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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2019.tde-09122021-110919
Document
Author
Full name
Cristine Marie Yde Ohki
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Braga, Patricia Cristina Baleeiro Beltrao (President)
Giordano, Ricardo José
Granato, Celso Francisco Hernandes
Zanotto, Paolo Marinho de Andrade
Title in Portuguese
Avaliação da infecção do Zika vírus em astrócitos.
Keywords in Portuguese
Astrócitos
Células-tronco pluripotentes induzidas
Microcefalia
Sistema nervoso central
Zika vírus
Abstract in Portuguese
O Zika vírus (ZIKV) é um flavivírus capaz de causar infecção em humanos após a contaminação através da picada por mosquitos fêmeas do gênero Aedes, por transmissão sexual ou vertical (da mãe para o feto). Em 2015, a linhagem brasileira deste vírus - ZIKVBR - produziu uma epidemia no Brasil, gerando diversos casos de infecção congênita por transmissão vertical, caracterizada por malformações cerebrais. Estudos in vitro em células progenitoras neurais, neurônios e organoides derivados de células-tronco pluripotentes induzidas (iPSC) revelaram o neurotropismo do ZIKVBR e ajudaram a clarificar sua relação com o Sistema Nervoso Central (SNC), mostrando que essas células são preferencialmente afetadas pela infecção. Neste projeto analisamos os efeitos da infecção do ZIKVBR em astrócitos, células da glia responsáveis pela homeostase e defesa do SNC. Para tanto, produzimos astrócitos a partir de duas linhagens de iPSC humanas para identificar aspectos moleculares e celulares decorrentes da infecção com o vírus. Os resultados mostraram que os astrócitos são susceptíveis e permissíveis à infecção por ZIKVBR e respondem à presença do vírus sofrendo apoptose e produzindo citocinas. Ademais, os astrócitos apresentaram aumento da expressão gênica de receptores TAM, receptores envolvidos com a regulação de resposta imune e também candidatos à entrada viral, e de transportadores de glutamato. Adicionalmente, a captação de glutamato foi prejudicada, bem como a sinaptogênese, quando cultivamos neurônios com astrócitos infectados ou com o sobrenadante de cultura de astrócitos livre de vírus, indicando que os produtos metabólicos gerados por astrócitos infectados afetam o fenótipo neuronal. Por fim, mostramos que mesmo infectadas, NPC se diferenciam em astrócitos. Os resultados gerados neste trabalho auxiliam a compreender os efeitos biológicos causados pelo ZIKVBR em astrócitos derivados de iPSC humanas, o que pode contribuir para o melhor entendimento da patogênese do ZIKV no SNC.
Title in English
Evaluation of Zika virus infection in astrocytes.
Keywords in English
Astrocytes
Central nervous system
Induced pluripotent stem cells
Microcephaly
Zika virus
Abstract in English
Zika Virus (ZIKV) is a flavivirus able to promote human infection through bites of Aedes female mosquitoes, sexual or vertical transmission (from expectant mothers to fetuses). In 2015, the Brazilian strain of this virus - ZIKVBR - caused a significant outbreak in Brazil, which led to numerous cases of congenital infection after vertical transmission. In vitro studies with neural progenitor cells (NPC), neurons and brain organoids derived from human induced pluripotent stem cells (iPSC), have reported ZIKVBR neurotropism and helped clarify its correlation with the Central Nervous System (CNS) by showing that these cells are significantly affected by infection. In this project, we analyzed the effects of ZIKVBR infection in astrocytes, glial cells responsible for CNS homeostasis and defense. In order to do so, we have generated astrocytes from two human iPSC lineages to verify molecular and cellular changes regarding virus infection. Our results have shown that astrocytes are susceptible and permissive to ZIKVBR and respond to viral presence by undergoing apoptosis and releasing cytokines. In addition, after infection, astrocytes had increased expression of TAM receptors, which are involved with suppression of immune response and also candidates for viral entry, and glutamate transporters. Additionally, we have also verified impaired glutamate uptake by these astrocytes, as well as synaptogenesis when mature neurons were cultivated in the presence of infected astrocytes or with their virus-free supernatant, indicating that metabolic products of infected astrocytes affected neuronal phenotype. At last, we have demonstrated that generation of astrocytes from infected NPC is possible. This work pursued to understand the biological effects caused by ZIKVBR in human iPSC-derived astrocytes, which could provide a better comprehension about ZIKV pathogenesis in CNS.
 
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Publishing Date
2022-01-13
 
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