Дисертації з теми "Danio rerio – Larves"

Les esters de phtalate constituent une famille de composés synthétiques très répandue du fait de leurs usages comme plastifiants. Ils entrent dans la composition de plusieurs produits plastiques tels que les emballages, les jouets, les produits cosmétiques, certains systèmes de toiture en plastique, ainsi que les matériaux de décoration de meubles. Les phtalates ne sont pas liés de manière covalente à la matrice des polymères et sont donc facilement rejetés dans l'environnement, entraînant par conséquent une exposition animale et humaine. En absence de produits de substitutions non-toxiques, les composés de type phtalate restent encore largement utilisés dans l'industrie en dépit de la classification de certains d'entre eux dans la catégorie des substances présumées toxiques par l'European Chemicals Agency (ECHA), en tant que perturbateurs endocriniens. De plus, ils sont cancérigènes et tératogènes. L'effet délétère des esters de phtalates sur les organismes est établi mais le caractère multiple des effets observés montre que les mécanismes d'action des phtalates ne sont que très partiellement élucidés. Nous avons utilisé deux approches de protéomique ciblée pour tenter d'éclairer nos connaissances sur les mécanismes d'actions des esters de phtalate. Pour cela, le dibutyl phtalate (DBP) a été utilisé comme phtalate modèle, et le poisson zèbre (D. rerio) comme organisme modèle. L'utilisation de la première approche de protéomique ciblée, le profilage protéique basé sur l'affinité (affinity-based protein profiling, AfBPP) a permis de montrer la perturbation fonctionnelle de protéines par le DBP avec des sondes photoactivables issues de la synthèse de types aryle azide. L'optimisation des conditions de fixation des sondes diazirine (Diazirine 2) devrait nous permettre de disposer d'une sonde pouvant être utilisée pour identifier les cibles protéiques du DBP dans le protéome du poisson zèbre. La deuxième approche, le profilage basé sur l'activité des enzymes (activity-based protein profiling, ABPP) a permis d'utiliser une sonde réactive spécifique des hydrolases à sérine (SHs) pour cartographier pour la première fois des SHs actives dans le protéome du poisson zèbre. L'identification des SHs dérégulées en présence de DBP chez les larves de poisson zèbre a également été rapportée dans cette étude. Nos résultats globaux indiquent que les approches de protéomiques ciblées telles que l'ABPP ou l'AfBPP peuvent être un atout pour comprendre les mécanismes d'action liés aux xénobiotiques en écotoxicologie
Phthalate esters are a family of synthetic compounds widely used as plasticisers. They are used in a number of plastic products such as packaging, toys, cosmetics, plastic roofing system and furniture decoration materials. Phthalates are not covalently bonded to the polymer matrix and are therefore easily released into the environment, resulting in animal and human exposure. In the absence of non-toxic substitutes, phthalate compounds are still widely used in industry, despite the classification of some of them by the European Chemicals Agency (ECHA) as suspected toxic substances and as endocrine disruptors. In addition, they are carcinogenic and teratogenic. The deleterious effect of phthalate esters on organisms is established, but the multiple nature of the effects observed shows that the mechanisms of action of phthalates are only partially elucidated. We used two targeted proteomics approaches to shed light on the mechanisms of action of phthalate esters. Dibutyl phthalate (DBP) was used as a model phthalate and zebrafish (D. rerio) as a model organism. Using the first targeted proteomics approach, affinity-based protein profiling (AfBPP), the functional disruption of proteins by DBP with photoaffinity probes from aryl azide synthesis was demonstrated. Optimisation of the binding conditions for diazirine probes (Diazirine 2) should provide us with a probe that can be used to identify DBP protein targets in the zebrafish proteome. The second approach, activity-based protein profiling (ABPP), used a reactive probe specific for serine hydrolases (SHs) to map active SHs in the zebrafish proteome for the first time. The identification of deregulated SHs in the presence of DBP in zebrafish larvae was also reported in this study. Overall, our results indicate that targeted proteomics approaches such as ABPP or AfBPP can be an asset for understanding xenobiotic-related mechanisms of action in ecotoxicology

Dans le but de comprendre les mécanismes relatifs à la mise en place des axes lors du développement embryonnaire précoce des lophotrochozoaires, les rôles morphogénétiques des récepteurs aux protéines de la superfamille des TGF- s ainsi que leurs implications en tant que cytokine ont été étudiés chez l'huître creuse C. Gigas. Après avoir cloné les transcrits et les gènes codant quatre de ces récepteurs (TGF- type I, activine type I et BMP types I et II) ainsi qu'une protéine de type BMP1/Tolloid, utilisant ensuite le poisson zèbre en tant " qu'organisme rapporteur ", la microinjection d'ARNm synthétisés in vitro dans ces embryons a démontré la capacité de ces molécules à jouer un rôle lors de l'embryogenèse précoce. Lors de la gastrulation, par interférence avec les molécules orthologues endogènes de poisson zèbre, la surexpression de ces transcrits résulte en la perturbation de la mise en place des axes dorsoventraux et antéropostérieurs via la modulation de l'induction du mésoderme.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The low-power LASER is a technology used in the area of ​​health as adjuvant therapy for various diseases. The use of conservative therapies as adjuncts to drug therapy, aim to potentiate therapeutic responses. There are currently some contraindications for the use of LASER in treatments of human patients with open wounds, tumor region and gravid uterus. Thus, zebrafish (D. rerio) is a consolidated organism as an experimental model in the biological, biomedical and environmental areas. The objective of the present work is to evaluate the development of embryos and larvae of zebrafish (D. rerio) exposed to low-power LASER. The LASER III flash with transverse beam output 0,028 cm2, power 100 mW, creep 35, 70 and 140 J / cm2, total energy of 1, 2 and 4 J (Joules), InGaAlP active medium, was used. The data were evaluated according to the normality behavior of Lilliefors using Fisher's exact test, t-test, Mann-Whitney, Kruskal-Wallis (post hoc-Dunn) and Linear Regression by the ORIGIN 7.0 program. The cumulative mortality rate and hatching in embryos (D. rerio) exposed to the LASER in the red spectrum 660 nm and infrared 830 nm 1, 2 and 4 J from 24 - 24 h to 96 hpf was not statistically significant in relation to the wavelength in energy doses in Joules (p ˃ 0,05), infrared laser 830 nm 4J, did not interfere in the development of embryos and larvae (D. rerio), when exposed from 24 - 24 h for a period of 216 hpf. The cumulative mortality rate in the control group was 23 % higher than in the LASER group and the hatching rate in the LASER group was statistically significant (p ˂ 0,05). The cumulative mortality rate in embryos and larvae (D. rerio) when exposed intensively to LASER was statistically significant, as was the hatching rate (p ˂ 0,05) for 96 hpf. There were no statistically significant differences for larval length between the control group compared to the LASER group 24 - 24 h and intensive LASER (p ˃ 0,05). The rate of heart rate in the LASER group 24 - 24 h was statistically significant in relation to the control group and the intensive LASER group (p ˂ 0,05). The rate of spontaneous movements was statistically significant when compared to the control group and the intensive LASER group (p ˂ 0,05). The rate of edema in the control group was statistically significant in relation to the LASER group 24 - 24 h for 96 hpf (p ˂ 0,05). The rate of edema in the intensive LASER group was not statistically significant; however, as in the LASER group 24 - 24 h, the edema in the control group was higher than in the LASER group. The scarcity of theoretical reference on this theme reinforces the pioneering of this work carried out in vivo. The results of this study highlight the importance of the use of embryos and larvae (D. rerio) as a model system in biological, environmental and biomedical research. Data analysis suggests that contraindications for low-power LASER irradiation in patients during the gestational period should be reviewed.
O LASER de baixa potência é uma tecnologia utilizada na área da saúde como terapia coadjuvante a diversas doenças. A utilização de terapias conservadoras como coadjuvantes a terapia medicamentosa, visam potencializar as repostas terapêuticas. Atualmente há algumas contraindicações para utilização do LASER em tratamentos de pacientes humanos com feridas abertas, região tumoral e útero gravídico. Sendo assim, o zebrafish é um organismo consolidado como modelo experimental nas áreas biológica, biomédica e ambiental. O objetivo do presente trabalho é avaliar o desenvolvimento de embriões e larvas do zebrafish (D. rerio) expostos ao LASER de baixa potência. Utilizou-se o flash LASERIII com saída transversa do feixe 0,028 cm2, potência 100 mW, fluência 35, 70 e 140 J/cm2, energia total de 1, 2 e 4 J(Joules), meio ativo InGaAlP. Os dados foram avaliados segundo o comportamento de normalidade Lilliefors através dos testes Exato de Fisher, teste t, Mann-Whitney, Kruskal-Wallis (post hoc-Dunn) e Regressão Linear pelo programa ORIGIN 7.0. A taxa de mortalidade acumulativa e eclosão em embriões (D. rerio) expostos ao LASER no espectro vermelho 660 nm e infravermelho 830 nm 1, 2 e 4 J de 24 - 24 h para 96 hpf não foi estatisticamente significativas em relação ao comprimento de onda em doses de energia em Joules (p ˃ 0,05), O LASER infravermelho 830 nm 4J, não interferiu no desenvolvimento de embriões e larvas (D. rerio), quando expostos de 24 - 24 h por um período de 216 hpf. A taxa de mortalidade acumulativa no grupo controle foi 23 % maior que no grupo LASER e a taxa de eclosão no grupo LASER foi estatisticamente significativa (p ˂ 0,05). A taxa de mortalidade acumulativa em embriões e larvas (D. rerio) quando expostos de forma intensiva ao LASER, foi estatisticamente significativa, assim como a taxa de eclosão (p ˂ 0,05) para 96 hpf. Não houve diferenças estatisticamente significativas para comprimento larval entre o grupo controle em relação ao grupo LASER 24 - 24 h e LASER intensivo (p ˃ 0,05). A taxa de batimentos cardíacos no grupo LASER 24 - 24 h foi estatisticamente significativo em relação ao grupo controle e ao grupo LASER intensivo (p ˂ 0,05). A taxa de movimentos espontâneos foi estatisticamente significativa, quando comparada ao grupo controle e ao grupo LASER intensivo (p ˂ 0,05). A taxa de edema no grupo controle foi estatisticamente significativa em relação ao grupo LASER 24 - 24 h para 96 hpf (p ˂ 0,05). A taxa de edema no grupo LASER intensivo não foi estatisticamente significativa, no entanto, assim como no grupo LASER 24 - 24 h, o edema no grupo controle foi maior que no grupo LASER. A escassez de referencial teórico sobre essa temática reforça o pioneirismo desse trabalho realizado in vivo. Os resultados desse estudo ressaltam a importância da utilização de embriões e larvas (D. rerio) como Sistema - modelo em pesquisas biológicas, ambientais e biomédicas. As análises dos dados sugerem que as contraindicações para irradiação com o LASER de baixa potência em pacientes durante o período gestacional sejam revistas.

As doenças inflamatórias intestinais são caracterizadas por uma desregulação na resposta imune contra a microbiota. O zebrafish, tem emergido como um novo modelo para o estudo de doenças inflamatórias. Os ácidos graxos de cadeia curta (AGCCs) são produtos da microbiota intestinal, que possuem papéis antiinflamatórios e aparecem como uma das possíveis terapias contra doenças inflamatórias. A implementação do modelo de inflamação intestinal induzido pelo ácido trinitrobenzenesulfônico (TNBS) em larvas de zebrafish aumentou a mortalidade de forma dose-dependente. Se observou dilatação do lúmen com alisamento das vilosidades intestinais. Não se detectaram mudanças na produção de muco nem no número de células caliciformes mas teve um leve aumento da apoptóse e diminuição da proliferação, nos expostos ao TNBS. Teve aumento de células mielóides infiltrantes e de citocinas inflamatórias, assim como disbiose da microbiota. O tratamento com AGCCs gero alta mortalidade a concentrações acima de 10 mM, mas existe ação anti-inflamatória a esta mesma concentração.
Inflammatory bowel diseases are characterized by a dysregulation in immune response against microbiota. The zebrafish has emerged as a new model for the study of inflammatory diseases. Short chain fatty acids (SCFAs) are products of the intestinal microflora, which have anti-inflammatory roles and appear as a possible therapy against inflammatory diseases. The implementation of intestinal inflammation model induced by trinitrobencenesulfonic acid (TNBS) in zebrafish larvae, increased mortality in a dose-dependent manner. Was observe dilatation lumen and straightening of the intestinal villi. No changes were detected in the production of mucus or in the number of goblet cells but had a slight increase in apoptosis and decreased proliferation in exposed to TNBS. Also has, increased cytokines and infiltrating myeloid cells, and dysbiosis of the microbiota. Treatment with SCFAs generate high mortality above 10 mM concentrations, but there anti-inflammatory action to this same concentration.

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Recent studies have shown several applications of graphene oxide (GO) in neurology in the imaging area as a contrast agent for diagnostic purposes, for drug delivery of anticarcinogenic drugs, proteins and peptides and in the field of tissue engineering for applications in the regeneration of nervous lesions. Advances in GO applications research in neurology require addressing its potential toxic mechanisms of action. Zebrafish (Danio rerio) has been widely used in neurotoxicological trials due to the homology of biochemical processes and neural structures with vertebrates. To investigate the potential neurotoxic effects of GO exposure in different concentrations at development period, we evaluated in vivo parameters for initial toxicological screening. We found that GO did not induce changes in survival, hatching and spontaneous movement. However, an increase in heart rate at 48 hpf was found and a reduction in body length without altering the ocular area at 5 dpf. Additionally, molecular gene expression analyses of nervous system-related proteins were performed, showing synapsin IIa expression is increased and dopamine transporter (dat) gene expression is reduced, suggesting a potential compensatory mechanism. The evaluation of the locomotion behavior of animals exposed at GO showed an increase in the absolute turn angle. Based on these initial parameters, we performed biochemical analyzes to determination of the enzyme acetylcholinesterase (AChE) activity and dopamine levels. The exposure at GO did not change AChE activity and decreased dopamine levels. Additionally, the gene expression of B cell lymphoma 2 (bcl2) gene and caspase 3 (casp3) gene were evaluated for the group of larvae exposed to GO, demonstrating an increase of bcl2 and unchanged casp3 expression, suggesting no apoptosis involvement. The tissue and cellular structure of zebrafish larvae brain exposed at GO was evaluated by transmission electron microscopy (TEM) and cell dead by autophagosome formation with loss of cellular architecture was observed most likely due to exposure to the nanomaterial. Further studies are necessary to the complete understanding of the neurological changes observed in zebrafish by exposure to GO and the mechanisms involved in this process, are necessary.
Estudos recentes t?m mostrado diversas aplica??es do ?xido de grafeno (GO) como agente de contraste para fins de diagn?stico, para o endere?amento de drogas (do ingl?s drug delivery) de f?rmacos anticarcinog?nicos, prote?nas e pept?deos e na ?rea de engenharia tecidual para aplica??es na regenera??o de les?es nervosas. O avan?o em pesquisas relacionadas a aplica??es do GO em neurologia exige a investiga??o dos mecanismos de a??o potencialmente t?xicos desencadeados pela exposi??o ao nanomaterial. O zebrafish (Danio rerio) tem sido amplamente utilizado em ensaios neurotoxicol?gicos devido ? homologia de processos bioqu?micos e estruturas neurais com outros vertebrados, vis?veis desde o in?cio de seu desenvolvimento externo. Para investigar os efeitos neurot?xicos da exposi??o a diferentes concentra??es do GO no per?odo de desenvolvimento, foram avaliados diversos par?metros in vivo para avalia??o toxicol?gica preliminar. Foi verificado que o GO n?o induziu altera??es na sobreviv?ncia, eclos?o e movimentos espont?neos. Contudo, o aumento dos batimentos card?acos em 48 hpf foi constatado e redu??o do tamanho do eixo axial, sem altera??o na ?rea ocular em 5 dpf. Adicionalmente, an?lises moleculares da express?o de genes espec?ficos para prote?nas do sistema nervoso foram realizadas mostrando que o GO aumenta a express?o de sinapsina IIa e reduz a express?o do transportador de dopamina (dat), sugerindo um prov?vel mecanismo compensat?rio. A avalia??o da locomo??o dos animais expostos ao GO mostra o aumento do ?ngulo absoluto de virada. Com base nestes par?metros iniciais, foram realizadas an?lises bioqu?micas para a determina??o da atividade da enzima acetilcolinesterase (AChE) e dosagem dos n?veis de dopamina. A exposi??o ao GO n?o alterou a atividade da AChE e reduziu os n?veis de dopamina. Adicionalmente, a express?o do gene de linfoma de c?lulas B2 (do ingl?s B cell lymphoma 2 (bcl2) ) e do gene que codifica para caspase 3 (casp3) foram avaliadas para o grupo de larvas expostas ao GO onde foi mostrado aumento da express?o de bcl2, enquanto que a express?o de casp3 permaneceu inalterada, sugerindo que o GO parece n?o induzir mecanismo de apoptose. A estrutura tecidual e celular do c?rebro de larvas de zebrafish expostas ao GO foi avaliada por microscopia eletr?nica de transmiss?o (TEM) onde morte celular por necrose com perda da arquitetura celular foi constatada. Estudos adicionais para o completo entendimento das altera??es neurol?gicas observadas pela exposi??o ao GO e dos mecanismos envolvidos nesse processo s?o necess?rios.

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CAPES
A WSMoL (do inglês water-soluble Moringa oleifera lectin) é uma lectina (proteína que reconhece carboidratos) proveniente das sementes da Moringa oleifera, as quais são utilizadas no tratamento de água. Algumas espécies de insetos podem atuar como vetores de doenças ou pragas agrícolas. O mosquito Aedes aegypti é o vetor da dengue e sabe-se que o extrato de sementes de M. oleifera e WSMoL são tóxicos para as larvas deste inseto. O extrato de sementes e WSMoL apresentaram uma concentração letal a 50% das larvas após 24 horas de exposição (CL₅₀) igual a 0,27 mg/mL e 0,197 mg/mL, respectivamente. Entretanto, para uma adequada utilização desse inseticida natural se faz necessário a realização de testes ecotoxicológicos a fim de avaliar os possíveis efeitos causados em organismos aquáticos não-alvo. Neste trabalho, foi avaliada a toxicidade de extrato de semente e WSMoL para larvas de Danio rerio, espécie de peixe muito utilizada na Ecotoxicologia devido à facilidade de observação de seu comportamento e desenvolvimento e serem sensíveis na fase larval. As larvas de D. rerio foram expostas a diferentes concentrações do extrato de sementes ou WSMoL por 168 horas (7 dias),tendo sido avaliados efeitos letais por meio da determinação da taxa de mortalidade. Além disso, efeitos sub-letais comportamentais e bioquímicos sobre a velocidade natatória das larvas e atividade da acetilcolinesterase, respectivamente,também foram avaliados. Foi observado um efeito dose dependente na taxa de mortalidade de D. rerio tanto para o extrato quanto para a lectina purificada. Após 24 horas, os valores de CL₅₀/₂₄h foram 0,365 e 0,21 mg/mL para extrato e WSMoL, respectivamente. Esses resultados mostram que a susceptibilidade das larvas de D. rerio a WSMoL foi similar a das larvas de A. aegypti (6% de diferença nos valores de LC₅₀/₂₄h) enquanto extrato é aproximadamente 34% mais potente para as larvas do mosquito. Após 96 horas, os valores de CL₅₀/₂₄h para extrato e WSMoL foram 0,031 e 0,135 mg/mL, respectivamente. Com relação à velocidade natatória dos indivíduos expostos ao extrato, houve um aumento significativo (p < 0,05) após incubação nas concentrações 0,03 e 0,27 mg/mL e uma diminuição para larvas incubadas com 0,81 mg/mL por esse mesmo período. Para incubação por 168 h, a velocidade foi maior que no grupo controle para os tratamentos a 0,27 e 0,81 mg/mL. Nos ensaios com WSMoL, a média da velocidade de natação de larvas expostas à lectina por 72 h foi significativamente (p < 0,05) menor que no controle nos tratamentos com 0,1 e 0,2 mg/mL. Para 168 h, a velocidade foi também menor que no controle no tratamento a 0,1 mg/mL enquanto para as concentrações de 0,02 e 0,05 mg/mL foi similar (p > 0,05) ao controle.Larvas expostas por 168 h ao extrato aquoso na concentração 0,81 mg/mL e a WSMoL na concentração de 0,1 mg/mL apresentaram uma redução significativa na atividade da acetilcolinesterase. Levando em consideração os resultados obtidos, esses inseticidas naturais devem ser usados cuidadosamente, uma vez que as concentrações necessárias para o controle de A. aegyptiforam podem também causar efeitos tóxicos letais e sub-letais para organismos não-alvo como o peixe D. rerio. Recomenda-se o uso em recipientes de armazenamento de água, e não em ecossistemas aquáticos como lagoas e lagos.
Some insect species can act as vectors of diseases or as agricultural pests. The WSMoL (water soluble Moringa oleifera lectin) is a lectin (protein that recognizes carbohydrate) derived from the seeds of Moringa oleifera, which are used in water treatment. The mosquito Aedes aegypti is the vector of dengue and it is known that M. oleifera seed extract and WSMoL are toxic to larvae of this insect. The seed extract and WSMoL showed a lethal concentration to 50% of the larvae after 24 hours of exposure (LC₅₀) equal to 0.27 mg/mL and 0.197 mg/mL, respectively. However, for proper use of these natural insecticides, it is necessary to carry out ecotoxicological tests in order to assess the possible effects on non-target aquatic organisms. In this work, the toxicity of seed extract and WSMoL to zebrafish (Danio rerio) larvae was evaluated. Danio rerio is a fish species very used in Ecotoxicology due to the ease of observation of its behavior and development and for being sensitive in the larval stage. D. rerio larvae were exposed to different concentrations of the seed extract or WSMoL for 168 h (7 days) and the lethal effects were assessed by determining the mortality rate. Additionally, behavioral and biochemical sublethal effects on larval swimming velocity and acetylcholinesterase activity, respectively, were also evaluated. It was observed a dose dependence effect on D. rerio mortality rate for both the extract and the purified lectin. After 24 h, the values of LC₅₀/₂₄h were 0.365 and 0.21 mg/mL to extract and WSMoL, respectively. These results show that the D. rerio larvae susceptibility to the WSMoL was similar to A. aegypti larvae (6% difference in LC₅₀/₂₄h values) while extract was about 34% more potent against mosquito larvae. After 96 h, the values of LC₅₀/₂₄h for extract and WSMoL were 0.031 and 0.135 mg/mL, respectively. Concerning the swimming velocity of individuals exposed to the extract there was a significant increase (p <0.05) after incubation for 72 h at 0.03 and 0.27 mg/mL and a decrease for larvae incubated at 0.81 mg/mL for this same period. For 168 h incubation, the velocity was higher than control group for treatments at 0.27 and 0.81 mg/mL. In tests with WSMoL, the average swimming velocity of larvae exposed to the lectin for 72 h was significantly (p <0.05) lower than the control on treatments with 0.1 and 0.2 mg/mL. For 168 h, the velocity was also lower than the control on treatment with 0.1 mg/mL, and with concentrations of 0.02 and 0.05 mg/mL the values were similar (p>0.05) to the control. Larvae exposed for 168 h to the aqueous extract at a concentration of 0.81 mg/mL and WSMoL at 0.1 mg/mL showed a significant reduction in the activity of acetylcholinesterase. Considering the results, these natural insecticides must be used carefully used since the concentrations necessary for the control of A. aegypti also can cause lethal toxic effects and sub-lethal to non-target organisms such as D. rerio fish. It is not recommended to use these natural insecticides in aquatic ecosystems such as ponds and lakes, but in water storage containers.

Superior swimming stamina in adult fish is presumably passed on to their offspring, but the ontogeny of the appearance of superior stamina and the requisite enhanced cardio-respiratory support for locomotion in larval fishes has not been determined. Is the expression of the suite of parental traits enabling superior swimming stamina in their offspring dependent upon their achieving juvenile/adult morphology, or does it appear earlier in their larvae? To answer this, adults were classified into three groups based on swimming stamina, followed by measurement of length, mass, and width. Larval offspring from the two parental groups -high stamina larvae (HSL) and low stamina larvae (LSL)- were reared at 27°C in aerated water (21% O2). Routine and active heart rate, routine and active mass specific oxygen consumption were recorded through 21dpf, and cost of transport (COT) and factorial aerobic scope were derived from oxygen consumption measurements. Routine heart rate at 2dpf of LSL was 164 ± 1 b·min-1, compared to only 125 ± 2 b·min-1 for HSL. Routine heart rate subsequently peaked at 203 ± 1 b·min-1 at 5dpf in the HSL group, compared to 207 ± 1 b·min-1, at 4dpf in the LSP larvae. Active heart rate at 5 dpf of LSL was 218 ± 2 b·min-1 compared to 216 ± 2 b·min-1 for HSL. Active heart rate increased slightly to 227 ± 2 b·min-1 for LSL before decreasing again, while active heart rate remained relatively constant for HSL. Routine O2 consumption at 2dpf of HSL was 0.09 μmol·mg-1·hr-1, compared to 0.03 μmol·mg-1·hr-1 in LSL. Routine O2 consumption subsequently peaked at 0.70 μmol·mg-1·hr-1 at 9dpf in the HSL, compared to 0.71 μmol·mg-1·hr-1, at 9dpf in the LSL. These values dramatically decreased before leveling off at around 0.20 μmol·mg-1·hr-1 and 0.15 μmol·mg-1·h-1, respectively. Active O2 consumption at 5dpf for HSL was 0.38 μmol·mg-1·hr-1, compared to 0.57 μmol·mg-1·hr-1 for LSL. Active O2 consumption subsequently peaked at 0.97 μmol·mg-1·hr-1 at 10dpf in HSL, compared to 1.19 μmol·mg-1·hr-1 at 7dpf in LSL. These values also dramatically decreased and leveled off. Significant differences (p < 0.05) in heart rate and oxygen consumption persisted through 21dpf. The onset of differences observed in routine and active heart rate in early larvae, correlated with parent stamina, show that juvenile or adult features are not required as a precondition for the emergence of phenotypic physiological differences.

Mestrado em Biologia Molecular e Celular
The scientific community has been concerned for several years about the presence of pharmaceuticals in the wild, since these compounds may have deleterious or unpredictable effects on living organisms. The scientific knowledge on the fate and effect of drug residues in the environment has evolved considerably in recent years, revealing that drug residues do indeed pose a threat to the aquatic ecosystem. Two examples of widely used pharmaceuticals that are present in the environment are paracetamol (acetaminophen) and ciprofloxacin, Paracetamol is a drug with analgesic properties, used for the temporary relief of mild to moderate pain associated with common colds, and in the reduction of fever. Ciprofloxacin is an antibiotic from the chemical class of the fluoroquinolones which presents a broad antibacterial spectrum. Firstly we decided the range of concentrations for each drug. In the case of paracetamol, the tested concentrations were 0.005 mg/L, 0.025 mg/L, 0.125 mg/L, 0.625 mg/L, and 3.125 mg/L, plus a control treatment with water from the facility system. And for ciprofloxacin, the tested concentrations were 0.005 μg/L, 0.013 μg/L, 0.031 μg/L, 0.078 μg/L, 0.195 μg/L, and 0.488 μg/L. These concentrations were chosen as for their environmental relevance since they are close to the real concentrations of these pharmaceuticals found in surface waters and effluents. This work aims to characterize the effects of both drugs in zebrafish embryos and larvae, not only in developmental and behaviour parameters, but also using a biomarker-based approach, namely by quantifying the activities of catalase (CAT), glutathione-S-transferase (GST’s), cholinesterases (ChE’s), glutathione peroxidase (GPx), and determining the TBARS level. Exposure to paracetamol caused an increase in the percentage of organisms with morphological deformations, but no morphological deformations were observed in organisms exposed to ciprofloxacin. Concerning larvae behavioural tests, significant differences were observed for larvae exposed to paracetamol but not for larvae exposed to ciprofloxacin. In the biomarker determination, both drugs caused a statistically significant increase in ChE activity; in CAT only ciprofloxacin caused a significant difference, a decrease in its activity. Paracetamol induced an increase in activity for total GPx and in GST’s. The TBARS levels significantly increased in the exposure to paracetamol, but they significantly decreased in the exposure to ciprofloxacin. In this work, we also prepared an immunohistochemical detection of global methylation, which allowed to observe that, in embryos, the highest concentration of paracetamol caused a slight increase in the intensity of the 5- mdC signal, which can be translated into a slight increase in the DNA methylation. On the other hand, the embryos exposed to ciprofloxacin did not appear to have any difference from control. The comparison of the here-obtained results for the two different drugs allows observing that zebrafish larvae were more sensitive to the exposure to paracetamol, being the most sensitive methodology the biomarker determination. We can also conclude that oxidative stress occurred as a consequence of the exposure to both pharmaceuticals, being more evident in the case of paracetamol. We also showed that the oxidative stress created by the two pharmaceuticals may be the cause of all the other observations.
A presença de produtos farmacêuticos na natureza é uma questão emergente para a comunidade científica uma vez que estes compostos podem ter efeitos deletérios ou imprevisíveis sobre os organismos vivos. O estudo dos efeitos dos resíduos de medicamentos no ambiente evoluiu consideravelmente nos últimos anos, revelando que estes resíduos são uma ameaça para o ecossistema aquático. Dois exemplos de produtos farmacêuticos amplamente utilizados por humanos e presentes no meio ambiente são o paracetamol (acetaminofeno) e a ciprofloxacina. O paracetamol é um medicamento com propriedades analgésicas, utilizado para o alívio temporário da dor leve a moderada associada a gripes comuns, e na redução da febre. A ciprofloxacina é um antibiótico da classe química das fluoroquinolonas que apresenta um amplo espectro antibacteriano. Este trabalho tem como objetivo utilizar larvas de peixe-zebra e caracterizar os efeitos de ambos os medicamentos, não apenas nos parâmetros de desenvolvimento e comportamento, mas também usando com uma abordagem baseada em biomarcadores, nomeadamente quantificando as atividades de catalase (CAT), glutationa-S-transferase (GST), colinesterases (ChE's), glutationa peroxidase (GPx) e determinação do nível TBARS, bem como uma abordagem epigenética. Neste trabalho, também avaliamos por técnicas histoquímicas a metilação global do ADN. No caso do paracetamol, as concentrações testadas foram 0.005 mg/L, 0.025 mg/L, 0.125 mg/L, 0.625 mg/L, e 3.125 mg/L, juntamente com o controlo que apenas continha água do sistema. Para a ciprofloxacina, as concentrações testadas foram 0.005 μg/L, 0.013 μg/L, 0.031 μg/L, 0.078 μg/L, 0.195 μg/L, e 0.488 μg/L. Estas concentrações foram escolhidas pela sua relevância ambiental, uma vez que são muito próximas às concentrações destes fármacos no ambiente. A exposição ao paracetamol causou um aumento na percentagem de organismos com deformações morfológicas, mas não foram observadas deformações morfológicas em organismos expostos à ciprofloxacina. Quanto aos testes comportamentais com larvas de peixe-zebra, observaram-se diferenças significativas nas larvas expostas ao paracetamol, que demonstraram uma maior distância de natação, mas o mesmo efeito não foi observado para as larvas expostas à ciprofloxacina. Os dados obtidos para os diferentes biomarcadores demonstraram que ambos os fármacos causaram um aumento estatisticamente significativo na atividade de ChE. Na actividade da CAT, apenas a ciprofloxacina causou diferença significativa, mais propriamente uma descida na atividade da CAT. O paracetamol induziu um aumento na atividade da GPx total e das GSTs. Os níveis de TBARS aumentaram significativamente após exposição ao paracetamol, mas diminuíram significativamente em organismos expostos à ciprofloxacina. Os dados relativos à quantificação da metilação global do ADN permitiram observar que, em embriões, a maior concentração de paracetamol causou um ligeiro aumento na intensidade do sinal do marcador de metilação de ADN (5- mdC,) quando observado por miscroscopia de confocal. Por outro lado, os embriões expostos à ciprofloxacina não aparentavam ter qualquer diferença no perfil deste marcador de epigenética comparativamente ao controlo. A comparação dos resultados aqui obtidos para os dois fármacos diferentes permite observar que as larvas de peixe zebra eram mais sensíveis à exposição ao paracetamol, sendo a metodologia mais sensível a determinação do biomarcador. Também podemos concluir que o estresse oxidativo ocorreu como conseqüência da exposição a ambos os produtos farmacêuticos, sendo mais evidente no caso do paracetamol. Também mostramos que o estresse oxidativo criado pelos dois produtos farmacêuticos pode ser a causa de todas as outras observações.

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants ubiquistes dont le niveau d’émission est en constante augmentation en raison de l’intensification des activités anthropiques. Ces composés semi-persistants dans l’environnement représentent une menace pour les organismes. La santé et l’équilibre des systèmes naturels sont essentiels au maintien de la vie et au fonctionnement de la société. L’évaluation de l’impact de ces composés est alors devenue un véritable enjeu sociétal, établissant ainsi des mesures européennes législatives (Directive Cadre sur l’Eau) et réglementaires (REACH, enregistrement, évaluation, autorisation et restriction de produits chimiques) pour une meilleure gestion des risques (éco)toxicologiques. Dans ce contexte, de nombreux biotests ont vu le jour afin d’évaluer la dangerosité des substances chimiques (ou de leurs mélanges), la qualité du milieu environnant et de comprendre les mécanismes d’action toxique sur les organismes. L’objectif de cette étude était alors de définir les risques (éco)toxicologiques liés à des expositions par les HAP à travers des tests embryo-larvaires de poisson, utilisant un modèle éprouvé, le poisson zèbre Danio rerio. Afin d’évaluer le transfert, le devenir et les effets toxiques de HAP, deux approches complémentaires (chimique et biologique) ont été utilisées. Les réponses biologiques induites ont été évaluées à différents niveaux d’intégration biologique, des réponses moléculaires (stress oxydatif, dommages à l’ADN, EROD) aux modifications comportementales (Réponse PhotoMotrice), en passant par des altérations morphologiques et physiologiques (rythme cardiaque). Parmi les trois voies de contamination utilisées, l’exposition par contact avec le sédiment ne s’est pas révélée appropriée pour l’évaluation de la toxicité de HAP, contrairement aux expositions par voie aqueuse à partir de fractions hydrosolubles de produits pétroliers, qui sont plus reproductibles, intégratives et révélatrices des effets toxiques à plusieurs échelles. Suite à ces expérimentations, le champ d’observation des effets induits devrait être élargi au-delà des temps normalisés par l’Organisation de Coopération et de Développement Économique afin de ne pas sous-estimer le caractère létal du ou des composé(s) exposés. De plus, notre étude multigénérationnelle a mis en évidence des perturbations physiologiques et comportementales chez la première génération de descendance issue de parents contaminés par voie trophique à trois extraits aromatiques d’origine pyrolytique et pétrogénique (lourd et léger). Bien que le transfert de la contamination n’a pas été révélé, les altérations observées (probablement dues à des modifications génétiques et épigénétiques) chez les stades précoces de poisson zèbre, pourraient avoir des conséquences néfastes sur la survie et le recrutement des populations. Les études multigénérationnelles constituent ainsi des approches intégrées pour l’évaluation de la toxicité des composés exposés et permettent de renforcer le caractère prédictif des effets. Ces études écotoxicologiques devraient être largement déployées afin d’évaluer le potentiel qu’une population exposée a pour se maintenir dans le futur
PAHs are ubiquitous widespread contaminants which emissions are overgrowing with increasing anthropogenic activities. These semi-persistent chemicals are threatening organisms in the environment. Ecosystems health and resilience are essential to life and societal functioning. Impact assessment of these chemicals is a real requirement for society thereby establishing the european legislative (DCE, Water Framework Directive) and regulations (REACH, Registration, Evaluation, Authorisation and Restriction of Chemicals) for better (eco)toxicological risk management. In this context, many bioassays have been developed to assess environmental quality, the toxicity of chemicals (including mixture) on organisms and its underlying mechanisms. This study aimed to identify (eco)toxicological risks in the context of fish embryo-larval assay, using a relevant and well-known model : the zebrafish Danio rerio. Two complementary approaches (chemical and biological) were used to assess transfer, fate and toxicity of PAHs. Induced-responses were evaluated at different levels of biological organization, from molecular (oxidative stress, DNA damage, EROD) to physiological (cardiac activity), behavioral (PhotoMotor Response) and morphological levels. Among the three exposure routes tested, the sediment contact exposure was not suitable for PAHs toxicity assessment. In contrast, exposures to water-accommodated fractions (WAF) of petroleum products represented a more reproducible, sensitive and integrative approach for testing multiscale toxic effects. Following these experiments, the observation scope of induced effects should be broadened beyond the standard duration recommended by the Organization for Economic Cooperation and Development in order not to underestimate the acute effect of the tested-compounds. Furthermore, our multigenerational study showed physiological and behavioral disturbances on the first generation of offspring providing from parents exposed to contaminated-food with three aromatic extracts pyrolytic and petrogenic origin (heavy and light). Although the contamination transfer of PAHs has not been revealed, the observed alterations (probably due to a transfer through genetic and epigenetic modifications) in the early stages of zebrafish could have adverse effects on survival and recruitment populations. Multigenerational studies prove to be an integrated approach for the toxicity assessment of chemicals and strengthen the predictive effects. These ecotoxicological studies should be widely undertaken to evaluate the potential for exposed-population to maintain in the future time

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Zebrafish (Danio rerio) is a bony fish originated in Pakistan and India with anatomical and physiological characteristics and its genome is homologous to the more developed organisms, which suggests that many genes involved in human diseases can be coincident to the zebrafish genome. Copper is a heavy metal involved in formation of ROS, responsible for the lipid peroxidation in the membrane, direct protein oxidation and cleavage of DNA and RNA molecules. Purines (ATP, ADP and adenosine) and pyrimidine (UTP and UDP) can act as extracellular signaling molecules, exerting different physiological effects when binding to purninergic receptors. There are two families of purinergic receptors: adenosine receptors or P1 (P1R), and P2 receptor (P2R). The P1R are a family of G protein-coupled receptors and are divided inA1, A2A, A2B, and A3 subtypes. Adenosine is the major ligand for P1R, and is involved in important physiological functions such as modulation of neurotransmitter release, synaptic plasticity, neuroprotection to ischemic events, hypoxia and oxidative stress. Studies suggest that drugs that interfere in adenosine signaling may weaken the protective mechanisms and lead to an exacerbation of the inflammatory response. Caffeine is the most widely consumed psychoactive substance in the world and is a nonselective antagonist of adenosine receptors. This study aimed to evaluate the effect of caffeine in copper-induced inflammation in zebrafish larvae. The animals were treated with copper at a concentration of 10 ?M and three different concentrations of caffeine (100 ?M, 500 ?M e 1 mM) to determine the survival rate and to define timepoints and concentrations for the following experiments. It was observed that high concentrations of caffeine combined with copper caused a significant decrease of larvae survival after 24 h of treatment. For the following experiments, we chose 4 and 24 h of exposure, 500 ?M caffeine and 10 ?M copper concentrations. After 4 h of treatment, caffeine was able to change the behavior parameters. It was shown that both copper and caffeine might have an anxiogenic effect on zebrafish larvae after 24 h exposure. For the evaluation of inflammatory profile, inflammation markers PGE2, COX-2, TNF, IL-6 and IL-10 were chosen. In 4 hours of exposure, PGE2 levels are increased in groups treated with copper and caffeine. Caffeine showed an additive effect on the PGE2 release. The gene expression of TNF, as well as COX-2 was increased by copper treatment, however, in treatments with caffeine there was a reduction of COX-2 gene expression. In 24 h of treatment, caffeine induced an increase in COX-2, TNF, IL-6 and decrease IL-10 expression. The treatment with copper and caffeine combined increased the expression of the four adenosine receptor subtypes, especially after 4 h of exposure. This may be a result of receptors blockade by caffeine, or a physiological response to inflammation induced by copper, since adenosine, in general, exerts an anti-inflammatory effect. Although caffeine markedly decreased animal survival when combined to copper, caffeine did not potentiate the inflammatory and behavioral effects induced by copper. Our results suggest that caffeine alone is able to exert inflammatory and anxiogenic actions in zebrafish larvae at the timepoints and concentrations assessed.
O zebrafish (Danio rerio) ? um peixe ?sseo origin?rio do Paquist?o e da ?ndia com caracter?sticas anat?micas e fisiol?gicas hom?logas aos organismos mais desenvolvidos. Possui o seu genoma mapeado, com homologia ao genoma humano, o que sugere que diversos genes envolvidos em doen?as humanas podem ser coincidentes ao do genoma do zebrafish. O cobre ? um metal pesado que est? envolvido na produ??o de esp?cies reativas de oxig?nio, respons?veis pela peroxida??o de l?pidios nas membranas, oxida??o direta de prote?nas, e clivagem de mol?culas de DNA e RNA. Purinas (ATP, ADP e adenosina) e pirimidinas (UTP e UDP) extracelulares podem atuar como mol?culas sinalizadoras end?genas exercendo diferentes efeitos fisiol?gicos. Para tais ligantes, existem duas fam?lias de receptores purin?rgicos: receptores de adenosina ou P1 (P1R), e receptores P2 (P2R). Os P1R t?m como ligante end?geno a adenosina, e s?o uma fam?lia de receptores metabotr?picos, acoplados ? prote?na G, e se dividem nos subtipos A1, A2A, A2B e A3. A sinaliza??o mediada pela adenosina est? envolvida em importantes fun??es fisiol?gicas, como a modula??o da libera??o de neurotransmissores, plasticidade sin?ptica, neuroprote??o em eventos isqu?micos, hip?xia e estresse oxidativo. Estudos sugerem que subst?ncias que interferem na sinaliza??o adenosin?rgica na hip?xia podem enfraquecer os mecanismos protetores, resultando no aumento da resposta inflamat?ria. A cafe?na (1,3,7 ? trimetilxantina) ? a subst?ncia psicoativa mais consumida no mundo e ? um antagonista n?o seletivo dos receptores de adenosina. Este trabalho teve como objetivo avaliar o efeito da cafe?na na inflama??o induzida por cobre em larvas de zebrafish. Os animais foram tratados com cobre na concentra??o de 10 ?M e com outras 3 concentra??es diferentes de cafe?na (100 ?M, 500 ?M e 1 mM). Foi observado, que altas concentra??es (500 ?M e 1 mM) de cafe?na juntamente com o cobre provocaram um aumento significativo na mortalidade das larvas ao final de 24 horas de tratamento. Para os experimentos seguintes, foram escolhidos os tempos 4 e 24 horas e a concentra??o de 500 ?M de cafe?na e 10 ?M de cobre. Em 4 horas de tratamento, a cafe?na foi capaz de alterar os par?metros de comportamento avaliados. Foi demonstrado que tanto o cobre quanto a cafe?na, podem exercer um efeito ansiog?nico nas larvas de zebrafish ap?s 24 horas. Para a avalia??o do perfil inflamat?rio, foram escolhidos os marcadores de inflama??o PGE2, COX-2, TNF, IL-6 e IL-10. Foi mostrado que em 4 horas de exposi??o h? um aumento dos n?veis de PGE2 nos grupos tratados com cobre e cafe?na, mostrando um efeito aditivo da cafe?na em rela??o ? inflama??o induzida por cobre. A express?o g?nica de TNF, bem como a de COX-2 foi aumentada pelo tratamento com cobre, por?m, nos tratamentos com cafe?na houve uma diminui??o da express?o g?nica de COX-2. Em 24 horas de tratamento, a cafe?na induziu um aumento na express?o de COX-2, TNF, IL-6 e diminui??o de IL-10. Os tratamentos com cobre e cafe?na aumentaram a express?o dos 4 subtipos de receptores de adenosina principalmente ap?s 4 horas de exposi??o. Isso pode ser devido ao bloqueio dos receptores pela cafe?na e por uma medida fisiol?gica de defesa em resposta ? inflama??o induzida pelo cobre, visto que a adenosina, de uma forma geral, exerce um efeito anti-inflamat?rio. Embora a cafe?na tenha induzido uma diminui??o significativa da sobrevida dos animais, quando adicionada ao tratamento com cobre, nos marcadores inflamat?rios e nos par?metros comportamentais estudados, n?o houve um efeito aditivo da cafe?na na inflama??o induzida pelo cobre. Nossos resultados sugerem que a cafe?na por si s? ? capaz de exercer uma a??o inflamat?ria e ansiog?nica em larvas de zebrafish nos tempos e concentra??es estudados.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Mosquitoes are responsible for the transmission of various pathogens. Over the past three years, the world has witnessed epidemics affecting all age groups, such as dengue fever, yellow fever, chikungunya and zika virus. To control and eliminate Aedes vectors responsible for the transmission of these diseases, it is necessary to use larvicides and insecticides such as pyriproxyfen. Pyriproxyfen is a larvicide used to control mosquitoes, mainly of the genus Aedes. This study evaluated the effects of pyriproxyfen on survival, morphology, behavior, and endocrine parameters at different stages of zebrafish development. We have demonstrated that pyriproxyfen can cause changes in the survival rate, behavior and morphology of exposed larvae, adults exposed at the larval stage, and adult offspring of exposed parents. Larvae exposed to pyriproxyfen for 1-96 hpf (hours post-fertilization) showed a decrease in survival rate, heart rate 3 dpf (days post-fertilization), and body length at 5 and 8 dpf. The distance traveled and the mean velocities of the exposed larvae were reduced in comparison to the vehicle group (1% DMSO). Therefore, pyriproxyfen changes the morphology and behavior of zebrafish in early stages of development and may affect the next generations. However, when the exposed larvae were maintained until the adult stage (6 months), we did not observe differences in locomotor parameters. Parental exposure to pyriproxyfen induced decrease in locomotion of zebrafish adult offspring as well as an anxiolytic-like behavior. We also tested pyriproxyfen exposure for 96 hours in adult zebrafish and the locomotion, anxiety, memory and endocrine parameters were analyzed as well as the gene expression of the glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF). These results showed that there was no significant difference in locomotion, anxiety and endocrine parameters. In addition, there was an impairment of habituation memory in adult zebrafish exposed to pyriproxyfen. Therefore, it is important to control the use of larvicides due to their toxic effects on non-target species. Our findings demonstrated the importance of studies related to the use of larvicides, since they are potential causes of morphological and behavioral alterations in aquatic species, such as zebrafish.
Mosquitos s?o respons?veis pela transmiss?o de v?rios agentes patog?nicos. Nos ?ltimos tr?s anos, o mundo testemunhou epidemias que afetam todas as faixas et?rias, tais como dengue, febre amarela, chikungunya e zika v?rus. Para controlar e eliminar vetores do g?nero Aedes, respons?vel pela transmiss?o destas doen?as, ? necess?rio usar larvicidas e inseticidas como o piriproxifeno. O piriproxifeno ? um larvicida usado para eliminar mosquitos, principalmente do g?nero Aedes. Este estudo avaliou os efeitos do piriproxifeno sobre a sobreviv?ncia, morfologia, comportamento e par?metros end?crinos em diferentes est?gios de desenvolvimento de peixe-zebra. N?s demonstramos que o piriproxifeno pode causar altera??es na taxa de sobreviv?ncia, comportamento e morfologia de larvas expostas, adultos expostos no est?gio larval e descendentes adultos de pais expostos. As larvas expostas ao piriproxifeno durante 1-96 hpf (horas p?s-fertiliza??o) mostraram uma diminui??o na taxa de sobreviv?ncia, frequ?ncia card?aca 3 dpf e comprimento corporal nos 5 e 8 dpf (dias p?s- fertiliza??o). A dist?ncia percorrida e a velocidade m?dia das larvas expostas foram reduzidas em rela??o ao grupo ve?culo (1% de DMSO). Portanto, o piriproxifeno altera a morfologia e o comportamento do peixe-zebra em est?gios iniciais de desenvolvimento e pode afetar as pr?ximas gera??es. No entanto, quando as larvas expostas foram mantidas at? o est?gio adulto (6 meses), n?o observamos diferen?as nos par?metros locomotores. A exposi??o parental ao piriproxifeno induziu uma diminui??o na locomo??o da prole adulta, bem como um comportamento do tipo ansiol?tico. N?s tamb?m avaliamos o efeito da exposi??o ao piriproxifeno em peixe-zebra na fase adulta durante 96 horas sobre a locomo??o, ansiedade, mem?ria e par?metros end?crinos, como express?o g?nica do receptor de glicocorticoide (GR) e fator de libera??o de corticotrofina (CRF). Os resultados demonstraram que n?o houve diferen?a significativa na locomo??o, ansiedade e par?metros end?crinos. Al?m disso, houve um preju?zo da mem?ria de habitua??o do peixe-zebra adulto exposto ? piriproxifeno. Portanto, ? importante controlar o uso de larvicidas devido aos seus efeitos t?xicos em esp?cies n?o-alvo. Nossos achados demonstram a import?ncia de estudos relacionados com a utiliza??o de larvicidas, uma vez que s?o potenciais causadores de altera??es morfol?gicas e comportamentais em esp?cies aqu?ticas, como o peixe-zebra.

Cardiac, metabolic and growth response of early-stage chicken embryos to perturbations in yolk environment was investigated. Also, effects of parental hypoxia exposure on hypoxia resistance, thermal tolerance and body length of zebrafish larvae were investigated. In the first study, thyroxine, triiodothyronine and testosterone produced differential effects on heart rate and development rate of chicken embryos during the first 4 days of development. Triiodothyronine caused a dose-dependent increase in heart rate when applied at 40 or 70 hours of age, while thyroxine caused a dose-dependent increase in heart rate when applied at 40 hours only. Testosterone and propyl-thiouracil (deiodinase antagonist) did not have an effect on heart rate. Development rate was not changed by thyroxine, triiodothyronine, testosterone or propyl-thiouracil, which suggested that heart rate changes did not result from changes in embryo maturity. In the second study, chicken embryos exposed to yolks of different bird species during early-stage embryonic development showed changes in heart rate, mass-specific oxygen consumption and body mass that scaled with the egg mass, incubation period length, and yolk triiodothyronine and testosterone levels of the species from which yolk was derived. In the third study, this phenomenon was investigated between layer and broiler chickens. Heart rate, oxygen consumption and body mass of broiler and layer embryos were significantly changed by a breed-specific change in yolk environment. Yolk triiodothyronine and testosterone concentrations of broiler and layer eggs did not suggest that these hormones were responsible for physiological and morphological changes observed. The final study demonstrated that hypoxia resistance and body lengths, but not thermal tolerance of zebrafish larvae was increased by parental hypoxia exposure.

Le comportement animal est souvent conçu comme résultant d'une association entre un stimulus et une réponse. Selon cette vision, comprendre le cerveau revient à dénouer les liens entre les entrées sensorielles et les sorties motrices. Toutefois, dans des conditions naturelles, l'influence entre l'action motrice et la perception sensorielle est réciproque. Les animaux utilisent constamment les rétroactions sensorielles causées par leurs actions pour ajuster les commandes motrices. Par ailleurs, le comportement n'est pas seulement une réponse à l'environnement sensoriel mais peut être généré par l'activité endogène du cerveau. Afin de comprendre le dialogue sensorimoteur en observant de larges régions cérébrales à une résolution cellulaire, j'ai étudié les comportements induits et spontanés chez la larve du poisson-zèbre. Les atouts de la larve du poisson zèbre sont sa petit taille et sa transparence. On peut utiliser des méthodes d'imagerie fonctionnelle optique, comme la microscopie par nappe laser, afin d'enregistrer l'activité dans une large portion des neurones. Afin d'étudier le comportement de navigation chez la larve dans des conditions compatibles avec l'observation du cerveau, j'ai développé un système de réalité virtuelle visuelle pour la larve du poisson zèbre. L'environnement visuel est mis à jour en fonction des mouvements du poisson. Cette rétroaction peut être choisie comme étant similaire à la rétroaction visuelle que le poisson expérimente en nage libre. En modifiant la rétroaction visuelle naturelle, on peut étudier la manière dont la larve s'adapte aux perturbations. Dans cette optique, j'ai d'abord généré une librairie de mouvements de nage libre. A partir de celle-ci, j'ai extrait la relation entre la trajectoire de la larve et la cinématique de ses mouvements de queue. Cette relation permet de décoder les intentions de déplacements chez une larve dont la tête est restreinte dans un gel et de mettre à jour un environnement visuel selon ses mouvements de queue. Dans un environnement virtuel, la larve parvient à contrôler son orientation et sa vitesse afin de suivre un mouvement d'ensemble ou bien à générer une séquence de mouvements nécessaires à atteindre une cible mobile. Lorsque la rétroaction visuelle n'est pas mise à jour continuellement mais à la fin de chaque mouvement, on observe que les mouvements sont alors plus longs. Cette faible perturbation réduit significativement le succès des déplacements du poisson vers des cibles virtuelles. Le comportement peut aussi résulter de l'activité endogène du cerveau. En absence de stimulus externe, la larve produit des mouvements stéréotypés similaires à ceux produits lorsqu'elle navigue en réponse à un stimulus. Après avoir établit une nouvelle méthode de classification des mouvements de queue, j'ai analysé la séquence des mouvements générés spontanément. Ces séquences sont composées de successions quasi rythmiques qui alternent avec de longues périodes de repos. Les mouvements consécutifs sont davantage similaires lorsqu'ils s’enchaînent rapidement (~10s). Afin d'étudier les mécanismes neuronaux responsables de la décision d'effectuer un mouvement spontané, j'ai couplé l’imagerie par nappe laser à l'analyse des mouvements. Des résultats préliminaires mettent en évidence des groupes de neurones dont l'activité prédit la direction des mouvements des virages. Deux groupes repartis bilatéralement oscillent en opposition de phase et l'état de cet oscillateur avant un mouvement prédit sa direction. Les neurones responsables de la décision du type de mouvement à effectuer spontanément sont différents des neurones qui contrôlent le timing de leur déclenchement. Ensemble, ces résultats éclairent les processus de rétroaction et de dynamique interne qui façonnent le comportement et ouvre la voie à l'étude de processus sensorimoteurs complexes dans des systèmes simples
Behavior is often conceived as resulting from a stimulus-response association. Under this paradigm, understanding the nervous system is reduced to finding the relation between a sensory input and a motor output. Yet, in naturally behaving animals, motor actions influence sensory perceptions just as much as the other way around. Animals are continuously relying on sensory feedback to adjust motor commands. On the other hand, behavior is not only induced by the sensory environment, but can be generated by the brain's rich internal dynamics. My goal is to understand the sensory-motor dialogue by monitoring large brain regions, yet, with a single-neuron resolution. To tackle this question, I have used zebrafish larva to study visually induced and internally driven motor behaviors. Zebrafish larvae have a small and transparent body. These features enable using large-scale optical methods, such as selective plane illumination microscopy (SPIM), to record brain dynamics. In order to study goal-driven navigation in conditions compatible with imaging, I developed a visual virtual reality system for zebrafish larva. The visual feedback can be chosen to be similar to what the animal experiences in natural conditions. Alternatively, alteration of the visual feedback can be used to study how the brain adapts to perturbations. For this purpose, I first generated a library of free-swimming behaviors from which I learned the relationship between the trajectory of the larva and the shape of its tail. I then use this technique to infer the intended displacements of head-fixed larvae. The visual environment was updated accordingly. In the virtual environment, larvae were capable of maintaining the proper speed and orientation in the presence of whole-field motion and produced fine changes in orientation and position required to capture virtual preys. I demonstrate the sensitivity of larvae to visual feedback by updating the visual world only after the discrete swimming episodes. This feedback perturbation induced a decay in the performance of prey capture behavior, suggesting that larva rely on real-time visual feedback during swimming. Behavior can also be induced by the internal dynamics of the brain. In the absence of salient sensory cues, zebrafish larva spontaneously produces stereotypical tail movements, similar to those produced during goal-driven navigation. After having developed a new method to classify tail movements, I analyzed the sequence of spontaneously generated tail movements. The latter switched between period of quasi-rhythmic activity and long episodes of rest. Moreover, consecutive movements were more similar when executed at short time intervals (~10s). In order to study the mechanisms responsible for the spontaneous decisions to move, I coupled SPIM to tail movement analysis. Using dimensionality reduction, I identified clusters of neurons predicting the direction of spontaneous turn movements but not their timings. This Preliminary result suggests that distinct pathways could be responsible for the timing (when) and the selection (what) of spontaneous actions. Together, the results shed light on the role of feedback and internal dynamics in shaping behaviors and open the avenue for investigating complex sensorimotor process in simple systems

Le transport moléculaire dans les neurones joue un rôle essentiel dans leur développement et la maintenance de leurs fonctions, en raison notamment de leurs longues arborescences. Les conséquences d’anomalies du transport axonal ont été associées de longue date à la maladie d’Alzheimer telle que le gonflement des axones consécutif à l’accumulation des organelles transportés, mais il y a peu d’outils permettant l’étude fine sur le long terme de ce transport en culture et encore moins in vivo.L’équipe dans laquelle cette thèse a été réalisée a développé une méthode de mesure du transport intraneuronal en culture de neurone de souris reposant sur le suivi d’endosomes ayant internalisé spontanément des nanodiamants fluorescents. Elle a également démontré la grande sensibilité de cette méthode à détecter des facteurs de risques génétiques de maladies neuropsychiatriques.L’objectif de ma thèse a été d’étendre cette méthode de mesure au circuit neuronal intègre du cerveau de larves de poisson zèbre. Pour ce faire j’ai utilisé des nanocristaux (NCs) à réponse optique non-linéaires, pouvant être excité dans une fenêtre de transparence des tissus, à une longueur d’onde ≈1 µm, et générant un signal de second harmonique (SHG). Nous avons utilisé le microscope multiphoton de la plateforme Emerg’In (INRAE, Jouy-en-Josas) qui est installée au sein d’une pisciculture et dispose de scanners galvanométriques résonants et de détecteurs hybrides, ce qui nous a permis de maintenir une cadence de 20 champs par seconde malgré la nécessité de balayer le faisceau laser, contrairement aux études en culture, menées en champ large.Nous avons mis au point un nouveau protocole de mesure, depuis l’injection des NC jusqu’à l’extraction des paramètres de transport à partir des données vidéos. Pour cela, j’ai optimisé et automatisé les analyses des données en développant deux programmes écrits en Python : l’un permettant de reconstruire automatiquement les trajectoires des NCs de manière la plus fidèle possible et l’autre permettant de segmenter les trajectoires en phases de mouvement et de pauses afin d’en extraire des paramètres de transport.J’ai appliqué ces nouveaux outils à l’étude, sur un modèle transgénique de poisson zèbre, de l’impact fonctionnel d’une mutation présente dans des neuropathies dont une paraplégie spastique héréditaire et la maladie de Charcot-Marie-Tooth périphérique. Chez des patients atteints par l’une de ces maladies, plusieurs mutations ont été identifiées sur le gène KIF5A codant pour la chaine lourde du moteur moléculaire kinésine 1 qui assure des déplacements antérogrades (du corps cellulaire vers la périphérie). Nous avons mesuré le transport d’endosomes dans les axones de neurones du cerveau de larve de poissons zèbres transgéniques portant une mutation sur kif5a, et avons observé des modifications subtiles de ce transport. Pour cela, plus de 500 trajectoires d’endosomes (acquises à une profondeur de ≈100 µm sous la surface de la tête) ont été analysées, issues de 40 larves.En guise de contrôle, nous avons également étudié le transport endosomal dans des mutants privés des moteurs dynéines, les seuls à assurer le transport rétrograde, et avons observé que le transport bidirectionnel était totalement arrêté dans les mutants.La méthodologie mise en place peut servir à cribler des anomalies fonctionnelles résultant de facteurs génétiques de troubles neurologiques ou neurodégénératifs
Molecular transport in neurons plays an essential role in their development and the maintenance of their functions, due in particular to their long branches. The consequences of axonal transport abnormalities have long been associated with Alzheimer's disease, such as the swelling of axons caused by the accumulation of transported organelles. However, there are few tools allowing the detailed long-term study of this transport in culture and even less in vivo.The team in which this work was conducted has developed a method for measuring intraneuronal transport in mouse cultured neurons based on the tracking of endosomes having spontaneously internalized fluorescent nanodiamonds. This technique was able to detect genetic risk factors of neuropsychiatric diseases, proving its high sensitivity.The objective of my thesis was to extend this measurement method to the neuronal circuit of the zebrafish larvae’s brain. For this purpose, I have used nanocrystals (NCs) with non-linear optical response, which can be excited in a tissue transparency window at a wavelength ≈1 µm, and generate a second harmonic signal. We have used the multiphoton microscope of the Emerg'In facility (INRAE, Jouy-en-Josas), which is installed next to a fish farming and has resonant galvanometric scanners and hybrid detectors, which allowed us to maintain a frame rate of 20 fps despite the need to scan the laser beam, unlike in culture studies conducted in wide field.We have developed a new measurement protocol, from the injection of the NC to the extraction of transport parameters from the videos data. To do this, I have optimized and automated the data analysis by developing two programs written in Python: one that automatically reconstructs the trajectories of the NCs as faithfully as possible and the other that segments the trajectories into motion and pause phases in order to extract transport parameters.I have applied these new tools to study, in a transgenic zebrafish model, the functional impact of a mutation present in neuropathies including hereditary spastic paraplegia and peripheral Charcot-Marie-Tooth disease. In patients suffering from one of these diseases, several mutations have been identified in the KIF5A gene coding for the heavy chain of the kinesin 1 molecular motor which ensures anterograde movements (from the cell body to the periphery). We have measured endosome transport in axons of neurons in the brain of transgenic zebrafish larvae bearing a mutation on kif5a, and we observed subtle changes in this transport. More than 500 endosome trajectories (acquired at a depth of ≈100 µm below the surface of the head) were analyzed from 40 larvae.As a control, we also studied endosomal transport in mutants deprived of dynein motors, the only ones to ensure retrograde transport, and observed that bi-directional transport was totally stopped in the mutants.This methodology can be used to screen for functional abnormalities resulting from genetic factors of neurological or neurodegenerative disorders

Au cours des dernières années, les plastiques ont attiré un intérêt particulier du public et des scientifiques. Les plastiques ont été classés parmi les polluants émergents, notamment dans le milieu marin de par leur présence ainsi que leur persistance, en particulier les microplastiques (< 5mm, MPs). De nombreuses études ont reportées la présence de MPs dans le milieu marin (eau de surface et sédiments). Cependant, il n’existe pas de méthodes standards pour évaluer leurs potentielle toxicités ainsi que leur rôle en tant que vecteur de polluants au sein des organismes. Cette thèse avait pour objectif, (1) l’étude de la sorption de composés chimiques modèles sur des MPs, (2) l’étude de la toxicité de ces composés adsorbés artificiellement sur le poisson zèbre (Danio rerio) ainsi que (3) la caractérisation et l’étude de MPs environnementaux. La première partie de cette thèse avait pour but d’étudier les processus de sorption de trois composés modèles ; l’acide perfluorooctanesulfonique (PFOS), benzo[a]pyrene (BaP) et l’oxybenzone (BP3) ; sur différentes tailles et différents polymères industriels. La seconde partie concernait l’étude de la vectorisation de ces 3 composés modèles adsorbés sur MPs en utilisant les stades précoces, juvéniles et adultes de poisson zèbre, au travers d’exposition directe ou via exposition trophique. La dernière partie évaluait la toxicité de MPs environnementaux provenant de deux plages (Guadeloupe, France), en utilisant les mêmes procédures que pour l’évaluation de la toxicité de MPs artificiels. La caractérisation polymérique et chimique des échantillons environnementaux a également été étudiée. L’étude de la sorption des polluants démontra des différences de réponses (cinétiques et efficacité de la sorption) dépendant de la taille des particules ainsi que des propriétés chimiques des composés. La toxicité des MPs artificiellement dopés avec des composés modèles a d’abord été évaluée selon la ligne directrice de l’OCDE 236 ainsi que via une exposition trophique chronique, débutant à l’état larvaire jusqu’à des poissons âgés de 5 mois. Des biomarqueurs à l’échelle individuelle et moléculaire ont été suivis au cours de l’exposition. Les résultats principaux correspondaient à une faible toxicité aiguë sur les stades précoces de développement (embryons et larves) exposés aux particules, aux extraits organiques mais également aux lixiviats, de MPs artificiels ou environnementaux. Néanmoins, l’ingestion des MPs au cours de l’exposition trophique a conduit à des effets à long-terme, avec des intensités différentes liées aux composés chimiques adsorbés aux MPs. Les effets toxiques incluaient des altérations de la croissance (taille et poids des poissons), des effets sur la reproduction, des modifications comportementales des adultes exposés ainsi que des changement d’activité natatoire des larves de la génération F1. En conclusion, cette thèse a permis de mettre en évidence le fait que (1) les MPs ont la capacité d’être des vecteurs de polluants mais qu’ils peuvent également (2) induire des effets sublétaux sur des juveniles et adultes de poisson exposés aux MPs via la nourriture mais également sur la progéniture F1
During the last decades, plastics have gained interest from media, public and scientists. Plastics have been categorized as emerging pollutants particularly in the marine environment due to their ubiquity and persistence, particularly microplastics (< 5 mm, MPs). Numerous studies reported the occurrence of MPs in the marine environment (surface water, sediments). However, neither there exist standardized nor harmonized methods to evaluate their potential toxicity and their role as vector of hazardous chemicals into organism. This thesis had three main objectives, (1) the investigation of sorption of chemicals models on MPs, (2) the study of the toxic potential of these sorbed chemicals on zebrafish and (3) the characterization and the study of the toxicity of environmental MPs. The first part of the thesis aimed at the investigation of the sorption processes of three model pollutants - perfluorooctanesulfonic acid (PFOS), benzo[a]pyrene (BaP) and oxybenzone (BP3) - on pristine MPs using different sizes and polymer types. The second part studied the vectorization of the three compounds sorbed on MPs toward zebrafish (Danio rerio) embryos, juveniles and adults through direct or trophic exposures, and their toxicities. The third part investigated the toxicity of environmental samples of MPs from two sandy beaches (Guadeloupe Island, France), using the same procedure as above, as well as the characterization of additives and adsorbed chemicals through a non-target approach. Study of pollutants sorption demonstrated differences in pattern (e.g. kinetics, sorption efficiency), depending on size particles and chemical used. Potential adverse effects of MPs associated with chemicals were investigated in zebrafish embryos using OECD 236 guideline and by using a chronic exposure from larvae or juveniles up to 5-months old adults through trophic exposure. Molecular and individual toxicological endpoints were monitored during exposure. Main findings were the low acute toxicity of MPs on early life stages (embryos and larvae) exposed to particles, organic extracts or leachates. Nevertheless, the ingestion of MPs by juveniles and adults led to a significant long-term toxicity for all tested MPs albeit with different intensity according to the spiked chemicals and to the environmental samples. Deleterious effects included growth alteration, reprotoxicity, behavioral disturbances as well as hyperactivity observed in offspring of exposed fish. In conclusion, the present work revealed that (1) MPs may play a role in the vectorization of pollutants and (2) may induce significant sublethal effects in juveniles of zebrafish chronically fed with pristine MPs or MPs artificially spiked with pollutants. Same conclusions were observed (3) with environmental MPs

A carboxilase dependente da vitamina K (VKD), permite que ocorra uma transformação dos resíduos de ácido glutamico (Glu) para ácido carboxiglutamico (Gla), que torna funcionais as proteínas dependentes da vitamina K (VKD), influenciando a fisiologia, a homeostasia, a apoptose, a transducção do sinal, o controlo do crescimento, a calcificação arterial e o desenvolvimento do esquelto. A proteina Gla da matriz (Mgp) e a osteocalcina (Oc ou proteína Gla do osso- Bgp), têm a capacidade de ligar o cálcio através do dominio contendo os resíduos gla, sendo uma função essencial para a sua actividade biológica como inibidor de calcificação (Mgp) e modulador dos cristais de hidroxiapatite (Oc). Usaram-se fases larvares de peixe zebra (Danio rerío), para compreender o efeito da varfarina no desenvolvimento esquelético. A varfarina é um conhecido anticoagulante, que inibe a acção da carboxilação da vitamina K (VKGC), inibindo a maturação da Oc e da Mgp e levando a um incorrecto resultado da sua função. Para avaliar os efeitos varfarina, recorreu-se à dupla coloração do esqueleto com azul de alcian e vermelho de alizarin, revelando uma inibição da mineralização. A imunodetecção da Oc e da Mgp revelaram uma acumulação acrescida em tecidos calcificados em desenvolvimento. A quantificação da expressão por PCR em tempo real revelou uma sobre-expressão de mgp e oc por resposta ao tratamento com varfarina. A varfarina aparenta afectar a correcta calcificação das larvas e na função da Oc e Mgp.
The Vitamin K-dependent (VKD) carboxylase allows the conversation of the glutamyl residues (Glu) to carbolxy glutamic acid (Gla), having an effect on the vitamin K dependente (VKD) proteins, and this go influence the physiology of the homeostasis, apoptosis, signal transduction, growth control, arterial calcification and skeletal development. Matrix Gla protein (MGP) and osteocalcin (Oc or bone Gla Protein- BGP) have the capacity to bind calcium trough the gla domain, something essential for biological activity as inhibitor of calcification (Mgp) and modulator of hydroxyapatite crystals. We used developing larvae of zebrafish (Danio rerío) to evaluate the effects of warfarin in skeletal development. Warfarin is a known anticoagulant, that inhibits the action of vitamin K carboxylation (VKGC), inhibiting the maturation of oc and mgp taking to an incorrect result of their function. For accessing the effects of warfarin we used whole mount double staining with Alcian Blue and Alizarin red revealing a decrease im mineralization. The imunodetection of Oc and Mgp showed an increased accumulation in calcified tissues and quantitative real time PCR showed a upregulation of both oc and mgp as response to warfarin treatment. Warfarin appears to affect the correct calcification in larvae and affect the function of Oc and Mgp.

Mercury (Hg) compounds are some of the most toxic compounds of any heavy metal on earth. Due to long-range transport from point sources Hg can be found world-wide in air, soil, water, and living organisms. Mercury compounds can cause a number of adverse effects, with the unborn fetus, infants, and children being most susceptible. Zebrafish (Danio rerio) are an excellent vertebrate model system for toxicological studies, including developmental effects. The overall objective of this research was to investigate the effects of inorganic forms of Hg in zebrafish larvae. Unique accumulation patterns were observed using synchrotron X-ray fluorescence imaging after zebrafish were exposed to one of four Hg compounds (i.e. mercuric chloride, mercury bis-L-cysteineate, methylmercury chloride, methylmercury L-cysteineate). Specifically, we noted chemical form dependant and tissue-specific Hg accumulation including the sensory cells of the olfactory epithelia and the neuromasts. Phenylthiourea (PTU) is commonly used to block zebrafish melanogenesis to generate transparent larvae to aid with enhanced visualization of immunohistochemical and vital stains. It was determined that PTU dramatically alters Hg toxicity through chemical interaction with Hg so that further studies were conducted in the absence of PTU. To investigate the effects of Hg on primary neurons, the immunohistochemistry protocol using anti-acetylated tubulin was performed and the results demonstrated that mercuric chloride damages primary neurons particularly in the olfactory pits. To study potential detoxification of Hg in zebrafish we examined the efficacy of two sequestration agents, dimercaptosuccinic acid and alpha lipoic acid, as well as endogenous selenium. The levels of Hg were not significantly lower following treatment with either sequestration agent under the conditions used in this research. Previous work examining the antagonistic relationship between Hg and selenium has been conducted by dosing animals with both Hg and selenium (Se). We discovered a mixed chalcogenide of the general form HgSxSe(1-x) forming in vivo following exposure to mercuric chloride without the addition of selenium. Indeed the selenium may have been remobilized from natural stores in the pigment spots. The research presented herein demonstrates that the target tissues for Hg depend strongly on chemical form. In particular inorganic Hg can accumulate in a number of important tissues including sensory systems. The formation of insoluble and non-toxic HgSxSe(1-x) in zebrafish larvae suggests that endogenous selenium may play critical roles in modulating toxicity.

Dissertação mest., Biologia Marinha, Universidade do Algarve, 2009
Zebrafish is a freshwater teleost which, due to several biological and genetic characters, has been widely used as a vertebrate model in a variety of research fields, with great potential in molecular biology. As a consequence of the increasing interest in zebrafish, the optimization of its rearing conditions becomes essential, such as the determination of a standardized feeding regime appropriate to this species’. Four different dietary regimes, composed of rotifers Brachionus sp., Artemia nauplii and inert food (Cyclop-eezeTM e BeneluxNV), were carried out. The effect of diet in zebrafish larvae and juvenile fish was evaluated by the determination of the survival and growth parameters as well as by the analysis of the development at the tissue, cellular and molecular levels in three distinct developmental stages. For that, histological, gene expression and immunohistochemical tools applied to molecular markers of the skeletal development (matrix Gla protein – mgp – and osteocalcin – oc) and digestive tract development (amylase and trypsin) were used. The assessment of bone remodeling regions and a histological analysis of the digestive system were also carried out. The obtained data suggest that rotifers are not appropriate for the larval requirements of this species, presenting the lowest survival and growth values. These data were also supported at the molecular level. Artemia nauplii and a weaning to inert food at 15 days post fertilization does not seem to be, also, an appropriate feeding regime. Comparatively, a diet composed exclusively by Artemia nauplii presented higher development-related parameters. These data suggest that this is a suitable food regime, as it has been argued by several authors. A daily mixture of foods also seems to be suitable to zebrafish healthy development. This work showed that, in zebrafish early development Artemia nauplii should used as food and could be, possibly, supplemented with other foods to obtain better results.