Felix Alexander Ayelazuno

• Malaria remains a significant global health threat, with an estimated 263 million cases and 597,000 related deaths annually, especially among children under five years old. It is caused by Plasmodium parasites, which are transmitted to vertebrates through the bite of infected female Anopheles mosquitoes. Of the five parasites known to cause malaria in humans, P. falciparum is the deadliest and accounts for most malaria-related illness and death. Although significant progress has been made in reducing malaria, challenges such as drug and insecticide resistance remain. Malaria parasites contain a small mitochondrion during the merozoite stage, which develops into an elongated tubular network in the schizont stage after invading red blood cells. This development requires the de novo synthesis of mitochondrial components, including the import and sorting of mitochondrial proteins. Although all living organisms have their own mitochondrial genome, nearly 99% of mitochondrial proteins are encoded in the nucleus, synthesized on cytosolic ribosomes, and then imported into the mitochondria. The process of importing proteins into mitochondria is believed to be highly conserved among eukaryotes. However, recent studies have revealed that the import machinery itself has undergone significant changes in various major eukaryotic lineages. The differences between the mitochondrial import machinery of malaria parasites and their hosts make mitochondria a promising target for drug development. Nevertheless, our understanding of mitochondrial protein import in malaria parasites and related apicomplexan parasites relies on poorly characterized components of the import machinery. To date, studies on the mitochondrial protein import machinery of malaria parasites have primarily relied on bioinformatics, with little experimental evidence validating their composition, sub-organellar localization, or interaction networks. This thesis examined the composition and localizations of the components of the mitochondrial protein import machineries in malaria parasites. It also analyzed the interactomes of validated core components of these machineries. The findings demonstrated, for the first time in malaria parasites, the production and mitochondrial localization of the core components of mitochondrial import machineries. Specifically, the core subunits of the TOM and SAM complexes, PfTom40 and PfSam50, respectively, were successfully confirmed through confocal microscopy and western blot analyses. Similarly, two subunits of the PAM complex (PfHsp70 and PfMge1) and the central subunit of the OXA pathway, PfOxa1, were shown to be produced and localized in the mitochondria of malaria parasites. Western blot analyses also suggested the possible formation of Tim8/Tim13 hexamers in P. falciparum. Confocal microscopy revealed that Tim8, Tim13, and Pam16 are localized to the mitochondria of malaria parasites. Interactome analysis of PfMge1 showed significant enrichment in the pulldown assay; however, PfMge1's interacting partners were not significantly enriched overall in this study. Additionally, the pulldown experiments for PfTom40 and PfSam50 did not show significant enrichment in the MS analyses. Future studies could explore chemical crosslinking and denaturing affinity purification methods to capture transient interacting partners and improve solubilization of integral membrane proteins. Gene knockout attempts for PFHSP70 and PFTOM40 indicated that transfection with circular plasmids could result in unwanted insertions or single crossovers in the parasite's genome; linearized plasmids are recommended.