Insights into human carboxylesterase 2 stability and activity in vivo and in vitro

Detalhes bibliográficos
Autor(a) principal: Ferreira, Pedro Garcia
Data de Publicação: 2012
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/8141
Resumo: Dissertation to obtain a Master Degree in Biotechnology
id RCAP_6fb387b2fdafff85a9dafbffd99abd53
oai_identifier_str oai:run.unl.pt:10362/8141
network_acronym_str RCAP
network_name_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository_id_str 7160
spelling Insights into human carboxylesterase 2 stability and activity in vivo and in vitroCarboxylesterase 2Enzymatic activityEnzymatic stabilityWhole living cellsDissertation to obtain a Master Degree in BiotechnologyDue to its role in ester containing xenobiotics metabolization, such as the activation of pro-drugs, carboxylesterase 2 (CES2) has become an attractive enzyme, with potential applications in the field of chemotherapy. Still, it hasn’t been as thoroughly characterized as the other main human carboxylesterase, the carboxylesterase 1 (CES1), having no described structure yet. The original hypothesis being tested in this work was the possibility of enzyme-enzyme interaction that had been suggested from previous work in the host laboratory. The initial task of this project was therefore to further assess CES2 stability and to test possible enzyme-enzyme interaction with CES1, by characterizing enzyme hydrolytic kinetics in different assay conditions and evaluating the behaviour of enzyme mixtures. Different CES2 activity rates were observed when tested in different reaction conditions suggesting a stabilizing/destabilizing effect on the enzyme, but evidence of in vitro interactions was not confirmed. Considering the dependence of conclusions on test methods as well as the extreme care measures that have to be taken for avoiding enzyme degradation during the preparation of cell extracts, a second goal was envisaged for this project. This consisted in the development of a new method capable of determining CES activity in conditions closer to those that are found physiologically. A new spectrofluorometric method able to assess carboxylesterase activity in intact living cells, using loperamide as a specific CES2 inhibitor and Bis(4-nitrophenyl) phosphate as a total carboxylesterase inhibitor was therefore developed and applied to Caco-2, HT-29. Hep G2 and HEK-293T cell lines. The method proved itself capable of detecting CES total activity in living cells, as well as to differentiate between CES2 and other CES activities. Differences were shown between this and the traditional methods using cell extracts, suggesting that the latter may not be the best option when trying to predict in vivo CES behavior.Fundação para a Ciência e Tecnologia - (PTDC/EBB-BIO/111530/2009, PEst-OE/EQB/LA0004/2011)Faculdade de Ciências e TecnologiaSimplício, AnaRUNFerreira, Pedro Garcia2012-11-15T15:30:33Z20122012-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/8141enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T03:40:37Zoai:run.unl.pt:10362/8141Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:17:58.371676Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
title Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
spellingShingle Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
Ferreira, Pedro Garcia
Carboxylesterase 2
Enzymatic activity
Enzymatic stability
Whole living cells
title_short Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
title_full Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
title_fullStr Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
title_full_unstemmed Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
title_sort Insights into human carboxylesterase 2 stability and activity in vivo and in vitro
author Ferreira, Pedro Garcia
author_facet Ferreira, Pedro Garcia
author_role author
dc.contributor.none.fl_str_mv Simplício, Ana
RUN
dc.contributor.author.fl_str_mv Ferreira, Pedro Garcia
dc.subject.por.fl_str_mv Carboxylesterase 2
Enzymatic activity
Enzymatic stability
Whole living cells
topic Carboxylesterase 2
Enzymatic activity
Enzymatic stability
Whole living cells
description Dissertation to obtain a Master Degree in Biotechnology
publishDate 2012
dc.date.none.fl_str_mv 2012-11-15T15:30:33Z
2012
2012-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10362/8141
url http://hdl.handle.net/10362/8141
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
_version_ 1799137826616377344

Registros relacionados