Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide
Autor(a) principal: | |
---|---|
Data de Publicação: | 2023 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/17506 |
Resumo: | Most neurodegenerative diseases are associated with abnormal protein aggregation. Amyloid proteins are highly prone to aggregation and, consequently, accumulate in the brain, disrupting synapses and causing neurotoxicity. The work of this thesis aims to optimize the methodology and expression of recombinant Aβ as well as to investigate the theranostic potential of Ru(II) polypyridine complexes as luminescent probes and as inhibitors of the Aβ aggregation process. Aβ expression and purification was optimized based on the protocol developed by Abelein and collaborators. With this methodology, the Aβ40 and Aβ42 isoforms were obtained with a high degree of purity, high yield and with kinetic properties equivalent to the commercially available Aβ. The electronic absorption spectrum of the complexes in PBS buffer is characterized by an intense absorption band in the ultraviolet region typical of intraligand charge transfer transitions (ILCT) from the unsaturated aromatic ligands and by a broad absorption band in the visible region attributed to metal to ligand charge transfer transitions. The complexes have a maximum emission at 655 nm with a Stokes shift (separation between emission and excitation maxima) in the order of 5800 cm-1. The complexes containing the 3,4-diaminopyridine ligand also showed high antioxidant activity against the ABTS.+ radical. The effect of the complexes on Aβ aggregation was evaluated using nephelometry, which monitors the turbidity of the solution during protein aggregation. The Ru3,4Apy, RuMe3,4Apy and RuMe4Apy complexes exhibited lower relative nephelometry units (URNs) values when compared to the control, indicating the formation of fewer aggregates. In particular, the RuMe3,4Apy and RuMe4Apy complexes showed the greatest difference in relation to the control in the proportions 1:2.5 and 1:5 Aβ:complex. The analysis of the morphology of the species formed by transmission electron microscopy showed that Ru3,4Apy and Ru4Apy did not influence the morphology of the fibrils formed while the RuMe3,4Apy and RuMe4Apy complexes induced the formation of amorphous aggregates, by which the absence of secondary structure was confirmed by circular dichroism. The transgenic nematodes C. elegans (Caenorhabditis elegans), in which human Aβ42 is expressed in their muscle tissue, were used to evaluate the effect of the complexes in an in vivo model of Alzheimer's disease. For these studies, RuMe3,4Apy and Ru4Apy were chosen given their in vitro results. The two complexes improved the motility of the animals, indicating an anti-amyloid effect, as observed in vitro. Visualization of amyloid aggregates was performed by staining C. elegans nematodes with the amyloid dye X34, which is specific for amyloid fibrils. The nematodes that were treated with the complexes do not show the amyloid deposits as seen in the control, indicating that they showed an anti-amyloid effect not only in vitro, but also in vivo. |
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Cali, Mariana PigozziCarlos, Rose Mariahttp://lattes.cnpq.br/1589143355309943http://lattes.cnpq.br/737945150583950709c0cfaa-6e04-4419-b629-f4a1c1f7bfee2023-03-16T13:56:18Z2023-03-16T13:56:18Z2023-02-27CALI, Mariana Pigozzi. Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide. 2023. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/17506.https://repositorio.ufscar.br/handle/ufscar/17506Most neurodegenerative diseases are associated with abnormal protein aggregation. Amyloid proteins are highly prone to aggregation and, consequently, accumulate in the brain, disrupting synapses and causing neurotoxicity. The work of this thesis aims to optimize the methodology and expression of recombinant Aβ as well as to investigate the theranostic potential of Ru(II) polypyridine complexes as luminescent probes and as inhibitors of the Aβ aggregation process. Aβ expression and purification was optimized based on the protocol developed by Abelein and collaborators. With this methodology, the Aβ40 and Aβ42 isoforms were obtained with a high degree of purity, high yield and with kinetic properties equivalent to the commercially available Aβ. The electronic absorption spectrum of the complexes in PBS buffer is characterized by an intense absorption band in the ultraviolet region typical of intraligand charge transfer transitions (ILCT) from the unsaturated aromatic ligands and by a broad absorption band in the visible region attributed to metal to ligand charge transfer transitions. The complexes have a maximum emission at 655 nm with a Stokes shift (separation between emission and excitation maxima) in the order of 5800 cm-1. The complexes containing the 3,4-diaminopyridine ligand also showed high antioxidant activity against the ABTS.+ radical. The effect of the complexes on Aβ aggregation was evaluated using nephelometry, which monitors the turbidity of the solution during protein aggregation. The Ru3,4Apy, RuMe3,4Apy and RuMe4Apy complexes exhibited lower relative nephelometry units (URNs) values when compared to the control, indicating the formation of fewer aggregates. In particular, the RuMe3,4Apy and RuMe4Apy complexes showed the greatest difference in relation to the control in the proportions 1:2.5 and 1:5 Aβ:complex. The analysis of the morphology of the species formed by transmission electron microscopy showed that Ru3,4Apy and Ru4Apy did not influence the morphology of the fibrils formed while the RuMe3,4Apy and RuMe4Apy complexes induced the formation of amorphous aggregates, by which the absence of secondary structure was confirmed by circular dichroism. The transgenic nematodes C. elegans (Caenorhabditis elegans), in which human Aβ42 is expressed in their muscle tissue, were used to evaluate the effect of the complexes in an in vivo model of Alzheimer's disease. For these studies, RuMe3,4Apy and Ru4Apy were chosen given their in vitro results. The two complexes improved the motility of the animals, indicating an anti-amyloid effect, as observed in vitro. Visualization of amyloid aggregates was performed by staining C. elegans nematodes with the amyloid dye X34, which is specific for amyloid fibrils. The nematodes that were treated with the complexes do not show the amyloid deposits as seen in the control, indicating that they showed an anti-amyloid effect not only in vitro, but also in vivo.A maioria das doenças neurodegenerativas estão associadas à agregação anormal de proteínas. Proteínas amiloides possuem alta propensão à agregação e, consequentemente, se acumulam no cérebro, interrompendo sinapses e causando neurotoxicidade. A presente tese tem como objetivos otimizar a metodologia e expressão do peptídeo beta amiloide recombinante bem como investigar o potencial teranóstico de complexos polipiridínicos de Ru(II) como sondas luminescentes e agentes inibidores do processo de agregação do Aβ. A expressão e purificação do Aβ foi otimizada a partir do protocolo desenvolvido por Abelein e colaboradores. Com essa metodologia as isoformas Aβ40 e Aβ42 foram obtidas com alto grau de pureza, alto rendimento e com propriedades cinéticas equivalentes ao Aβ vendido comercialmente. O espectro de absorção eletrônica dos complexos em solução tampão PBS é caracterizado por uma banda de absorção intensa na região de ultravioleta típica das transições internas de transferência de carga de ligantes aromáticos insaturados (ILCT) e por uma banda de absorção larga na região do visível atribuída às transições de transferência de carga do metal para ligante. Os complexos apresentaram máximo de emissão em 655 nm apresentando deslocamento de Stokes (separação entre o máximo de emissão e de excitação) na ordem de 5800 cm-1. Os complexos contendo o ligante 3,4-diaminopiridina também apresentaram alta atividade antioxidante frente ao radical ABTS.+. O efeito dos complexos na agregação do Aβ foi avaliado através da técnica de nefelometria, a qual monitora a turbidez da solução durante a agregação da proteína. Os complexos Ru3,4Apy, RuMe3,4Apy e RuMe4Apy exibiram valores de unidades relativas de nefelometria (URNs) menores quando comparadas ao controle, indicando a formação de um menor número de agregados. Em especial, os complexos RuMe3,4Apy e RuMe4Apy apresentaram a maior diferença em relação ao controle nas proporções 1:2.5 e 1:5 Aβ:complexo. A análise da morfologia das espécies formadas por microscopia de transmissão eletrônica mostrou que os complexos Ru3,4Apy e Ru4Apy não influenciam na morfologia das fibrilas formadas enquanto os complexos RuMe3,4Apy e RuMe4Apy induzem a formação de agregados amorfos, cuja ausência de estrutura secundária foi confirmada por dicroísmo circular. Os nematoides transgênicos C. elegans (Caenorhabditis elegans), nos quais Aβ42 humano é expresso no tecido muscular dos animais, foram utilizados para avaliar o efeito dos complexos em um modelo in vivo da doença de Alzheimer. Para estes estudos foram escolhidos os complexos RuMe3,4Apy e Ru4Apy, os quais apresentaram melhor resultado in vitro. Os complexos RuMe3,4Apy e Ru4Apy melhoraram a motilidade dos animais, indicando um efeito anti-amiloide, assim como observado in vitro. A visualização dos agregados amiloides foi realizada corando os nematoides C. elegans com o corante X34, o qual é específico para fibrilas amiloides. Os nematoides que foram tratados com os complexos não apresentam os depósitos amiloides como foi visualizado para o controle, indicando que os mesmos apresentaram efeito anti-amiloide não só in vitro, como também in vivo.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Processo 141137/2019-3, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Processo 88887.569985/2020-00, Coordenação de Aperfeiçoamento de Pessoa de Nível Superior - Programa Nacional de Internacionalização (CAPES-PrInt)porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessDoença de AlzheimerComplexos de RutênioPeptídeo Beta AmilóideAlzheimer's diseaseRuthenium ComplexesAmyloid Beta PeptideCIENCIAS BIOLOGICAS::BIOFISICA::BIOFISICA MOLECULARCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARCIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA::QUIMICA BIO-INORGANICACIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICAInfluência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloideInfluence of Ru(II) polypyridinic complexes on amyloid beta aggregationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis60060081ea1b7e-7dcf-438f-a839-b8cab12c5740reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARCC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8810https://repositorio.ufscar.br/bitstream/ufscar/17506/3/license_rdff337d95da1fce0a22c77480e5e9a7aecMD53ORIGINALTese_MarianaCali_final.pdfTese_MarianaCali_final.pdfTese de Doutoradoapplication/pdf7779800https://repositorio.ufscar.br/bitstream/ufscar/17506/1/Tese_MarianaCali_final.pdfde296e15a4266146715c55c2728c097eMD51TEXTTese_MarianaCali_final.pdf.txtTese_MarianaCali_final.pdf.txtExtracted texttext/plain248953https://repositorio.ufscar.br/bitstream/ufscar/17506/4/Tese_MarianaCali_final.pdf.txt29bed730b3721e9766631b6b2fb0e271MD54THUMBNAILTese_MarianaCali_final.pdf.jpgTese_MarianaCali_final.pdf.jpgIM Thumbnailimage/jpeg9405https://repositorio.ufscar.br/bitstream/ufscar/17506/5/Tese_MarianaCali_final.pdf.jpg1da6887fb146205a35865d188e5ecbd1MD55ufscar/175062023-09-18 18:32:33.149oai:repositorio.ufscar.br:ufscar/17506Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:32:33Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
dc.title.alternative.eng.fl_str_mv |
Influence of Ru(II) polypyridinic complexes on amyloid beta aggregation |
title |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
spellingShingle |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide Cali, Mariana Pigozzi Doença de Alzheimer Complexos de Rutênio Peptídeo Beta Amilóide Alzheimer's disease Ruthenium Complexes Amyloid Beta Peptide CIENCIAS BIOLOGICAS::BIOFISICA::BIOFISICA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA::QUIMICA BIO-INORGANICA CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA |
title_short |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
title_full |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
title_fullStr |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
title_full_unstemmed |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
title_sort |
Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide |
author |
Cali, Mariana Pigozzi |
author_facet |
Cali, Mariana Pigozzi |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/7379451505839507 |
dc.contributor.author.fl_str_mv |
Cali, Mariana Pigozzi |
dc.contributor.advisor1.fl_str_mv |
Carlos, Rose Maria |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/1589143355309943 |
dc.contributor.authorID.fl_str_mv |
09c0cfaa-6e04-4419-b629-f4a1c1f7bfee |
contributor_str_mv |
Carlos, Rose Maria |
dc.subject.por.fl_str_mv |
Doença de Alzheimer Complexos de Rutênio Peptídeo Beta Amilóide |
topic |
Doença de Alzheimer Complexos de Rutênio Peptídeo Beta Amilóide Alzheimer's disease Ruthenium Complexes Amyloid Beta Peptide CIENCIAS BIOLOGICAS::BIOFISICA::BIOFISICA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA::QUIMICA BIO-INORGANICA CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA |
dc.subject.eng.fl_str_mv |
Alzheimer's disease Ruthenium Complexes Amyloid Beta Peptide |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOFISICA::BIOFISICA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA::QUIMICA BIO-INORGANICA CIENCIAS EXATAS E DA TERRA::QUIMICA::QUIMICA INORGANICA |
description |
Most neurodegenerative diseases are associated with abnormal protein aggregation. Amyloid proteins are highly prone to aggregation and, consequently, accumulate in the brain, disrupting synapses and causing neurotoxicity. The work of this thesis aims to optimize the methodology and expression of recombinant Aβ as well as to investigate the theranostic potential of Ru(II) polypyridine complexes as luminescent probes and as inhibitors of the Aβ aggregation process. Aβ expression and purification was optimized based on the protocol developed by Abelein and collaborators. With this methodology, the Aβ40 and Aβ42 isoforms were obtained with a high degree of purity, high yield and with kinetic properties equivalent to the commercially available Aβ. The electronic absorption spectrum of the complexes in PBS buffer is characterized by an intense absorption band in the ultraviolet region typical of intraligand charge transfer transitions (ILCT) from the unsaturated aromatic ligands and by a broad absorption band in the visible region attributed to metal to ligand charge transfer transitions. The complexes have a maximum emission at 655 nm with a Stokes shift (separation between emission and excitation maxima) in the order of 5800 cm-1. The complexes containing the 3,4-diaminopyridine ligand also showed high antioxidant activity against the ABTS.+ radical. The effect of the complexes on Aβ aggregation was evaluated using nephelometry, which monitors the turbidity of the solution during protein aggregation. The Ru3,4Apy, RuMe3,4Apy and RuMe4Apy complexes exhibited lower relative nephelometry units (URNs) values when compared to the control, indicating the formation of fewer aggregates. In particular, the RuMe3,4Apy and RuMe4Apy complexes showed the greatest difference in relation to the control in the proportions 1:2.5 and 1:5 Aβ:complex. The analysis of the morphology of the species formed by transmission electron microscopy showed that Ru3,4Apy and Ru4Apy did not influence the morphology of the fibrils formed while the RuMe3,4Apy and RuMe4Apy complexes induced the formation of amorphous aggregates, by which the absence of secondary structure was confirmed by circular dichroism. The transgenic nematodes C. elegans (Caenorhabditis elegans), in which human Aβ42 is expressed in their muscle tissue, were used to evaluate the effect of the complexes in an in vivo model of Alzheimer's disease. For these studies, RuMe3,4Apy and Ru4Apy were chosen given their in vitro results. The two complexes improved the motility of the animals, indicating an anti-amyloid effect, as observed in vitro. Visualization of amyloid aggregates was performed by staining C. elegans nematodes with the amyloid dye X34, which is specific for amyloid fibrils. The nematodes that were treated with the complexes do not show the amyloid deposits as seen in the control, indicating that they showed an anti-amyloid effect not only in vitro, but also in vivo. |
publishDate |
2023 |
dc.date.accessioned.fl_str_mv |
2023-03-16T13:56:18Z |
dc.date.available.fl_str_mv |
2023-03-16T13:56:18Z |
dc.date.issued.fl_str_mv |
2023-02-27 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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dc.identifier.citation.fl_str_mv |
CALI, Mariana Pigozzi. Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide. 2023. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/17506. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/17506 |
identifier_str_mv |
CALI, Mariana Pigozzi. Influência de complexos polipiridínicos de Ru(II) no processo de agregação do peptídeo beta amiloide. 2023. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2023. Disponível em: https://repositorio.ufscar.br/handle/ufscar/17506. |
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https://repositorio.ufscar.br/handle/ufscar/17506 |
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Universidade Federal de São Carlos Câmpus São Carlos |
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