Submitted by André Chamelete null (andre_ecco@hotmail.com) on 2016-02-12T14:02:00Z No. of bitstreams: 1 Dissertação de Mestrado - FINAL.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-12T17:20:57Z (GMT) No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Made available in DSpace on 2016-02-12T17:20:57Z (GMT). No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) Previous issue date: 2016-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O BaP é um contaminante ambiental capaz de causar inflamação e desregulação de vias celulares. Pela ação da CYP1a1 e CYP1b1, é convertido a metabólitos mais reativos. A literatura mostra que o BaP aumenta a expressão de algumas citocinas próinflamatórias, como a IL-1, porém, são bem contraditórios os relatos sobre o efeito do BaP no cFOS, o qual apresenta papel importante na proliferação, na formação de tumores e, possivelmente, na inflamação. O objetivo deste estudo foi de elucidar a participação do receptor purinérgico P2X7 sobre a expressão dos genes IL-1 e cFOS, durante exposição ao BaP. Foi empregado as técnicas de qPCR para quantificação de expressão gênica, e testes de correlação e regressão entre IL-1 e cFOS. A exposição ao BaP induziu a expressão dos dois genes, além das enzimas do seu metabolismo. Quando bloqueado o receptor P2X7, além de uma menor indução das CYPs, os níveis de IL-1 e cFOS caíram abaixo dos níveis controle, sugerindo a participação do P2X7. Os testes de correlação e regressão mostraram uma relação forte direta entre IL-1 e cFOS, reforçando o papel do cFOS na inflamação. / BaP is an environmental contaminant capable to cause inflammation and impair cellular pathways. CYP1a1 and CYP1b1 convert it to more reactive metabolites. Studies show that BaP enhances some proinflammatory citokines expression, like IL-1, yet reports about BaP affecting cFOS, which plays important role in proliferation, tumor formation and inflammation, are controversial. This work aimed to elucidate whether P2X7 purinergic receptor plays a role in IL-1 and cFOS expression during BaP exposure. We applied qPCR techniques to quantify gene expression, correlation and regression assays. Our results showed that BaP raised both IL-1 and cFOS genes expression, besides CYPs ones. Morevoer, when blocking P2X7 receptor, IL-1 and cFOS expression dropped under normal levels, which suggest P2X7 participation, in addition to a smaller enzymes induction. Correlation and regression assays exhibited a strong straight relationship between IL-1 and cFOS expression, reinforcing the role of cFOS in inflammation.

Submitted by PATRÍCIA CANOSSA GAGLIARDI null (p.canossa@hotmail.com) on 2017-06-08T17:20:30Z No. of bitstreams: 1 Diss_Mest_PCG.pdf: 2551839 bytes, checksum: ae03233945fd3b7993bb5baed2aac057 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-13T13:35:52Z (GMT) No. of bitstreams: 1 gagliardi_pc_me_jabo.pdf: 2551839 bytes, checksum: ae03233945fd3b7993bb5baed2aac057 (MD5) / Made available in DSpace on 2017-06-13T13:35:52Z (GMT). No. of bitstreams: 1 gagliardi_pc_me_jabo.pdf: 2551839 bytes, checksum: ae03233945fd3b7993bb5baed2aac057 (MD5) Previous issue date: 2017-04-28 / O zebrafish representa um importante candidato, apresentando alta homologia genética com seres humanos e respostas, porém, são poucas as informações sobre seus mecanismos eritropoiéticos frente ao e leucopoiéticos. Assim o presente estudo buscou investigar o efeito da administração exógena de eritropoietina e da dexametasona sobre a resposta hematopoiética e leucocitária durante estímulo inflamatório através da indução de aerocistite infecciosa. Para isto, realizou-se indução de aerosciste através de inóculo de A. hydrophila, seguida da aplicação por via subcutânea de dexametasona (DEX) na dose de 25µg por unidade animal e 2UI de eritropoietina recombinante humana (EPO), constituindo os seguintes tratamentos: CC+ = não tratada com EPO e não tratado com DEX (n=10); DEX+= não tratada com EPO e tratada com DEX (n=10); EPO+ = tratada com EPO e não tratada com DEX (n=10); EPO/DEX+= tratada com EPO e tratada com DEX (n=10) e PF= (padrão fisiológico) onde os animais não receberam nenhuma medicação e não foi induzido a aeroscistite (n=10). para posterior coleta sanguínea e do exsudato inflamatório da bexiga natatória no período de 2 e 4 dias após inoculação (2DPI e 4DPI) e contagem eritrocitária total, contagem de reticulócitos, leucocitária total, diferencial leucocitário e contagem total e diferencial celular do exsudato da bexiga natatória. Na análise dos dados obtidos, observou-se um aumento geral dos parâmetros eritrocitários e de reticulócitos nos grupos EPO+ e DEX+, bem como leucocitários no grupo EPO+, o que indica possível resposta eritropoiética e leucopoiética positiva proveniente do uso da EPO. Na análise leucocitária foi possível observar também significante redução de valor total, granulócitos, monócitos e linfócitos nos grupos de tratamento com uso de DEX, indicando um presente efeito imunossupressor derivado do uso do glicocorticoide na espécie, demonstrando efeitos similares à mamíferos da EPO e da DEX. / Zebrafish represents an important candidate, presenting high genetic homology with humans and answers, however, there is little information about its erythropoietic mechanisms against leukopoietic. Thus the present study sought to investigate the effect of exogenous administration of erythropoietin and dexamethasone on the hematopoietic and leukocyte response during inflammatory stimulation through the induction of infectious aerocystitis. For this, an aerosol induction was performed through A. hydrophila inoculum, followed by subcutaneous application of dexamethasone (DEX) at the dose of 25 μg per animal and 2UI of recombinant human erythropoietin (EPO), constituting the following treatments: CC + = untreated with EPO and not treated with DEX (n = 10); DEX + = untreated with EPO and treated with DEX (n = 10); EPO + = treated with EPO and not treated with DEX (n = 10); EPO / DEX + treated with EPO and treated with DEX (n = 10) E And PF = (physiological standard) where the animals received no medication and were not induced to aerosctitis (n = 10), for subsequent blood collection and inflammatory exudate of the swimming bladder at 2 and 4 days after inoculation (2DPI and 4DPI) and total erythrocyte count, reticulocyte count , Total leukocytes, differential leukocytes, and total and differential cell count of the swim bladder exudate. In the analysis of the obtained data, a general increase of erythrocyte and reticulocyte parameters in the EPO + and DEX + groups was observed, as well as leukocytes in the EPO + group, indicating a positive erythropoietic and leukopoietic response from EPO use. In the leukocyte analysis, it was also possible to observe a significant reduction of total value, granulocytes, monocytes and lymphocytes in the treatment groups with DEX, indicating a present immunosuppressive effect derived from glucocorticoid use in the species, demonstrating similar effects to the EPO and DEX mammals.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-05-16T13:16:47Z No. of bitstreams: 1 Sergio Clementino da Costa.pdf: 1278139 bytes, checksum: 9762e5c57a26ef77c09e890e773a4e28 (MD5) / Made available in DSpace on 2018-05-16T13:17:57Z (GMT). No. of bitstreams: 1 Sergio Clementino da Costa.pdf: 1278139 bytes, checksum: 9762e5c57a26ef77c09e890e773a4e28 (MD5) Previous issue date: 2018-02-28 / Finasteride and testosterone are hormones that have anti-androgenic action (inhibition of 5α-reductase) and androgen respectively, which can act as endocrine disruptors causing negative effects on aquatic animals and their offspring. Finasteride was initially developed to treat benign prostatic hyperplasia, however, was presented as a major discovery in the treatment of androgenetic alopecia (baldness) already the testosterone is the hormone responsible for secondary sexual characteristics and has the most active metabolite the dihydrotestosterone, main responsible for baldness. However, finasteride as well as testosterone are commonly used in various situations, both by men in the treatment against hair loss (finasteride) or sexual reversal of fish (testosterone), which increases the presence of these hormones in the aquatic environment. Once in the environment both finasteride and testosterone begin to have action of endocrine disruptors, being one of the most powerful aquatic pollutants. Because it has hydrophobic properties which reduces their bioavailability, such hormones can be complexed to cyclodextrins (CDs) to reduce this limitation, therefore its use in inclusion complexes would increase its solubility and absorption, and may change one or more hormonal pathways. In view of the foregoing, the present study aims to evaluate the toxic effects of the inclusion complex Finasteride β-cyclodextrin (β-CD) and Testosterone β-cyclodextrin (β-CD), through chronic exposure to sublethal concentrations under the embryos and adults of the zebrafish (Danio rerio). The work was aimed to evaluate the toxic effects of finasteride and testosterone free and complexed into cyclodextrin on the physiological and behavioral parameters of embryos and adults of the Danio rerio fish. The embryos were exposed for periods of 96 hours post-fertilization for the evaluation of mortality, teratogenic effects, and heart rate. The groups were divided into control, free finasteride (FIN) and testosterone (T) at the concentrations of 280, 560, 1120, 2240, 4480 ng/L, and finasteride (FIN-β-CD) and testosterone (T-β-CD) complexed into β-CD at concentrations of 280 and 4480 ng/L. Adults were exposed for a period of 60 days to evaluate behavioral changes and daily survival related to exposure to free finasteride (FIN) and testosterone (T) complexed into β-CD at concentrations 280 and 4480 ng/L. The embryos did not exhibit mortality at concentrations of 280 - 4480 ng/L in the all groups exposed. Similarly, it was not observed teratogenic effects in the groups exposed to different concentrations. In the heart frequencies of the embryos, significant variations were observed (p < 0 .05) when compared to the control group. Adults exposed to free andcomplexed finasteride and testosterone had significant alterations (p < 0.05) in the most active behaviors such as slow swim and fast swim, as well as in agonistic behaviors. It can be concluded that in tests with embryos, FIN and FIN-β-CD as well T and T- β-CD, may interfere in the heart rate and the exposure of adults to the same hormones promoted a reduction in their locomotor capacity and can increase in the aggressiveness of the animals. / A finasterida e a testosterona são hormônios que possuem ação anti-androgênica (inibição da 5α-redutase) e androgênica respectivamente, que podem agir como disruptores endócrinos causando efeitos negativos aos animais aquáticos e suas proles. A finasterida foi desenvolvida inicialmente para tratar a hiperplasia prostática benigna, porém, foi apresentada como uma grande descoberta no tratamento da Alopecia androgenética (calvície), já a testosterona é o hormônio responsável pelas características sexuais secundárias e tem como metabólito mais ativo a dihidrotestosterona, principal responsável pela calvície. Entretanto, a finasterida assim como a testosterona são comumente usadas em diversas situações, tanto por homens no tratamento contra a perda de cabelo (finasterida) ou na reversão sexual de peixes (testosterona), o que eleva as presenças desses hormônios no ambiente aquático. Uma vez no ambiente tanto a finasterida como a testosterona passam a ter ação de disruptores endócrinos, sendo um dos mais potentes poluentes aquáticos. Por possui propriedades hidrofóbicas que reduz suas biodisponibilidades, tais hormônios podem ser complexados a ciclodextrinas (CDs) o que reduzir esta limitação, logo sua utilização em complexos de inclusão aumentaria sua solubilidade e absorção, podendo alterar uma ou mais vias hormonais. Diante do exposto, o presente trabalho tem como objetivo avaliar os efeitos tóxicos do complexo de inclusão Finasterida β-ciclodextrina (β-CD) e Testosterona β-ciclodextrina (β-CD), através da exposição crônica em concentrações subletais sob os e embriões e adultos de zebrafish (Danio rerio). Os embriões foram expostos por períodos de 96 horas pós-fertilização para a avaliação da mortalidade, efeitos teratogênicos e frequência cardíaca. Os grupos foram divididos em: controle, finasterida (FIN) e testosterona (T) livres nas concentrações de 280, 560, 1120, 2240, 4480 ng/L, e finasterida (FIN-β-CD) e testosterona (T-β-CD) complexadas a β-CD nas concentrações de 280 e 4480 ng/L. Os adultos foram expostos por um período de 60 dias para a avaliar alterações comportamentais e sobrevivência diária relacionadas a exposição de finasterida (FIN) e testosterona (T) livres e complexadas (β-CD) nas concentrações 280 e 4480 ng/L. Os embriões, não apresentaram mortalidade nas concentrações de 280 - 4480 ng/L em todo os todos os grupos expostos. Efeitos teratogênicos, de forma semelhante também não foram observados nos grupos nas diferentes concentrações. Nas frequências cardíacas dos embriões, foram constatadas variações significativas (p <0,05) quando comparadas ao grupo controle em todos os grupos expostos. Os adultos expostos às concentrações de finasterida e testosterona livres e complexadas apresentaram modificações (p < 0,05) na frequência de exibição de comportamentos ativos como Nadar lento e Nadar rápido, como também nos comportamentos agonísticos. Conclui-se então, que em testes com os embriões, FIN e FIN-β-CD assim como T e T- β-CD, podem modificar suas frequências cardíacas e que a exposição dos adultos aos mesmo hormônios promoveram uma redução na sua capacidade locomotora e um aumento na agressividade dos animais.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-06-18T13:53:36Z No. of bitstreams: 1 Tiago Queiroz da Mota Bittencourt.pdf: 3519794 bytes, checksum: 6c8d40c94975ee0c835f086dd5e829ec (MD5) / Made available in DSpace on 2018-06-18T13:54:56Z (GMT). No. of bitstreams: 1 Tiago Queiroz da Mota Bittencourt.pdf: 3519794 bytes, checksum: 6c8d40c94975ee0c835f086dd5e829ec (MD5) Previous issue date: 2017-02-24 / The toxic effects of sodium metavanadate (MV), vanadium pentoxide (PV) and oxovanadium sulfate (SV) on embryos and adults of zebrafish (D. rerio) were evaluated. Embryos were exposed to concentrations of 10-1000 μg/mL for evaluation of 96-h LC50 and their teratogenic effects. Adults were exposed to 20 μg/mL to evaluate behavioral changes related to vanadium compounds exposure and daily survival. Embryos showed a 96-h LC50 of 74.14 for PV and 100% mortality at the concentrations of 400-1000 μg/mL of the three compounds. The most significant teratogenic effects were pericardial edema and yolk sac (p < 0.05). The adults exposed to the vanadium compounds presented higher opercular beating and darkening of the gill arches. Floating and Resting behaviors in exposed adults were significant (p < 0.05), as well as the behavior of air breathing behavior. It can be concluded that in tests with the embryos the vanadium pentoxide is the most toxic compound and that pericardial edema and yolk sac were the most significant teratogenic effects and that the exposure of adults to vanadium compounds promoted an increase in the frequency of behaviors Inactive and stress response. / Foram avaliados os efeitos tóxicos do metavanadato de sódio (MV), pentóxido de vanádio (PV) e sulfato de oxovanádio (SV) em embriões e adultos de zebrafish (D. rerio). Os embriões foram expostos a concentrações de 10-1000 μg/mL para avaliação da CL50 96 h e seus efeitos teratogênicos. Os adultos foram expostos a 20 μg/mL para avaliar alterações comportamentais relacionadas à exposição dos compostos de vanádio e sobrevivência diária. Os embriões apresentaram uma CL50 96 h de 74,14 para o PV e 100% de mortalidade nas concentrações de 400-1000 μg/mL dos três compostos. Os efeitos teratogênicos mais significativos foram edemas de pericárdio e saco vitelínico (p < 0,05). Os adultos expostos aos compostos de vanádio apresentaram maior batimento opercular e escurecimento dos arcos branquiais. A exibição dos comportamentos Flutuar e Descansar nos adultos expostos foram significativos (p < 0,05), como também a exibição do comportamento Respiração aérea. Pode-se concluir que nos testes com os embriões o pentóxido de vanádio é o composto mais tóxico e que edemas de pericárdio e saco vitelínico foram os efeitos teratogênicos mais significativo e que a exposição dos adultos aos compostos de vanádio promoveram um aumento na frequência dos comportamentos inativos e de resposta ao estresse.

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

Lors des infections bactériennes, selon les tissus infectés, et selon la nature des pathogènes, l’organisme répond en mobilisant différents acteurs. Nous avons décidé d’utiliser le modèle du zebrafish ou Danio rério pour étudier la réponse immunitaire innée dans les situations d’infection bactérienne où les phagocytes professionnels ne peuvent pas venir au contact direct des bactéries. Pour cela, j’ai développé un modèle d’infection de la notochorde del’embryon de zebrafish. Lors de l’injection des bactéries dans ce compartiment, les bactéries se retrouvent protégées par une épaisse gaine de collagènes que les phagocytes ne peuvent pas pénétrer. Alors que les mycobactéries,protégées par la gaine de collagène ne sont pas détectées par les phagocytes, les bactéries E. coli sont immédiatement détectées ce qui déclenche une importante inflammation locale autour de la notochorde. Alors que les bactéries E. coli, bien qu’inaccessibles à la phagocytose sont éliminées dans les première 24 heures qui suivent l’injection, l’inflammation dure plusieurs jours.J’ai étudié les mécanismes qui conduisent à cette inflammation persistante et ses conséquences à long terme sur le développement du poisson. J’ai montré le rôle central de la cytokine IL1b dans ce processus, et j’ai développé une lignée transgénique qui permet d’étudier l’induction de cette cytokine in vivo chez le poisson.J’ai ensuite étudié le rôle des deux principales populations de phagocytes dans l’élimination des bactéries E coli. J’ai montré que les macrophages ne sont pas impliqués dans la disparition des bactéries alors que les neutrophiles, bien qu’incapable de pénétrer à l’intérieur de la gaine de collagène sont nécessaires à l’élimination des bactéries.J’ai ensuite montré que la myelopéroxidase et le monoxyde d’azote ne sont pas impliqués dans l’élimination des bactéries alors que les espèces réactives de l’oxygène produites par les neutrophiles sont nécessaires pour éradiquer l’infection. / In bacterial infections, according to the infected tissue and the nature of pathogens, the body responds by mobilizing various actors. I decided to use zebrafish or Danio rerio model to study the innate immune response to bacterial infection in the situations that professional phagocytes cannot come in direct contact with the bacteria. For this, I developed a model of infection in the notochord of zebrafish embryo. Upon injection of bacteria in this compartment, the microbes find themselves protected by the thick collagensheath where the phagocytes cannot penetrate. While mycobacteria are not detected by phagocytes; E. coli bacteria are sensed and a significant local inflammation around the notochord is mounted. The E. coli, although inaccessible to phagocytosis are eliminated within the first 24 hours after injection, the inflammation lasts several days.I studied the mechanisms that lead to this persistent inflammation and its long term consequences on the development of the fish. I showed the central role of the cytokine IL1B in this process, and I developed a transgenic line that allows studying in vivo the induction of this cytokine in fish.I then studied the roles of the two main populations of phagocytes in the elimination of E. coli. I revealed that macrophages are not involved in the removal of bacteria but neutrophils, although unable to penetrate inside the collagen casing, are necessary for the bacterial elimination. I also confirmed that myeloperoxidase and nitrogen monoxide are not involved in the removal of bacteria, rather the reactive oxygen species produced by neutrophils are needed to eradicate the infection.

Since their first discovery in the 19th century, microsporidian species have been found to be successful obligate intracellular parasites capable of infecting a wide variety of hosts including economically and ecologically important organisms as well as model organisms for biomedical research. Recently, cases of infection of the widely used animal model Danio rerio, commonly known as the zebrafish, by the newly described microsporidium Pseudoloma neurophilia have been reported in an increasing number of research facilities. Current knowledge of the biology of this parasite found in 75% of the Zebrafish Resource Center facilities is limited to microscopic analyses on its lifecycle as well as its physical, behavioral and psychic impact on its hosts. Despite the growing relevance of this parasite in biomedical research no current data is available on its genome. In this dissertation, I provide additional knowledge on the basic biology of P. neurophilia by acquiring and exploring the content and structure of the first genome draft of the zebrafish parasite. My findings reveal that the 5.25 Mb genome of P. neurophilia harbors an unusually high amount of transposable elements as well as numerous inserts found in coding regions typically conserved in microsporidia and other organisms. This peculiar obligate parasite demonstrates strong phylogenetic and genetic relationships with other fish-mosquito microsporidia. Similar to what is observed in closely related species, intra-genomic analyses of P. neurophilia’s genome suggest that it is diploid and possesses a large repertoire of over a thousand putative genes unique to this specie. Overall, my findings provide new insights into the basic biology of this parasite and represent a milestone in the understanding of P. neurophilia and D. rerio host-parasite interaction and ultimately in the development of treatments against this parasite that has been infecting the zebrafish research industry for the past decades.

Memoria para optar al Título Profesional de: Ingeniero Agrónomo / El pez cebra (Danio rerio) es un organismo modelo ampliamente utilizado en estudios genéticos debido a características únicas como su pequeño tamaño, corto tiempo generacional y transparencia de embriones entre otras, todas ellas facilitan los estudios realizados en esta especie y cuyos resultados pueden ser extrapolados a la gran mayoría de los vertebrados. Se ha logrado modificar genéticamente para utilizarlo en diversas áreas, desde biomedicina al monitoreo medioambiental y también como peces ornamentales. Estudios citogenéticos en esta especie existen pocos, mientras que en peces cebra transgénicos no existen. Bajo esta directriz se desarrolló esta investigación, donde se compara el cariotipo de dos cepas silvestres “wild type” obtenidas en laboratorios de las Facultades de Ciencias Agronómicas y de Ciencias de la Universidad de Chile y una línea ornamental transgénica comercial para definir si existen diferencias a nivel cariotípico entre ellas. Para esto se obtuvieron placas metafásicas mediante el método de goteo de suspensión celular de riñón, las cuales se sometieron a análisis cariotípico con tinción con Giemsa y tinción con ioduro de propidio para la obtención del bandeo C fluorescente. Se midió cada cromosoma del cariotipo y se calculó su índice centromérico para finalmente poder confeccionar el idiograma. Todas las cepas analizadas, incluyendo la línea ornamental transgénica presentaron un número cromosómico modal de 2n = 50, todos de morfología submetacéntrica. En general, el bandeo C reveló presencia de heterocromatina constitutiva principalmente en la zona pericentromérica de los cromosomas, se detectaron brazos cromosómicos bandeados completamente y algunas bandas a nivel telomérico. Bajo este análisis, la cepa silvestre de Agronomía presentó algunas diferencias con respecto a la cepa silvestre de Ciencias. Las placas metafásicas revelaron una variabilidad muy marcada en el número y morfología de los cromosomas de los peces transgénicos. Los resultados de esta memoria son un aporte al conocimiento de las cepas de pez cebra mantenidas en cautiverio y abren una ventana para el desarrollo de nuevos estudios en pez cebra transgénicos. / The zebrafish (Danio rerio) is a model organism, widely used in genetic studies due to unique features such as their small size, short generation time and transparency of embryos, among others, all of them provide great advantages to work with this fish, and results of research performed on this species can be extrapolated to the majority of vertebrates. Zebrafish has been genetically modified to be used in diverse areas, such as biomedicine, monitoring environmental and also as ornamental fish. Few cytogenetic studies have been developed on this species, while in transgenic zebrafish there is not cytogenetic research yet. Under this guideline is performed this research, which compare the karyotype of two strains of wild type obtained from laboratories of hatcheries of Agronomy Sciences and Sciences Faculties of University of Chile, and a commercial transgenic ornamental line to define if there are karyotypic differences between them. To do this, metaphases plates were obtained from cells from kidney, which were subjected to karyotypic analysis with staining with Giemsa and staining with propidium iodine to obtain the fluorescent C-banding. Each chromosome karyotype was measured and calculated centromeric index, to be able to finally make the idiogram. All analyzed strains, including the ornamental transgenic line, had the same chromosomal modal numbers 2n = 50, all of them with submetacentric morphology. In general, the C-banding revealed presence of constitutive heterochromatin, mainly in the pericentromeric areas of chromosomes. After this analysis, the wild type strain from Agronomy Faculty presented differences with respect to the wild type of Science Faculty. Metaphases revealed a marked variability in the chromosomes number and morphology of transgenic fish. The results of this report are a contribution to the knowledge of the strains of zebrafish in captivity and open a window for the development of new transgenic zebrafish studies.

Atrazine (ATZ) is an agricultural herbicide. The US Environmental Protection Agency has set the maximum contaminant level at 3 μg/l in potable water, though concentrations can greatly exceed this amount depending on the time of year. Epidemiological studies report associations with developmental health outcomes with potable water exposure. Studies in model organisms identify ATZ as a neurotoxicant and endocrine disrupting chemical. The zebrafish model system was used to test the hypothesis that developmental ATZ exposure has immediate health consequences as well as in the subsequent generation. It was first hypothesized that developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in larvae. In the exposed generation, targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals. The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group. These findings suggest that developmental ATZ exposure generates metabolite profiles similar to mammals leading to behavioral alterations supporting ATZ as a neurodevelopmental toxicant. In the subsequent generation (F1), it was hypothesized that parental ATZ exposure altered protein expression leading to modifications in morphology and behavior in developing progeny. Proteomic analysis identified differential expression associated with neurological development and disease and organ and organismal morphology, specifically the skeletomuscular system. Head length and the ratio of head length to total length was significantly increased in the F1 in the 0.3 and 30 ppb ATZ treated groups. Craniofacial morphology was assessed based on molecular pathway analysis and revealed decreased cartilaginous structure size, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord, indicating delayed ossification. The visual motor response assay showed hyperactivity in the F1 of the 30 ppb treatment group for total distance and time spent moving in the F1 of the 0.3 and 30 ppb treatment groups for all phases. Collectively, these results demonstrate persistent ATZ developmental toxicity in this multigeneration study.

No description available.