No description available.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

Submitted by Andreza Leão (andrezaflh@gmail.com) on 2018-08-08T17:51:39Z No. of bitstreams: 1 Tese_ValidacaoFamacologicaEsquiva.pdf: 1022849 bytes, checksum: 36438bca0570b50b2e2b23a230037656 (MD5) / Approved for entry into archive by Celia Santana (celiasantana@ufpa.br) on 2018-12-12T17:51:04Z (GMT) No. of bitstreams: 1 Tese_ValidacaoFamacologicaEsquiva.pdf: 1022849 bytes, checksum: 36438bca0570b50b2e2b23a230037656 (MD5) / Made available in DSpace on 2018-12-12T17:51:04Z (GMT). No. of bitstreams: 1 Tese_ValidacaoFamacologicaEsquiva.pdf: 1022849 bytes, checksum: 36438bca0570b50b2e2b23a230037656 (MD5) Previous issue date: 2013-06-12 / O Danio rerio é uma espécie de peixe muito utilizada em modelos de ansiedade devido ao fato que seu comportamento, anatomia, neuroanatomia e bioquímica estarem bem descritos na literatura. O Teste de esquiva inibitória é um modelo de ansiedade ainda em desenvolvimento para esta espécie, o qual consiste na supressão de um comportamento exploratório com a finalidade de evitação de um estímulo aversivo. Este experimento realizou o teste do lado claro como aversivo e a validação farmacológica da esquiva inibitória utilizando as seguintes drogas: antidepressivos – fluoxetina (5, 10 e 20 mg/kg) e imipramina (4, 8 e 16 mg/kg); ansiolíticos – diazepam (0,06, 1,25 e 2,5 mg/kg) e clonazepam (0,02, 0,05 e 1,10 mg/kg); e estimulantes – dietilpropiona (2,5, 5 e 10 mg/kg) e cafeína (10, 20 e 40 mg/kg). Os resultados confirmam a aversividade do lado claro no teste de esquiva inibitória. A fluoxetina, imipramina, cafeína e dietilpropina possuem efeito ansiogênico, assim impedindo a aquisição da esquiva inibitória, com exceção da imipramina 8 mg/kg que facilitou a esquiva. O diazepam também facilitou a aquisição da esquiva, enquanto que o clonazepam, embora não tenha alterado a aquisição da esquiva, apresenta efeito ansiolítico. Conclui-se que o teste de esquiva inibitória em Danio rerio é um efetivo modelo para o estudo da ansiedade. / The Danio rerio is a fish species widely used in emerging models of anxiety due to well described behavior, anatomy, neuroanatomy and biochemistry. The inhibitory avoidance test is an anxiety-related parameters in the model development that described a suppressed behavior emitted to avoid an aversive stimulus. This experiment aimed to describe the inhibitory avoidance averssiveness of white side and the pharmacological validation of this test by using the following drugs: antidepressants - fluoxetine (5, 10 and 20 mg/kg) and imipramine (4, 8 and 16 mg/kg), anxiolytics - diazepam (0.06, 1.25 and 2 , 5 mg/kg) and clonazepam (0.02, 0.05 and 1.10 mg/kg), and stimulants - diethylpropion (2.5, 5 and 10 mg/kg) and caffeine (10, 20 and 40 mg/kg). The data confirm the aversiveness of the white side in inhibitory avoidance test. Flouxetine, imipramine, caffeine and dietilpropione have anxiogenic-like effect and imapaired inhibitory avoidance, excluding imipramine 8 mg/kg who facilitates its acquisition. Diazepam also facilitates inhibitory avoidance acquisition and clonazepam show anxiolytic-like effect on this test. These data show the effectiveness of inhibitory avoidance test as a model to study axiety.

Submitted by JACIARA CRISTINA ALMEIDA DO AMARAL (jaciaramaral@ufpa.br) on 2018-09-19T17:12:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_versão_final_abstract.pdf: 1425516 bytes, checksum: b01d9b3b8d0c8f847bc74f73d1c54b31 (MD5) / Approved for entry into archive by JACIARA CRISTINA ALMEIDA DO AMARAL (jaciaramaral@ufpa.br) on 2018-09-19T17:14:04Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_versão_final_abstract.pdf: 1425516 bytes, checksum: b01d9b3b8d0c8f847bc74f73d1c54b31 (MD5) / Made available in DSpace on 2018-09-19T17:14:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_versão_final_abstract.pdf: 1425516 bytes, checksum: b01d9b3b8d0c8f847bc74f73d1c54b31 (MD5) Previous issue date: 2018-07-13 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O comportamento tipo ansiedade pode ser definido como um estado de apreensão, onde o perigo é iminente, podendo ocorrer a partir da exposição a ambientes novos ou a estímulos aversivos incontroláveis. Muitos sistemas de neurotransmissão podem estar envolvidos na modulação dos estados de ansiedade em mamíferos assim como em teleóteos. Dentre estes, os sistemas GABAérgico e glutamatérgico, apresentam-se como as principais vias de modulação do comportamento tipo ansiedade. Assim, o presente trabalho tem por objetivo avaliar os níveis extracelulares de GABA e glutamato ao longo do processo de geração da ansiedade nos testes de Preferência Claro/Escuro (PCE) e Distribuição Vertical Eliciada pela Novidade (DVN) em Danio rerio. Foram utilizados 60 animais da espécie Danio rerio (selvagem, longfinn), os quais foram expostos aos Testes PCE e DVN nos tempos 5, 10 e 15 min. Os parâmetros analisados para o PCE foram: tempo no compartimento claro, número de quadrantes cruzados no compartimento claro e cruzamentos entre os compartimentos; para o DVN, os parâmetros foram: Tempo na região superior do aquário, número de quadrantes cruzados, entradas no topo, velocidade máxima, velocidade média e Distância total percorrida. Em seguida, os cérebros foram dissecados e incubados com Hank/Na+ para quantificação de GABA e Glutamato por Cromatografia Líquida de Alta Eficácia (CLAE). Todos os testes foram filmados e os vídeos avaliados utilizando o software Zebtrack. Foi aplicado o teste ANOVA de uma via com pós-teste Tukey, considerando significativos valores com p<0,05. Os dados foram expressos em média ± erro padrão e todos os experimentos foram previamente aprovados pelo comitê de ética local (CEPAE UFPA 213-14). Nossos resultados demonstram exploração do compartimento claro no teste PCE, sem diferença entre os tempos (F[3, 20]= 17.10) e sem alteração entre número de cruzamentos entre os minutos 5, e 10 (F[3, 20]= 6.28; p<0.05). Há aumento do número de cruzamento entre os quadrantes no tempo de 15 minutos em relação aos demais tempos de exploração (F[3, 20]= 6.28; p<0.05). Além disso, há aumento de conteúdo extracelular de glutamato no decorrer do teste (F [4, 10] = 8.98) e diminuição de GABA nos últimos minutos em comparação aos animais não expostos ao teste (F [4, 9] = 20.83; p<0.05). Os padrões comportamentais dos animais expostos ao teste DVN também variam de acordo com o tempo de exposição, onde, conforme há aumento do tempo, há aumento do tempo de exploração do topo (F[3, 28]= 15.99; p<0.01), aumento do número de transições para o topo (F[3, 22]= 16.86 p<0.05), aumento do número de quadrantes cruzados (F[3, 21]= 38.70; p<0.01), aumento da distância total percorrida (F[3, 27]= 61.44; p<0.01), sem alteração das velocidades máximas (F[3, 28]= 19.73; p<0.01) e média em todos os tempos. Os níveis de glutamato aumentam na exposição ao teste (F [4, 10] = 24.62) e os níveis de GABA permanecem inalterados (F [4, 9] = 1.76). Concluímos, assim, que os sistemas glutamatérgicos e gabaérgicos modulam de maneira diferente o comportamento tipo ansiedade em Danio rerio. / Anxiety-like behavior can be defined as a state of apprehension where the danger is imminent and may occur from exposure to new environments or uncontrollable aversive stimuli. Many neurotransmission systems may be involved in the modulation of anxiety states in mammals as well as in teleosts. Among these, the GABAergic and glutamatergic systems are the main modulation pathways of anxiety-like behavior. Therefore, the present work aims to evaluate the extracellular levels of GABA and glutamate throughout the process of anxiety generation in Danio rerio exposed in Dark/light Preference (DLP) and Novel Tank (NT) tests. Sixty animals (Danio rerio, (wild type, longfinn) were used, which were exposed to DLP and NT at times 5, 10 and 15 minutes. The parameters analyzed for DLP were: time in the white compartment, number of quadrants crossed in the white compartment and transitions between compartments; for the NT, the parameters were: Time in the upper half, number of squares crossed, entrances at the top, maximum speed, average speed and total distance traveled. Then, the brains were dissected and incubated with Hank/Na+ for further quantification of GABA and Glutamate by High Performance Liquid Chromatography (HPLC). All tests were filmed and videos evaluated using Zebtrack software. One-way ANOVA test with Tukey post-test was applied, considering significant values p<0.05. Data were expressed as mean ± standard error mean and all experiments were previously approved by the local ethics committee (CEPAE UFPA 213-14). Our results demonstrate exploration of the white compartment in the LDT test, with no difference between the times (F [3, 20] = 17.10) and no change in the number of midleline crossings between the compartments between minutes 5 and 10 (F [3,20] = 6.28, p <0.05). There was an increase in the number of squares crossed in the time of 15 minutes in relation to the other exploration times (F [3, 20] = 13.04, p <0.03).In addition, there was an increase in extracellular glutamate content during the test (F [4, 10] = 8.98) and decrease of GABA in the last minutes compared to animals not exposed to the test (F [4,9] = 20,83; <0.05). The behavioral patterns of the animals exposed to the NT test also vary according to the time of exposure, where, as time increases, there is an increase in the time of the top exploration (F [3, 28] = 15.99, p <0.01), (F [3, 22] = 16.86 p <0.05), increase in the number of squares crossed (F [3, 21] = 38.70, p <0.01), increase in the total distance traveled [3, 27] = 61.44, p <0.01), with no change in maximum speed (F [3, 28] = 19.73, p <0.01) and mean speed at all times. Glutamate levels increase on exposure to the test (F [4, 10] = 24.62) and GABA levels remain unchanged (F [4,9] = 1.76). We conclude, therefore, that glutamatergic and gabaergic systems modulate the anxiety-like behavior in Danio rerio differently.

Submitted by Andreza Leão (andrezaflh@gmail.com) on 2018-09-11T17:08:37Z No. of bitstreams: 1 Tese_DesamparoAprendidoZebrafish.pdf: 1298867 bytes, checksum: 346722cdaba63e27b71acc96438f38f1 (MD5) / Approved for entry into archive by Celia Santana (celiasantana@ufpa.br) on 2018-12-12T18:03:06Z (GMT) No. of bitstreams: 1 Tese_DesamparoAprendidoZebrafish.pdf: 1298867 bytes, checksum: 346722cdaba63e27b71acc96438f38f1 (MD5) / Made available in DSpace on 2018-12-12T18:03:06Z (GMT). No. of bitstreams: 1 Tese_DesamparoAprendidoZebrafish.pdf: 1298867 bytes, checksum: 346722cdaba63e27b71acc96438f38f1 (MD5) Previous issue date: 2014-05-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Exposição a eventos aversivos incontroláveis gera dificuldade de aprendizagem para relações de contingência, fuga e/ou esquiva. Tal fenômeno foi denominado de desamparo aprendido e tem sido relacionado à depressão e transtorno de estresse póstraumático. Este trabalho é composto de três estudos com os objetivos de: produzir desamparo aprendido em Danio rerio (Estudo I), investigar os efeitos da luz sobre o teste de fuga (Estudo II) e avaliar os efeitos da imipramina administrada em regime agudo sobre o desamparo aprendido em Danio rerio (Estudo III). Em todos os experimentos foi utilizada uma shuttlebox de acrílico (INSIGHT Equipamentos), que continham uma área central (removível) que permitia o isolamento dos sujeitos. O protocolo geral consistiu de duas fases: 1) Tratamento, onde cada peixe do tratamento choque incontrolável (CHI) foi submetido a 60 choques aleatórios e incontroláveis (0.7- 0.9 V, com duração de 30 segundos), nesta fase os sujeitos do grupo que não receberam tratamento com choque elétrico (NCH) apenas permaneceram na área central por 60 minutos; e 2) Teste, onde cada sujeito, indiferente do tratamento recebido, foi submetido a uma sessão de fuga com 30 choques (0.7 a 0.9V, duração máxima de 30 segundos). No estudo I, foram utilizados 3 grupos, um experimental (GE-CHI) e dois controles, um permanecendo no aquário experimental por 60 minutos sem choque (GCNCH) e outro que permaneceu no aquário viveiro durante a fase I (GF-NCH). No estudo II foram utilizados 4 grupos, um par de NCH e CHI submetidos ao teste com luz, outro par (NCH e CHI) que realizaram o teste na condição sem luz. Já no estudo III foram utilizados 6 grupos de acordo com a concentração de imipramina administrada por 10 minutos, antes da sessão teste: 0,0 mg/l NCH; 0,0 mg/CHI; 1,0 mg/l NCH; 1,0 mg/l CHI; 2,0 mg/l NCH; e 2,0 mg/l CHI. Os principais resultados mostraram que é possível gerar desamparo aprendido em Danio rerio (estudo I), sendo que a luz é uma variável que pode interferir diretamente na aquisição do fenômeno (estudo II) e a imipramina aguda, nas doses aqui administradas não reverteram o desamparo aprendido em Danio rerio. / Exposure to uncontrollable aversive events leads to difficulty in learning contingency relations, escape and/or avoidance. Such phenomenon was named learned helplessness and it has been correlated to depression and post-traumatic stress disorder. This thesis is composed by three studies that together have the aim of: producing learned helplessness in Danio rerio (Study I); investigating the effects of light on the escape test (Study II) and evaluating the effects imipramine have on learned helplessness when applied acutely in Danio rerio (Study III). In every experiment it was used an acrylic shutllebox (INSIGHT equipment) which contained a central area (removable) that enabled the isolation of the subjects. The general protocol consisted in two phases: 1) Treatment, each subject of the Uncontrollable shock (USH) treatment was subjected to 60 random and uncontrollable shocks (0.7 – 0.9V, lasting 30 seconds each), in this phase the subjects of the group that didn't receive treatment with electric shock (NSH) only remained in the central area for 60 minutes; and 2) Test, each subject from both groups was subjected to an escape session with 30 shocks (0.7 – 0.9V, maximum duration of 30 seconds each). In Study I, 3 groups were used, one experimental (EGUSH) and two controls, one remaining in the experimental aquarium for 60 minutes without shock (CG-NSH). In Study II, 4 groups were used, a pair from NSH and USH subjected to the light test, another pair (NSH and USH) underwent the test no light condition. In Study III 6 groups were used according to the concentration of imipramine administered during 10 minutes before the test session: 0.0 mg/l NCH; 0.0 mg/CHI; 1.0 mg/l NCH; 1.0 mg/l CHI; 2.0 mg/l NCH; e 2.0 mg/l CHI. The main results show that it is possible to create learned helplessness in Danio rerio (study I), considering light as a variable that can interfere directly in the acquisition of the phenomenon (study II), in addition, it was showed that acute imipramine in the doses administered here did not revert the learned helplessness in Danio rerio.

L'objectif de ces travaux de thèse était de caractériser la toxicité de l'uranium sur le métabolisme du poisson zèbre, Danio rerio. Puisque les effets de l'uranium se traduisent par des modifications de la performance du métabolisme, la question suivante se pose: que savons-nous du métabolisme du poisson zèbre témoin? Très peu de chose. En effet, nos connaissances à ce sujet sont assez limitées en dépit d'un grand nombre de travaux sur le développement de ce poisson. C'est pourquoi les trois premiers chapitres de ce manuscrit sont dédiés à la caractérisation du métabolisme témoin du Danio. J'ai utilisé la théorie des bilans d'énergie dynamique (DEB) pour procéder à cette caractérisation; à l'heure actuelle c'est la seule théorie qui quantifie l'ingestion, l'assimilation, la croissance, la reproduction, la maturation, la maintenance et le vieillissement pendant le cycle de vie entier d'un organisme. L'effet de l'uranium sur l'organisme implique un effet sur au moins une des processus cités ci-avant. Étant donné que la longévité du poisson zèbre est d'environ quatre ans et demi, et que l'intensité des effets liés à un stress chimique est inversement proportionnel à la taille, nous avons centré nos efforts sur les stades de vie précoces (embryon, juvénile et reproduction adulte). De surcroît, les stades de vies précoces semblent plus sensibles aux effets de l'uranium surtout au niveau des effets sur la croissance. <br /><br /> D'importants progrès ont été réalisés dans le domaine de la quantification du développent, de la croissance et de la reproduction du poisson zèbre. Il s'est avéré que le poisson zèbre accélère son développent après la naissance (c'est-à-dire l'instant où l'individu commence à se nourrir), jusqu'à la métamorphose, où l'accélération cesse. Ce processus a été constaté chez d'autre espèces de poissons, mais pas toutes. Une autre conclusion surprenante était que la maintenance somatique est beaucoup plus élevée que la valeur typique d'un poisson. Nous n'arrivons pas encore à expliquer pourquoi. De plus nous avons découvert que les détails sur la physiologie reproductive sont importants pour caractériser les effets de l'uranium: chez l'adulte les ressources allouées à la reproduction sont stockées dans un compartiment où siègent les processus de préparation de "batch " d'œufs (=buffer de reproduction). Il est donc important de comprendre ce processus pour comprendre comment le poisson zèbre élimine l'uranium via les œufs. <br /><br /> La théorie DEB spécifie que l'individu atteint un stade de développement à un niveau de maturité donné. Selon la température et/ou la nourriture, ce niveau de maturité peut être atteint à des tailles ou des âges différents. Nous avons élargi le concept pour inclure tous les stades de développement (définis sur la base de critères morphologiques) publiés dans les atlas de développement. Ce travail nous a permis d'expliquer par la théorie DEB à présent la variabilité en termes de taille et d'âge. <br/><br /> Dans le but de tester si la théorie DEB peut expliquer des perturbations au niveau de la maturation, nous avons étudié le développement de deux espèces de grenouilles taxonomiquement proches et de taille similaires. Une des espèces possède un développement typique comprenant un stade embryonnaire, un stade têtard qui se nourrit et puis un stade juvénile avec la morphologie typique d'une grenouille. Par contre la deuxième espèce témoigne d'une accélération du développement après l'éclosion mais avant la naissance- qui correspond au stade de développement où l'individu commence à se nourrir. Cette accélération est trahie par une augmentation de la respiration et un retard de la croissance avec au final une diminution de la taille à chaque stade de développement par rapport à la première espèce. Cette accélération s'estompe après la métamorphose (le moment où les jeunes grenouilles quittent l'eau). Toutes les différences entre les deux types de développement ont été expliquées par la théorie DEB en considérant qu'un seul paramètre changeait temporairement de valeur: la fraction de la réserve mobilisée vers la croissance et la maintenance somatique. La conclusion est que les perturbations observées au niveau de la maturation et de la variabilité de l'âge et la taille entre les différents stades de développement soutiennent empiriquement la façon que la théorie DEB incorpore la maturation. <br /><br /> Non seulement notre étude requérait une quantification détaillée de la maturation, mais elle requérait aussi la prise en compte de périodes (prolongées) de jeune, et ce plus particulièrement pour les stades précoces. Selon la théorie DEB la maintenance est alimentée avec l'énergie mobilisée de la réserve. Dès lors que la nourriture devient rare ou disparait cette dernière ne suffit plus pour alimenter la maintenance somatique. Nous avons détaillé ce cas de figure en modélisant le lien entre les processus de rajeunissement et d'amaigrissement extrême et la probabilité de survie. Les prédictions du modèle sont en accord avec les trajectoires de survie de larves obtenues en conditions de laboratoire. Certaines poissons libèrent plus d'un million d'œufs par événement de ponte et pourtant, si la dynamique de la population est stable, à chaque génération chaque poisson n'est remplacé que par un seul individu. Le processus de survie des larves représente une grande énigme irrésolue dans le domaine de la dynamique de populations de poisson. <br /><br /> Par le biais de ces travaux de doctorat, nous disposons à présent d'un outil permettant de comprendre, et de prédire, la manière dont la performance physiologique du poisson zèbre dépend de son niveau de nutrition. Le modèle a été utilisé pour détecter les modifications induites par l'uranium sur la performance physiologique d'un individu exposé par rapport à celle du témoin. A cette fin, nous avons développé un modèle dynamique qui spécifie la manière dont l'uranium s'accumule et s'élimine chez un individu qui se nourrit, grandit et se reproduit. Nous avions imaginé que l'uranium pourrait affecter le système immunitaire ainsi que d'autres mécanismes de défense cellulaire (e.g. système antioxydant). Selon la théorie DEB, l'allocation des ressources à la maturation comprend une fraction fixe de la réserve mobilisée auquel est soustrait le coût de maintenance de la maturité. Notre idée est que les coûts du système immunitaire et de défense cellulaire contribuent à la maintenance de la maturité. Si l'uranium augmentait les coûts de ce dernier alors la maturation ralentira, ainsi j'ai porté une attention soutenu aux taux de maturation. <br /><br /> Nous avons montré que l'uranium altère l'histologie de la paroi intestinale (acteur majeure dans l'assimilation des nutriments) et pourrait potentiellement modifier l'homéostasie des interactions hôte-bactérienne (acteur majeur dans l'assimilation et l'immunité inné). De plus nos travaux suggèrent que l'uranium augmenterait les coûts de synthèse de la structure et diminuerait l'assimilation et/ou augmenterait le coût de la maintenance somatique. Chose étonnante, malgré ce que nous pensions, nous n'avons pas pu détecter d'effets notables sur la maturation à ces faibles concentrations. Puisque la maturation interagit avec la croissance, la reproduction et la maintenance, je considère néanmoins que les travaux que j'ai pu mener sur la maturation sont pertinents. La toxicité de l'uranium est telle que les effets sur le coût de la synthèse de la structure et de la maintenance somatique sont estimés proches de 0 nM d'uranium dans l'eau. <br /><br /> Un résultat très important se dégageant de ces travaux est que la condition des poissons (structure, maturité, réserve, buffer de reproduction, stade de préparation des "batch") au début de l'expérience dépend de l'individu et conditionne la réponse de celui-ci au stress pendant (toute) l'expérience. Ce problème s'aggrave lorsque nous travaillons avec des poissons zèbres adultes car la contribution de la masse du buffer de reproduction par rapport à la masse totale diffère de manière important entre chaque individu. Ceci affecte alors non seulement les trajectoires de masse dans le temps, mais aussi la concentration interne, car la reproduction représente une voie importante d'élimination de l'uranium. La quantité totale de réserve (à savoir : réserve + buffer de reproduction) conditionne la sévérité de l'effet toxique contribuant ainsi à la variabilité dans les données. En prenant en compte les différences entre les conditions initiales de chaque individu, j'ai pu expliquer les résultats contradictoires publiés dans la littérature ainsi qu'expliquer mes propres résultats sur les effets de l'uranium. La leçon à retenir est que des données acquises sur des individus ne devraient pas être moyennées sur des groupes d'individus. <br /><br />

Tese(doutorado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2011. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-06-23T16:27:42Z No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-07-27T20:22:46Z (GMT) No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Made available in DSpace on 2012-07-27T20:22:46Z (GMT). No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) Previous issue date: 2011 / A aquicultura tem crescido significativamente nas últimas décadas devido ao aumento da demanda de pescado no mundo e à estagnação do setor pesqueiro. Porém, este crescimento depende do desenvolvimento de novos pacotes tecnológicos que visem o aumento da produtividade. Uma alternativa é a manipulação genética (transgenia), sendo que o hormônio do crescimento (GH) tem sido o principal alvo das pesquisas com peixes transgênicos. Entretanto, está comprovado que o excesso de GH acarreta uma série de efeitos adversos devido a sua ação pleiotrópica. A ativação do eixo somatotrófico de forma tecido-específica e independente do excesso de hormônio circulante pode contornar estes problemas. Neste contexto, o objetivo desta tese foi superexpressar o gene do receptor do GH (GHR) no tecido muscular esquelético do zebrafish (Danio rerio) e estudar os efeitos desta manipulação sobre os mecanismos envolvidos na regulação gênica do crescimento muscular. A linhagem transgênica estável obtida expressa o GHR especificamente no tecido muscular 100 vezes mais do que os não transgênicos. Estes transgênicos não apresentaram aumento significativo no crescimento, provavelmente devido à queda na expressão do fator de crescimento tipo insulina I (IGF-I). Esta queda foi, provavelmente, relacionada à ação dos principais moduladores da sinalização do GH (SOCS1 e 3), os quais apresentaram-se aumentados nos transgênicos. Ainda, foi observada uma queda na expressão das principais proteínas musculares estruturais (Acta1, myhc4 e mylz2), o que explica a ausência de hipertrofia nos transgênicos. Por outro lado, o aumento na expressão dos principais fatores reguladores miogênicos (myod, myf5 e myog) explica a hiperplasia observada nas análises histológicas. Para verificar como a superexpressão do GHR ativou a transcrição dos fatores reguladores miogênicos (MRFs) e, por consequência a hiperplasia, foram estudados os possíveis mecanismos envolvidos neste processo. Dentre estes, tanto a via proliferativa (MEK/ERK) quanto à via relacionada com a síntese protéica (PI3K/Akt), não tiveram alteração na expressão de seus genes. Entretanto, foi observado aumento na expressão das proteínas de transporte para o núcleo (importinas 1, 3 e 1), podendo-se concluir que a ativação dos MRFs está relacionada ao transporte do GHR para o núcleo das células musculares. Desta forma, pode se concluir que hiperplasia e hipertrofia seguem duas vias de sinalização intracelular distintas, ambas desencadeadas pelo GH, mas reguladas por mecanismos diferentes. / Aquiculture practice has been significantly increasing during the last decades due to the fish rising demand and to fishery activity stagnation. However, such increase depends on new technological packages development aiming to productivity rises. Genetic manipulation is an alternative, once growth hormone (GH) has been the main target on transgenic fish researches. Nevertheless, it has been proved that GH excess leads to many adverse effects due to its pleiotropic action. The somatotropic axis activation in a tissue-specific manner and independent on the circulating hormone excess may bypass these problems. Following these ideas, the present thesis objective was overexpressing GH receptor’s gene (GHR), in zebrafish (Danio rerio) skeletal muscular tissue, and studying such manipulation effects over the mechanisms involved in muscular growth gene regulation. The stable transgenic lineage obtained expresses GHR, specifically in muscular tissue, 100 times more than non-transgenic. These transgenic did not present significant growth, possibly due to a gene expression fall in insulin-like growth factor I (IGF-I). The mentioned fall is, probably, related to GH main signaling modulators (SOCS1 and 3) action, which were increased in transgenic. Also, a gene expression fall from the main structural muscle proteins (Acta1, myhc4 and mylz2) was observed, explaining the transgenic hypertrophy absence. However, the main myogenic regulatory factors (myod, myf5 and myog) expression rising explains the observed hyperplasia in histological analysis. Intending to verify how GHR overexpression has activated the myogenic regulatory factors (MRFs) transcription and, consequently, the hyperplasia, the mechanisms possibly involved in this process were studied. Among these, even the proliferative pathway (MEK/ERK) or the pathway related to protein synthesis (PI3K/Akt), did not presented gene expression altering. However, a gene expression rise in transporting proteins into nucleus (importins 1, 3 and 1) was observed, which may be understood as a correlation between MRFs activation and GHR transport into muscular cell’s nucleus. Therefore, it may be understood that hyperplasia and hypertrophy follow two distinct intracellular signaling pathways, both triggered by GH, but regulated by different mechanisms. These data may be important for aquiculture new transgenic lineages development.

Tissue-resident stem cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells self-renew or differentiate are highly context dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem cells (McSCs) are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified adult McSCs in the zebrafish. To elucidate mechanisms governing McSC self-renewal and differentiation fates we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for McSCs, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of McSC direct differentiation. Analysis of the scRNAseq dataset also revealed a population of mitfa/aox5 co-expressing cells that divides following melanocyte destruction, likely corresponding to cells that undergo self-renewal. Our findings show how different subpopulations of mitfa-positive cells underlie regeneration and differentiation of at least one subpopulation requires reactivation of developmental KIT signaling to properly reconstitute the melanocyte stripe.

No description available.

Nous proposons dans cette thèse de caractériser quantitativement la variabilité à différentes échelles au cours de l'embryogenèse. Pour ce faire, nous utilisons une combinaison de modèles mathématiques et de résultats expérimentaux. Dans la première partie, nous utilisons une petite cohorte d'oursins digitaux pour construire une représentation prototypique du lignage cellulaire, reliant les caractéristiques des cellules individuelles avec les dynamiques à l'échelle de l'embryon tout entier. Ce modèle probabiliste multi-niveau et empirique repose sur les symétries des embryons et sur les identités cellulaires; cela permet d'identifier un niveau de granularité générique pour observer les distributions de caractéristiques cellulaires individuelles. Le prototype est défini comme le barycentre de la cohorte dans la variété statistique correspondante. Parmi plusieurs résultats, nous montrons que la variabilité intra-individuelle est impliquée dans la reproductibilité du développement embryonnaire. Dans la seconde partie, nous considérons les mécanismes sources de variabilité au cours du développement et leurs relations à l'évolution. En nous appuyant sur des résultats expérimentaux montrant une pénétrance incomplète et une expressivité variable de phénotype dans une lignée mutante du poisson zèbre, nous proposons une clarification des différents niveaux de variabilité biologique reposant sur une analogie formelle avec le cadre mathématique de la mécanique quantique. Nous trouvons notamment une analogie formelle entre l'intrication quantique et le schéma Mendélien de transmission héréditaire. Dans la troisième partie, nous étudions l'organisation biologique et ses relations aux trajectoires développementales. En adaptant les outils de la topologie algébrique, nous caractérisons des invariants du réseaux de contacts cellulaires extrait d'images de microscopie confocale d'épithéliums de différentes espèces et de différents fonds génétiques. En particulier, nous montrons l'influence des histoires individuelles sur la distribution spatiales des cellules dans un tissu épithélial. / We propose in this thesis to characterize variability quantitatively at various scales during embryogenesis. We use a combination of mathematical models and experimental results. In the first part, we use a small cohort of digital sea urchin embryos to construct a prototypical representation of the cell lineage, which relates individual cell features with embryo-level dynamics. This multi-level data-driven probabilistic model relies on symmetries of the embryo and known cell types, which provide a generic coarse-grained level of observation for distributions of individual cell features. The prototype is defined as the centroid of the cohort in the corresponding statistical manifold. Among several results, we show that intra-individual variability is involved in the reproducibility of the developmental process. In the second part, we consider the mechanisms sources of variability during development and their relations to evolution. Building on experimental results showing variable phenotypic expression and incomplete penetrance in a zebrafish mutant line, we propose a clarification of the various levels of biological variability using a formal analogy with quantum mechanics mathematical framework. Surprisingly, we find a formal analogy between quantum entanglement and Mendel’s idealized scheme of inheritance. In the third part, we study biological organization and its relations to developmental paths. By adapting the tools of algebraic topology, we compute invariants of the network of cellular contacts extracted from confocal microscopy images of epithelia from different species and genetic backgrounds. In particular, we show the influence of individual histories on the spatial distribution of cells in epithelial tissues.