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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2016.tde-17082016-151526
Document
Author
Full name
Lucas Borges Pereira
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Garcia, Celia Regina da Silva (President)
Cardoso, Alexandre Bruni
Schechtman, Deborah
Soares, Irene da Silva
Wunderlich, Gerhard
Title in Portuguese
Caracterização da apirase do parasita P. falciparum e análise do papel do Ca2+ no egresso de T. gondii.
Keywords in Portuguese
Plasmodium falciparum
Toxoplasma gondii
Apirase
Cálcio
Egresso
GCaMP3
Abstract in Portuguese
Plasmodium falciparum e Toxoplasma gondii são protozoários parasitas pertencentes ao filo Apicomplexa. Apirases são enzimas metabolizadoras de nucleotídeos extracelulares. Nesta tese mostramos pela primeira vez a presença de um membro desta família de enzimas em P. falciparum, o qual foi capaz de degradar ATP extracelular. Análises por RT-qPCR revelaram a expressão da apirase durante todo o ciclo intraeritrocítico. A adição de inibidores desta classe de enzimas foi capaz de prejudicar o desenvolvimento dos parasitas e a invasão de novas hemácias pelos merozoitos, sugerindo assim um papel da apirase nestes processos. A via de sinalização por Ca2+ é universal e vital para todas as células. Para melhor entender a fisiologia celular de P. falciparum construímos uma nova linhagem de parasitas transgênicos, PfGCaMP3, que nos tornam capazes de monitorar a dinâmica de Ca2+ sem o uso de protocolos invasivos de marcação. De modo semelhante utilizamos uma nova linhagem de T. gondii expressando de forma estável o indicador de Ca2+ GCaMP3 para estudar o papel deste íon na saída da célula. T. gondii possui o Ca2+ necessário para promover este processo, entretanto Ca2+ extracelular age como um fator intensificador neste passo essencial do ciclo lítico.
Title in English
Characterization of P. falciparum apyrase and analysis of the role of Ca2+ in T. gondii egress.
Keywords in English
Plasmodium falciparum
Toxoplasma gondii
Apyrase
Calcium
Egress
GCaMP3
Abstract in English
Plasmodium falciparum and Toxoplasma gondii are protozoan parasites that belong to phylum Apicomplexa. Apirases are metabolizing enzymes of extracellular nucleotides. In this work we show for the first time the presence of an apyrase in P. falciparum, which was able to degrade extracellular ATP. RTqPCR analysis revealed the expression of apyrase throughout the intraerythrocytic cycle. Addition of apyrase inhibitors was able to impair the development of the parasites and the invasion of new erythrocytes by merozoites, thus suggesting a role of apyrase in these processes. Calcium signaling is universal and vital to all cells. To better understand the cellular physiology of P. falciparum we construct a new strain of transgenic parasites, PfGCaMP3, which enable us to monitor the Ca2+ dynamics without using invasive protocols. Similarly we use a new strain of T. gondii that stably express the Ca2+ indicator GCaMP3 to study the role Ca2+ in parasite egress. T. gondii has the Ca2+ required to promote this process, however extracellular Ca2+ acts as an enhancer factor in this crucial step of the lytic cycle.
 
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Publishing Date
2016-08-17
 
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