High quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish.

In aquaculture, short term chilled storage has been used to transport brood stock fish embryos for genetic improvement programmes. It is therefore important to understand the effect of chilling on embryos at both developmental and molecular levels. In the present study, gene expression patterns in zebrafish embryos were studied before investigations were carried out on the effect of chilling on gene and protein expression in these embryos. The gene expression results obtained in different developmental stages using conventional PCR showed that, only sox genes were expressed throughout the tested developmental stages from 30% epiboly to 6 somites. Quantitative RT-PCR was then used to investigate sox gene expression patterns during chilling of 50% epiboly stage embryos at 0°C for up to 180 min and also after warming. Significant decreases in sox2 and sox3 expressions were observed when compared to those of controls following chilling whilst significant increases of expressions of the two genes were observed after warming in the embryos chilled for 30 and 60 min. Studies on the impact of cryoprotectant MeOH on sox genes and protein expression showed that 50% epiboly stage zebrafish embryos could tolerate chilling for up to 6 h with or without MeOH. It was observed that expression of all three sox genes were significantly decreased following chilling for 3 h at 0°C. However the degree of decrease was less pronounced in embryos chilled with different concentrations of MeOH. Significant increases in sox genes were observed in hatching stage embryos chilled with 1 M MeOH for 3h but subsequent sox2 and sox19a protein expression was not affected. The effect of long term chilling (18h) on sox gene and protein expression in 50% epiboly stage embryos was also investigated. Improved hatching rates (56% ± 5) were achieved when embryos were chilled with 1 M MeOH + 0.1 M sucrose. Results from gene expression studies showed a stable sox2 gene expression in 18 h chilled embryos in cryoprotectant mixture when compared to that of embryos chilled without cryoprotectant mixture. Similar patterns were observed when the expression of sox2 and sox3 protein was investigated. This is the first study carried out on the effect of chilling in early stage zebrafish embryos at the molecular level. The results obtained from the present study provided useful information on the molecular mechanisms of the effect of chilling on zebrafish embryos and will have important implications in designing chilled storage protocols for fish embryos.

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.

Made available in DSpace on 2016-06-02T19:29:00Z (GMT). No. of bitstreams: 1 TeseRF.pdf: 4503749 bytes, checksum: 9ddaa154f7e50a29fcda9669257e2a6e (MD5) Previous issue date: 2006-06-05 / In order to evaluate the physical, chemical, biological and ecotoxicological parameters of Monjolinho River system (in laboratory and in situ), water and sediment samples (from the river and from eight of its tributaries) we obtained in july/03, October/03, January/04 and April/04. Juveniles and adult individuals of Danio rerio and Poecilia reticulata were used in the ecotoxicological studies. Mortality, juvenile biometry, and deletery effects observed on the gills tissue were considered in the tests. The limnological data revealed the higher concentrations of heavy metals and organochlorides in the dry season, being over the limits established for aquatic organisms protection. The concentrations of zinc and heptachlore found in the sediments of the last study sites resulted in deletery effects. The higher concentrations of the analyzed parameters were found in the study sites UFSCar, Confluência and Córrego Tijuco, probably due to the higher concentrations of clay and silt found in these areas. The partially chronic tests performed using samples obtained from the natural environment revealed significant toxicity for at least one fish species and one parameter. Tests in situ showed more drastic effects, especially in July/03. P. reticulata was more vulnerable when compared to D. rerio in all of the analyses, especially considering the juvenile stages. Two contaminants were used in the laboratory ecotoxicological analyzes, using D. rerio juveniles as a test organism. Cadmium showed a CL(I)50, 96h, of 1894,73µg/L. In the tests of static partial chronic toxicity, the chronic value obtained was 189,73µg/L, for both survival and growth, indicating that in the same concentration, the effects on the survival and biometry were similar. Gills morphological alterations increased with the increasing concentrations: 0.6 µg/L, 6 µg/L, 60 µg/L and 600 µg/L, being the fusion of the secondary lamellaes observed in the last concentration. Regarding to the endossulfan sulphate, the semi-static CL(I)50, 96h, was 7.24 µg/L and the chronic value, considering survival rates, was 0.948 µg/L. Histological effects were observed in all of the concentrations (0.03 µg/L, 0,3 µg/L and 3 µg/L), being the hyperplasia and secondary lamellae fusions more drastic in the last concentration. The toxicity tests, as well as the limnological analyses, showed that the Monjolinho system in highly disturbed, with inadequate conditions for the conservation of the aquatic organisms and may represent risks for the human health. / Com o objetivo de avaliar as características físicas, químicas, biológicas e ecotoxicologicas (laboratoriais e in situ) da água e sedimento, do sistema Monjolinho, foram realizadas coletas em julho/03, outubro/03, janeiro/04 e abril/04 (rio e 8 tributários). Na avaliação ecotoxicológica, utilizouse Danio rerio e P. reticulata, juvenis e adultos. A mortalidade, biometria final (dos juvenis) e efeitos deletérios nos tecidos branquiais foram as variáveis consideradas em testes. Os resultados limnológicos revelaram que, em geral, o período seco apresentou as maiores concentrações das diferentes variáveis analisadas, incluindo-se os metais e organoclorados, acima dos limites recomendados para proteção da vida aquática. Nos sedimentos, o zinco e o heptacloro apresentaram-se em concentrações capazes de provocar efeitos deletérios nos organismos biológicos, nos últimos pontos do sistema. O sedimento dos pontos UFSCar, Confluência e córrego Tijuco, apresentaram as maiores concentrações das variáveis analisadas, possivelmente devido à fração de silte e argila detectadas. Nos testes crônicos parciais com amostras ambientais, pelo menos uma das espécies e variáveis analisadas, indicaram a toxicidade do rio Monjolinho. Nos testes in situ, os efeitos foram mais pronunciados para ambas as espécies, especialmente em julho/03. P. reticulata demonstrou a maior sensibilidade comparando-se à espécie D. rerio, em todas as situações avaliadas, principalmente considerando-se a fase juvenil. Foram escolhidos dois poluentes para avaliação ecotoxicológica em laboratório, utilizando-se D. rerio juvenis como organismos-teste. O cádmio apresentou uma CL(I)50,96h de 1894,77µg/L. Nos testes de toxicidade crônica parcial estático, obteve-se um valor crônico de 189,73µg/L tanto para sobrevivência como para a biometria final, indicando que na mesma concentração os efeitos na sobrevivência e biometria foram semelhantes. As alterações branquiais foram progressivas nas concentrações de 0,6µg/L, 6µg/L, 60µg/L e 600µg/L, sendo detectado na última, a junção de várias lamelas secundárias. Considerando-se o endossulfan sulfato, a CL(I)50, 96h, semi-estático foi de 7,24µg/L e o valor crônico, considerando-se a sobrevivência foi de 0,948µg/L os efeitos histológicos foram obtidos em todas as concentrações (0,03µg/L, 0,3µg/L e 3µg/L), com o efeito mais pronunciado na última concentração, com hiperplasia e conseqüente junção de lamelas secundárias. Os testes de toxicidade refletiram o estado de degradação instalado em todo o sistema Monjolinho, confirmada pelas análises limnológicas, as quais em última instância comprometem também a saúde da população.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-01T13:47:03Z No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-07T13:37:56Z (GMT) No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) / Made available in DSpace on 2017-08-07T13:37:56Z (GMT). No. of bitstreams: 1 RicardoRodriguesAmorim_DISSERT.pdf: 1875129 bytes, checksum: 59df4759f100ee4855427e495c7bf6b8 (MD5) Previous issue date: 2017-04-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Bebidas alco?licas s?o popularmente consumidas em diversas culturas humanas. Por?m, o consumo alco?lico excessivo e a longo prazo pode promover danos sociais, f?sicos e psicol?gicos de dif?cil revers?o. De fato, o ?lcool exerce diferentes efeitos no organismo. Baixas concentra??es promovem euforia, relaxamento, e al?vio do estresse/ansiedade (efeito ansiol?tico), e a abstin?ncia, ap?s doses m?dias cr?nicas, aumentam o estresse/ansiedade (efeito ansiog?nico), podendo interferir na aprendizagem. No entanto, h? poucas informa??es sobre como essa droga altera aspectos cognitivos e psicol?gicos em situa??es de estresse/ansiedade que envolvam aprendizagem. O que demanda investiga??es aprofundadas com a proposi??o de modelos animais para uso translacional. Diante disso, caracter?sticas como: f?cil manuten??o e reprodu??o em laborat?rio, homologia gen?tica superior a 70% ? do genoma humano, e facilidade na administra??o de f?rmacos, tornam o peixe paulistinha (Danio rerio) um modelo ideal em pesquisas translacionais que envolvam drogas de abuso. Nesse sentido, o presente trabalho buscou investigar o efeito de diferentes tratamentos alco?licos (cr?nico e agudo) no desdobramento da resposta de ansiedade e na aprendizagem associativa aversiva do peixe paulistinha. Para isso, foram testados os efeitos do ?lcool na aprendizagem associativa aversiva do paulistinha (artigo 1) e os efeitos do ?lcool e do enriquecimento ambiental (EE) na impossibilidade do peixe evitar um estressor (artigo 2). Os resultados indicaram que comportamentos tipo-ansiosos em paulistinha s?o alterados por mudan?as nas concentra??es alco?licas, no regime de exposi??o ou no ambiente. ?lcool agudo aumenta ansiedade e potencializa a percep??o em rela??o ao eletrochoque. J? o EE promove efeito ansiol?tico e diminui a percep??o do eletrochoque. Por fim, sugerimos que esse trabalho serve de base para pesquisas neurofisiol?gicas gen?ticas e comportamentais sobre os efeitos da intera??o entre drogas de abuso e ambiente. / Alcoholic beverages are popularly consumed in various human cultures. However, excessive and long-term alcohol consumption can promote social, physical and psychological damages that are difficult to reverse. In fact, alcohol has different effects on the body. Low concentrations promote euphoria, relaxation, and stress/anxiety relief (anxiolytic effect), and abstinence after chronic medial doses increases stress / anxiety (anxiety) and may interfere with learning. However, there is little information on how this drug alters cognitive and psychological aspects in stress/anxiety situations involving learning. Therefore, in-depth investigations are required with the proposition of translational animal models. As a result, characteristics such as: easy maintenance and reproduction in the laboratory, genetic homology up to 70% to that of the human genome, and ease of administration of drugs, make zebrafish (Danio rerio) an ideal model in translational research involving drugs of abuse. In this sense, the present study sought to investigate the effect of different alcoholic treatments (chronic and acute) in the unfolding of the anxiety response and in the aversive associative learning of zebrafish.For this, the effects of alcohol in the aversive associative learning of zebrafish (manuscript 1) and the effects of alcohol and environmental enrichment (EE) on the impossibility of the fish to avoid a stressor were tested (manuscript 2). The results indicated that anxiety-like behaviors in zebrafish are altered by changes in alcoholic concentrations, in the exposure regime or in the environment. Acute alcohol increases anxiety and potentiates the electroshock perception. While EE promotes anxiolytic effect and decreases the perception of electroshock. Finally, we suggest that this work serves as a basis for genetic and behavioral neurophysiological research regarding the effects of the interaction between drugs of abuse and environment.

Orientador: Cláudia Vianna Maurer-Morelli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T13:52:07Z (GMT). No. of bitstreams: 1 Barbalho_PatriciaGoncalves_M.pdf: 8177547 bytes, checksum: 9a3923a19cb47c5609f933774ee68213 (MD5) Previous issue date: 2013 / Resumo: O Danio rerio é um peixe teleósteo popularmente conhecido como zebrafish que têm se destacado como modelo animal favorável para investigações genéticas devido à facilidade de manipulação in vivo, por sua transparência nas fases embrionária e larval e por seu desenvolvimento externo. Recentemente, foi demonstrado que o zebrafish é capaz de exibir padrão comportamental e alteração da atividade eletrográfica durante crise epiléptica como visto em roedores, tornando-o um modelo promissor para as investigações moleculares das epilepsias. Os estudos sobre os diferentes eventos já conhecidos das epilepsias no zebrafish estão apenas começando e, portanto, há ainda muito que ser investigado para uma melhor caracterização deste modelo para estudos em epilepsia. Estudos clínicos e em modelos animais mostraram que a crise epiléptica eleva os níveis da interleucina-1 beta e induz morte neuronal. Nesse sentido, o presente trabalho se propôs a investigar (i) o perfil temporal de expressão do transcrito do gene da interleucina-1 beta (il1b) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol por transcriptase reversa-PCR quantitativa e também, sua relação da expressão com a idade em que é realizada a indução da crise no cérebro imaturo e (ii) a morte neuronal pela histoquímica do Fluoro-Jade B (FJB) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol. Neste trabalho conseguimos estabelecer com sucesso as condições ideais para o acasalamento desta espécie e obtenção de embriões e, para a criação de larvas. Além disso, padronizamos o processamento histológico para a criosecção do cérebro do zebrafish. A análise temporal do transcrito do gene il1b revelou um padrão de expressão similar ao observado em roedores. A marcação por FJB não identificou inequivocadamente a presença de morte neuronal após crise epiléptica / Abstract: Danio rerio is a teleost fish popular named as zebrafish that has emerged as a suitable animal model for genetic investigations due to its features such as: facility for in vivo manipulation, transparency during embryonic and larval stages and external development. Recently, It was demonstrated that larvae and adult zebrafish (Danio rerio) seizure model mimics the behavior, eletrographic and c-fos features that are well established in rodent models of epilepsy, making the zebrafish as a promising model for studying the molecular mechanisms underlying epilepsies. However, the potential of the zebrafish model for epilepsy studies has been partly described since the molecular changes and neuronal loss after seizure has not yet been investigated. Proinflammatory cytokines, such as interleukin-1beta (il1b), has shown to be upregulated in surgical specimens of pharmacoresistent patients and in experimental rodent models of seizure, however the profile of this cytokine in zebrafish model for epilepsy is unknown. For this reason, and in view of the importance of inflammatory response in the pathophysiology of epilepsy, we sought to investigate the temporal transcript profile of interleukin-1b (il1b), a proinflammatory cytokine, in adult and immature (larvae) zebrafish brain after Pentylenetetrazole-evoked seizure, as well its aged-related expression in the developing brain by reverse transcriptase-quantitative PCR and neuronal death by Fluoro-Jade B (FJB) staining in adult zebrafish brain. Zebrafish breeding was successfully established during this work as well as the protocol for histological procedures using the zebrafish brain. Our results showed a short up-regulation of il1b mRNA levels after seizure in immature and adult zebrafish brain similar as with those patterns observed in rodent models. FJB did not show a reliable neuronal death staining in the zebrafish brain after seizure / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.