Orientadores: Fernando Ferreira Costa, Anderson Ferreira da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T17:06:48Z (GMT). No. of bitstreams: 1 Branco_DianaSantos_D.pdf: 12860329 bytes, checksum: 5c5da209d2a41c9596907283c3c9e5e8 (MD5) Previous issue date: 2013 / Resumo: Os mecanismos moleculares envolvidos no perfil de expressão durante a eritropoese tem sido objeto de numerosas investigações como, por exemplo, o estudo da regulação gênica em células de linhagem eritroide. Esses estudos permitem a identificação de novos genes com potencial participação nesse processo e, adicionalmente, possibilitam um melhor entendimento dos genes já identificados na maturação das células eritroides e que possam estar envolvidos na produção de hemoglobinas. Nosso grupo de pesquisa identificou os diversos genes diferencialmente expressos durante a eritropoese. Dentre eles, os fatores de transcrição, EYA3 e HES6 e a latexina, LX, apresentaram maior expressão na fase final da eritropoese in vitro. Nossos dados sugerem participação dos genes EYA3 e LX, nas fases intermediaria e final da diferenciação eritroide, na expressão dos genes das globinas alfa e gama e na produção de HbF in vitro. Adicionalmente, no modelo in vivo zebrafish, os genes eya1, eya2, eya3, eya4, hes6 e hes13 apresentaram padrão de expressão ubíquo, enquanto que o gene lxn apresentou expressão especifica na ICM, tornando-o o candidato mais promissor para ser silenciado. O silenciamento desse gene em zebrafish apresentou fenótipo anêmico em embriões 72hpf, mas não em 48hpf, sugerindo que a anemia e decorrente de um processo no final da diferenciação eritroide, corroborando os dados encontrados em cultura in vitro. Estudos adicionais são necessários para compreensão dos mecanismos e vias envolvidos na participação do gene LX, durante o processo de diferenciação eritroide. Outros genes com potencial participação no processo de eritropoese são CLPX, TRAK2 E GFI. O gene CLPX codifica a caseinolytic peptidase X, uma proteína xvii altamente conservada durante a evolução e que apresenta função de chaperona dependente de ATP. Os dados deste trabalho mostram que o silenciamento do gene clpx1 reduziu significativamente os níveis de hemoglobinizacao e produção de eritrócitos em zebrafish. Contudo, estudos adicionais para o gene clpx2 precisam ser realizados para melhor compreender a possível função desses genes na produção de Heme. O gene TRAK2, por sua vez, e uma Trafficking Protein, Kinesin-Binding 2, envolvida no movimento da mitocôndria ao longo dos microtubulos. Os resultados obtidos em colaboração com o pesquisador Jeffrey Miller, M.D. (NIH/NIDDK) mostraram envolvimento desse gene na eritropoese em modelo in vitro de cultura primaria. No presente estudo, dentre os ortologos para o gene TRAK2 humano avaliados, apenas o trak1.1 parece ter sua função conservada nos teleósteos. O silenciamento desse gene gerou fenótipo anêmico nos embriões avaliados, corroborando os dados obtidos originalmente em cultura de células primaria. Finalmente, os fatores de transcrição de zebrafish gfi1aa, gfi1ab e gfi1b, ortologos aos fatores de transcrição da família Grow Factor Independence (GFI) em humanos também tiveram seu papel avaliado na hematopoese. Nossos dados mostram participação de gf1aa fase inicial de hematopoese e de gf1b na hematopoese definitiva. Também foi determinada a relação epistática entre os fatores gfi e os fatores de transcrição chave hematopoiéticos, mostrando que gfi1aa e gfi1b, juntamente com lmo2, scl, runx-1 e c-myb atuam como reguladores de HSPC em teleósteos / Abstract: Molecular mechanisms involved in expression profile during erythropoiesis have been the subject of numerous investigations such study of gene regulation in erythroid cell culture. These studies allow us to identify new genes potentially involved in erythroid differentiation and additionally to investigate genes already known as regulators of red blood cells and hemoglobin production. Our research group identified several genes differentially expressed during erythropoiesis. Among them, the transcription factors, EYA3 and HES6 and the latexin, LX, were found to have higher expression in the final phase of the in vitro erythropoiesis. Our data suggest that EYA3 and LX, are involved in the intermediate and final stages of erythropoiesis, expression pattern of alfa and gama globin and HbF production in vitro. Additionally in zebrafish model, eya1, eya2, eya3, eya4, hes6 and hes13 showed a ubiquitous expression pattern, while lxn showed specific expression in the ICM, making it the most promising candidate to be knockdowned. lxn knockdown in zebrafish showed anemic phenotype at 72hpf embryos, but not at 48hpf, suggesting that the anemia results is due to a process in the end of the erythroid differentiation, corroborating the results found for in vitro cultures. Additional studies are necessary to understand the mechanisms and pathways involved in the participation of the LX, gene during the process of erythroid differentiation. CLPX, TRAK2 and GFI transcription factors are also potentially candidates to be involved in erythropoiesis. CLPX gene codes for caseinolytic peptidase X, a protein highly conserved during evolution, which presents an ATP-dependent chaperone function. Data from this study showed that clpx1 knockdown reduced significantly hemoglobinization levels and erythroid production in zebrafish. However, future studies xv for the clpx2 gene is needed to better understand the function of these genes in the heme production. TRAK2 gene, in turn, is a Trafficking Protein, Kinesin-Binding 2, involved in mitochondrial movement along microtubules. Results obtained in collaboration with the researcher Jeffrey Miller, M.D. (NIH/NIDDK), showed the involvement of this gene in erythropoiesis in primary culture in vitro models. In this study, from the orthologs for the human TRAK2 gene analyzed, only trak1.1 appears to have its function conserved in teleosts. The silencing of this gene generated anemic phenotype in the embryos tested, corroborating the original results obtained in primary cell culture. Finally, gfi1aa, gfi1ab and gfi1b zebrafish transcription factors, orthologous to the Grow Factor Independence (GFI) family transcription factors in humans, also had their function evaluated in hematopoiesis. Our data suggest is involved in the initial phase of hematopoiesis while gf1b has a role in the definite hematopoiesis. The epistatic relation between the gfi and the hematopoietic key transcription factors was also determined, showing that gfi1aa and gfi1b, together with lmo2, scl, runx-1 and c-myb also act as regulators of HSPC in teleosts / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

The toxicity of 36 aliphatic amines on the embryos of the zebrafish Danio rerio were investigated. The DarT (Danio rerio Toxicity assay) was used to determine the lethal concentrations within a 48 h static acute toxicity test. A QSAR (Quantitative Structure-Activity Relationship) was performed using the LC50 values and molecular descriptors such as lipophilicity, maximum positive charge on hydrogen atom and the effective diameter of the molecule. In general, the toxicity of primary and secondary amines could be described by the lipophilicity as descriptor. The toxicity of the tertiary amines tested could be only described by a bilinear regression model. Further, regression models for other aquatic species such as the fathead minnow Pimephales promelas, Daphnia magna and Tetrahymena pyriformis showed that the toxicity of each species is a good predictor for each other.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.

Ce travail de thèse a visé à étudier les paramètres toxicocinétiques et toxicologiques de l'uranium chez le poisson. Il apparaît que l'uranium est hautement bioaccumulé et bioconcentré par les poissons. Sa répartition, bien que généralisée à l'ensemble de l'organisme, est néanmoins très hétérogène (branchies et foie constituant des sites majeurs d'accumulation). On note une perturbation du système antioxydant hépatique (inhibition des activités SOD, CAT et GPx ; déplétion du GSHtot) et du système cholinergique cérébral (inhibition/suractivation de l'AChE). Des atteintes génotoxiques surviennent également au niveau sanguin, hépatique et gonadique. L'activité radiologique de l'uranium influe sur les cinétiques d'apparition de ces perturbations biochimiques, celles-ci apparaissant d'autant plus précocement que l'activité délivrée est importante. Des atteintes histologiques (de différentes natures selon l'activité radiologique mise en jeu) sont également constatées (branchies, muscle).

Cryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. The success of cryopreservation is usually analysed in terms of cell survival, although there are other potential adverse effects that don’t necessarily result in cell death. These include DNA damage, which could result in altered gene expression. The aim of this study is to discover if cryopreservation has an impact on gene expression using zebrafish (Danio rerio) as the model organism. As the whole embryo cannot yet be successfully cryopreserved, isolated blastomeres from the embryos were used to investigate the impact of cryo-treatment on gene expression. This study sets out firstly to determine an optimum cryopreservation protocol for 50% epiboly blastomeres (Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole). Blastomeres had the highest survival level (70.2 ± 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. As quantitative analysis of gene expression using real-time PCR requires the use of a housekeeping gene as an internal control to normalize date, the second study aimed to identify and validate housekeeping genes for use in cryopreservation studies of zebrafish blastomeres. Seven potential housekeeping genes were analysed across a range of embryo stages and isolated blastomeres using the GeNorm and NormFinder software packages. Results indicated β-actin and EF1α housekeeping genes to be the most appropriate for cryopreservation studies on zebrafish embryos and blastomeres, and these housekeeping genes were used in the third study, the effect of cryopreservation on Pax gene expression. The results indicated that the trends (profile changes) in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryos which could have a detrimental effect on embryo development. This is the first study on the stability of housekeeping and transcription factor genes in chilled and cryopreserved embryonic cells of the zebrafish. This work will significantly enhance future studies investigating the impact of cryopreservation on gene expression in zebrafish embryos.

Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.

Submitted by Andreza Leão (andrezaflh@gmail.com) on 2018-08-20T19:16:26Z No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) / Approved for entry into archive by Celia Santana (celiasantana@ufpa.br) on 2018-12-12T18:01:20Z (GMT) No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) / Made available in DSpace on 2018-12-12T18:01:20Z (GMT). No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) Previous issue date: 2014-12-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Danio rerio é um modelo animal amplamente utilizado e com protocolos bem estabelecidos para experimentos comportamentais em aquários branco/preto. O lado branco do aquário parece causar aversão e a preferência parece ser por permanecer maior tempo no lado escuro. A melatonina é um indolamina com influência no funcionamento fisiológico de animais. Os objetivos deste trabalho são: fazer um levantamento na literatura sobre melatonina e sua correlação com peixes e com o Danio rerio; comparar a eficiência de dois aparatos de isolamento em testes comportamentais; testar a influência de quatro períodos do dia e de cinco fotoperíodos na variação da resposta em Zebrafish submetidos ao teste claro-escuro; comparar o tempo de permanência em cada lado do aquário teste no período noturno, com sujeitos isolados em fotoperíodo de 12/12 horas; comparar o tempo de permanência em cada lado do aquário teste no período, com sujeitos isolados em fotoperíodo de 12/12 horas, com exposição a sete diferentes concentrações de melatonina; comparar estatisticamente a amostra em quartis dentro de cada concentração; e comparar estatisticamente a amostra em quartis entre os primeiros, segundos e terceiros quartis de cada concentração. O Zebrafish e os peixes parecem ter sua fisiologia e comportamento influenciados pela melatonina (tanto endógena quanto exógena). Ambos os aparatos de isolamento demonstraram ser igualmente eficazes para testes comportamentais. A luminosidade parece arrastar o ciclo circadiano do Zebrafish, diminuindo sua aversão ao lado branco do aquário, no fotoperíodo 12/12 horas e período de coleta noturno. Ela parece influir na alteração causada pelo fotoperíodo, diminuindo ou anulando o arrastamento. Na amostra analisada parece haver subpopulações que respondem de maneira diferente a exposição à mesma concentração de melatonina. Estudos com cepas específicas de Zebrafish e com um leque maior de concentrações de melatonina parecem ser necessários para identificar a possível dose específica para contrabalancear a atuação luminosa no arrastamento do ciclo circadiano em Danio rerio. / Danio rerio is a widely used animal model for behavioral experiments in white / black tanks. The white side of the aquarium seems to cause aversion and the preference seems to be to stay longer on the dark side. Melatonin is a indoleamine which influence the physiological functioning of fish. The aims of this work are: to survey the literature on melatonin and its correlation with fishes and Danio rerio; compare the efficiency of two apparatuses insulation in behavioral tests; testing the influence of four periods of the day and five photoperiods response variation in Zebrafish to light-dark test; compare the time spent on each side of the aquarium test period, subjects isolated on a photoperiod of 12/12 hours with exposure to seven different concentrations of melatonin; statistically comparing the sample into quartiles within each concentration; and statistically compare the sample into quartiles among the first, second and third quartiles of each concentration. The Zebrafish and the fishes seem to have their physiology and behavior influenced by melatonin (both endogenous as exogenous). Both apparatuses isolation proved to be equally effective for behavioral tests. Luminosity seems to drag the Zebrafish circadian cycle, reducing their aversion to the white side of the aquarium, photoperiod 12/12 hour period and night collection. Melatonin seems to influence the change caused by photoperiod, decreasing or canceling the dragging. In our sample seems to be subpopulations that respond differently to exposure to the same concentration of melatonin. Studies with specific strains of Zebrafish and with a wider range of concentrations of melatonin appear to be necessary to identify the specific dosage possible to counteract the performance in light entrainment of the circadian cycle in Danio rerio.

Loss of hair cells due to acoustic trauma results in the loss of hearing. In humans, unlike other vertebrates, the mechanism of hair cell regeneration is not possible. The molecular mechanisms that underlie this regeneration in nonmammalian vertebrates remain elusive. To understand the gene regulation during hair cell regeneration, our previous microarray study on zebrafish inner ears found that growth hormone (GH) was significantly upregulated after noise exposure. In this current study, we utilized Next Generation Sequencing (NGS) to examine the genes and pathways that are significantly regulated in the zebrafish inner ear following sound exposure and GH injection. Four groups of 20 zebrafish each were exposed to a 150 Hz tone at 179 dB re 1μPa RMS for 40 h. Zebrafish were injected with either salmon GH, phosphate buffer or zebrafish GH antagonist following acoustic exposure, and one baseline group received no acoustic stimulus or injection. RNA was extracted from ear tissues at 1 and 2 days post-trauma, and cDNA was synthesized for NGS. The reads from Illumina Pipeline version SCS 2.8.0 were aligned using TopHat and annotated using Cufflinks. The statistically significant differentially expressed transcripts were identified using Cuffdiff for six different pairwise comparisons and were analyzed using Ingenuity Pathway Analysis. I found significant regulation of growth factors such as GH, prolactin and fibroblast growth factor receptor 2, different families of solute carrier molecules, cell adhesion molecules such as CDH17 and CDH23, and other transcription factors such as Fos, FosB, Jun that regulate apoptosis. Analysis of the cell proliferation network in the GH-injected condition compared to buffer-injected day 1 showed significant up-regulation of GH while downregulation of apoptotic transcription factors was found. In contrast, the antagonist-injected condition compared to the GH-injected condition showed an opposite pattern in which up-regulation of apoptotic transcription factors were found while GH was down-regulated. A number of other transcripts (e.g., POMC, SLC6A12, TMEM27, HNF4A, CDH17 and FGFR2) that showed up-regulation in GH-injected condition showed down-regulation in antagonist-injected condition. These results strongly suggest that injection of exogenous GH potentially has a protective role in the zebrafish inner ear following acoustic trauma.

Les PCB (polychlorobiphényles) sont des molécules connues pour leur longue demi-vie et leur forte liposolubilité qui conduisent à une bioaccumulation et une bioamplification dans les réseaux trophiques, menant à un potentiel risque pour les prédateurs de haut niveau tel que l'Homme. De plus, il a été démontré que leur affinité avec les composés lipidiques conduisaient à une transmission de la femelle à l'œuf chez les poissons. Dans ce contexte, et comme d'autres travaux ont déjà montré des effets des PCB sur la physiologie et le comportement d'animaux contaminés de différentes façons, nous avons observé les effets de ces molécules chez deux espèces, la sole commune et le poisson zèbre. La contamination a été réalisée via l'alimentation avec deux mélanges de PCB et deux concentrations qui correspondent à des situations environnementales, en termes de dose ou de choix et de proportion des congénères retenus. La dose la plus haute est équivalente à celle mesurée dans de la chair de molusques en baie de Seine et la dose intermédiaire à celle mesurée en estuaire de Loire. Les soles contaminées ont montré une diminution du niveau d'activité locomoteur après 30 jours (j) de contamination et une altération des capacités cryptiques après 60 j de contamination. Les poissons zèbre contaminés ont montré une augmentation de l'activité locomotrice après 250 j de contamination. La génération issue de cette génération de poisson zèbre contaminée a elle aussi montré une augmentation de l'activité locomotrice au stade larvaire et adulte. Chez les adultes, cela s'est traduit par une diminution de l'utilisation de la zone de fond des bacs et une augmentation du nombre de transition de zones, ce qui s'explique par une perte d'inhibition comportementale. Dans les deux cas, les phénotypes comportementaux observés chez les groupes PCB sont associés à une altération de la locomotion dans le sens d'une baisse d'activité pour une espèce placide comme la sole et dans le sens de l'augmentation pour une espèce mobile comme le poisson zèbre.

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, the sensitivity to chilling and toxicity of cryoprotectants of early stage zebrafish ovarian follicles were studied before designing protocols for their cryopreservation using controlled slow cooling. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. In vitro culture method for early stage zebrafish ovarian follicles were also developed. The studies showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles and methanol was the least toxic cryoprotectant. 4M methanol in potassium chloride (KCl) buffer was found to be the optimal cryoprotective solution and the optimum cooling rate was 4 °C/min for stage I and II follicles. Although the highest survivals after 2 h post-thawed incubation were 50.7 ± 4.0% for stage II ovarian follicles obtained with FDA+PI staining, ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell death. Furthermore, in vitro culture experiments showed that there was no growth for stage I and II ovarian follicles after cryopreservation, indicating that successful cryopreservation of early stage zebrafish ovarian follicles at liquid nitrogen still remains elusive. From in vitro culture study, 90% L-15 medium at pH 9.0 containing 10 IU/ml hCG was effective for in vitro culture of stage I and II ovarian follicles. Systematic study on cryopreservation of early stage fish ovarian follicles at liquid nitrogen temperature is reported ii here for the first time. The results will provide useful information on the future development of protocol design for successful cryopreservation of early stage fish ovarian follicles.