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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2020.tde-12022020-170537
Document
Author
Full name
André Luiz Brandão Bitencourt
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Sebollela, Adriano Silva (President)
Borges, Júlio César
Cordeiro, Yraima Moura Lopes
Oliveira, Eduardo Brandt de
Title in Portuguese
Isolamento de oligômeros tóxicos do peptídeo B-amiloide usando o anticorpo de cadeia única NUsc1
Keywords in Portuguese
Doença de Alzheimer
Oligômeros de b-amiloide
scFv
Abstract in Portuguese
A doença de Alzheimer é a principal cauda de demência em todo o mundo, e é caracterizada pelo acúmulo cerebral de diferentes agregados proteicos, incluindo aqueles formados pelo peptídeo A?. Embora a patogênese da doença de Alzheimer não seja completamente compreendida, evidências crescentes sugerem que os oligômeros solúveis do peptídeo A? desempenham um papel chave no início e progresso da doença. Neste trabalho desenvolvemos e otimizamos um método de isolamento de oligômeros de ?-amiloide tóxicos, no qual utilizamos o fragmento de anticorpo de cadeia única NUsc1 como ferramenta de captura. NUsc1 foi descrito pelo nosso grupo como um anticorpo seletivo para oligômeros do peptídeo ?-amiloide, sendo capaz de inibir sua citotoxicidade in vitro e in vivo. Fomos capazes de confirmar a especificidade de NUsc1 por uma subpopulação de oligômeros de ?-amiloide tóxicos, e de mostrar que estes desempenham um papel chave no processo de agregação para formação de fibras amiloides. Também, fomos bem-sucedidos no desenvolvimento de um método de isolamento de oligômeros de ?-amiloide alvos de NUsc1 utilizando beads magnéticas. Este método permitiu o isolamento de oligômeros de ?-amiloide de diferentes fontes, incluindo extrato de cérebro de camundongo transgênico. Além disso, avançamos na caracterização destas espécies tóxicas, sendo capazes de estabilizar o complexo formado entre A?Os-NUsc1 através de crosslinking. Nossos achados confirmaram a interação preferencial de NUsc1 por oligômeros de ?-amiloide de alto peso molecular, auxiliando assim a revelar a identidade destes agregados tóxicos.
Title in English
Isolation of toxic oligomers formed by the B-amyloid peptide using the single-chain antibody NUsc1
Keywords in English
Alzheimer's disease
b-amyloid oligomers
scFv
Abstract in English
Alzheimer disease (AD), the leading cause of dementia worldwide, is characterized by the brain accumulation of different protein aggregates, including those formed by the ?-amyloid peptide (A?). Although the pathogenesis of AD is not completely understood, mounting evidence implicates soluble A? oligomers (A?Os) as key players in disease onset and progression. In this work, we developed and optimized a method for the isolation of toxic amyloid ?-oligomers, in which the single chain antibody fragment NUsc1 was used as a capturing tool. NUsc1 has been described by our group as a selective antibody against amyloid ?-oligomers, being able to inhibit A?O cytotoxicity both in vitro and in vivo. Her, we were able to confirm the preference of NUsc1 for a subpopulation of toxic amyloid ?-oligomers of >= 100kDa, and show that these species play a key role in the aggregation leading to fiber formation. Also, we have successfully developed a method to isolate amyloid ?-oligomers targeted by NUsc1 using magnetic beads. This method allowed us to isolate of amyloid ?-oligomers from different sources, including transgenic mouse brain extracts, thus helping in the characterization of these toxic amyloid ?-oligomers. In addition, we have advanced in the characterization of these toxic species, as we were able to stabilize the complex formed between A?Os-NUsc1 through crosslinking. Our findings confirmed the preference of NUsc1 by high molecular weight amyloid ?-oligomers, contributing to the field to determine the identity of these toxic aggregates.
 
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Publishing Date
2020-04-28
 
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