockout-Linien für die Gene tcf12, twist1a und twist1b generiert. Analysen der Knockoutmutanten zeigten, dass ein heterozygoter Verlust von tcf12 und twist1b in seltenen Fällen zu partiellen Fusionen der Koronarnähte im Zebrafisch führt. Des Weiteren konnte bei tcf12 und twist1b Einzel- und Doppelmutanten ein abnormes Wachstum der Schädelplatten im Bereich der Suturen beobachtet werden. Die Expressionsstudien und die Analysen der Knockoutmutanten deuten auf eine Regulation von TCF12 bei der Differenzierung der Stammzellen sowie der Proliferation der Osteoblasten innerhalb der Schädelnähte hin. Um die Auswirkung von TCF12 Mutationen auf funktioneller Ebene zu untersuchen wurden im Verlauf dieser Arbeit Luciferase-Reporter Assays durchgeführt. Anhand dieser konnte nachgewiesen werden, dass Mutationen, die die basic helix-loop-helix (bHLH)-Domäne beeinträchtigen, die Transaktivierungsfähigkeit von TCF12 aufheben. Co-Transfektions-Experimente mit TWIST1 offenbarten eine Regulation der Transaktivierung von TCF12 durch TWIST1, sowohl im Menschen, als auch im Zebrafisch. Im Rahmen dieser Arbeit konnten die genauen Expressionsorte von TCF12 während der Morphogenese des Schädeldachs nachgwiesen und die Funktion von TCF12 und seinem Interaktionspartner TWIST1 bei der Entstehung von Kraniosynostosen weiter aufgeklärt werden. / The morphogenesis of the calvaria is initiated during early embryogenesis and completed during adulthood. The growth of the skull must continuously adapt to the growth of the developing brain. Where two cranial bones meet, fibrous sutures form. The cranial sutures consist of connective tissue and serve as growth sites of the skull. A premature closure (fusion) of one or several of the cranial sutures is a condition called craniosynostosis. Further bone growth in this area is prevented and the neurocranium cannot adapt to the expansive growth of the brain. The result is a compensatory growth of the skull leading to craniofacial dysmorphisms and also, in more severe cases, to an increased intracranial pressure. Clinical studies and research on model organisms have been able to identify a large number of genes involved in suture development and craniosynostosis, including the transcription factors TCF12 and TWIST1. In humans, heterozygous mutations in both, TCF12 and TWIST1, are associated with craniosynostosis. In mice, haploinsufficiency of Tcf12 alone does not lead to coronal suture fusion. Only loss of Twist1 along with loss of Tcf12 results in craniosynostosis of the coronal suture. Zebrafish (Danio rerio) show a remarkable similarity regarding the anatomy and morphology of the skull vault to that of humans. To unravel the function of tcf12 in cranial suture development, this study aimed to establish a zebrafish in vivo model for tcf12 induced craniosynostosis. First, the expression pattern of tcf12 was analyzed throughout zebrafish development. Special focus was placed on examining the expression of tcf12 during development of the skull plates and the cranial sutures. PCR-analysis and whole-mount RNA in-situ hybridization revealed a broad tcf12 expression in different tissues beginning from the 1-3-somites stage. For more in-depth in vivo analyses, transgenic tcf12:EGFP reporter lines were generated. During cranial vault development, the transgenic fish showed a high amount of tcf12 expressing cells along the growth fronts of the skull plates, within the cranial sutures as well as in the periosteum and the Dura mater. In addition, with the tcf12:EGFP fish as a reference, we tested the transcriptional activity of three highly conserved non-coding elements (CNEs) in zebrafish in vivo. We could validate two of the CNEs as tcf12 enhancer elements driving gene expression in the central nervous system during neurogenesis. The two CNEs show a high conservation between humans and zebrafish. Due to the different sensitivities to loss of TCF12 and TWIST1 in humans and mice, the effect of a gene knockout of the orthologous genes on the development of the sutures should be examined in zebrafish. Therefore, various knockout lines for the genes tcf12, twist1a and twist1b were generated using CRISPR/Cas9. Analyses of the knockout mutants showed that, in a few cases, a heterozygous loss of tcf12 or twist1b led to partial fusions of the coronal sutures in zebrafish. Furthermore, abnormal growth of the skull plates in the area of the sutures could be observed in tcf12 and twist1b single and double knockout mutants. The expression studies and the analyses of the knockout mutants indicate a regulation of TCF12 in the differentiation of stem cells and in the proliferation of osteoblasts within the cranial sutures. In order to investigate the effects of TCF12 mutations on a functional level, luciferase reporter assays were performed. Based on the reporter assays it was demonstrated that mutations impairing the basic helix-loop-helix (bHLH) domain compromise the transactivation ability of TCF12 remarkably. Co-transfection experiments with TWIST1 revealed regulation of the transactivation of TCF12 by TWIST1, both in humans and in zebrafish. Within the scope of this work, the exact expression patterns of TCF12 could be demonstrated during the morphogenesis of the cranial vault. Moreover, the function of TCF12 and its interaction partner TWIST1 could be further clarified in the development of craniosynostosis.

Les effets de la contamination au méthylmercure chez le poisson zèbre Danio rerio ont été interrogés au sein de deux tissus, le muscle squelettique et le système nerveux central, dont le choix fut motivé par leur fort potentiel bioaccumulateur de cet organométallique. Après contamination par voie trophique, à un niveau d’exposition représentatif de ce qui peut advenir dans certains écosystèmes aquatiques, nos observations expérimentales indiquent que ce métal perturbe fortement le métabolisme mitochondrial dans les muscles, mais épargne celui du cerveau ; en outre, l’analyse de l’expression génique suggère une perturbation de l’homéostasie du calcium dans ces deux tissus. Dans le système nerveux central, parmi les synapses glutamatergique et GABAnergique, seule la voie métabolique du GABA semble montrer une adaptation face au MeHg. Concernant le muscle squelettique, l’analyse SAGE a permis d’appréhender les impacts du MeHg à l’échelle cellulaire, révélant également une perturbation de la synthèse protéique, l’induction d’un stress au niveau du reticulum endoplasmique ainsi que l’induction de plusieurs gènes impliqués dans les processus de détoxication et les voies de réponse générale au stress. Notre étude a également mis en évidence la surexpression du gène de la vitellogénine dans le muscle chez des poissons mâles désignant ainsi ce métal comme un perturbateur endocrinien. Enfin, nous avons également révélé une perturbation importante de l’éclosion des œufs associée à un transfert maternel de ce toxique. / The effects of methylmercury contamination on the zebrafish, Danio rerio, were assessed in two tissues, the skeletal muscle and the central nervous system, whose choice was motivated by their high potential to bioaccumulate this organometallic. After contamination by dietary, at a representative exposure level of which may arise in some aquatic ecosystems, our experimental observations indicate that metal strongly disrupts the mitochondrial metabolism in the muscles, but savings that the brain; in addition, the gene expression analysis suggests a disruption of the calcium homeostasis in these two tissues. In the central nervous system among glutamatergique and GABAnergique synapses, only the metabolic pathway of the GABA seems to show an adaptation to the MeHg. Concerning the skeletal muscle scanning SAGE analysis helped to understand the impacts of the MeHg at the cellular scale. It is also revealing a disruption of the protein synthesis, the induction of a stress at the endoplasmic reticulum level as well as the induction of several genes involved in detoxification process and general stress response. Our study has also highlighted the induction of the vitellogenin gene in the muscle of male fish designating this metal as an endocrine disruptor. Finally, we have also revealed a significant disruption of hatching eggs associated with maternal transfer of this toxic.

O resveratrol, encontrando principalmente sob as formas trans-3,5,4\',5-trihidroxiestilbeno e cis-3,5,4\'-trihidroxiestilbeno é uma substância que possui muitas propriedades benéficas a saúde, entre elas, vasodilatação, metabolismo de lipoproteínas, inibição da agregação de plaquetas e até mesmo possui ação terapêutica e preventiva de câncer. Um importante destaque é a sua capacidade de agir como antioxidante, o tornando atraente para uso como substância radiomodificadora. A radiação ionizante (RI), que é utilizada na radioterapia, pode ocasionar diversos danos a células saudáveis, seus componentes e estruturas moleculares, dependendo da dose absorvida e do tipo de célula. O Danio rerio é um modelo animal ideal para avaliação dos efeitos biológicos da RI. Neste trabalho, o resveratrol foi avaliado como potencial radiomodificador em ensaios com embriões de Danio rerio expostos em diferentes horas pós fertilização(hpf), com e sem o córion, a radiação gama. Todos os ensaios foram baseados no protocolo da OECD 236. Para entender os efeitos na mortalidade dos embriões expostos ao resveratrol a radiação separados, foram obtidos valores de CL50 e DL50, respectivamente. Os resultados foram: CL50 de 154 μM (para embriões de 2hpf), 283 μM (para embriões de 12hpf), 699 μM (para embriões de 24 hpf sem córion). E DL50 de 11,50Gy e 12,18Gy (embriões de 4 e 6hpf), 24,8Gy (embriões de 24hpf com córion) e 16,99(embriões de 24 hpf sem o córion). A partir destes resultados foram escolhidas as concentrações de 15, 30 e 60 μM de resveratrol e as doses 5 e 15 Gy (para embriões de 4 e 6 e 24 hpf sem córion) 10 e 20 Gy (para embriões de 24hpf com córion) de radiação gama para observar a atividade radiomodificadora do resveratrol em ensaios com duração de 144 horas, a cada 24 horas foram contabilizados os embriões mortos para comparação estatística por meio do teste Z. Houve diferença na porcentagem de mortalidade em todos os ensaios, mas para embriões expostos a radiação com 24hpf sem córion na presença de 30μM de resveratrol essa diferença foi estatisticamente significativa apontando um indício de radioproteção. Nos embriões de 24hpf sem córion irradiados na presença de 60μM a mortalidade dos embriões irradiados é foi maior quando comparada apenas aos irradiados, indicando que em concentrações mais altas o resveratrol pode exercer a função de radiosensibilizador, embora neste caso não tenha sido estatisticamente significativo, o resultado corrobora com dados na literatura que indicam ação sensibilizadora do composto em células expostas a radiação nessa mesma concentração. / Resveratrol, mainly found in the forms trans-3,5,4 \', 5-trihydroxystilbene and cis-3,5,4\'-trihydroxystilbene is a substance that has many beneficial health properties, among them, vasodilation, lipoprotein metabolism, inhibition of platelet aggregation and even has therapeutic and preventive action of cancer. An important highlight is its ability to act as an antioxidant, making it attractive for use as a radiomodifying substance. Ionizing radiation (IR), which is used in radiotherapy, can cause various damage to healthy cells, their components and molecular structures, depending on the absorbed dose and the cell type. Danio rerio (paulistinha)is an ideal animal model to evaluate the biological effects of IR. In this work, resveratrol was evaluated as a potential radiomodifier in assays with embryos exposed at different hours post-fertilization (hpf), with and without the chorion, to different doses of gamma radiation. All assays were based on the protocol of OECD 236. To understand the effects on mortality of embryos exposed to resveratrol and radiation apart, values of LC50 and LD50, respectively, were obtained. The results were: LC50 of 154 μM (for 2hpf embryos), 283 μM (for 12hpf embryos), 699 μM (for embryos of 24 hpf without chorion). And DL50 of 11.50Gy and 12.18Gy (embryos of 4 and 6hpf), 24.8Gy (embryos of 24hpf with chorion) and 16.99 (embryos of 24 hpf without the chorion). From these results, concentrations of 15, 30 and 60 μM of resveratrol and doses 5 and 15 Gy (for embryos of 4 and 6 and 24 hpf without chorion) 10 and 20 Gy (for 24hpf embryos with chorion) were chosen from gamma radiation to observe the radiomodifying activity of resveratrol in trials with a duration of 144 hours, the dead embryos were counted every 24 hours and these results were compared with a Z test. There was difference in the percentage of mortality in all the trials, but for the exposed to radiation with 24hpf without chorion in the presence of 30μM of resveratrol the number of dead embryos was less and statistically significant indicating radioprotection of resveratrol. In the 24hpf embryos irradiated in the presence of 60μM, the mortality of irradiated embryos was higher when compared to irradiated embryos, indicating that in higher concentrations resveratrol may exert the radiosensitizer function, although in this case it was not statistically significant, these result corroborates with data in the literature that indicate the sensitizing action of the compound in cells exposed to radiation in this same concentration.

Relógios biológicos são marcapassos endógenos presentes tanto em eucariotos quanto em procariotos. Relógios diferentes possuem períodos distintos, e aqueles que se aproximam de 24h de oscilação são chamados circadianos. Em mamíferos, o primeiro relógio circadiano identificado situa-se no núcleo supraquiasmático, localizado no hipotálamo. O funcionamento do relógio circadiano envolve mecanismos de retroalimentação positiva e negativa, em geral tendo início com a ativação dos genes Per e Cry por CLOCK e BMAL1. Atualmente sabe-se que os relógios estão presentes em áreas do cérebro fora do núcleo supraquiasmático e em muitos tecidos periféricos. Em Drosophila e Danio rerio, os osciladores periféricos podem ser sincronizados diretamente por luz, enquanto em mamíferos o reinício de fase dos mesmos parece ser controlado por sinais regulados pelo marcapasso do núcleo supraquiasmático. Uma nova opsina, denominada melanopsina, foi recentemente descoberta na retina de todos os vertebrados estudados, em uma subpopulação de células ganglionares intrinsecamente fotossensíveis. Ela é responsável pela captura de luz e envio dessa informação para o núcleo supraquiasmático. A endotelina (ET) é um peptídeo vasoconstritor composto por 21 resíduos de aminoácidos. Existem três isoformas endógenas de ETs, designadas ET-1, ET-2 e ET-3. Três tipos de receptores para endotelinas já foram clonados, sendo eles designados ETA, ETB e ETC. Todos pertencem à família dos receptores acoplados à proteína G. Órgãos, tecidos e células de Danio rerio constituem um excelente modelo para o estudo dos genes de relógio e de ritmos in vitro. Em células embrionárias ZEM 2S deste teleósteo, constatamos a presença de melanopsina, do receptor ETA para endotelina, e dos seis genes Cry através de PCR. A presença de melanopsina também foi confirmada por imunocitoquímica. Foram realizadas curvas de crescimento em células ZEM 2S previamente mantidas por cinco dias em regime de 14C:10E (luz acesa às 9:00h). No 6º. dia, as células foram transferidas para as seguintes condições: escuro constante; 14C:10E; 10C:14E e luz constante. Houve inibição da proliferação celular por luz. O padrão de expressão temporal dos genes Per1, Cry1b, Clock e da melanopsina foi estudado, assim como sua modulação por ET-1. Células ZEM 2S foram mantidas em fotoperíodo 12C:12E (luz acesa às 9:00h) durante cinco dias, após o que foram tratadas com ET-1 nas concentrações 10-11M, 10-10M, 10-9M e 10-8M, durante 24h. O RNA extraído a cada 3h foi submetido a RT-PCR para posterior análise por PCR quantitativo. RNA ribossômico 18S foi utilizado como normalizador do experimento. Melanopsina não apresentou ritmicidade de expressão em fotoperíodo 12C:12E. ET-1 exerceu efeito bifásico, aumentando a expressão nas menores concentrações de hormônio utilizadas e diminuindo nas maiores. Na concentração 10-10M, ET-1 aparentemente estabeleceu uma oscilação ao longo das 24 horas, com crescente expressão na fase de escuro, atingindo um pico em ZT21 e decrescente durante o período de luz, com o mínimo em ZTs 6 e 9. A expressão do gene Clock é rítmica em regime fotoperíodo 12C:12E, com valores significativamente maiores em ZT12 a ZT21 do que em ZT0, ZT3 e ZT9, indicando um aumento de expressão coincidente com o período de escuro. Foi observado um pico de expressão em ZT6, durante a fase de luz. ET-1 nas concentrações de 10-11 e 10-10M aboliu o ritmo de expressão de Clock, e inibiu o pico de expressão em ZT6. Expressão de Clock permaneceu elevada somente em ZT18. Nas maiores concentrações (10-9M e 10-8M), a inibição ocorreu em todos os ZTs, abolindo completamente o ritmo e atenuando qualquer variação previamente observada entre os ZTs. A expressão do gene Per1 é rítmica em regime fotoperíodo 12C:12E, com valores significativamente maiores nos ZTs 21, 0, 3, 6 e 9 do que nos ZTs 12, 15 e 18, indicando um aumento de expressão na fase de claro. Vale mencionar que já em ZT21, há um aumento significativo antecipatório da fase de luz. Nas concentrações de 10-11 e 10-10M, ET-1 não alterou o período ou a amplitude desse ritmo. A ação evidente de ET-1 foi a inibição da expressão de Per1 na fase de luz (ZT0, ZT3, ZT6 e ZT9), e também em ZT21 (fase de escuro) nas maiores concentrações (10-9M e 10-8M) não afetando o período da oscilação, mas diminuindo marcadamente sua amplitude. A expressão de Cry1b foi rítmica durante o ciclo claro:escuro, com aumento na fase de claro e diminuição na fase de escuro. Novamente a ET-1 apresentou um efeito bifásico sobre a expressão deste gene, aumentando a mesma durante a fase de luz na concentração de 10-11M, e em ZT6 e ZT9 na concentração 10-10M. No entanto, não alterou o período ou a amplitude do ritmo. Por outro lado, durante toda a fase de luz houve inibição deste gene na presença de ET-1 10-9 e 10-8M, diminuindo a amplitude observada nas células controle. / Biological clocks are endogenous timekeepers that are present both in eukaryotic as in prokaryotic organisms. Different clocks have different periods, and those that have about 24h of oscillation are called circadian clocks. In mammals, the first identified circadian clock is located in the suprachiasmatic nucleus, in the hipothalamus. It is now well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In Drosophila and Danio rerio, peripheral oscillators can be synchronized directly by light, while in mammals the reset of the phase seems to be controlled by signals regulated by the suprachiasmatic timekeepers. The maintenance of the circadian clock is governed by positive and negative feedback loops, in general starting with the activation of Per and Cry genes by CLOCK and BMAL1. A new opsin called melanopsin, was recently discovered in the retina of all studied vertebrates, in a subset of intrinsically photosensitive ganglion cells. This photopigment is responsible for capturing light and sending this information to the suprachiasmatic nucleus. Endothelin (ET) is a 21-amino acid residue vasoconstrictor peptide. There are three endogenous isoforms of ETs, ET1, ET2 and ET3. Three subtypes of endothelin receptors have already been cloned: ETA, ETB and ETC, all members of the family of G protein -coupled receptors. Organs, tissues and cells of Danio rerio constitute an excellent model for the study of clock genes and rhythms in vitro. In ZEM 2S embryonic cells of this teleost, we demonstrated the presence of melanopsin, the endothelin receptor ETA, and the six Cry genes by PCR. The presence of melanopsin was also confirmed by immunohistochemistry. ZEM 2S cells previously kept for five days in 14L:10D (lights on 9:00am) were transferred in the sixth day to the following conditions: constant darkness, 14L:10D, 10L:14D and constant light, and growth curves were determined. ZEM 2S showed inhibition of proliferation by light. The temporal expression pattern of the genes Per1, Cry1b, Clock and of melanopsin and their modulation by ET-1 were studied. ZEM 2S cells were kept in 12D:12L photoperiod (lights on 9:00am) for five days, and then treated with 10-11M, 10-10M, 10-9M and 10-8M ET-1, for 24h. RNA extracted every 3 hours was submitted to RT-PCR for subsequent analysis by Real Time-PCR. 18S ribosomal RNA was used to normalize the results. Melanopsin did not show rhythmicity of expression in 12D:12L photoperiod. ET-1 exhibited a biphasic effect, increasing the expression in the lower concentrations, and reducing at the higher concentrations. At 10-10M, ET-1 apparently established an oscillation along the 24h-period, with increasing expression in the dark phase, reaching a peak at ZT2, and decreasing during the light phase, with the minimum at ZT6 and 9. The expression of Clock gene was rhythmic in 12D:12L photoperiod, with significant higher values in ZT12 to ZT21 than ZT0, ZT3 e ZT9, indicating an increase of expression coincident with the dark period. A peak of expression was observed at ZT6, during the light phase. At 10-11 and 10-10M, ET-1 abolished the rhythm of expression of Clock, and inhibited the peak of expression at ZT6. Expression of Clock remained high only at ZT18. At the higher concentrations (10-9M e 10-8M), the inhibition occurred at all ZTs, completely abolishing the rhythm and attenuating any variation previously observed among ZTs. The expression of Per1 gene was rhythmic in 12D:12L photoperiod, with significant higher values at ZTs 21, 0, 3, 6 and 9 than at ZTs 12, 15 and 18, indicating an increase of expression in the light phase. It is important to mention that at ZT21 there was already a significant increase, anticipatory of the light phase. At 10-11 e 10-10M, ET-1 did not alter neither the period nor the amplitude of this rhythm. The evident action of ET-1 was the inhibition of Per1 expression in the light phase (ZT0, ZT3, ZT6 e ZT9), and also at ZT21 (dark phase), at the higher concentrations (10-9M e 10-8M), with no change in the oscillation period, but markedly reducing its amplitude. The expression of Cry1b was rhythimic during the light:dark cycle, with increase in the light phase and reduction in the dark phase. Again, ET-1 showed a biphasic effect on this gene expression, increasing it during the light phase at the concentration of 10-11M, and at ZT6 and 9 at 10-10M. However, the hormone did not affect either the period or the amplitude of the rhythm. On the other hand, along the light phase, there was inhibition of Cry1b in the presence of ET-1 10-9 and 10-8M, reducing the amplitude observed in the control cells.

O estudo da expressão circadiana de genes de relógio tem sugerido hormônios como potenciais agentes sincronizadores. A regulação da ritmicidade de relógios periféricos é, aparentemente, mais um dentre os diferentes efeitos fisiológicos atribuídos aos glicocorticoides (GCs), que se constituem como candidatos a zeitgeber dado ao fato de que seus níveis circulantes apresentam padrões diários robustos e são uma das principais eferências do relógio central em mamíferos. Entretanto, em outros vertebrados, o papel dessa classe hormonal em relação a este aspecto ainda é pouco explorado e se a regulação é direta ou indireta, quais suas consequências e de que maneira ela ocorre são indagações a serem respondidas. Empregando a técnica do PCR quantitativo, avaliamos o perfil de expressão dos genes da alça negativa do núcleo da maquinaria molecular do relógio, per1b e cry1b, em células ZEM-2S do teleósteo Danio rerio expostas ao regime fotoperiódico 12:12 CE ou mantidas em escuro constante (EE) e mantidas em EE, mas condicionadas a trocas diárias de meio de cultura ou a pulsos diários de dexametasona a 10-7 M (DEXA), agonista sintético de glicocorticoide. Em 12:12 CE, ambos os genes apresentaram variação temporal, com picos de expressão observados na fase clara do ciclo. Em EE, per1b mostrou um perfil oscilatório de amplitude atenuada, enquanto para cry1b, não foi detectada oscilação ao longo das 24 h. Trocas de meio em EE alteraram significativamente o padrão de expressão de per1b e cry1b, no entanto, os pulsos diários de DEXA promoveram uma oscilação temporal muito mais pronunciada para per1b, modulando positiva ou negativamente sua expressão nos diferentes pontos temporais. O emprego do antagonista RU 486 na concentração de 10-5 M aboliu o pico de expressão detectado no ZT 16, porém, na concentração de 10-6 M, a expressão gênica foi aumentada em cerca de 3 vezes comparado ao tratamento apenas com DEXA. O gene cry1b, por sua vez, não se apresentou suscetível aos pulsos diários de DEXA. Estes resultados permitem confirmar as células ZEM-2S como modelo de relógios periféricos em cultura dada a fotossensibilidade intrínseca das mesmas, reafirmando o papel do ciclo CE como principal agente sincronizador. Os glicocorticoides certamente exercem modulação de per1b por meio da via genômica direta, sem, no entanto excluir uma possível ação via receptor de membrana, tendo em vista a atividade agonista de RU 486, podendo se constituir em um dos fatores que regulam o complexo funcionamento da maquinaria molecular do relógio biológico de zebrafish / Studies on the circadian expression of clock genes have suggested hormones as potential synchronizing agents. Regulation of rhythmicity of peripheral clocks is among the various physiological effects of glucocorticoids (GCs), which are candidate to zeitgeber, since their circulating levels present robust daily patterns and are one of the major outputs of the mammalian central pacemaker. However, in other vertebrates, such a feature has yet to be clarified as well as if the hormonal regulation is direct or indirect, what are its mechanisms and consequences. Applying qPCR technique, we evaluated the gene expression profile of per1b and cry1b, negative feedback loop members of the molecular machinery of biological clock, in ZEM-2S cells of the teleost Danio rerio. The cells were exposed to photoperiod regimen of 12:12 LD; kept in constant darkness (DD); kept in DD but subject to medium changes or treated with daily pulses of 10-7 M dexamethasone (10-7 M DEXA), a glucocorticoid synthetic analogue. In 12:12 LD, both genes presented temporal variation, with peaks of expression in the light phase. In DD, per1b showed an oscillatory profile with attenuated amplitude, whereas cry1b did not oscillate throughout 24 h. DEXA-free medium changes in constant DD conditions altered significantly per1b and cry1b expression profiles. Nevertheless, DEXA daily pulses promoted a temporal oscillation much more pronounced for per1b, modulating positively or negatively its expression at different time points, whereas cry1b was not susceptible to the hormone. RU 486 at 10-5 M abolished the peak of expression of per1b previously observed at ZT 16. On the other hand, RU 486 at 10-6 M increased the gene expression around 3-fold in comparison to just DEXA treatment. These results confirm ZEM-2S cells as a model of peripheral clocks in culture due to their intrinsec photosensitivity, emphasizing the light/dark cycle as a major synchronizing agent. The glucocorticoid modulates per1b expression, probably through a direct genomic pathway, without excluding however its possible action through membrane receptors, since RU 486 exerted agonistic activity. Glucocorticoids could, therefore, be one of the factors that regulate the complex molecular machinery of zebrafish biological clock

Nos últimos anos, a prescrição e o consumo de medicamentos psicotrópicos vêm aumentando ao redor do mundo, crescendo assim a presença destes compostos em efluentes. Embora os esgotos domésticos e hospitalares em geral sejam encaminhados para as Estações de Tratamento de Esgotos (ETEs), o tratamento convencional aplicado não é eficiente na remoção da maioria dos fármacos e, consequentemente, estes poluentes alcançam os corpos hídricos. Frente a essas informações, é importante entender os possíveis danos causados tanto para seres humanos, como para o ecossistema como um todo, após a exposição a esses fármacos, por meio da água contaminada. Desta maneira, nossa hipótese é que os fármacos psicotrópicos presentes em amostras de água possam induzir a danos neuronais e no desenvolvimento de organismos não alvo, como peixes. Para responder a esta hipótese, avaliamos os efeitos teratogêncios e neurotóxicos de fármacos psicotrópicos como venlafaxina, haloperidol, carbamazepina e fluoxetina, por meio da análise de parâmetros de letalidade e sub-letalidade nos em Danio rerio, visando a determinação dos possíveis efeitos teratogênicos. Adicionalmente, utilizamos a determinação da atividade da enzima acetilcolinesterase como biomarcador de neurotoxicidade em larvas, após a exposição dos embriões e a avaliação do padrão de movimentação. Os fármacos foram testados individualmente e em misturas, para avaliar os efeitos da co-exposição, simulando um cenário mais real de contaminação aquática. Os resultados mostraram que os fármacos carbamazepina e venlafaxina não apresentaram nenhum dos efeitos analizados de forma estatisticamente significativa. Já os fármacos haloperidol e fluoxetina, mesmo não apresentando efeito sobre a teratogenicidade e movimentação, provocaram um aumento na atividade enzimática, em doses consideradas bastante baixas. Considerando que esta estimulação inicial voltou a níveis próximos ao controle negativo e, no caso da fluoxetina, um estudo inibição da atividade em doses mais altas, sugerimos a manifestação de hormesis. A mistura dos fármacos não apresentou efeito significativo nas avaliações realizadas, mostrando que o aumento da atividade enzimática dfoi antagonizado na quando em mistura. Nosso trabalho mostra a importância do estudo de toxicidade em doses baixas, para que uma correta avaliação do risco seja possível. / In the last years, the prescription and use of psychotropic drugs have being increasing around the world, consequently the discharge of these drugs and their metabolites have also increased in wastewater. Considering the presence in the environment and the toxic effects to target and non target species, these compounds were classified as emerging water contaminants. The main sources of these compounds are the domestic and hospital treated effluents, because the conventional treatment applied by effluent treatment plants is not efficient to remove most of drugs, and consequently these pollutants reach the water bodies. In this context, it is important to understand the possible damages to humans and ecosystems after exposure to these drugs through contaminated water. In that way, the hypothesis of this research is that psychotropic drugs present in water samples could induce neurologic and development damages in non-target organisms, such as fish. Therefore, we propose the evaluation of teratogenic and neurotoxic effects of the psychotropic drugs Fluoxetine, Carbamazepine, Venlafaxine and Haloperidol using the determination of the activity of cholinesterase enzyme as biomarker of neurotoxicity in larvae, after the exposure of Danio rerio eggs. Lethality and sub-lethality parameters will be also analyzed in the same organisms to establish the potential of teratogenic effects, the movement analysis was also performed. These drugs will be tested individually and in mixtures to evaluate the effects of coexposure simulating a real scenario of water pollution. Carbamazepine and venlafaxine don\'t responde with significant results in any of these evaluations. Haloperidol and Fluoxetine presented negative results in teratogenic and moviment effects, but they incresed the activity of acetylcholinesterase enzime, this result was different from another study, that these drugs in highest concentrations decrease this activity, so it can indicate the presence of hormesis effect. However, the mixtures of drugs don\'t present any significant effect on all evaluations, this result can suggest that these effects caused by haloperidol and fluoxetine on the enzime activity were canceled, it can be a interaction of antagonist effects on the mixtures.