Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans
Autor(a) principal: | |
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Data de Publicação: | 2007 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10216/22064 |
Resumo: | 2. Abstract Several resistance mechanisms are associated to antifungal resistance in pathogenic fungi, namely C. albicans: over-expression of CDR1, CDR2 and MDR1 genes encoding for eflux pumps; alterations in gene expression levels or point mutations in ERG11 gene, encoding for the azoles target enzyme lanosterol 14 - demethylase, associated to ergosterol biosynthesis. The aim of the present work was to uncover the resistance mechanisms in a large group of clinical C. albicans strains, performing phenotypic and gene expression studies. Five control strains and 62 clinical strains of C. albicans were used. Standard protocol from CLSI M27-A2 was used to determine the susceptibility phenotype calculating minimal inhibitory concentration for the antifungals fluconazole, itraconazole and voriconazole, with and without ibuprofen (100µg/ml). Also, reversion by efflux hypothesis was tested by flow cytometry, using as fluorescent marker FUN-1, known as an efflux marker. Next, real time PCR protocols for detection and quantification of the target genes CDR1, CDR2, MDR1 e ERG11, were optimized. Different reaction parameters were tested, to achieve values of efficiency and error obtained from standard curves, according to reference values, to ensure accuracy. ACT1 gene, which encodes for actin was used as normalizing gene for the gene expression levels. Most clinical strains resistant to all tested azoles showed a significant over-expression of CDR1 and specially CDR2 when compared to ERG11; in these strains ibuprofen induced the reversion of resistance and an increase in FUN-1 fluorescence. The 2 strains that did not revert their phenotype, ibuprofen did not increase the fluorescence of cells stained with FUN-1 and gene over-expression was not only for CDR1 and CDR2 but mostly for ERG11 gene. It should be stressed that C. albicans control strain 12-99 with known multiple resistance mechanisms did not revert the resistance phenotype in the presence of ibuprofen. MDR1 expression was not detected in none of the C. albicans clinical strains. Strains with multiple resistance mechanisms did not revert, wich might indicate that ibuprofen could be a potential antifungal efflux blocker. Real Time PCR is easily available and a sensitive method for gene expression studies, and also SYBR Green is acceptable, although being an unspecific marker, when a melting curve is added in each assay. |
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Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicansMedicina e Oncologia MolecularPorto2. Abstract Several resistance mechanisms are associated to antifungal resistance in pathogenic fungi, namely C. albicans: over-expression of CDR1, CDR2 and MDR1 genes encoding for eflux pumps; alterations in gene expression levels or point mutations in ERG11 gene, encoding for the azoles target enzyme lanosterol 14 - demethylase, associated to ergosterol biosynthesis. The aim of the present work was to uncover the resistance mechanisms in a large group of clinical C. albicans strains, performing phenotypic and gene expression studies. Five control strains and 62 clinical strains of C. albicans were used. Standard protocol from CLSI M27-A2 was used to determine the susceptibility phenotype calculating minimal inhibitory concentration for the antifungals fluconazole, itraconazole and voriconazole, with and without ibuprofen (100µg/ml). Also, reversion by efflux hypothesis was tested by flow cytometry, using as fluorescent marker FUN-1, known as an efflux marker. Next, real time PCR protocols for detection and quantification of the target genes CDR1, CDR2, MDR1 e ERG11, were optimized. Different reaction parameters were tested, to achieve values of efficiency and error obtained from standard curves, according to reference values, to ensure accuracy. ACT1 gene, which encodes for actin was used as normalizing gene for the gene expression levels. Most clinical strains resistant to all tested azoles showed a significant over-expression of CDR1 and specially CDR2 when compared to ERG11; in these strains ibuprofen induced the reversion of resistance and an increase in FUN-1 fluorescence. The 2 strains that did not revert their phenotype, ibuprofen did not increase the fluorescence of cells stained with FUN-1 and gene over-expression was not only for CDR1 and CDR2 but mostly for ERG11 gene. It should be stressed that C. albicans control strain 12-99 with known multiple resistance mechanisms did not revert the resistance phenotype in the presence of ibuprofen. MDR1 expression was not detected in none of the C. albicans clinical strains. Strains with multiple resistance mechanisms did not revert, wich might indicate that ibuprofen could be a potential antifungal efflux blocker. Real Time PCR is easily available and a sensitive method for gene expression studies, and also SYBR Green is acceptable, although being an unspecific marker, when a melting curve is added in each assay.Faculdade de Medicina da Universidade do PortoFMUP20072011-02-07T00:00:00Z2011-02-07info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/mswordhttp://hdl.handle.net/10216/22064porRicardo, Elisabete Travassos Araújoinfo: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:RCAAP2023-11-29T15:30:11Zoai:repositorio-aberto.up.pt:10216/22064Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:25:08.256426Repositório 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 |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
title |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
spellingShingle |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans Ricardo, Elisabete Travassos Araújo Medicina e Oncologia Molecular Porto |
title_short |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
title_full |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
title_fullStr |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
title_full_unstemmed |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
title_sort |
Detecção e quantificação da expressão de genes de resistência aos azoles em Candida albicans |
author |
Ricardo, Elisabete Travassos Araújo |
author_facet |
Ricardo, Elisabete Travassos Araújo |
author_role |
author |
dc.contributor.author.fl_str_mv |
Ricardo, Elisabete Travassos Araújo |
dc.subject.por.fl_str_mv |
Medicina e Oncologia Molecular Porto |
topic |
Medicina e Oncologia Molecular Porto |
description |
2. Abstract Several resistance mechanisms are associated to antifungal resistance in pathogenic fungi, namely C. albicans: over-expression of CDR1, CDR2 and MDR1 genes encoding for eflux pumps; alterations in gene expression levels or point mutations in ERG11 gene, encoding for the azoles target enzyme lanosterol 14 - demethylase, associated to ergosterol biosynthesis. The aim of the present work was to uncover the resistance mechanisms in a large group of clinical C. albicans strains, performing phenotypic and gene expression studies. Five control strains and 62 clinical strains of C. albicans were used. Standard protocol from CLSI M27-A2 was used to determine the susceptibility phenotype calculating minimal inhibitory concentration for the antifungals fluconazole, itraconazole and voriconazole, with and without ibuprofen (100µg/ml). Also, reversion by efflux hypothesis was tested by flow cytometry, using as fluorescent marker FUN-1, known as an efflux marker. Next, real time PCR protocols for detection and quantification of the target genes CDR1, CDR2, MDR1 e ERG11, were optimized. Different reaction parameters were tested, to achieve values of efficiency and error obtained from standard curves, according to reference values, to ensure accuracy. ACT1 gene, which encodes for actin was used as normalizing gene for the gene expression levels. Most clinical strains resistant to all tested azoles showed a significant over-expression of CDR1 and specially CDR2 when compared to ERG11; in these strains ibuprofen induced the reversion of resistance and an increase in FUN-1 fluorescence. The 2 strains that did not revert their phenotype, ibuprofen did not increase the fluorescence of cells stained with FUN-1 and gene over-expression was not only for CDR1 and CDR2 but mostly for ERG11 gene. It should be stressed that C. albicans control strain 12-99 with known multiple resistance mechanisms did not revert the resistance phenotype in the presence of ibuprofen. MDR1 expression was not detected in none of the C. albicans clinical strains. Strains with multiple resistance mechanisms did not revert, wich might indicate that ibuprofen could be a potential antifungal efflux blocker. Real Time PCR is easily available and a sensitive method for gene expression studies, and also SYBR Green is acceptable, although being an unspecific marker, when a melting curve is added in each assay. |
publishDate |
2007 |
dc.date.none.fl_str_mv |
2007 2011-02-07T00:00:00Z 2011-02-07 |
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/10216/22064 |
url |
http://hdl.handle.net/10216/22064 |
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por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/msword |
dc.publisher.none.fl_str_mv |
Faculdade de Medicina da Universidade do Porto FMUP |
publisher.none.fl_str_mv |
Faculdade de Medicina da Universidade do Porto FMUP |
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 |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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