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Authors
Advisor(s)
Abstract(s)
Os organóides de intestino são estruturas celulares semelhantes ao epitélio intestinal in vivo, podendo ser utilizados para estudar protozoários como o Toxoplasma gondii. O T. gondii é um parasita intracelular obrigatório capaz de infetar várias espécies, incluindo humanos. O ciclo de vida do parasita apresenta duas fases – assexuada e sexuada. Esta última só ocorre no intestino dos felinos (hospedeiro definitivo). Recentemente um estudo utilizando organóides de intestino provou que são as altas concentrações de ácido linoleico no intestino dos felinos que despoletam o desenvolvimento sexuado deste parasita e que inibindo a enzima que o degrada é possível despoletar este desenvolvimento em células intestino de ratinho (Mus musculus). O nosso trabalho teve como principal objetivo estabelecer culturas de organóides de intestino de gato (Felis silvestres catus) e de ratinho para estudar a expressão de genes específicos da fase sexuada. Estabelecemos organóides de intestino de ratinho e monocamadas derivadas destes, confirmando a existência de enterócitos nestas estruturas. Inoculámos taquizoítos de T. gondii em monocamadas derivadas de organóides e em células HFF suplementadas com ácido linoleico e inibidor SC-26196, para testar a existência de desenvolvimento sexuado nas células de organóides de intestino de ratinho e em células distintas destas, na presença de elevadas concentrações de ácido linoleico e do inibidor da enzima que o degrada. Analisámos a existência de toxicidade causada nas células de organóides de intestino e em células HFF e nos taquizoítos por microscopia ótica de campo claro e por imunofluorescência indireta. Não observámos indícios de toxicidade. Analisámos a existência de desenvolvimento sexuado dos taquizoítos usando como marcadores deste desenvolvimento o aumento da expressão dos genes TgMob1 e TgLats. Não observámos aumento da expressão destes genes indicando que não houve desenvolvimento sexuado dos taquizoítos.
ABSTRACT - Intestinal organoids are cellular structures similar to the intestinal epithelium in vivo and can be used to study protozoa such as Toxoplasma gondii. T. gondii is an obligate intracellular parasite capable of infecting several species, including humans. The life cycle of the parasite has two phases – asexual and sexual. The latter only occurs in the intestine of felines (definitive host). Recently, a study using intestinal organoids proved that it is the high concentrations of linoleic acid in the intestine of felines that trigger the sexual development of this parasite and that by inhibiting the enzyme that degrades it, it is possible to trigger this development in mice intestine cells (Mus musculus). The main objective of our work was to establish cultures of cats (Felis silvestres catus) and mouse intestinal organoids to study the expression of specific genes of the sexual phase. We established mouse intestinal organoids and monolayers derived therefrom, confirming the existence of enterocytes in these cell structures. We inoculated T. gondii tachyzoites into organoid-derived monolayers and into HFF cells supplemented with linoleic acid and SC-26196 inhibitor, to test for sexual development in mouse intestinal organoid cells and cells other than these, in the presence of high concentrations of linoleic acid and the inhibitor of the enzyme that degrades it. We analyzed the existence of toxicity caused in these intestinal organoid cells and HFF cells and in tachyzoites by bright field optical microscopy and by indirect immunofluorescence. We observed no signs of toxicity. We analyzed the existence of sexual development of tachyzoites using the increase in the expression of TgMob1 and TgLats genes as markers of this development. We did not observe an increase in these genes’ expression, indicating no sexual development of the tachyzoites.
ABSTRACT - Intestinal organoids are cellular structures similar to the intestinal epithelium in vivo and can be used to study protozoa such as Toxoplasma gondii. T. gondii is an obligate intracellular parasite capable of infecting several species, including humans. The life cycle of the parasite has two phases – asexual and sexual. The latter only occurs in the intestine of felines (definitive host). Recently, a study using intestinal organoids proved that it is the high concentrations of linoleic acid in the intestine of felines that trigger the sexual development of this parasite and that by inhibiting the enzyme that degrades it, it is possible to trigger this development in mice intestine cells (Mus musculus). The main objective of our work was to establish cultures of cats (Felis silvestres catus) and mouse intestinal organoids to study the expression of specific genes of the sexual phase. We established mouse intestinal organoids and monolayers derived therefrom, confirming the existence of enterocytes in these cell structures. We inoculated T. gondii tachyzoites into organoid-derived monolayers and into HFF cells supplemented with linoleic acid and SC-26196 inhibitor, to test for sexual development in mouse intestinal organoid cells and cells other than these, in the presence of high concentrations of linoleic acid and the inhibitor of the enzyme that degrades it. We analyzed the existence of toxicity caused in these intestinal organoid cells and HFF cells and in tachyzoites by bright field optical microscopy and by indirect immunofluorescence. We observed no signs of toxicity. We analyzed the existence of sexual development of tachyzoites using the increase in the expression of TgMob1 and TgLats genes as markers of this development. We did not observe an increase in these genes’ expression, indicating no sexual development of the tachyzoites.
Description
Mestrado em Tecnologias Moleculares em Saúde
Keywords
Toxoplasma gondii Organoides de intestino Desenvolvimento sexuado Taquizoítos Intestinal organoids Sexual development Tachyzoites
Citation
Esteves MF. Estabelecer culturas de organoides de intestino como modelo para estudar a expressão de genes da fase sexuada de Toxoplasma gondii [dissertation]. Lisboa: Escola Superior de Tecnologia da Saúde de Lisboa/Instituto Politécnico de Lisboa; 2022.
Publisher
Instituto Politécnico de Lisboa, Escola Superior de Tecnologia da Saúde de Lisboa