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Title: Understanding the role of unusual dynamin-related proteins in the lizard pathogen Entamoeba invadens
Author: Turfah, M.
ISNI:       0000 0004 8510 748X
Awarding Body: University of Exeter
Current Institution: University of Exeter
Date of Award: 2020
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Amoebiasis is the third most common cause of death due to parasitic diseases in the world after malaria and schistosomiasis. In developing countries, it infects more than 50 million individuals annually causing 50,000 -100,000 deaths. Entamoeba. histolytica, an intestinal protozoan parasite, is the causative agent of amoebiasis in humans. The cyst is responsible for the transmission of the disease, in which the host gets infected through the ingestion of cyst-contaminated foods and water. About 90% of infections are asymptomatic cyst carriers and spreaders, in which each individual shed up to 45 million cysts per day. The process of conversion from motile trophozoite to dormant cyst is called encystation. However, little known about the molecular and cellular events that trigger this encystation. Numerous studies have attempted to investigate the molecules and genes involved in encystation. Dynamin, which is a conserved family of large GTPase, were suggested to play a role during encystation in Entamoeba. The unusual dynamin-related proteins Drp3 and Drp4 were revealed to be upregulated during stage transition. Despite the fact that all eukaryotes contain at least one dynamin protein, no homologues of Drp3 and Drp4 were identified in mammals. In this study, I attempted to study potential role of Drp3 and Drp4 during encystation in the laboratory model Entamoeba invadens. Furthermore, study the effect of the overexpression of these dynamins in mammalian cells. Using molecular biology techniques, immunofluorescence microscopy, transmission electron microscopy, and immunoelectron microscopy. In this study, we conducted stage conversion experiments to understand the importance of dynamins during cyst formation. Localization experiments showed that Drp3 and Drp4 have dual localization to both nucleus and cytoplasm. Drp3 existed as punctate structures localized to the cytoplasm and the nucleus in trophozoites and elongated structures localized to the nucleus in the cyst. The localization of the mutated form of Drp3 lacking the key residues in the GTPase domain (Drp3-mutant) was mainly nuclear and to a lesser extent cytoplasmic. The nuclear localization revealed the presence of Drp3 on the nucleus, the nuclear vesicles, and the nuclear envelope. Interestingly, cells overexpressing Drp3-mutant exhibited cytokinesis failure and multinucleation, suggesting a possible role in cytokinesis. In the mammalian cells, both Drp3 and Drp4 displayed the negative dominant effect on COS-7 cells. Drp3 localized to the nucleus and Drp4 localized to the mitochondria. However, both dynamins induced membranous tubulation in COS-7 cells. The work presented in this study demonstrated that Drp3 and Drp4 have nuclear and cytoplasmic functions. Drp3 is associated with the nucleus in mammalian cells and with the nucleus and the cytoplasm in E. invadens. The effect of Drp3 mutant on E. invadens cells indicates a potential role in cytokinesis and nuclear division. Drp4 is associated with Mitochondria in mammalian cells and some cytoplasmic compartments in E. invadens might involve the mitosomes.
Supervisor: Van Der Giezen, M. ; Schrader, M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Entamoeba histolytica ; Entamoeba invadens ; Entamoeba encystation ; Dynamin related proteins ; Mammalian dynamins