Navegando por Autor "Almeida, Daniela Volcan"

Agora exibindo 1 - 18 de 18

Item
Anti-MDR and antitumoral action of acetylsalicylic acid on leukaemic cells
(Biochemical Society, 2011) Garcia, Michele Carrett Dias; Votto, Ana Paula de Souza; Filgueira, Daza de Moraes Vaz Batista; Almeida, Daniela Volcan; Vallochi, Adriana Lima; D'Oca, Marcelo Gonçalves Montes; Marins, Luis Fernando Fernandes; Trindade, Gilma Santos
ASA (acetylsalicylic acid) is an NSAID (non-steroidal anti-inflammatory drug). ASA has gained attention as a potential chemopreventive and chemotherapeutic agent for several neoplasms. The aim of this study was to analyse the possible antitumoural effects of ASA in two erythroleukaemic cell lines, with or without the MDR (multidrug resistance) phenotype. The mechanism of action of different concentrations of ASA were compared in K562 (non-MDR) and Lucena (MDR) cells by analysing cell viability, apoptosis and necrosis, intracellular ROS (reactive oxygen species) formation and bcl-2, p53 and cox-2 gene expression. ASA inhibited the cellular proliferation or induced toxicity in K562 and Lucena cell lines, irrespective of the MDR phenotype. The ASA treatment provoked death by apoptosis and necrosis in K562 cells and only by necrosis in Lucena cells. ASA also showed antioxidant activity in both cell lines. The bcl-2, p53 and cox-2 genes in both cell lines treated with ASA seem to exhibit different patterns of expression. However, normal lymphocytes treated with the same ASA concentrations were more resistant than tumoral cells. The results of this work show that both cell lines responded to treatment with ASA, demonstrating a possible antitumoral and anti-MDR role for this drug.
Item
Anti-MDR and antitumoral action of acetylsalicylic acid on leukaemic cells
(2011) Garcia, Michele Carrett Dias; Votto, Ana Paula de Souza; Filgueira, Daza de Moraes Vaz Batista; Almeida, Daniela Volcan; Vallochi, Adriana Lima; D'Oca, Marcelo Gonçalves Montes; Marins, Luis Fernando Fernandes; Trindade, Gilma Santos
ASA (acetylsalicylic acid) is an NSAID (non-steroidal anti-inﬂammatory drug). ASA has gained attention as a potential chemopreventive and chemotherapeutic agent for several neoplasms. The aim of this study was to analyse the possible antitumoural effects of ASA in two erythroleukaemic cell lines, with or without the MDR (multidrug resistance) phenotype. The mechanism of action of different concentrations of ASA were compared in K562 (non-MDR) and Lucena (MDR) cells by analysing cell viability, apoptosis and necrosis, intracellular ROS (reactive oxygen species) formation and bcl-2, p53 and cox-2 gene expression. ASA inhibited the cellular proliferation or induced toxicity in K562 and Lucena cell lines, irrespective of the MDR phenotype. The ASA treatment provoked death by apoptosis and necrosis in K562 cells and only by necrosis in Lucena cells. ASA also showed antioxidant activity in both cell lines. The bcl-2, p53 and cox-2 genes in both cell lines treated with ASA seem to exhibit different patterns of expression. However, normal lymphocytes treated with the same ASA concentrations were more resistant than tumoral cells. The results of this work show that both cell lines responded to treatment with ASA, demonstrating a possible antitumoral and anti-MDR role for this drug
Item
A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach
(2012) Alves-Costa, Fernanda Antunes; Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Wasko, Adriane Pinto
The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.
Item
Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model
(2011) Rosa, Carlos Eduardo da; Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes
The aim of this study was to evaluate the effects of growth hormone (GH)overexpression on the gene expression profile of multiple components of the antioxidant defense system(ADS)of different genotypes of a GH transgenic zebrafish (Danio rerio)model. Several ADS-related genes were analyzed by semiquantitative reverse transcription–PCR in the liver of hemizygous (HE) and homozygous (HO)transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotypedependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse,which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.
Item
GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model
(2011) Kuradomi, Rafael Yutaka; Figueiredo, Márcio de Azevedo; Lanes, Carlos Frederico Ceccon; Rosa, Carlos Eduardo da; Almeida, Daniela Volcan; Maggioni, Rodrigo; Silva, Maeli Dal Pai; Marins, Luis Fernando Fernandes
The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish(Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the a-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.
Item
GH overexpression modifies muscle expression of anti-oxidant enzymes and increases spinal curvature of old zebrafish
(2010) Fonseca, Duane Barros da; Rosa, Carlos Eduardo da; Kuradomi, Rafael Yutaka; Almeida, Daniela Volcan; Lanes, Carlos Frederico Ceccon; Figueiredo, Marcio de Azevedo; Dytz, Aline Guerra; Marins, Luis Fernando Fernandes
Growth hormone (GH) excess causes an increment in the metabolic rate and in reactive oxygen species generation, which accelerate the ageing process in mammals. Considering that there is no information on this subject in fish, the aim of the present study was to evaluate the excess GH effect on senescence in a zebrafish (Danio rerio) transgenic model. In order to reach this objective, we analyzed the phenotype of spinal curvature and expression of genes related to the anti-oxidant defense system and myogenesis in muscle of 8 and 30 months old GH-transgenic males. Gene expression analyses revealed that both superoxide dismutase isoforms were down-regulated only in 30 months old animals, while glutamate cysteine igase was down-regulated in GH-transgenic zebrafish. Acceleration of the spinal curvature and a reduction in the expression of miogenin at both ages and MyoD in the old fish were also observed. Although neurolipofuscin accumulation was not significant in GH-transgenic zebrafish, the estimation of maximum longevity based on the von Bertalanffy growth function was significantly lower in this group. The results obtained here indicate that GH overexpression reduces the transcription of anti-oxidant defense system and myogenesis-related genes, which probably accelerates senescence in the zebrafish transgenic model used
Item
Improving the production of transgenic fish germlines: in vivo evaluation of mosaicism in zebrafish (Danio rerio) using a green fluorescent protein (GFP) and growth hormone cDNA transgene co-injection strategy
(2007) Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes
In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio) were produced using two transgenes, both comprised of the carp β-actin promoter driving the expression of either the green fluorescent protein (GFP) reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.
Item
Induction of phase II enzymes and hsp70 genes by copper sulfate through the electrophile-responsive element (EpRE): insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin
(2010) Almeida, Daniela Volcan; Nornberg, Bruna Félix da Silva; Monserrat, Laura Alicia Geracitano; Barros, Daniela Marti; Monserrat, José María; Marins, Luis Fernando Fernandes
We have evaluated the homology of the electrophile-responsive element (EpRE) core sequence, a binding site for the Nrf2 transcription factor, in the proximal promoters of the mouse and zebrafish glutathione-S-transferase (gst), glutamate cysteine ligase catalytic subunit (gclc) and heat shock protein 70 (hsp70) genes. The EpRE sites identified for both species in the three analyzed genes showed a high similarity with the putative EpRE core sequence. We also produced a transgenic zebrafish model carrying a transgene comprised of the luciferase (luc) reporter gene under transcriptional control of a mouse EpRE sequence. This transgenic model was exposed to copper sulfate, and the reporter gene was significantly activated. The endogenous gst, gclc and hsp70 zebrafish genes were analyzed in the EpRELuc transgenic zebrafish and showed an expression pattern similar to that of the reporter transgene used. Our results demonstrate that EpRE is conserved between mouse and zebrafish for detoxificationrelated genes and that the development of genetically modified models using this responsive element to drive the expression of reporter genes can be an important tool in understanding the action mechanism of aquatic pollutants.
Item
Induction of phase II enzymes and hsp70 genes by copper sulfate through the electrophile-responsive element (EpRE): insights obtained from a transgenic zebrafish model carrying an orthologous EpRE sequence of mammalian origin.
(2009) Almeida, Daniela Volcan; Nornberg, Bruna Félix da Silva; Monserrat, Laura Alicia Geracitano; Barros, Daniela Marti; Monserrat, José María; Marins, Luis Fernando Fernandes
We have evaluated the homology of the electrophile-responsive element (EpRE) core sequence, a binding site for the Nrf2 transcription factor, in the proximal promoters of the mouse and zebrafish glutathione-S-transferase (gst), glutamate cysteine ligase catalytic subunit (gclc) and heat shock protein 70 (hsp70) genes. The EpRE sites identified for both species in the three analyzed genes showed a high similarity with the putative EpRE core sequence. We also produced a transgenic zebrafish model carrying a transgene comprised of the luciferase (luc) reporter gene under transcriptional control of a mouse EpRE sequence. This transgenic model was exposed to copper sulfate, and the reporter gene was significantly activated. The endogenous gst, gclc and hsp70 zebrafish genes were analyzed in the EpRELuc transgenic zebrafish and showed an expression pattern similar to that of the reporter transgene used. Our results demonstrate that EpRE is conserved between mouse and zebrafish for detoxificationrelated genes and that the development of genetically modified models using this responsive element to drive the expression of reporter genes can be an important tool in understanding the action mechanism of aquatic pollutants.
Item
Initial results in the development of a reporter cell line for toxicology studies at gene expression level: Activation of the electrophile-responsive element by copper and methyl parathion
(2008) Almeida, Daniela Volcan; Trindade, Gilma Santos; Monserrat, Laura Alicia Geracitano; Barros, Daniela Marti; Monserrat, José María; Marins, Luis Fernando Fernandes
Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE)within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1)electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt–GFP construct and successfully selected with G418 antibiotic. EpREmt–GFP HTC cells were treated with 0.002 mg L 1, 0.02 mg L 1, 0.2 mg L 1 and 2mg L 1 copper sulfate and 0.001 mg L 1,0.01 mg L 1, 0.1 mg L 1 and 1 mg L 1 methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2 mg copper L 1 and 1 mg methyl parathion L 1, respectively (p < 0.01). The results obtained in the present study demonstrate that the EpREmt–GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.
Item
Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish
(2008) Rosa, Carlos Eduardo da; Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Monserrat, José María; Marins, Luis Fernando Fernandes
Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.
Item
Modelos in vivo e in vitro geneticamente modificados como biomarcadores de poluição aquática
(2007) Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes
A indução de vários genes que codificam para enzimas envolvidas no processo de detoxificação celular e resposta antioxidante é mediada através de um mecanismo comum, o qual depende da presença dos elementos de resposta eletrofílicos (EpRE) na região promotora destes genes. No presente estudo foi analisada a homologia da seqüência central do EpRE, a qual representa um sítio de ligação para o fator de transcrição Nrf2, de regiões conservadas de promotores das enzimas glutationa S-transferase (Gsta1), subunidade catalítica da glutamato cisteína ligase (Gclc) e proteína de choque térmico (Hsp70) de zebrafish (Danio rerio) e camundongo (Mus musculus). A seqüência EpRE foi identificada para ambas as espécies em todos os genes comparados, mostrando uma alta similaridade com a seqüência central do EpRE. Adicionalmente, foi produzido um modelo de zebrafish transgênico carregando um transgene que contém um EpRE obtido do promotor da Gsta1 de camundongo fusionado ao promotor mínimo da metalotioneína (mt1) do camundongo direcionando a expressão do gene repórter da luciferase (luc). Este modelo in vivo foi exposto ao sulfato de cobre e o gene repórter foi significativamente ativado. Os genes gclc e hsp70 do zebrafish foram analisados nos peixes transgênicos carregando a construção EpRE-Luc e mostraram um padrão similar de expressão com o gene da luciferase. Também foi produzido um modelo alternativo carregando o transgene EpRE-GFP. Nesta construção genética o gene repórter da luc foi substituído pelo gene da proteína verde fluorescente (gfp). Os peixes carregando o transgene EpRE-GFP foram expostos ao cobre e a expressão da Gfp foi diretamente quantificada nas larvas vivas em fluorímetro. Neste caso, um aumento na expressão da Gfp só foi detectado na maior concentração de cobre, provavelmente, devido ao efeito do mosaismo ou da fluorescência basal das larvas. Na última etapa deste trabalho, foi produzida uma linhagem estável de hepatoma (HTC) carregando o transgene EpRE-GFP. A linhagem EpRE-GFP HTC foi tratada com sulfato de cobre e metil paration por 48 h. Para avaliar as respostas genéticas produzidas por estes poluentes, a expressão da Gfp foi diretamente quantificada e os resultados mostraram que a expressão da Gfp é significativamente aumentada somente nas maiores concentrações de ambos os poluentes. Desta forma, os resultados obtidos neste estudo demonstram que o uso de EpRE associado a genes repórteres pode ser um modelo interessante para estudo de respostas genéticas das células frente a poluentes aquáticos, bem como uma ferramenta adequada para estudos toxicológicos in vivo e in vitro.
Item
mRNA expression and activity of ion-transporting proteins in gills of the blue crab callinectes sapidus: effects of waterborne copper
(2011) Martins, Camila De Martinez Gaspar; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Bianchini, Adalto
Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2%) and full (30%) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 mM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodiumpotassium adenosine triphosphatase [Naþ/Kþ-ATPase], hydrogen adenosine triphosphatase [Hþ-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Naþ/Kþ/2Cl_) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Naþ/Kþ-ATPase and the Naþ/Kþ/2Cl_ cotransporter. However, Cu did not affect Naþ/Kþ-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30%). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30%) than in diluted seawater (2%).
Item
Nova variante da proteína fluorescente DSRED2 com potencial aplicação na piscicultura ornamental
(2016) Hernández, Natalia Ossa; Martins, Luis Fernando; Almeida, Daniela Volcan
A rentabilidade econômica gerada pela aquicultura ornamental a torna um dos importantes componentes da indústria aquícola. O aumento da demanda de peixes ornamentais gera a necessidade da utilização de técnicas que permitam a produção de novas variedades. Neste cenário, genes codificantes para proteínas fluorescentes têm sido inseridos com êxito no genoma de diversas espécies de peixes, gerando novas variedades ornamentais. Porém, a disponibilidade de proteínas fluorescentes no mercado é limitada. Desse modo, este trabalho teve como objetivo desenvolver novas variantes de proteínas fluorescentes para aplicação na aquicultura ornamental. Para isso, o gene DsRed2 que codifica para a proteína vermelha fluorescente do coral Discosoma sp. foi utilizado como base para a produção de novas variantes através de mutagênese randômica induzida por error-prone PCR (epPCR). Um total de 12 genes variantes foram isolados, sequenciados e analisados. A variante denominada Mutante #10 apresentou alterações interessantes nas suas propriedades espectrais, possuindo xxxx emissão de fluorescência quando excitada nos comprimentos de onda de xxx e xxxxx nm, respectivamente. Esta nova variante foi expressa em Escherichia coli e também no tecido muscular do zebrafish (Danio rerio), apresentando a xxxxxx emissão de fluorescência em ambos modelos. Uma proteína com xxxxx emissão de fluorescência pode ter aplicações em diversas áreas, desde as ciências básicas até a aquicultura ornamental.
Item
Nova variante da proteína fluorescente DsRed2 com potencial aplicação na piscicultura ornamental
(2016) Hernández, Natalia Ossa; Marins, Luis Fernando Fernandes; Almeida, Daniela Volcan
A rentabilidade econômica gerada pela aquicultura ornamental a torna um dos importantes componentes da indústria aquícola. O aumento da demanda de peixes ornamentais gera a necessidade da utilização de técnicas que permitam a produção de novas variedades. Neste cenário, genes codificantes para proteínas fluorescentes têm sido inseridos com êxito no genoma de diversas espécies de peixes, gerando novas variedades ornamentais. Porém, a disponibilidade de proteínas fluorescentes no mercado é limitada. Desse modo, este trabalho teve como objetivo desenvolver novas variantes de proteínas fluorescentes para aplicação na aquicultura ornamental. Para isso, o gene DsRed2 que codifica para a proteína vermelha fluorescente do coral Discosoma sp. foi utilizado como base para a produção de novas variantes através de mutagênese randômica induzida por error-prone PCR (epPCR). Um total de 12 genes variantes foram isolados, sequenciados e analisados. A variante denominada Mutante #10 apresentou alterações interessantes nas suas propriedades espectrais, possuindo xxxx emissão de fluorescência quando excitada nos comprimentos de onda de xxx e xxxxx nm, respectivamente. Esta nova variante foi expressa em Escherichia coli e também no tecido muscular do zebrafish (Danio rerio), apresentando a xxxxxx emissão de fluorescência em ambos modelos. Uma proteína com xxxxx emissão de fluorescência pode ter aplicações em diversas áreas, desde as ciências básicas até a aquicultura ornamental.
Item
SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous gh-transgenic zebrafish (danio rerio)
(2009) Studzinski, Ana Lupe Motta; Almeida, Daniela Volcan; Lanes, Carlos Frederico Ceccon; Figueiredo, Marcio de Azevedo; Marins, Luis Fernando Fernandes
Homozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NTand HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO(p < 0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p < 0.01) and 4.3 times (p < 0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins.
Item
Superexpressão do hormônio do crescimento (GH) e as respostas ao estresse abiótico em um modelo de zebrafish (Danio rerio) transgênico
(2013) Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes
Peixes transgênicos que superexpressam o gene do hormônio do crescimento (GH) podem apresentar taxas de crescimento almejadas para a produção na aquicultura. No entanto, o aumento do nível de GH causado pela transgenia pode modificar as respostas biológicas frente à presença de estressores bióticos e abióticos. Desta forma, o principal objetivo deste estudo foi analisar os efeitos do excesso de GH promovido pela transgenia sobre a tolerância a diferentes estressores abióticos, em um modelo de peixe (Danio rerio) transgênico para o GH. A exposição às variações de salinidade, temperatura e oxigênio foram os parâmetros considerados como estressores abióticos. A alteração da tolerância a estes fatores, promovida pela superexpressão do GH foi avaliada em nível de expressão de genes, atividades enzimáticas e produtos metabólicos, com a finalidade de entender os mecanismos destas modificações. No primeiro estudo, peixes transgênicos adultos foram expostos à salinidade hiperosmótica de 11 ppt e os resultados demonstraram que o excesso de GH aumenta a atividade da enzima Na+,K+14 ,ATPase e aumenta, consequentemente, a absorção de Na+. O excesso de Na+ 15 nos peixes transgênicos pode justificar a maior taxa de mortalidade destes animais. No segundo estudo, as respostas ao estresse térmico foram analisadas em peixes transgênicos adultos e juvenis expostos à alta temperatura (39o17 C) e baixa temperatura (13o18 C). Estes experimentos demonstraram um aumento na sobrevivência dos peixes transgênicos juvenis expostos à 13o19 C, e uma diminuição nos adultos e juvenis transgênicos expostos à 39 o20 C. Associado a estes resultados, houve uma alteração no padrão de expressão das proteínas de choque térmico (HSPs), relacionado à idade e à temperatura. No último estudo, peixes transgênicos e não-transgênicos foram expostos ao estresse hipóxico (1.5 mgO2/L). Os resultados indicam que os peixes transgênicos são mais suscetíveis à baixa concentração de oxigênio. Adicionalmente, foi observado um aumento na atividade da enzima lactato desidrogenase (LDH) e na concentração de lactato nos peixes transgênicos. Assim estes resultados sugerem que o excesso de GH promovido pela transgenia prejudica a habilidade de manter o metabolismo aeróbico em baixos níveis de oxigênio, ativando o metabolismo anaeróbico nestes animais. Em conclusão, a exposição a distintos estressores abióticos mostrou que a superexpressão do GH pode alterar, de forma diferenciada, a tolerância termal, osmótica e hipóxica, de acordo com a idade e ao tipo de estresse.
Item
The effect of GH overexpression on GHR and IGF-I gene regulation in different genotypes of GH-transgenic zebrafish
(2007) Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Proietti, Maíra Carneiro; Marins, Luis Fernando Fernandes
Most biological actions of growth hormone (GH) are mediated by the insulin-like growth factor I (IGF-I) that is produced after the interaction of the hormone with a specific cell surface receptor, the GH receptor (GHR). Even though the GH excess on fish metabolism is poorly known, several species have been genetically engineered for this hormone in order to improve growth for aquaculture. In some GH-transgenic fish growth has been dramatically increased, while in others high levels of transgene expression have shown inhibition of the growth response. In this study, we used for the first time different genotypes (hemizygous and homozygous) of a GH transgenic zebrafish (Danio rerio) lineage as a model for studying the GH resistance induced by different GH transgene expression levels. The results obtained here demonstrated that homozygous fish did not grow as expected and have a lower condition factor, which implies a catabolic state. These findings are explained by a decreased IGF-I and GHR gene expression as a consequence of GH resistance. Together, our results demonstrated that homozygous GH-transgenic fish showed similar characteristics to the starvation-induced fish and could be an interesting model for studying the regulation of the GH/GHR/IGF-I axis in fish.