Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/6275 |
Resumo: | The development of ligand screening assays plays a very important role in the discovery of biologically active compounds. In this context, the use of immobilized capillary reactors has been adopted as a new technique for high throughput screening assays, which furnishes high selectivity, reproducibility. This work describes the covalent immobilization of electric fish (Electrophorus electricus) and human acetylcholinesterases (AChE) on fused silica capillaries and magnetic beads. The selected enzyme acts on the central nervous system and is a validated target for the treatment of Alzheimer s disease as for new insecticides. Zonal chromatography with the acetylcholinesterase capillary bioreactors was employed to determine kinetics constants, to a ligand screening assay of 79 compounds, and to inhibition mechanisms determination of the 05 identified ligands. A new approach to the screening of natural product extracts was developed based on ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The later was employed also under nonlinear condition to determine the affinity constant of the fished ligand. This work reports also the identification by a solution assay of a new acetylcholinesterase substrate and, the studies by zonal bioaffinity chromatography to identify the bi-substrate mechanism. |
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Vanzolini, Kenia LourençoCass, Quezia Bezerrahttp://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4781559E6http://lattes.cnpq.br/8140494122257511b9ffc67d-1ef9-410a-86cc-1e946921da9e2016-06-02T20:34:45Z2013-09-032016-06-02T20:34:45Z2013-05-03VANZOLINI, Kenia Lourenço. Acetylcholinesterase bioreactors: study of conditions for ligand screening. 2013. 133 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2013.https://repositorio.ufscar.br/handle/ufscar/6275The development of ligand screening assays plays a very important role in the discovery of biologically active compounds. In this context, the use of immobilized capillary reactors has been adopted as a new technique for high throughput screening assays, which furnishes high selectivity, reproducibility. This work describes the covalent immobilization of electric fish (Electrophorus electricus) and human acetylcholinesterases (AChE) on fused silica capillaries and magnetic beads. The selected enzyme acts on the central nervous system and is a validated target for the treatment of Alzheimer s disease as for new insecticides. Zonal chromatography with the acetylcholinesterase capillary bioreactors was employed to determine kinetics constants, to a ligand screening assay of 79 compounds, and to inhibition mechanisms determination of the 05 identified ligands. A new approach to the screening of natural product extracts was developed based on ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The later was employed also under nonlinear condition to determine the affinity constant of the fished ligand. This work reports also the identification by a solution assay of a new acetylcholinesterase substrate and, the studies by zonal bioaffinity chromatography to identify the bi-substrate mechanism.O desenvolvimento de ensaios para a triagem de ligantes é de grande importância para a descoberta de compostos biologicamente ativos. Neste contexto, o emprego de biorreatores enzimáticos tem sido adotado em ensaios em massa para triagem de ligantes, uma vez que possuem elevada seletividade com alta reprodutibilidade e produtividade. Este trabalho descreve a imobilização covalente da acetilcolinesterase de peixe-elétrico (Electrophorus electricus) e humana em capilares de sílica fundida e partículas magnéticas. A enzima selecionada justifica-se uma vez que atua no sistema nervoso central e é um alvo validado para o tratamento da doença de Alzheimer e desenvolvimento de inseticidas. Os biorreatores capilares de acetilcolinesterase foram empregados por cromatografia zonal para determinação das constantes cinéticas, e também para a triagem de 79 compostos com determinação do mecanismo de ação dos 05 ligantes identificados. Um novo modelo de triagem de extratos de produtos naturais foi desenvolvido usando os biorreatores de partículas magnéticas para pescar ligantes e os biorreatores capilares para determinar o percentual de atividade. O uso dos biorreatores capilares em cromatografia zonal não-linear permitiu caracterizar a afinidade do ligante selecionado. Este trabalho reporta também, um novo substrato identificado através de ensaios em solução para a acetilcolinesterase e, os estudos por cromatografia zonal de bioafinidade, para o mecanismo bi-substrato.Universidade Federal de Sao Carlosapplication/pdfporUniversidade Federal de São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarBRAnálise cromatográficaCromatografia líquida de alta eficiênciaCromatografia de bioafinidadeEnzimasImobilização de enzimasCIENCIAS EXATAS E DA TERRA::QUIMICABiorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantesAcetylcholinesterase bioreactors: study of conditions for ligand screeninginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis-1-1867fc213-f339-49e1-a669-a1e54cf68bf1info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL5327.pdfapplication/pdf3614226https://repositorio.ufscar.br/bitstream/ufscar/6275/1/5327.pdf2b258357050da3177584310ef5f5ac3aMD51TEXT5327.pdf.txt5327.pdf.txtExtracted texttext/plain0https://repositorio.ufscar.br/bitstream/ufscar/6275/4/5327.pdf.txtd41d8cd98f00b204e9800998ecf8427eMD54THUMBNAIL5327.pdf.jpg5327.pdf.jpgIM Thumbnailimage/jpeg10520https://repositorio.ufscar.br/bitstream/ufscar/6275/5/5327.pdf.jpgbbf2b1acc54cca5043a9841056ffa09dMD55ufscar/62752023-09-18 18:31:52.228oai:repositorio.ufscar.br:ufscar/6275Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:52Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
dc.title.alternative.eng.fl_str_mv |
Acetylcholinesterase bioreactors: study of conditions for ligand screening |
title |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
spellingShingle |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes Vanzolini, Kenia Lourenço Análise cromatográfica Cromatografia líquida de alta eficiência Cromatografia de bioafinidade Enzimas Imobilização de enzimas CIENCIAS EXATAS E DA TERRA::QUIMICA |
title_short |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
title_full |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
title_fullStr |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
title_full_unstemmed |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
title_sort |
Biorreatores de acetilcolinesterase: estudo de condições para a triagem de ligantes |
author |
Vanzolini, Kenia Lourenço |
author_facet |
Vanzolini, Kenia Lourenço |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/8140494122257511 |
dc.contributor.author.fl_str_mv |
Vanzolini, Kenia Lourenço |
dc.contributor.advisor1.fl_str_mv |
Cass, Quezia Bezerra |
dc.contributor.advisor1Lattes.fl_str_mv |
http://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4781559E6 |
dc.contributor.authorID.fl_str_mv |
b9ffc67d-1ef9-410a-86cc-1e946921da9e |
contributor_str_mv |
Cass, Quezia Bezerra |
dc.subject.por.fl_str_mv |
Análise cromatográfica Cromatografia líquida de alta eficiência Cromatografia de bioafinidade Enzimas Imobilização de enzimas |
topic |
Análise cromatográfica Cromatografia líquida de alta eficiência Cromatografia de bioafinidade Enzimas Imobilização de enzimas CIENCIAS EXATAS E DA TERRA::QUIMICA |
dc.subject.cnpq.fl_str_mv |
CIENCIAS EXATAS E DA TERRA::QUIMICA |
description |
The development of ligand screening assays plays a very important role in the discovery of biologically active compounds. In this context, the use of immobilized capillary reactors has been adopted as a new technique for high throughput screening assays, which furnishes high selectivity, reproducibility. This work describes the covalent immobilization of electric fish (Electrophorus electricus) and human acetylcholinesterases (AChE) on fused silica capillaries and magnetic beads. The selected enzyme acts on the central nervous system and is a validated target for the treatment of Alzheimer s disease as for new insecticides. Zonal chromatography with the acetylcholinesterase capillary bioreactors was employed to determine kinetics constants, to a ligand screening assay of 79 compounds, and to inhibition mechanisms determination of the 05 identified ligands. A new approach to the screening of natural product extracts was developed based on ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The later was employed also under nonlinear condition to determine the affinity constant of the fished ligand. This work reports also the identification by a solution assay of a new acetylcholinesterase substrate and, the studies by zonal bioaffinity chromatography to identify the bi-substrate mechanism. |
publishDate |
2013 |
dc.date.available.fl_str_mv |
2013-09-03 2016-06-02T20:34:45Z |
dc.date.issued.fl_str_mv |
2013-05-03 |
dc.date.accessioned.fl_str_mv |
2016-06-02T20:34:45Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
VANZOLINI, Kenia Lourenço. Acetylcholinesterase bioreactors: study of conditions for ligand screening. 2013. 133 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2013. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/6275 |
identifier_str_mv |
VANZOLINI, Kenia Lourenço. Acetylcholinesterase bioreactors: study of conditions for ligand screening. 2013. 133 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2013. |
url |
https://repositorio.ufscar.br/handle/ufscar/6275 |
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openAccess |
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Universidade Federal de São Carlos |
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Programa de Pós-Graduação em Química - PPGQ |
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UFSCar |
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BR |
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Universidade Federal de São Carlos |
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