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Doctoral Thesis
DOI
10.11606/T.42.2017.tde-18052017-132950
Document
Author
Full name
Flávia Menezes Zimbres
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Wrenger, Carsten (President)
Barboza, Renato
Bargieri, Daniel Youssef
Boscardin, Silvia Beatriz
Cardoso, Josiane
Title in Portuguese
Identificação e interferência de proteínas integradas na superfície do eritrócito infectado por Plasmoidum falciparum.
Keywords in Portuguese
Malária
Piridoxal Quinase
Proteínas de superfície eritrocítica
Tecnologia SELEX
Terapia
Abstract in Portuguese
A malaria humana é uma doença infecciosa transmitida por um mosquito que está atrelada a uma alta taxa de mortalidade. Dentre os parasitas que causam a malaria humana a espécie Plasmodium falciparum é responsável por 90% dos casos letais. Sua virulência é ocasionada por modificações na célula hospedeira provenientes do tráfego de proteínas para a superfície de eritrócitos infectados. Com o intuito de interferir com estas proteínas, aplicamos a técnica Cell- SELEX (do inglês: Systematic Evolution of Ligands by Exponential Enrichment). Após ciclos iterativos de seleção obtivemos moléculas de DNA simples- fita, conhecidas como aptâmeros, que possuem alta afinidade e especificidade contra proteínas alvo presentes na superfície de eritrócitos infectados por P. falciparum. Ensaios de pull- down usando aptâmeros selecionados em sistema de purificação por streptavidina-biotina seguidos por análises de espectrometria de massa revelaram a interação destas moléculas de DNA com proteínas como RIFIN, PfEMP, RAP1, entre outras incorporadas na superfície de eritrócitos infectados. Como consequência destas interações, os aptâmeros selecionados demostraram ser inibidores potenciais na proliferação dos parasitas, como demonstrado por testes in vitro. Outra estratégia usada foi a produção de aptâmeros contra a piridoxal quinase de P. falciparum (PfPdxK) expressa. Usando tanto SELEX-proteína, bem como, ensaios de eletroforese capilar, selecionamos aptâmeros com capacidade para inibir a atividade enzimática da PfPdxK. Nossos dados constituem evidências sobre as aplicações da tecnologia SELEX no desenho racional de compostos contra a malária humana.
Title in English
Identification and interference of integrated proteins in the infected-erythrocytes surface by Plasmodium falciparum.
Keywords in English
Erythrocyte surface proteíns
Malaria
Pyridoxal Kinase
SELEX technology
Therapy
Abstract in English
The human malaria is a mosquito-borne infectious disease associated with a high risk of mortality. Among the parasite species that causes human malaria, Plasmodium falciparum is responsible by 90% of the lethal cases. The virulence of this pathogen is associated with host cell modifications by trafficking proteins to the surface of infected erythrocytes. Aiming to interfere with these proteins we have applied the Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. After iterative cycles of selection we obtained single stranded DNA molecules, known as aptamers, with high binding affinity and specificity against protein targets present in the surface of erythrocytes infected with P. falciparum. Pull-down assays using the selected aptamers in a streptavidin-biotin affinity assay followed by mass spectrometry analysis revealed the interaction of these DNA molecules with proteins such as RIFIN, PfEMP, RAP1, among others embedded in the surface of infected erythrocytes. As consequence of these interactions, the selected aptamers demonstrated to be potent inhibitors of parasite proliferation, as validated by in vitro tests. Another strategy used was the production of aptamers against the recombinantly expressed plasmodial pyridoxal kinase (PfPdxK). Using both protein-SELEX as well as capillary electrophoresis assays, we selected aptamers with capacity to inhibit the enzymatic activity of PfPdxK. Our data constitute evidences about the application of SELEX technology in the rationale design of inhibitors against human malaria.
 
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Release Date
2019-05-18
Publishing Date
2017-05-18
 
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