O objetivo do estudo foi avaliar a qualidade de efluentes líquidos industriais, pela caracterização físico-química e ensaios ecotoxicológicos agudos com Danio rerio, Daphnia similis e Vibrio fischeri. Foram comparadas as sensibilidades dos organismos-teste aos variados tipos de efluentes (indústrias alimentícias, papeleiras, bebidas, petroquímicas e farmacêutica), sendo que estes organismos pertencem a três níveis tróficos diferentes. Além disso, foi implementado o método de ensaio com bactérias luminescentes, o Microtox, de acordo com a NBR 15411 (ABNT, 2006). Na maioria dos ensaios, os efluentes apresentaram parâmetros físico-químicos dentro dos limites permitidos pela legislação. Mesmo assim, algumas vezes foram observados efeitos nos organismos-teste. Foram utilizados efluentes tratados das ETEI, podendo ser avaliada a sensibilidade de cada organismo a cada efluente do estudo. Para a Indústria Alimentícia 1 foram realizadas coletas de efluente bruto e primário, que se mostraram tóxicos aos três organismos. Essa coleta também foi realizada para a Indústria Farmacêutica, na qual o efluente bruto foi tóxico aos três organismos e o efluente do tratamento primário, foi tóxico à Daphnia similis e à Vibrio fischeri. O efluente bruto da Indústria Alimentícia 2, da Indústria de Papel e Celulose 2 e da Indústria de Bebidas foram coletados e avaliados ecotoxicologicamente por meio do ensaio Microtox, demonstrando toxicidade aguda com baixos valores de CE(I)50 para todas as indústrias. Alguns parâmetros físicoquímicos das indústrias foram correlacionados com a toxicidade do efluente final para Daphnia similis, Danio rerio e Vibrio fischeri por meio da correlação de Spearman. O teste não paramétrico Mann-Whitney foi usado para comparar grupos de parâmetros físico-químicos que apresentaram presença ou ausência de toxicidade. Em alguns efluentes tratados das ETEI das Indústrias Alimentícia 1, Alimentícia 2, de Papel e Celulose 2 e Petroquímica 1, foram observadas respostas biológicas das bactérias aos efluentes, o efeito hormesis, que indica que a amostra não possui toxicidade aguda, mas é muito provável que apresente toxicidade crônica. / The aim of the study was to evaluate the quality of industrial effluents by physicochemical characterization and ecotoxicological acute assays with Danio rerio, Daphnia similis and Vibrio fischeri. We compared the sensitivities of organisms to test various types of industrial effluent (food, paper, beverage, petrochemical and pharmaceutical industries), and these organisms belong to three different trophic levels. Furthermore, the Microtox method with luminescent bacteria has been implemented, according to NBR 15411 (ABNT, 2006). In most tests, the effluent presented physicochemical parameters within the limits allowed by law. Still, sometimes sensitivities were caused in the test-organisms. Were used ETEI treated effluents and could be assessed the sensitivity of each organism in each industrial effluent. For the Food Industry 1, collections were made of raw and primary effluent which proved toxicity for the three test-organisms. This collection was also realized for the pharmaceutical industry, in which the raw effluent was toxic to the three organisms and the primary effluent was toxic to Daphnia similis and Vibrio fischeri. For the effluent from the Food Industry 2, Pulp and Paper Industry 2 and Beverage Industry were collected raw effluents and evaluated the toxicity by Microtox assay, demonstrating acute toxicity with low values of EC(I)50 for all industries. Some physicochemical parameters of the industries were correlated with the toxicity of the final effluent to Daphnia similis, Danio rerio and Vibrio fischeri by Spearman Correlation. The nonparametric Mann-Whitney test was used to compare groups of physico-chemical parameters that showed the presence or absence of toxicity. In some of the treated effluent, Food Industry 1, Food Industry 2, Pulp and Paper Industry 2 and Petrochemical Industry 1 were observed biological responses of bacteria, the hormesis effect, which indicates that the sample does not have acute toxicity but is very likely presenting chronic toxicity.

Neste trabalho foi avaliada a qualidade das águas da Bacia do Rio Morto, localizado na Baixada de Jacarepaguá Rio de Janeiro, com base em análise físicoquímicas e ensaios ecotoxicológicos agudos com Danio rerio, Daphnia similis e Aliivibrio fischeri e ensaios ecotoxicológicos crônicos referentes à reprodução com Daphnia similis. Foram comparadas as sensibilidades dos organismos-teste, que pertencem a níveis tróficos diferentes, nos quatro pontos selecionados para coleta de amostras de água no Rio Morto e seus principais tributários: Rio Branco, Rio Sacarrão e canal do Morro do Bruno. Além disso, foi implementado no laboratório o método de ensaio crônico com o microcrustáceo Daphnia similis. As amostras, em sua maioria, apresentaram parâmetros físico-químicos dentro dos limites permitidos pela legislação nacional para a classe de águas doces em que a Bacia estudada está inserida. Não foram observados efeitos agudos nos organismos-teste, não sendo possível o cálculo da CE50 ou CL50, por conseqüência, o FT ficou fixado em 1. No teste agudo para Aliivibrio fischeri, para algumas amostras, foi constatado efeito Hormesis. O mesmo foi verificado em algumas amostras submetidas aos testes crônicos com Daphnia similis. / The quality of Rio Morto watershed, located in Jacarepagua district, Rio de Janeiro city, was investigated performing physicochemical determinations of water samples, as well as, acute ecotoxicity assays with three organisms (Danio rerio, Daphnia similes, Aliivibrio fischeri) and chronic ecotoxicity assays with Daphnia similis. The responses of the three organisms, belonging to different trophic levels, were compared for samples collected from four selected places in River Morto and its tributaries: River Branco, River Sacarrao and Morro do Bruno channel. In addition, the method of determination of chronic ecotoxicity using Daphnia similis was successfully implanted in the laboratory. Most samples presented physicochemical parameters that fulfill the requirements of the national standards for the River Morto watershed. Acute ecotoxicity was not observed for the tested organisms, not allowing the determination of the parameters EC50 and LC50 and imposing a FT value equal to one. Acute toxicity assays with Aliivibrio fischeri revealed, for some samples, the Hormesis effect. The same was observed for some samples submitted to chronic ecotoxicity determination with Daphnia similis.

A adaptação temporal é fundamental para a sobrevivência de espécies que precisam coordenar sua fisiologia e comportamentos ajustando-se a sinais externos. Ritmos biológicos não são simplesmente uma resposta às mudanças de 24 horas no ambiente físico impostas pela rotação da Terra sobre o seu próprio eixo, ao contrário, surgem a partir de um sistema de cronometragem endógeno. No teleósteo Danio rerio, ainda não foi identificada a presença de uma região que atue como relógio central; alguns estudos têm evidenciado a existência de células e tecidos que contêm relógios circadianos autônomos, fotossensíveis, comprovando um outro tipo de regulação dos ritmos circadianos onde a percepção do ambiente e o ajuste do período circadiano são efetivados diretamente em nível celular. As consequências deletérias do aumento da temperatura são impedidas, em certa medida, por uma resposta adaptativa que assegura a sobrevivência celular na presença de calor. Esta via de sobrevivência ativada por calor, conhecida como resposta ao choque térmico, é composta por uma cascata de eventos que conduzem à indução de proteínas de choque térmico (HSPs) que minimizam a lesão celular aguda. Acredita-se que os sistemas de percepção dos ciclos diários de temperatura e luminosidade sofreram as mesmas pressões seletivas em sua co-evolução, resultando em sua associação. As bases da sensação térmica estão em um grupo de canais altamente conservados, presente em todos os metazoários estudados até o momento e envolvidos em uma série de modalidades sensoriais, os canais de potencial receptor transiente (TRP); os que respondem a estímulos térmicos foram agrupados em uma subfamília e denominados termoTRPs. O objetivo deste trabalho foi investigar a influência do pulso de temperatura (33 ºC) na expressão de genes de relógio e de proteínas de choque térmico, bem como o papel do canal TRPV1, em células embrionárias de blástula de Danio rerio, denominadas ZEM-2S, submetidas a escuro constante (DD) ou ciclos claro-escuro (LD 12:12). Através de PCR em tempo real (quantitativo) demonstrou-se que as células ZEM-2S expressam os genes dos seguintes canais TRP: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c e trpM5. Após um pulso de temperatura, observou-se um aumento no transcrito de hsp90 aa1 em células mantidas tanto em DD como em LD, sendo a expressão de hsp90 aa1 em LD, no ponto uma hora, duas vezes menor quando comparada a sua expressão no mesmo ponto temporal em DD. O pulso de temperatura não promoveu efeito em nenhum dos genes do relógio estudados (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2) quando as células foram mantidas em DD. Porém, o transcrito de per2 aumentou em resposta ao pulso de temperatura quando as células foram sincronizadas pelos ciclos claro-escuro. A inibição do canal TRPV1 não alterou o efeito induzido pelo pulso de temperatura na expressão do gene hsp90 aa1 em células ZEM-2S mantidas em DD. Por outro lado, nossos dados permitem afirmar que o mesmo participa parcialmente na indução do aumento da expressão do gene per2 pelo estímulo térmico em células mantidas em LD, tendo em vista um decaimento significativo na resposta deste gene. Os dados obtidos neste trabalho abrem uma nova perspectiva sobre a investigação da relação temperatura e genes de relógio, colocando um novo “ator” na regulação deste fenômeno: o canal TRPV1 / Temporal adaptation is essential for the survival of species which need to coordinately adjust their physiology and behavior to external signals. Biological rhythms are not just a response to the 24 hour changes in the physical environment imposed by the rotation of the Earth around its own axis, but they arise from an endogenous timing system. In the teleost Danio rerio, there has not been identified so far a region in the nervous system that could act as a central clock; some studies have reported the existence of cells and tissues which contain photosensitive, autonomous circadian clocks, demonstrating the existence of another type of circadian rhythm regulation in which environment perception and entrainment of the circadian period are directly effected at cell level. The deleterious consequences of temperature increase are prevented by an adaptive response which assures cell survival in the presence of heat. This survival pathway activated by heat, known as response to temperature shock, is signaled by a cascade of events leading to the induction of thermal shock proteins (HSPs) which attenuate the acute cell lesion. It is believed that the systems perceiving temperature and light daily cycles were subject to the same selective pressures during their co-evolution, resulting in their association. The base of thermal sensation is a family of highly conserved channels, present in all metazoans studied to date, and involved in a variety of sensorial modalities, the transient receptor potential channels (TRP); those responding to thermal stimuli were grouped in a sub-family named thermo-TRPs. The aim of this work was to investigate the influence of a temperature pulse (33 ºC) on the expression of clock and heat shock protein genes, as well as the role of TRPV1 channel, in blastula embryonic cells of Danio rerio, named ZEM-2S, subject to constant dark (DD) or light-dark cycles (LD). Using quantitative PCR, we demonstrated that ZEM-2S cells express genes for the following TRP channels: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c and trpM5. After the pulse of temperature, we observed an increase of hsp90 aa1 transcripts in DD as well as in LD; hsp90 aa1 expression 1 hour after the stimulus was two-fold lower in LD than in DD. Temperature pulse did not affect the expression of any of the studied clock genes (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2), when the cells were kept in DD. However, per2 transcript increased in response to the temperature pulse when the cells were synchronized by light-dark cycles. Inhibition of TRPV1 channel did not change the effect induced by the temperature pulse on hsp90 aa1 in ZEM-2S cells kept in DD. On the other hand, our data suggest that this channel participates, at least partially, in the temperature-induced increase of per2 in cells maintained in LD, as indicated by the significant decay observed in the gene response in the presence of the inhibitor. Our results open new investigative perspective about the relationship between temperature and clock genes, placing a new “actor” in the regulation of the phenomenon: the TRPV1 channel

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.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as global endocrine and physiological effectors. In recent years the zebrafish has been utilized as a favorable animal model as a step towards dissecting and better understanding these postembryonic developmental processes. One of the more powerful methods utilized in zebrafish has been the identification of new gene functions through the use of mutant screens. The nutria mutant was recovered from one such screen to identify postembryonic defects in pigment pattern, growth and metamorphosis. These mutants exhibited a pigment cell defect, touch unresponsiveness and severe growth retardation. Here I will discuss my work towards dissecting the underlying developmental processes governing the phenotypic changes in nutria mutants. I characterize gross alterations in skeletal development in nutria mutants that lead to accelerated endochondral ossification but delayed intramembranous ossification. I show that the nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and a kinase. I find trpm7 expression in the fish-specific, ion homeostasis-regulating gland known as the corpuscles of Stannius (CS), and in the mesonephric kidney. I show that mutants also develop kidney stones. Together these results suggest a role for trpm7 activity in regulation of physiological ion homeostasis. Next I confirm that role by identifying late-embryonic and early larval defects in the CS and the kidney, two organs that regulate physiological ion homeostasis. I demonstrate the early larval detection of kidney stones in trpm7 mutants and show that their appearance is presaged by decreased levels of total calcium and magnesium. Furthermore I establish a link between trpm7 function in the CS and stanniocalcin1 (stc1), a potent molecular regulator of calcium homeostasis. Finally, using transgenic overexpression and morpholino-oligonucleotide knockdown, I demonstrate that stc1 modulates calcium and magnesium levels in trpm7 mutant and wild-type backgrounds. Together these analyses establish postembryonic roles for trpm7 function in growth, skeletogenesis, kidney function, and physiological ion homeostasis. / text

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.