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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Triagem de compostos com base na ativação do receptor de ecdisteroides em culturas celulares e planejamento racional de moléculas inseticidas associado a ferramentas computacionais / Cell-based screening system for ecdysone receptor activation and rational iseticide design with association of computational approaches

Pinto, Ciro Pedro Guidotti 23 February 2017 (has links)
Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-07-11T18:09:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Ciro Pinto- pré textual.pdf: 2735772 bytes, checksum: c9b21bacb392400253c3094a61ad6427 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-07-17T20:35:34Z (GMT) No. of bitstreams: 2 Dissertação Ciro Pinto- pré textual.pdf: 2735772 bytes, checksum: c9b21bacb392400253c3094a61ad6427 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-17T20:35:34Z (GMT). No. of bitstreams: 2 Dissertação Ciro Pinto- pré textual.pdf: 2735772 bytes, checksum: c9b21bacb392400253c3094a61ad6427 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A biotecnologia celular aplicada é essencial para o Manejo Integrado de Pragas, através da aplicação de várias técnicas de alto desempenho, possibilita o planejamento racional de novos inseticidas. Os receptores nucleares são importantes alvos na busca de novos inseticidas, destacando-se o receptor de ecdisteroides como um dos alvos mais importantes atualmente. Este receptor é uma proteína cuja ativação artificial é mediada por moléculas (ligantes) que se ligam ao seu sítio ativo, causando assim um efeito agonista ou antagonista ao hormônio natural, o 20-Hidroxiecdisônio. Ferramentas computacionais são de fundamental importância para o entendimento da interação receptor/ligante, possibilitando assim, o design de novas moléculas. Com isto, esta pesquisa teve como objetivo estabelecer um protocolo para transfecção de células de insetos com um plasmídeo sensível à atividade de receptor de ecdisteroides, comparar a atividade de ecdisteroides e dibenzoilhidrazinas, e novos compostos organocalcogênicos (estruturalmente semelhantes a dibenzoilhidrazinas), entendendo seu mecanismo de ação com o uso de ferramentas computacionais. Os bioensaios consistiram na transfecção de células das linhagens S2 (Diptera) e SF9 (Lepidoptera) com o plasmídeo B.act.ere.luc., planejado para expressar a enzima luciferase frente à ativação do receptor de ecdisteroides das células. Foram avaliadas a atividade dos hormônios 20-hidroxiecdisônio e PonasteronaA, do inseticida comercial tebufenozide (dibenzoilhidrazina) e de onze novas moléculas organocalcogênicas, em linhagens celulares. Foram preparadas curvas de dose resposta com os dois hormônios e a tebufenozida. Com as novas moléculas, foi investigada a citotoxicidade, efeito agonista e antagonista. Para a atividade antagonista, o parâmetro estabelecido para uma molécula possuir considerável efeito foi a redução de >50% da atividade, comparando à ação do hormônio isolado. Posteriormente foram usados modelos tridimensionais para investigar as interações entre os ligantes e o sitio ativo do receptor de ecdisteroides das ordens Diptera e Lepidoptera. Concluiu-se que é possível utilizar o presente sistema de triagem em células, na busca de novas moléculas com atividade no receptor de ecdisteroides. Em ambas as linhagens celulares, Ponasterona-A foi a molécula mais ativa, sendo que na linhagem SF9, a tebufenozida se mostrou mais ativa quando comparada a 20-Hidroxiecdisônio. Das novas moléculas testadas, nenhuma apresentou efeito agonista, entretanto, efeito antagonista foi observado apenas em células de Lepidoptera, com padrões diferentes conforme o hormônio utilizado. As interações observadas entre o receptor e as moléculas antagonistas servirão como base para o design racional de novas moléculas inseticidas. / Applied cellular biotechnology is essential for Integrated Pest Management, because through several high performance techniques, it allows the rational planning of new insecticides. Nuclear receptors are important targets in the research for new insecticides, and the ecdysteroid receptor is one of the most important targets nowadays. This receptor is a protein whose artificial transactivation is mediated by molecules (ligands) that bind to its active site, thus causing an agonist or antagonistic effect on the hormone 20-hydroxyecdysone. Computational tools are of fundamental importance for the understanding of receptor/ligand interactions, thus enabling the design of new molecules. The aim of this research was to establish a protocol for transfection of insect cells with a plasmid sensitive to the activity of the ecdysone receptor, to compare the activity of ecdysteroids and dibenzoylhydrazines, and new organocalcogenic molecules (similar to dibenzoylhidrizines) understanding its mechanism of action with computational tools. Bioassays consisted of the transfection of S2 (Diptera) and SF9 (Lepidoptera) cell lines with the plasmid B.act.ere.luc., designed to express the luciferase enzyme against the activation of the receptor. The activity of the hormones 20-hydroxyecdysone and Ponasterone-A, the insecticide tebufenozide (dibenzoylhydrazine) and eleven new molecules of dibenzoylhydrazines in cell lines were evaluated. Dose response curves were prepared with the two hormones and tebufenozide. With the new molecules, the cytotoxicity, agonist and antagonist effect on ecdysteroids was investigated. To investigate the antagonistic activity of the new molecules on the ecdysteroids, the parameter established for a molecule have a considerable effect was the reduction of >50% of the activity, comparing to the action of the isolated hormone. Finally, using three-dimensional models, the interactions between ligands and the active site of the Diptera and Lepidoptera ecdysone receptor were investigated. It was concluded that it is possible to find new molecules with activity in the insect ecdysone receptor. In both cell lines, Ponasterone-A was the most active molecule, and in SF9, tebufenozide was more active than 20-Hydroxyecdysone. Among the new dibenzoylhydrazines tested, none showed an agonist effect. Antagonistic effect was observed only in Lepidoptera cells, but with different patterns according to the hormone used. The antagonistic activity observed from some new molecules can be due to the fitting of them in the binding site from the ecdysone rceptor, blocking important ligation among receptor and hormone.
2

Identification of Heat Shock Factor Binding Sites in the Drosophila Genome

Gonsalves, Sarah E. 12 December 2012 (has links)
The heat shock response (HSR) is a highly conserved mechanism that enables organisms to survive environmental and pathophysiological stress. In Drosophila, the HSR is regulated by a single transcription factor, heat shock factor (HSF). During stress, HSF trimerizes and binds to over 200 loci on Drosophila polytene chromosomes with only nine mapping to major heat shock (HS) inducible gene loci. The function of HSF binding to the other sites in the genome is currently unknown. Some of these sites may contain yet unidentified “minor” HS genes. Interestingly, the binding of HSF also coincides with puff regression at some sites. Two such sites contain the major developmentally regulated genes Eip74 and Eip75: key regulators in the response to 20-hydroxyecdysone (20E), the main hormone responsible for the temporal co-ordination of post-embryonic development in Drosophila. Previous work in our and other labs indicates that the regression of non-HS puffs during the HSR is dependent on the presence of functional HSF. Using chromatin immunoprecipitation (ChIP) followed by hybridization to genome tiling arrays (Chip), I have identified 434 regions in the Drosophila Kc cell genome that are bound by HSF during HS, and have determined that 57% of these sites are located within the transcribed regions of genes. By examining the transcriptional response to HS in Kc cells and third instar larvae using expression microarrays, I found that only about 10% of all genes within 1250 bp of an HSF binding site are transcriptionally regulated by HS and many genes whose transcript levels change during HS do not appear to be near an HSF binding site. Furthermore, genes with an HSF binding site within their introns are significantly enriched (modified Fisher Exact p-value between 2.0x10-3 and 1.5x10-6) in gene ontology terms related to developmental processes and reproduction. Using expression microarray technology, I characterized the transcriptional response to 20E and its structural analog ponasterone A. I have identified multiple HSF binding sites within Eip74 and Eip75, and show that induction of the HSR correlates with repression of these genes and all other 20E-inducible genes. Taken together, this work provides a basis for further investigation into the role of HSF binding to sites not associated with HS genes and its possible function as a repressor of gene transcription during conditions of stress and as a regulator of developmental genes under stress and non-stress conditions.
3

Identification of Heat Shock Factor Binding Sites in the Drosophila Genome

Gonsalves, Sarah E. 12 December 2012 (has links)
The heat shock response (HSR) is a highly conserved mechanism that enables organisms to survive environmental and pathophysiological stress. In Drosophila, the HSR is regulated by a single transcription factor, heat shock factor (HSF). During stress, HSF trimerizes and binds to over 200 loci on Drosophila polytene chromosomes with only nine mapping to major heat shock (HS) inducible gene loci. The function of HSF binding to the other sites in the genome is currently unknown. Some of these sites may contain yet unidentified “minor” HS genes. Interestingly, the binding of HSF also coincides with puff regression at some sites. Two such sites contain the major developmentally regulated genes Eip74 and Eip75: key regulators in the response to 20-hydroxyecdysone (20E), the main hormone responsible for the temporal co-ordination of post-embryonic development in Drosophila. Previous work in our and other labs indicates that the regression of non-HS puffs during the HSR is dependent on the presence of functional HSF. Using chromatin immunoprecipitation (ChIP) followed by hybridization to genome tiling arrays (Chip), I have identified 434 regions in the Drosophila Kc cell genome that are bound by HSF during HS, and have determined that 57% of these sites are located within the transcribed regions of genes. By examining the transcriptional response to HS in Kc cells and third instar larvae using expression microarrays, I found that only about 10% of all genes within 1250 bp of an HSF binding site are transcriptionally regulated by HS and many genes whose transcript levels change during HS do not appear to be near an HSF binding site. Furthermore, genes with an HSF binding site within their introns are significantly enriched (modified Fisher Exact p-value between 2.0x10-3 and 1.5x10-6) in gene ontology terms related to developmental processes and reproduction. Using expression microarray technology, I characterized the transcriptional response to 20E and its structural analog ponasterone A. I have identified multiple HSF binding sites within Eip74 and Eip75, and show that induction of the HSR correlates with repression of these genes and all other 20E-inducible genes. Taken together, this work provides a basis for further investigation into the role of HSF binding to sites not associated with HS genes and its possible function as a repressor of gene transcription during conditions of stress and as a regulator of developmental genes under stress and non-stress conditions.

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