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Double expression systems with respect to parasitic protozoa
Gromyko, Anastasiia ; Verner, Zdeněk (vedoucí práce) ; Kraeva, Natalia (oponent)
Infekce parazitickými prvoky nadále představují významní zdravotní riziko v rozvojových zemích a vedou ke stovkám tisíc úmrtí každý rok. Tito parazité vykazují složitý vícehostitelský životní cyklus a mají jedinečné buněčné struktury. Mnohé z jejich biologických procesů však zůstávají nedostatečně prozkoumány. Multigenová exprese je slibným přístupem k řešení této mezery ve znalostech, protože umožňuje expresi funkčních proteinových komplexů in vivo, přidání fluorescenčních proteinových značek pro vizualizaci lokalizace proteinu v buňce a studiu interakcí protein-protein. Tato bakalářská práce shrnuje současné poznatky o dostupných systémech a přístupech ke studiu klíčových modelových parazitických druhů prvoků. Klíčová slova: expresní systémy, Trypanosoma brucei, Leishmania tarentolae, Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Toxoplasma gondii, Plasmodium falciparum, pET-Duet, CRISPR-Cas9
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Characterisation of novel mitochondrial proteins in \kur{Trypanosoma brucei}
RAŠKOVÁ, Vendula
This study analyses and characterises novel mitochondrial proteins in the parasitic protist Trypanosoma brucei. Applying phylogenetic analysis was described the evolutionary origin of ZapE protein in eukaryotes, using a newly developed proximity-dependent biotinylation approach (BioID2) we identified ZapE interaction partners like Oxa1. We also discovered a relationship when distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. TbPams were detected by phylogenetic analyses as orthologs of corresponding proteins in Opistokonts. We analyse the function of TbPam18 and TbPam16 in the replication of the mitochondrial DNA and determine, how the TMDs of TbPam18 and TbPam16 are essential for their functions. Finally, we evaluated a set of putative mitochondrial proteins of the heterolobosean N. gruberi defined by Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and analyse the origin of mtFfh and mtFtsY.
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The Mitochondrial Contact Site and Cristae Organization System and F1FO-ATP Synthase Crosstalk is a Fundamental Property of Mitochondrial Cristae
CADENA, Lawrence Rudy
The acquisition of mitochondria from an endosymbiont closely related to extant alphaproteobacteria occurred prior to the divergence of modern eukaryotes. Since then, diverse eukaryotes have not only developed a number of different mechanisms to adapt to their environment regarding growth and proliferation, but perpetuated certain traits that have persisted for eons. This thesis postulates an ancestral mechanism for cristae development in mitochondria involving interplay between two cristae shaping protein complexes, the Mitochondrial Contact Site and Cristae Organization System and F1FO-ATP Synthase, that has remained conserved throughout eukaryotic diversification for over 2 billion years.
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Elucidating the source of bloodstream \kur{Trypanosoma brucei} mitochondrial ATP
HUSOVÁ, Michaela
For decades, it has been assumed that the reduced function and structure of the bloodstream form Trypanosoma brucei mitochondrion renders it a strictly ATP consuming organelle. Emerging evidence from refocused studies suggest that the bloodstream mitochondrion retains complex bioenergetic pathways that allow the parasite to adapt to various environments. This thesis is focused on the source of bloodstream mitochondrial ATP, with a special emphasis on the role of succinyl-CoA to produce ATP via mitochondrial substrate phosphorylation. We will also discuss alternative bioenergetic pathways present in this life stage of a human pathogen.
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Preparation of antibodies to determine the association of mitoribosomal complexes with mitochondrial membrane
ZELLNER, Laura
The assembly pathways of mitoribosomes, descendants of bacterial ancestors producing proteins encoded by vestigial mitochondrial genomes, remains largely unknown. To shed light on structural features of a precursor of the highly divergent small mitoribosomal subunits (mtSSU) from Trypanosoma brucei, called mtSSU assemblosome, recently characterized by cryoEM, we raised antibodies against two assembly factors present in the complex and against two subunits of the mature mtSSU. We used the antibodies in pilot experiments to determine whether the assemblosome associates with the inner mitochondrial membrane.
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Post-transcriptional regulation of TbIF1 in life cycle of Trypanosoma brucei
GRATZL, Sascha
TbIF1, a protein Inhibitor of F1-ATPase in Trypanosoma brucei, is expressed exclusively in the insect stage of the parasite. In the bloodstream form, TbIF1 is switched off, because its activity interferes with the essential role of the ATP synthase in the maintenance of the mitochondrial membrane potential. Here, we employ a series of reporter genes to study the impact of 3'UTR of TbIF1 on mRNA stability and translatability to get insight into the tight post-transcriptional control of TbIF1. We provide evidence that developmentally regulated RNA binding protein Rbp10 is critical for downregulation of TbIF1 on translation level in bloodstream-form trypanosomes.
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