No description available.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

Melanoma is the most lethal type of skin cancer with over 132,000 cases occurring globally each year and continually rising incidence. BRAFV600E inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance. Evidence from human melanoma indicates that the melanocyte lineage is critical for melanoma survival and contributes to therapeutic resistance. MITF is a highly conserved “master melanocyte transcription factor” with a complex role in melanoma. Our lab has previously developed a temperature sensitive BRAFV600E mitfavc7 zebrafish melanoma model carrying a human oncogene and mitfavc7 splice site mutation that enables the conditional control of its endogenous activity by changes to water temperature. As part of my PhD project, I characterized and compared two new models developed since then: a very aggressive BRAFV600E mitfavc7p53M214K melanoma model with three driving mutations and a slower developing BRAF-independent mitfavc7p53M214K. I showed that the MITF activity is crucial for melanocyte survival in both models and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in fish nevi and melanoma resembling the pathology of human disease. Both models are also relevant to a low-MITF subclass of human melanomas that emerged from a recent classification by The Cancer Genome Atlas Network. In addition, I established that, similarly to the BRAFV600Emitfavc7, complete inhibition of MITF activity leads to rapid tumour regression, but once its activity is restored the melanomas recur at the same site as the original tumour. I used histopathology studies and melanocyte lineage transgenes to identify and visualize subpopulations of cells remaining at the site of regression in these new zebrafish melanoma models. I hypothesised that these are the cells of origin for tumour recurrence (melanoma stem or progenitor cells), showed that some of them express a cancer stem cell marker aldehyde dehydrogenase, and attempted to target these subpopulations using 5-nitrofurans (a prodrug NFN1, shown previously by our lab to target ALDHhigh subpopulations in context of melanoma) in fish after melanoma regression. Finally, I also developed and described a new primary zebrafish melanoma cell line that I derived from one of these zebrafish tumours. This study is still in progress, but the cell line will be a useful tool for further investigation of these proposed melanoma progenitor cells in vitro, with potential applications for lineage tracing and transplantations.

Submitted by ANA CERREJÓN PALANCO null (anacerrejon@live.com) on 2016-09-29T21:40:59Z No. of bitstreams: 1 Mestrado-Ana-Cerrejón-Palanco.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-10-04T19:11:41Z (GMT) No. of bitstreams: 1 carrejonpalanco_a_me_arafcf.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) / Made available in DSpace on 2016-10-04T19:11:41Z (GMT). No. of bitstreams: 1 carrejonpalanco_a_me_arafcf.pdf: 2202083 bytes, checksum: 3d49fc49aae6a457cad301dea9e43240 (MD5) Previous issue date: 2016-09-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A criptococose é uma infecção fúngica sistêmica e oportunista, causada pelas leveduras capsuladas das espécies Cryptococcus neoformans e Cryptococcus gattii, afetando tanto pacientes imunocompetentes quanto imunocomprometidos. Uma propriedade importante do gênero Cryptococcus é a capacidade de formação de biofilmes. A resistência dos biofilmes aos agentes antifúngicos traz a necessidade da busca de novas terapias. Desta forma, o nosso grupo vem trabalhando com moléculas antifúngicas derivadas de fontes naturais, como as chalconas, onde foi verificado que C. neoformans é sensível a algumas destas moléculas. Assim, o objetivo desse trabalho foi testar a 3’- chalcona como antifúngico contra C. gattii em situação planctônica e biofilme, como também a eficiência e toxicidade em modelos animais alternativos como Danio rerio (Zebrafish) e Galleria mellonella, propondo uma nova alternativa para o tratamento. In vitro, os resultados para a forma planctônica do fungo mostraram que a 3’- chalcona foi mais eficaz para a cepa 118, com concentração inibitória mínima (CIM) de 0,96 µg/mL e para a cepa 56990 de C. gattii, a molécula mostrou ação potente com CIM entre 1,95 e 3,90 µg/mL. No ensaio de formação do biofilme com as duas cepas, observou-se uma atividade metabólica crescente até 72 horas e uma biomassa e matriz extracelular que alcançou seu máximo nas 48 horas. Em seguida, foi feita a avaliação da atividade da 3’- chalcona contra a formação do biofilme maduro após 48 horas, apresentando uma inibição a uma concentração de 62,5 µg/mL para cepa ATCC e de 31,2 µg/mL frente ao isolado 118. Também foi avaliado o crescimento do biofilme frente à anfotericina B (AmB), obtendo como resultado, inibição em todas as concentrações por parte das duas cepas e, frente ao fluconazol, apresentaram-se resistentes. In vivo, os resultados de toxicidade em embriões de Zebrafish revelaram uma concentração letal 50% (CL50) de 3,42±0,6 µg/mL em 48 horas pós-fertilização (hpf). No modelo G. mellonella as doses testadas de 3’- chalcona (2 a 160 mg/Kg) não foram tóxicas. Entretanto, nas larvas infectadas com as duas cepas de C. gattii, a molécula não apresentou eficácia antifúngica. Assim, considerando o potencial antifúngico in vitro, inclusive contra o biofilme de C. gattii, a 3’- chalcona pode passar por modificações moleculares que aumentem sua distribuição e eficácia in vivo. / Criptococose is a fungal, systemic and opportunistic infection mainly caused by capsulated yeast of the species Cryptococcus neoformans e Cryptococcus gattii, affecting immunocompetent and immunocompromised patients. An important property of the genus Cryptococcus is the biofilm formation, which can be considered a virulence factor and resistance. The resistance of biofilms to antifungal agents brings the need to search new therapies. Thus, our group has been working with antifungal molecules derived from natural sources such as chalcones, where it was found that C. neoformans is sensitive to some of these molecules. For this fact, the objective of this study was to test the 3'- chalcone as antifungal against C. gattii in planktonic and biofilm forms, as well as efficiency and toxicity in alternative animal models like Danio rerio (Zebrafish) and Galleria mellonella, proposing a new alternative for treatment. In vitro, the results for the planktonic form of the fungus showed that the 3'- chalcone was more effective for the 118 strain, the minimum inhibitory concentration (MIC) was 0.96 µg/mL. For the strain 56990 of C. gattii the molecule showed potent activity with MIC values between 1.95 and 3.90 µg/mL. Biofilm formation assay for both strains had an increased metabolic activity within 72 hours and a biomass and extracellular matrix which reached its maximum in 48 hours. Then, the evaluation of the activity of the 3'-chalcone against the formation of mature biofilm was made after 48 hours, showing inhibition at a concentration of 62.5 µg/mL for ATCC strain and 31.2 µg/mL against the isolated 118. Also, the growth of biofilm against to amphotericin B (AmB) was evaluated, obtained as a result of inhibition at all concentrations for the two strains and against fluconazole were resistant. In vivo, the results of toxicity in zebrafish embryos revealed a lethal concentration 50% (LC50) of 3.42 ± 0.6 µg/mL at 48 hours post-fertilization (hpf). In the model G. mellonella tested doses of 3’- chalcona (2 to 160 mg/kg) were not toxic. However, in larvae infected with the two strains of C. gattii, the molecule did not show efficacy of antifungal agents. Thus, considering the potential antifungal in vitro, including against biofilms of C. gattii, 3'- chalcone can experiment molecular changes that increase their distribution and efficacy in vivo.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

The exoskeleton of the fins comprises fin rays and actinotrichia; the latter are small unmineralized fibrils found at the distal margin of fin rays. Actinotrichia play a role in the growth and structure of the fins during fin development and regeneration. Our lab has previously identified the actinodin (and) gene family, which codes for structural proteins in actinotrichia. Interestingly, the loss of this gene family has been proposed to be involved in the loss of fin rays, an important step in the fin-to-limb transition during evolution. Furthermore, the and genes are expressed in the epithelial cells and in the migrating mesenchymal cells of the zebrafish embryonic pectoral and median fin fold. The presence of tissue-specific cis-acting regulatory elements were found within the 2 kilobase pair genomic region (2P) located upstream of and1’s first untranslated exon by performing analyses of the expression of a fluorescent reporter (EGFP) placed under the control of fragments of various lengths originating from the 2P genomic fragment in zebrafish transgenic lines. Using these various and1 reporter lines, tissue-specific and1 expression was previously characterized during the embryonic stage of zebrafish development. However, these transgenic reporter lines were not analyzed throughout important fin morphogenesis events occurring during fin development, such as the initial formation of lepidotrichia and the resorption of the median fin fold, and throughout fin regeneration as well. This study mainly enabled us to characterize in great details and1 expression throughout fin development and regeneration using the various tissue-specific and1 reporter lines by performing time course analyses. In doing so, we were able to demonstrate that these reporter lines recapitulate endogenous and1 expression through in iii situ hybridization and RT-PCR experiments. Furthermore, the distinct transgene expression patterns observed during lepidotrichia formation/regeneration in the various and1 reporter lines supports previous research that proposes and1-expressing cells may indirectly contribute to lepidotrichia formation not only during fin regeneration but during fin development as well. Furthermore, the characterization of the tissue-specific and1 reporter lines throughout development allowed us to characterize specific changes in the cis-acting regulation of and1 in the fins of adult fish when compared with the tissue-specific and1 reporter expression patterns characterized during the embryonic stage. All in all, this study provides further clues on the contribution of and1-expressing cells throughout fin development and regeneration.

Contrairement aux mammifères, le poisson zèbre a la capacité de régénérer son coeur après une blessure importante. Une meilleure compréhension de ce phénomène pourrait contribuer à la conception de thérapies cliniques pour améliorer la régénération cardiaque des mammifères. Dans cette étude, nous avons identifié des voies de signalisation de la régénération cardiaque du poisson zèbre utilisant un criblage génétique chimique. De plus, nous avons identifier la présence d'un facteur de transcription bHLH, TAL1 et nous avons montrer son importance au cours de la régénération cardiaque du poisson zèbre. Enfin, nous avons examiner le rôle du système immunitaire lors de la régénération cardiaque du poisson zèbre et nous avons montrer que la sécrétion des metalloprotéinases (MMP14) par les macrophages permettait la dissolution du caillot sanguin, processus nécessaire pour la réussite de la régénération cardiaque. / Unlike mammals, zebrafish have the ability to regenerate their heart after substantial injury. A deeper understanding of this phenomenon could aid in the design of clinical therapies to enhance mammalian cardiac regeneration. In this study, we have identified signaling pathways in zebrafish heart regeneration using a chemical genetic screen. Furthermore, we identify the presence of a bHLH transcription factor, Tal1 and show its requirement during zebrafish cardiac regeneration. Finally, we examine the role of the immune system during zebrafish heart regeneration and demonstrate a model of scar removal by MMP14 positive macrophages and show that this process is required for successful heart regeneration to occur.

No description available.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

Many studies have reported individual differences in resting metabolic rate (RMR), the energetic cost of self-maintenance. Differences among individuals in the energetic cost of self-maintenance may influence life-history decisions and hence, fitness. In this study, we examined potential causes of intra-specific variation in RMR in zebrafish, Danio rerio. First, the repeatability of RMR was determined to check whether a single measure was reflective of future physiological performance. As predicted, RMR was repeatable over a period of three weeks. However, none of stress-coping style, baseline cortisol levels, metabolically-active organ (gill, heart, intestine and liver) mass, aggression or activity levels were correlated with RMR, i.e. none of these factors were significant contributors to individual variation in RMR. These results imply that other factors must be sought to explain the inter-individual variation in RMR observed in zebrafish.