PARP1 and PARP2 are the most extensively studied proteins of the poly(ADP-ribose) polymerase (PARP) family. They share partially overlapping functions. These two proteins are best known for their roles in DNA repair. The DNA damage response is actually the most active area of research involving the PARP proteins given the success of PARP inhibitors for cancer therapy. However, PARPs possess many other functions. PARP3, a very little characterized protein, appears to be somewhat involved in the response to DNA damage by genotoxic agents but its physiological function is unknown. Recent evidence indicated that PARP3 is involved in the epigenetic regulation of transcription. For this reason, our collaborators identified PARP3-bound genes by screening the genomic occupancy of PARP3 and found that PARP3-bound genes associate with developmental transcription factors especially involved in neurogenesis. We used zebrafish, a well established vertebrate model in developmental biology, to study the role of PARP3 in development. By knocking-down Parp3 in zebrafish, we found that the loss of Parp3 function reduces the expression of neural crest “specifier” sox9a and of dlx3b/dlx4b. It impairs the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud. In parallel, the reduced expression of Parp3 leads to a massive increase in apoptosis. I also knocked-down Parp1 and Parp2 in zebrafish. Results suggest that the function of Parp1 is different from that of Parp3 in zebrafish while the data from Parp2 were inconclusive. Our findings demonstrate that Parp3 is essential during early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions during the specification of the neural plate border and by mediating cell survival during the early stages of development.

PARP1 and PARP2 are the most extensively studied proteins of the poly(ADP-ribose) polymerase (PARP) family. They share partially overlapping functions. These two proteins are best known for their roles in DNA repair. The DNA damage response is actually the most active area of research involving the PARP proteins given the success of PARP inhibitors for cancer therapy. However, PARPs possess many other functions. PARP3, a very little characterized protein, appears to be somewhat involved in the response to DNA damage by genotoxic agents but its physiological function is unknown. Recent evidence indicated that PARP3 is involved in the epigenetic regulation of transcription. For this reason, our collaborators identified PARP3-bound genes by screening the genomic occupancy of PARP3 and found that PARP3-bound genes associate with developmental transcription factors especially involved in neurogenesis. We used zebrafish, a well established vertebrate model in developmental biology, to study the role of PARP3 in development. By knocking-down Parp3 in zebrafish, we found that the loss of Parp3 function reduces the expression of neural crest “specifier” sox9a and of dlx3b/dlx4b. It impairs the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud. In parallel, the reduced expression of Parp3 leads to a massive increase in apoptosis. I also knocked-down Parp1 and Parp2 in zebrafish. Results suggest that the function of Parp1 is different from that of Parp3 in zebrafish while the data from Parp2 were inconclusive. Our findings demonstrate that Parp3 is essential during early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions during the specification of the neural plate border and by mediating cell survival during the early stages of development.

PARP1 and PARP2 are the most extensively studied proteins of the poly(ADP-ribose) polymerase (PARP) family. They share partially overlapping functions. These two proteins are best known for their roles in DNA repair. The DNA damage response is actually the most active area of research involving the PARP proteins given the success of PARP inhibitors for cancer therapy. However, PARPs possess many other functions. PARP3, a very little characterized protein, appears to be somewhat involved in the response to DNA damage by genotoxic agents but its physiological function is unknown. Recent evidence indicated that PARP3 is involved in the epigenetic regulation of transcription. For this reason, our collaborators identified PARP3-bound genes by screening the genomic occupancy of PARP3 and found that PARP3-bound genes associate with developmental transcription factors especially involved in neurogenesis. We used zebrafish, a well established vertebrate model in developmental biology, to study the role of PARP3 in development. By knocking-down Parp3 in zebrafish, we found that the loss of Parp3 function reduces the expression of neural crest “specifier” sox9a and of dlx3b/dlx4b. It impairs the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud. In parallel, the reduced expression of Parp3 leads to a massive increase in apoptosis. I also knocked-down Parp1 and Parp2 in zebrafish. Results suggest that the function of Parp1 is different from that of Parp3 in zebrafish while the data from Parp2 were inconclusive. Our findings demonstrate that Parp3 is essential during early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions during the specification of the neural plate border and by mediating cell survival during the early stages of development.

Mycobacterium abscessus (Mabs) est un pathogène émergent entrainant de graves infections pulmonaires, notamment chez les patients mucoviscidosiques. L'expression différentielle des glycopeptidolipides (GPLs) permet de distinguer le morphotype rugueux (R), présentant un défaut de synthèse des GPLs, du morphotype lisse (S) exprimant les GPLs. Différents modèles ex vivo et in vivo rapportent que le variant R est impliqué dans des manifestations plus sévères associées à une réponse inflammatoire intense. Cependant, ces modèles d'étude restent particulièrement limités pour élucider les caractéristiques de cette infection. L'embryon de zebrafish (ZF) offre de nombreux avantages motivant et validant son utilisation pour une meilleure compréhension des maladies infectieuses. Ce travail de thèse a pour objectif de développer un modèle d'infection de Mabs dans l'embryon de ZF.Pour étudier la physiopathologie de l'infection de Mabs dans ce modèle, l'élaboration d'un protocole de microinjection des bactéries et des méthodes de suivi de la charge bactérienne ont été réalisés. Les techniques d'imagerie ont été employées pour déterminer la chronologie de l'infection au sein des embryons infectés. Les techniques de qRT-PCR, l'utilisation de lignées de ZF transgéniques et la technologie antisens (morpholinos) ont été utilisées pour déterminer le rôle du système immunitaire (Si) inné et de l'inflammation dans la physiopathologie infectieuse. Par ailleurs, le potentiel du ZF en tant qu'organisme modèle en pharmacologie a été mis à profit pour étudier l'activité in vivo d'antibiotiques (ATB) sur Mabs.Le variant R induit une infection létale plus robuste que le S, caractérisée par le développement d'abcès au niveau du système nerveux central (SNC) associés à une réponse inflammatoire intense et au recrutement de neutrophiles. Le suivi des infections a révélé que les bactéries étaient rapidement phagocytées par les macrophages au niveau du site d'injection. Une fois infectés, ces derniers traversent la barrière endothéliale et transportent les mycobactéries dans les tissus du SNC, soulignant leur rôle clé dans la dissémination du pathogène. Des expériences menées dans des embryons dépourvus de macrophages ont validé ces observations en montrant que les bactéries étaient incapables de rejoindre le SNC et restaient confinées dans le système vasculaire. Implanté au sein du tissu nerveux, le macrophage infecté entre en apoptose, libérant ainsi le pathogène dans le milieu extracellulaire. Une fois libéré, à la différence du variant S, la morphotype R forme des cordes augmentant rapidement de taille et capables d'initier le développement d'abcès volumineux. La taille démesurée de ces cordes par rapport à celle des phagocytes professionnels représenterait une stratégie permettant au variant R d'échapper à la phagocytose et donc de promouvoir sa multiplication extracellulaire et d'assurer la progression létale du processus infectieux. Enfin, ce modèle nous a permis de déterminer, en temps réel, l'efficacité thérapeutique de plusieurs ATBs sur les embryons infectés,qui s'accompagne d'une forte réduction de mortalité des embryons et d'une importante diminution des signes physiopathologiques au niveau du SNC. Ces résultats indiquent que l'embryon de ZF représente un modèle d'infection prometteur et pertinent pour 1) l'étude de la virulence de Mabs 2) l'évaluation de la contribution du SI innée au cours de l'infection et 3) le suivi directe de l'effet d'ATBs. Ce nouveau modèle, combiné aux outils déjà disponibles chez le ZF, devrait permettre de mieux caractériser la relation entre Mabs et mucoviscidose, notamment l'implication éventuelle de la protéine CFTR dans la résistance à cette bactérie. Par ailleurs, l'embryon étant particulièrement propice au criblage à haut débit, l'optimisation de ce système biologique pourrait être exploitée dans le cadre d'approches thérapeutiques innovantes pour identifier de nouveaux agents anti-infectieux contre Mabs. / The emerging pathogen Mycobacterium abscessus causes severe lung infections particularly in cystic fibrosis (CF) patients. The Smooth (S) morphotype displays surface expression of glycopeptidolipids (GPL) while the Rough (R) morphotype is characterized by the loss of surface-associated GPL. Previous studies suggested that the R variant is involved in more severe clinical forms, associated with a hyper-proinflammatory response. However, the molecular mechanisms responsible for the virulence and physiopathology associated to the Rvariant remain unknown. The zebrafish embryo offers many advantages that motivated and validated its use for a better understanding of infectious diseases. In this study, a zebrafish model of infection was developed toinvestigate and compare the pathogenesis of R and S variants.A microinjection protocol was first developed and the fate and progression of the infection was monitored at a spatiotemporal level by videomicroscopy. A transcriptomic approach by qRT-PCR, an antisense technology using morpholinos and transgenic zebrafish lines were used to evaluate the contribution of theinnate immune system and the role of inflammation during infection. In addition, the potential of the embryo has been used to study the in vivo pharmacological activity of antibiotics during M. abscessus infection. In contrast to the S variant, the R morphotype induced a more robust and lethal infection in zebrafish embryos, characterized by the rapid development of bacterial foci within the central nervous system (CNS). The use of a mpx:GFP zebrafish transgenic line, exhibiting green fluorescent granulocytes, indicated that neutrophils are actively recruited to CNS infection foci. An intense pro-inflammatory response with production of TNFα was measured by qRT-PCR. Next, the use of a mpeg1:mCherry transgenic zebrafish line, exhibiting red fluorescent macrophages, demonstrated the presence of isolated or small aggregated bacilli within macrophages during early infection. In contrast, later stages were characterized by the presence of large aggregates of extensively replicating extracellularly that enables mycobacteria to induce a strong inflammatoryresponse, leading to rapid tissue damage (abscess) and to larval death. In addition, the high propensity of the R variant to form cords in vivo may, represent a strategy evolved by the R (but not S) M. abscessus, to escape themacrophage or avoid being phagocytosed by macrophages or granulocytes. The role of macrophages in the diffusion of bacteria to the CNS was evaluated in macrophage-depleted embryos. Here, M. abscessus failed to disseminate from vasculature to CNS as shown by infections performed in KDR:GFP transgenic line, exhibiting green vascular endothelium. In addition, we also showed that the activity of antibiotics on infected-embryos is associated with a strong reduction of embryonic mortality, reduction in the bacterial burden and a significant decrease in physiopathological signs in the CNS, which could be imaged in real-time and at high resolution.These results propose the zebrafish embryo as a suitable model, particularly relevant to 1) the study of M. abscessus virulence, 2) the evaluation of role of innate immune system during infection process and to 3)monitor, at spatiotemporal level, the effects of antibiotics in an infected vertebrate. In addition, the antisense technology allowing knocking-down cftr expression can now be optimized to mimic a CF environment. This should greatly help to define the relationship between M. abscessus in CF patients. Moreover, the embryo isparticularly conducive to high-throughput screening, thus allowing this biological system to be exploited in the search for new therapeutic molecules against M. abscessus and other CF-associated patients

A busca pelo homem por uma vida saudável tem impulsionado pesquisas por novas substâncias capazes de atender tal desejo. O composto fenólico resveratrol (3, 4\', 5- trihidroxiestilbeno) é uma dessas substâncias que apresenta uma variedade de ações farmacológicas, como potencial antioxidante, capacidade antiinflamatória, proteção contra doenças cardíacas e câncer. Apesar dos inúmeros estudos sobre os benefícios do resveratrol à saúde, há poucos dados na literatura sobre sua toxicidade em organismos aquáticos, e principalmente sua concentração no ambiente, tornando o presente estudo fundamental para a contribuição de informações sobre a ecotoxicidade do resveratrol no ambiente aquático. O presente estudo avaliou a toxicidade do resveratrol em embriões e larvas de Danio rerio (zebrafish). Para isso foi realizado o ensaio in vitro de citotoxicidade do resveratrol, ensaios de ecotoxicidade e ensaio de biomarcadores enzimáticos. A avaliação do resveratrol por cromatografia líquida de alta pressão (HPLC) também foi realizada. De acordo com os resultados obtidos, o índice de citotoxicidade (IC50), concentração do resveratrol que causou a morte de 50% das células da linhagem NCTC-L929 foi de 39 mg L-1. A concentração de resveratrol que causa mortalidade em 50% dos organismos expostos (CL50), nos ensaios de ecotoxicidade crônica de curta duração com larvas do peixe Danio rerio foi de 51,37 mg L-1. A CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 96 h de duração foi de 75,33 mg L-1 e a CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 168 h de duração foi de 50,87 mg L-1. Nas concentrações mais elevadas de resveratrol foram observadas deformidades em embriões e larvas. O resveratrol alterou as atividades das enzimas LDH e ChE no estágio embriolarval de Danio rerio. Na análise do resveratrol por HPLC não foi observado degradação do composto. / The concern about human being healthy life has driven researchers to study new compounds capable of reaching that desire. Resveratrol (3, 4\', 5 trihydroxystilbene) a phenolic compound, is one of these substances which presents a variety of pharmacological actions, as antioxidant potential, antiinflammatory capacity, protection against heart and cancer diseases. Despite the numerous studies on the benefits of resveratrol to health, there is little evidence in the literature of its toxicity to aquatic organisms, and especially its concentration in the environment, making the present study fundamental for the contribution of information on the ecotoxicity of resveratrol in the aquatic environment. The present study evaluated the toxicity of resveratrol in embryos and larvae of Danio rerio (zebrafish). For this purpose the in vitro cytotoxicity of resveratrol assay, ecotoxicity assays and enzyme biomarker assay were performed. The evaluation of resveratrol by high pressure liquid chromatography (HPLC) was also performed. According to the results, the cytotoxicity index (IC50), concentration of resveratrol that caused the death of 50% of the cells of the NCTC-L929 lineage was 39 mg L-1. The concentration of resveratrol that causes mortality in 50% of exposed organisms (LC50) in the short-lived chronic ecotoxicity assays with larvae of the Danio rerio fish was 51.37 mg L-1. The LC50 obtained in the embryo-active acute ecotoxicity test of the Danio rerio fish with 96 h duration was 75.33 mg L-1 and the LC50 obtained in the embryo-active acute ecotoxicity assay of the Danio rerio fish with 168 h duration was 50.87 mg L-1. At higher concentrations of resveratrol deformities were observed in embryos and larvae. Resveratrol altered the activities of LDH and ChE enzymes in the embryonal stage of Danio rerio. No degradation of resveratrol was observed in the HPLC analysis of compound.

A melanopsina foi descoberta em 1998 por Ignacio Provencio e colaboradores em melanóforos de Xenopus leavis. Desde sua descoberta, esse fotopigmento surgiu como um possível candidato a intermediar os fenômenos de sincronização nos vertebrados. Nos mamíferos, a melanopsina é encontrada num pequeno subgrupo de células ganglionares da retina, conhecido como células ganglionares retinianas intrinsecamente fotossensíveis (ipRGCs) e o seu papel como fotopigmento responsável pela percepção luminosa, que leva à sincronização das espécies dessa classe aos ciclos de claro e escuro, já foi estabelecido. A melanopsina está presente na retina de todas as classes de vertebrados estudadas até o momento, mas, em contraposição a essa afirmação, a sua estrutura tem maior semelhança com opsina de invertebrados do que com opsina de vertebrados, sugerindo que sua fototransdução ocorra através da via dos fosfoinositídeos. Essa hipótese foi confirmada por diversos trabalhos na literatura e estudos posteriores demonstraram que, em vertebrados não mamíferos, a melanopsina é codificada por dois genes: um ortólogo ao de mamíferos, Opn4m, e um ortólogo ao de X. leavis, Opn4x, levantando diversas questões a respeito da funcionalidade dessa opsina. Nosso grupo vem estudando esse fotopigmento nos tecidos periféricos de vertebrados desde 2001, sendo que foi pioneiro em demonstrar, em melanóforos de Xenopus laevis, que a dispersão dos grânulos de melanina se dá através da fotoativação da melanopsina que desencadeia a cascata de fosfoinositídeos. E estudos mais recentes vêm colocando a melanopsina como um dos possíveis fotopigmentos responsáveis pela sincronização de relógios periféricos em organismos como peixes e anfíbios. Nesse sentido, a linhagem de células ZEM-2S do peixe teleósteo Danio rerio é um ótimo modelo para o estudo das vias de fototransdução em relógios periféricos. Já foi demonstrado que essa linhagem de células é responsiva a estímulos luminosos, exibindo uma proliferação diferencial frente a diferentes regimes de claro e escuro, e ativando a expressão de genes de relógio como clock, per1 e cry1b, que conhecidamente são responsáveis por sincronizar os ritmos biológicos ao fotoperíodo ambiental. Nossos experimentos de imunocitoquímica detectaram a presença das duas proteínas codificadas pelos genes opn4m-1 e opn4m-2 da melanopsina, e mostraram uma significativa diferença na distribuição das proteínas Opn4m-1 e Opn4m-2. Análises de PCR quantitativo mostraram que um pulso de luz azul de 10 min é capaz de alterar a expressão dos genes de relógio per1b, per2, cry1a e cry1b, e que essa alteração ocorre através da via dos fosfoinositídeos em células embrionárias ZEM-2S de Danio rerio. Em adição mostramos que para promover a alteração dos genes de relógio, a via dos fosfoinositídeos interage com outras vias de sinalização como a via do óxido nítrico (NO) e a via das proteína quinases ativadas por mitógenos (MAPKs). Esses dados sugerem que a melanopsina seja um dos principais candidatos a intermediar os processos de sincronização nessas células, pois a somatória dos resultados de detecção da melanopsina, estimulação dentro de seu espectro de absorção e ativação da via dos fosfoinositídeos, a coloca a frente de outras opsinas como vertebrate ancient opsin (Va-opsin) e teleost multiple tissue opsin (Tmt-opsin) e de outros candidatos como Crys fotossensíveis e mecanismos de estresse oxidativo. No curso deste trabalho também conseguimos definir metodologias eficientes de transfecção de RNA de interferência e de DNA plasmidial em células ZEM-2S de D. rerio, que são ferramentas fundamentais nos estudos de expressão gênica nesse modelo / Melanopsin was discovered in 1998 by Ignacio Provencio and colleagues in Xenopus leavis melanophores. Since its discovery, this photopigment has emerged as a possible candidate to mediate synchronization in vertebrates. In mammals the melanopsin is found in a subset of retinal ganglion cells, known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and their role as the photopigment responsible for photoentrainment in mammals has already been established. Melanopsin is present in the retina of all vertebrate classes studied to date, nevertheless, its structure is more similar to invertebrate than to vertebrates opsins, suggesting that their phototransduction pathway occurs through the phosphoinositide pathway. This hypothesis has been confirmed by several studies in the literature. Later studies showed that melanopsin is encoded by two genes in non-mammalian vertebrates, Opn4m orthologous to mammalian and Opn4x orthologous to X. leavis, raising new questions about the functionality of this opsin. Our group has studied this photopigment in vertebrate peripheral tissues since 2001 and, in Xenopus laevis melanophores, we demonstrated that pigment granule dispersion occurs through photoactivation of melanopsin and triggering of phosphoinositide pathway. More recent studies have put melanopsin as a possible photoreceptor responsible for peripheral clocks entrainment in organisms like fish and amphibians. In this context, the ZEM-2S cell line of the teleost fish Danio rerio is a good model to study the mechanism of phototransduction in peripheral clocks. It has been previously demonstrated that this cell line is responsive to light stimuli, exhibiting a differential proliferation when submitted to different light/dark regimes and activating the expression of clock genes such as clock, per1 and cry1b, known to synchronize the biological rhythms to environmental photoperiod. Our immunocytochemistry experiments detected the presence of two proteins encoded by the melanopsin genes opn4m-1 and opn4m-2, and showed a significant difference in the distribution of proteins Opn4m-1 Opn4m-2. Quantitative PCR analyses showed that a 10-min blue light pulse is able to change the expression of the clock genes per1b, per2, cry1b and cry1a, and that this change occurred through the phosphoinositide cascade in embryonic ZEM-2S cells of D. rerio. In addition we showed that, to promote the change in clock gene expression, the phosphoinositide pathway interacts with other signaling pathways such as the nitric oxide (NO) and the mitogen-activated protein kinase (MAPK) pathways. These data suggest that melanopsin is a major candidate to mediate the photoentrainment in these cells, because taken together, the detection of melanopsin, stimulation within its absorption spectrum and activation of the phosphoinositide cascade, puts it ahead of other opsins, as the vertebrate ancient opsin (Va-opsin) and teleost multiple tissue opsin (Tmt-opsin), and other candidates, as photosensitive Crys and mechanisms of oxidative stress. In the course of this work, we could also define efficient methods for transfection of interference RNA and plasmidial DNA in ZEM-2S cells of D. rerio, which are fundamental tools in studies of gene expression in this model

A melanopsina foi descoberta em 1998 por Ignacio Provencio e colaboradores em melanóforos de Xenopus leavis. Desde sua descoberta, esse fotopigmento surgiu como um possível candidato a intermediar os fenômenos de sincronização nos vertebrados. Nos mamíferos, a melanopsina é encontrada num pequeno subgrupo de células ganglionares da retina, conhecido como células ganglionares retinianas intrinsecamente fotossensíveis (ipRGCs) e o seu papel como fotopigmento responsável pela percepção luminosa, que leva à sincronização das espécies dessa classe aos ciclos de claro e escuro, já foi estabelecido. A melanopsina está presente na retina de todas as classes de vertebrados estudadas até o momento, mas, em contraposição a essa afirmação, a sua estrutura tem maior semelhança com opsina de invertebrados do que com opsina de vertebrados, sugerindo que sua fototransdução ocorra através da via dos fosfoinositídeos. Essa hipótese foi confirmada por diversos trabalhos na literatura e estudos posteriores demonstraram que, em vertebrados não mamíferos, a melanopsina é codificada por dois genes: um ortólogo ao de mamíferos, Opn4m, e um ortólogo ao de X. leavis, Opn4x, levantando diversas questões a respeito da funcionalidade dessa opsina. Nosso grupo vem estudando esse fotopigmento nos tecidos periféricos de vertebrados desde 2001, sendo que foi pioneiro em demonstrar, em melanóforos de Xenopus laevis, que a dispersão dos grânulos de melanina se dá através da fotoativação da melanopsina que desencadeia a cascata de fosfoinositídeos. E estudos mais recentes vêm colocando a melanopsina como um dos possíveis fotopigmentos responsáveis pela sincronização de relógios periféricos em organismos como peixes e anfíbios. Nesse sentido, a linhagem de células ZEM-2S do peixe teleósteo Danio rerio é um ótimo modelo para o estudo das vias de fototransdução em relógios periféricos. Já foi demonstrado que essa linhagem de células é responsiva a estímulos luminosos, exibindo uma proliferação diferencial frente a diferentes regimes de claro e escuro, e ativando a expressão de genes de relógio como clock, per1 e cry1b, que conhecidamente são responsáveis por sincronizar os ritmos biológicos ao fotoperíodo ambiental. Nossos experimentos de imunocitoquímica detectaram a presença das duas proteínas codificadas pelos genes opn4m-1 e opn4m-2 da melanopsina, e mostraram uma significativa diferença na distribuição das proteínas Opn4m-1 e Opn4m-2. Análises de PCR quantitativo mostraram que um pulso de luz azul de 10 min é capaz de alterar a expressão dos genes de relógio per1b, per2, cry1a e cry1b, e que essa alteração ocorre através da via dos fosfoinositídeos em células embrionárias ZEM-2S de Danio rerio. Em adição mostramos que para promover a alteração dos genes de relógio, a via dos fosfoinositídeos interage com outras vias de sinalização como a via do óxido nítrico (NO) e a via das proteína quinases ativadas por mitógenos (MAPKs). Esses dados sugerem que a melanopsina seja um dos principais candidatos a intermediar os processos de sincronização nessas células, pois a somatória dos resultados de detecção da melanopsina, estimulação dentro de seu espectro de absorção e ativação da via dos fosfoinositídeos, a coloca a frente de outras opsinas como vertebrate ancient opsin (Va-opsin) e teleost multiple tissue opsin (Tmt-opsin) e de outros candidatos como Crys fotossensíveis e mecanismos de estresse oxidativo. No curso deste trabalho também conseguimos definir metodologias eficientes de transfecção de RNA de interferência e de DNA plasmidial em células ZEM-2S de D. rerio, que são ferramentas fundamentais nos estudos de expressão gênica nesse modelo / Melanopsin was discovered in 1998 by Ignacio Provencio and colleagues in Xenopus leavis melanophores. Since its discovery, this photopigment has emerged as a possible candidate to mediate synchronization in vertebrates. In mammals the melanopsin is found in a subset of retinal ganglion cells, known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and their role as the photopigment responsible for photoentrainment in mammals has already been established. Melanopsin is present in the retina of all vertebrate classes studied to date, nevertheless, its structure is more similar to invertebrate than to vertebrates opsins, suggesting that their phototransduction pathway occurs through the phosphoinositide pathway. This hypothesis has been confirmed by several studies in the literature. Later studies showed that melanopsin is encoded by two genes in non-mammalian vertebrates, Opn4m orthologous to mammalian and Opn4x orthologous to X. leavis, raising new questions about the functionality of this opsin. Our group has studied this photopigment in vertebrate peripheral tissues since 2001 and, in Xenopus laevis melanophores, we demonstrated that pigment granule dispersion occurs through photoactivation of melanopsin and triggering of phosphoinositide pathway. More recent studies have put melanopsin as a possible photoreceptor responsible for peripheral clocks entrainment in organisms like fish and amphibians. In this context, the ZEM-2S cell line of the teleost fish Danio rerio is a good model to study the mechanism of phototransduction in peripheral clocks. It has been previously demonstrated that this cell line is responsive to light stimuli, exhibiting a differential proliferation when submitted to different light/dark regimes and activating the expression of clock genes such as clock, per1 and cry1b, known to synchronize the biological rhythms to environmental photoperiod. Our immunocytochemistry experiments detected the presence of two proteins encoded by the melanopsin genes opn4m-1 and opn4m-2, and showed a significant difference in the distribution of proteins Opn4m-1 Opn4m-2. Quantitative PCR analyses showed that a 10-min blue light pulse is able to change the expression of the clock genes per1b, per2, cry1b and cry1a, and that this change occurred through the phosphoinositide cascade in embryonic ZEM-2S cells of D. rerio. In addition we showed that, to promote the change in clock gene expression, the phosphoinositide pathway interacts with other signaling pathways such as the nitric oxide (NO) and the mitogen-activated protein kinase (MAPK) pathways. These data suggest that melanopsin is a major candidate to mediate the photoentrainment in these cells, because taken together, the detection of melanopsin, stimulation within its absorption spectrum and activation of the phosphoinositide cascade, puts it ahead of other opsins, as the vertebrate ancient opsin (Va-opsin) and teleost multiple tissue opsin (Tmt-opsin), and other candidates, as photosensitive Crys and mechanisms of oxidative stress. In the course of this work, we could also define efficient methods for transfection of interference RNA and plasmidial DNA in ZEM-2S cells of D. rerio, which are fundamental tools in studies of gene expression in this model

A busca pelo homem por uma vida saudável tem impulsionado pesquisas por novas substâncias capazes de atender tal desejo. O composto fenólico resveratrol (3, 4\', 5- trihidroxiestilbeno) é uma dessas substâncias que apresenta uma variedade de ações farmacológicas, como potencial antioxidante, capacidade antiinflamatória, proteção contra doenças cardíacas e câncer. Apesar dos inúmeros estudos sobre os benefícios do resveratrol à saúde, há poucos dados na literatura sobre sua toxicidade em organismos aquáticos, e principalmente sua concentração no ambiente, tornando o presente estudo fundamental para a contribuição de informações sobre a ecotoxicidade do resveratrol no ambiente aquático. O presente estudo avaliou a toxicidade do resveratrol em embriões e larvas de Danio rerio (zebrafish). Para isso foi realizado o ensaio in vitro de citotoxicidade do resveratrol, ensaios de ecotoxicidade e ensaio de biomarcadores enzimáticos. A avaliação do resveratrol por cromatografia líquida de alta pressão (HPLC) também foi realizada. De acordo com os resultados obtidos, o índice de citotoxicidade (IC50), concentração do resveratrol que causou a morte de 50% das células da linhagem NCTC-L929 foi de 39 mg L-1. A concentração de resveratrol que causa mortalidade em 50% dos organismos expostos (CL50), nos ensaios de ecotoxicidade crônica de curta duração com larvas do peixe Danio rerio foi de 51,37 mg L-1. A CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 96 h de duração foi de 75,33 mg L-1 e a CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 168 h de duração foi de 50,87 mg L-1. Nas concentrações mais elevadas de resveratrol foram observadas deformidades em embriões e larvas. O resveratrol alterou as atividades das enzimas LDH e ChE no estágio embriolarval de Danio rerio. Na análise do resveratrol por HPLC não foi observado degradação do composto. / The concern about human being healthy life has driven researchers to study new compounds capable of reaching that desire. Resveratrol (3, 4\', 5 trihydroxystilbene) a phenolic compound, is one of these substances which presents a variety of pharmacological actions, as antioxidant potential, antiinflammatory capacity, protection against heart and cancer diseases. Despite the numerous studies on the benefits of resveratrol to health, there is little evidence in the literature of its toxicity to aquatic organisms, and especially its concentration in the environment, making the present study fundamental for the contribution of information on the ecotoxicity of resveratrol in the aquatic environment. The present study evaluated the toxicity of resveratrol in embryos and larvae of Danio rerio (zebrafish). For this purpose the in vitro cytotoxicity of resveratrol assay, ecotoxicity assays and enzyme biomarker assay were performed. The evaluation of resveratrol by high pressure liquid chromatography (HPLC) was also performed. According to the results, the cytotoxicity index (IC50), concentration of resveratrol that caused the death of 50% of the cells of the NCTC-L929 lineage was 39 mg L-1. The concentration of resveratrol that causes mortality in 50% of exposed organisms (LC50) in the short-lived chronic ecotoxicity assays with larvae of the Danio rerio fish was 51.37 mg L-1. The LC50 obtained in the embryo-active acute ecotoxicity test of the Danio rerio fish with 96 h duration was 75.33 mg L-1 and the LC50 obtained in the embryo-active acute ecotoxicity assay of the Danio rerio fish with 168 h duration was 50.87 mg L-1. At higher concentrations of resveratrol deformities were observed in embryos and larvae. Resveratrol altered the activities of LDH and ChE enzymes in the embryonal stage of Danio rerio. No degradation of resveratrol was observed in the HPLC analysis of compound.

CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...

Boston University. University Professors Program Senior theses. / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-02