Previous research showed mutations in muscle sarcoplasmic reticulum-bound calcium handler proteins cause swimming defects in embryonic zebrafish. CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed in all vertebrates has been defined to activate and inactivate multiple Ca2+ handler proteins involved in excitation- contraction coupling and relaxation of cardiac and skeletal muscle. In this study, evidence is provided through pharmacological and genetic intervention that CaMK-II inhibition and overexpression causes swimming defects, particularly response to stimuli and swimming ability, reinforced by immunolocalization of skeletal muscle. Transient CaMK-II inactivation does not have any long-term defects to swimming behavior. Overexpression of wild-type, constitutively active, and dominant-negative CaMK-II-GFP in embryos tended to co-localize in fast muscle which led to defects in swimming behavior. This study concludes that inhibition or overexpression of CaMK-II in skeletal muscle diminishes normal swimming behavior specifically in response to mechanical stimulation and swimming ability.

Os rins são órgãos responsáveis por gerir um conjunto de tarefas fisiológicas que mantém a homeostase do organismo, dentre elas a remoção de metabólitos tóxicos do sangue, produção de hormônios e a regulação do balanço de eletrólitos. As lesões renais agudas (LRA) levam a uma rápida perda das funções do órgão, em horas ou dias. Durante o processo de lesão tecidual, o sistema imune atua, muitas vezes, de forma prejudicial ao órgão. Uma das principais células envolvidas nesse processo de lesão tecidual são os macrófagos, que participam durante a primeira fase da LRA, já diferenciados em um perfil mais pró-inflamatório, descritos como macrófagos M1, e, também, durante a fase de reparo do tecido, diferenciados em macrófagos M2. Produtos como o acetato, um dos os ácidos graxos de cadeia curta (AGCC) mais abundantes, oriundos do metabolismo de bactérias pertencentes à microbiota intestinal são capazes de modular a resposta inflamatória em modelos de lesão tecidual, porém a influência desses produtos, na função, migração e diferenciação dos macrófagos no contexto da LRA ainda não foi bem descrita. Desta forma o objetivo deste trabalho foi avaliar o efeito do acetato nas funções biológicas dos macrófagos na LRA. Para isso, desenvolvemos um modelo de LRA induzida por cisplatina em peixes Danio rerio adultos, injetando intraperitonealmente (ip) diferentes doses de cisplatina e avaliando sobrevida e progressão da doença por histologia, imunofluorescência e análise do infiltrado celular. Em seguida para avaliar o efeito do acetato na LRA, os animais foram submetidos ao tratamento com acetato, por gavagem, e depois, injetados com cisplatina para analise de sobrevida, extensão da lesão renal por histologia e infiltrado de células do sistema imune no rim. Os resultados mostraram que a injeção de cisplatina (0,1275 mg/g) induz lesão tecidual no rim 24 hrs pós-injeção com perda de estrutura tubular, levando a morte de 80% dos animais. Porém, os animais sobreviventes foram capazes de regenerar o tecido lesado oito dias após a injeção. Também se observou aumento do infiltrado inflamatório e alta taxa de morte celular, concomitantes a altas taxas de proliferação de células não imunes. O acetato foi capaz de aumentar a sobrevida dos animais injetados com cisplatina e reduzir o dano induzido pela droga no rim, por outro lado, não alterou a atuação das células inflamatórias do sistema imune. Em conclusão, o modelo de injeção intraperitoneal de cisplatina foi capaz de induzir uma LRA em zebrafish adulto, com o máximo de prejuízo às 24 hpi e com recuperação e regeneração do tecido renal em uma semana, demonstrando ser um ótimo modelo para o estudo da regeneração no rim. O acetato parece ser promissor no tratamento da LRA experimental, pois diminui a taxa de morte induzida pela cisplatina. / The kidneys are organs responsible for managing a set of physiological tasks that maintain the homeostasis of the organism, among them the removal of toxic metabolites from the blood, production of hormones and the regulation of electrolyte balance. Acute kidney injury (AKI) leads to a rapid loss of organ function in hours or days. During the process of tissue injury, the immune system often initially acts in a detrimental way to the organ. One of the main cells involved in this process of tissue injury are macrophages, which participate during the first phase of AKI, already differentiated in a more proinflammatory profile, described as macrophages M1, and then during the tissue repair phase, differentiated into macrophages M2. Products derived from the metabolism of bacteria belonging to the intestinal microbiota, such as acetate, one of the main short chain fatty acids (SCFA), are able ofmodulating the inflammatory response in tissue injury models, but the influence of these products on the function, migration and differentiation of macrophages in the context of the AKI has not yet been well described. Thus the objective of this work was to evaluate the effect of acetate on the biological functions of macrophages in AKI. First, we developed a cisplatin-induced AKI model in Danio rerio adult fish, injecting different doses of cisplatin intraperitoneally (ip) and evaluating the survival and progression of the disease by histology, immunofluorescence and analysis of the cellular infiltrate. Subsequently, the animals were treated with acetate by gavage and then injected with cisplatin and analyzed for survival, the extent of the renal lesion by histology and the immune cell infiltrate in the kidney. The results showed that the injection of cisplatin (0,1275 mg/g) induced tissue damage in the kidney 24 h post-injection with loss of tubular structure, leading to the death of 80% of the animals. However, the surviving animals were able to regenerate the injured tissue eight days after the injection. There was also an increase in inflammatory infiltrate and a high rate of cell death, concomitant with high rates of proliferation of non-immune cells. The acetate was able to increase the survival of the animals injected with cisplatin and reduce the damage induced by the drug in the kidney, on the other hand, did not alter the performance of inflammatory cells of the immune system. In conclusion, the intraperitoneal cisplatin injection model was able to induce AKI in adult zebrafish, with maximum damage at 24 hpi and recovery and regeneration of renal tissue in one week, proving to be a good model for the study of regeneration in the kidney. Acetate appears to be promising in the treatment of experimental AKI, as it decreases the rate of cisplatin-induced death.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-09-06T23:00:46Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Susi Mara Soecki Sborgi.pdf: 1825313 bytes, checksum: b59ed76e89fc9e7c7cf0e1480e2a5f9f (MD5) / Made available in DSpace on 2018-09-06T23:00:46Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Susi Mara Soecki Sborgi.pdf: 1825313 bytes, checksum: b59ed76e89fc9e7c7cf0e1480e2a5f9f (MD5) Previous issue date: 2018-02-23 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Os distúrbios de ansiedade pertencem a um grupo de transtornos mentais onde os pacientes apresentam medo e preocupação excessivos. A prevalência dessa patologia vem aumentando no decorrer dos anos e tende a aumentar ainda mais nos próximos. Estudos afirmam que o estresse oxidativo é um processo fisiopatológico importante envolvido nesses transtornos, assim como a desregulamentação do sistema gabaérgico. Dessa forma, a pesquisa de substâncias que possam reduzir esses sintomas torna-se interessante, uma vez que os tratamentos já existentes nem sempre são eficazes aos pacientes. Estudos com ácido ferúlico têm demonstrado resultados positivos para tratar sintomas depressivos e por esse motivo, o fármaco foi escolhido para ter a atividade ansiolítica e seu possível mecanismo de ação avaliados em zebrafish. Para isso, foi realizado o teste de preferência claro/escuro após exposição dos animais ao ácido ferúlico, clonazepam ou fluoxetina, a fim de comparar o comportamento dos animais e verificar a ação ansiolítica dessa substância. Já para sugerir o possível mecanismo de ação, foi realizado o pré-tratamento com flumazenil, seguido do tratamento com o ácido ferúlico e/ou controle positivo, com posterior realização do mesmo teste. No teste de preferência claro/escuro, os animais tratados com clonazepam 0,75 mg/L, fluoxetina 10 mg/L, ácido ferúlico 250 e 500 mg/L, permaneceram mais tempo no lado claro em comparação aos animais não tratados. A latência para a primeira entrada no compartimento escuro também foi maior nos grupos tratados com fluoxetina 10 mg/L, ácido ferúlico 250 e 500 mg/L, quando comparados ao grupo controle. Na avaliação do possível mecanismo de ação, o tempo de permanência no compartimento claro dos animais pré-tratados com flumazenil 1,25 mg/L seguido de tratamento com ácido ferúlico 500 mg/L, diminuiu significativamente se comparado ao grupo sem pré-tratamento. Os resultados encontrados sugerem atividade ansiolítica e possível mecanismo de ação ligado ao sítio de ligação benzodiazepínico do receptor GABAA. / Anxiety disorders belong to a group of mental disorders in which the patients present excessive fear and worry. The prevalence of this pathology has been increasing over the years and tends to increase even more in the coming years. Studies have stated that oxidative stress is an important pathophysiological process involved in these disorders, as well as the deregulation of the gabaergic system. In this way, the research for substances that can reduce these symptoms becomes interesting, since the existing treatments are not always effective for the patients. Studies with ferulic acid have shown positive results to treat depressive symptoms and for this reason, the drug was chosen to have the anxiolytic activity and its possible mechanism of action evaluated in zebrafish. For this, the light/dark preference test was performed after exposure of the animals to ferulic acid, clonazepam or fluoxetine, in order to compare the behavior of the animals and verify the anxiolytic action of this substance. In order to suggest the possible mechanism of action, pre-treatment with flumazenil was performed, followed by treatment with ferulic acid and/or positive control, after whichthe same test was performed. In the light/dark preference test, the animals treated with clonazepam 0.75 mg/L, fluoxetine 10 mg/L, ferulic acid 250 and 500 mg/L, remained more time on the light side compared with untreated animals. The latency for the first entry into the dark compartment was also longer in the groups treated with fluoxetine 10 mg/L, ferulic acid 250 and 500 mg/L, when compared with the control group. In the mechanism of action test, the dwell time in the clear compartment of the animals pretreated with flumazenil 1.25 mg/L followed by treatment with ferulic acid 500 mg/L, decreased significantly compared with the group without pre-treatment. The results suggest an anxiolytic activity and mechanism of action linked to the benzodiazepine binding site of the GABAA receptor.

In the present study using zebrafish as the model organism, we have investigated the function of UII/UII-receptor (UIIR) signaling pathway during early embryogenesis. Herein we presented five lines of evidence supporting the hypothesis that UII/ UIIR signaling pathway is required for normal determination of asymmetric axis during early embryogenesis. First, function-loss of UII results in a concordant randomization of viscus asymmetries in embryos, including abnormalities in cardiac looping and positioning of visceral organs. Second, knockdown of UII randomizes the left-sided expression of asymmetrical genes including lefty2, spaw and pitx2c in the lateral plate mesoderm (LPM) and bmp4 in the developing heart domain and the LPM. Third, reduced UII levels interfere with the normal organogenesis of Kupffer's vesicle (KV), an organ implicated in the early steps of left-right (L-R) patterning of embryos. Fourth, repression of UII function perturbs the asymmetrical distribution of free Ca2+ (intracellular Ca2+) at the region surrounding embryo KV during early somitogenesis, which is one of the signaling mechanisms that propagandize and amplify the early clue of left-right (L-R) asymmetry. Fifth, depressing UII levels alters the normal pattern of Bmp and Nodal signaling, which modulate the establishment of L-R axis of developmental embryo. Collectively, these observations support a model in which UII/UIIR signal system takes part in the early molecular events of L-R asymmetry patterning of embryo by modulating Bmp and Nodal signaling, regulating KV normal morphogenesis, so then, maintaining the asymmetrical distribution of free intracellular Ca2+ at the peripheral region surrounding embryo KV. This study documents a role of UII/UIIR signaling pathway in the establishment of L-R axis of embryos which promises to reveal the molecular mechanisms responsible for human congenital diseases with heterotaxy. / Urotensin II (UII) is the most potent vasoconstrictor identified so far. This cyclic peptide stimulates its G protein-coupled receptor (GPR) to modulate cardiovascular system function in humans and in other animal species. / Li, Jun. / Advisers: Christopher HK Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 143-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

During vertebrate gastrulation, a well-orchestrated series of cell movements leads to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, a model organism for vertebrate development, the mesendodermal progenitor cells separate from the ectodermal cells and migrate towards the animal pole. To identify proteins controlling these processes, I used a comparative proteomic approach following two alternative strategies: (1) Based on the notion that Wnt11 regulates cell movement and morphology during gastrulation independent of transcriptional regulation, I performed a screen aimed at the identification of proteins phosphorylated upon Wnt11 signalling. To regulate Wnt11 expression tightly, I engineered a transgenic slb/wnt11-/- fish line expressing wnt11 under the control of a heat shock promoter. Using this line, I performed a quantitative comparison of protein phosphorylation with or without Wnt11 pathway activation by analysing 32P-labelled embryo extracts on 2D gels. (2) Since these experiments did not reveal any Wnt11 targets, I addressed, in the second approach, proteomic differences causal for the changes in cell adhesion and motility observed in mesendodermal cells upon involution. Quantitative 2D gel analysis comparing ectodermal and mesendodermal cells revealed 37 significantly regulated spots, 36 of which I identified by mass spectrometry. Interestingly, the majority of these proteins were not regulated on a transcriptional level as determined by an accompanying microarray analysis confirming the complementary nature of proteomics and transcriptomics. Among the identified targets, several proteins, including Ezrin2, had previously been assigned a cytoskeleton-related function. I characterised Ezrin2 in more detail showing that Ezrin2 is specifically activated by phosphorylation in mesendodermal cells and that it is required for proper gastrulation movements. In the course of this study, I developed techniques for proteomic analysis of early zebrafish embryos, including a protocol to remove the yolk. I identified several cytoskeleton-related proteins in a comparative proteomic screen for regulators of gastrulation movements. The subsequent characterisation of Ezrin2 confirmed the power of proteomics for the analysis of developmental processes. In conclusion, this work provides a foundation to study developmental and cell biological questions in early zebrafish embryos using proteomics.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 115-128).

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.

Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed 2008/07/23. Includes bibliographical references.