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FoF1-ATP synthase/ATPase in the parasitic protist, \kur{Trypanosoma brucei}
ŠUBRTOVÁ, Karolína
This thesis primarily focuses on the FoF1-ATP synthase/ATPase complex in the parasitic protist, Trypanosoma brucei. Instead of its normal aerobic function to synthesize ATP, it is required to hydrolyze ATP to maintain the m in the infective bloodstream stage of T. brucei and the related parasite, T. b. evansi. To better understand the composition, structure and function of this druggable target, my work focused on deciphering the function of three of the unique Euglenozoa specific subunits that comprise this complex molecular machine. Furthermore, the ADP/ATP carrier, which provides substrates for the FoF1-ATP synthase/ATPase, was functionally characterized and evaluated if it is physically associated with the complexes of the oxidative phosphorylation pathway.
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Iron-Sulfur Cluster Assembly in Trypanosoma brucei
HAINDRICH, Alexander Christoph
In this thesis we investigated genes of the Cytosolic Iron sulfur cluster assembly (CIA) pathway in T. brucei procyclic and blood-stream form for their possible functional redundancy. For this, RNAi double knockdown plasmids were generated containing knockdown partners which were selected based on the proposed model of the CIA pathway in S. cerevisiae. The generated plasmids were transfected into T. brucei cells, and growth effects on the transfectants upon tetracycline induced RNAi was measured.
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Interactions of Trypanosoma brucei FOF1 ATP Synthase Subunits - An Application of Yeast Two Hybrid
GUGGENBERGER, Matthias
The FOF1 -ATP synthase has a unique composition and function in Trypanosoma brucei, a flagellated parasite endemic to developing regions of the world where it causes Human African Trypanosomiasis (HAT) and Nangana in cattle. Currently there are no good treatments to cure the disease as most of the medicine is antiquated, difficult to administer and highly toxic. Because of the unique properties of the T. brucei FOF1-ATP synthase, this enzyme is considered a possible drug target. This final component of the oxidative phosphorylation pathway actually works in reverse during the infectious stage of this parasite as it needs to hydrolyze ATP to maintain the essential mitochondrial membrane potential in the absence of a cytochrome mediated respiratory chain. In addition to the well conserved eukaryotic subunits, this large complex is also comprised of several subunits that have no known homology outside of kinetoplastids, an ancient group of protists with a unique mitochondrial DNA structure. To further explore the composition and organization of this potential drug target, we employed the technique of yeast two-hybrid to map the protein-protein interactions of the individual subunits. Our preliminary results suggest that important information about the organization of the T. brucei FOF1-ATP synthase can be gleaned from this experimental approach.
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Functional analysis of Ssc1 and Iba57 proteins in \kur{Trypanosoma brucei}
SKALICKÝ, Tomáš
Aim of this thesis was to shed light on the function(s) of Iba57 and Ssc1 proteins in both life cycle stages of T. brucei using RNA interference. Depletion of Ssc1 resulted in severe grow phenotype, decrease in activities of iron-sulphur cluster-containing enzyme aconitase but no increase in oxidative stress sensitivity or accumulation of ROS in mitochondrion. Down regulation of Iba57, specialized maturation factor of aconitase and homoaconitase, lead to depletion of aconitase, destabilization of Isa1 and increased sensitivity to oxidative stress and accumulation of ROS in both stages.
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The impact of iron-sulfur assembly on the mitochondrial tRNA import in \kur{Trypanosoma brucei}
PARIS, Zdeněk
This thesis addresses several aspects of mitochondrial iron sulfur (Fe-S) cluster biogenesis and mitochondrial tRNA import and modifications in Trypanosoma brucei. Using RNA interference it uncovers essential role of Fe-S cluster assembly in tRNA(s) thiolation in both the cytosol and the mitochondrion of T. brucei. Further, this thesis describes the role of modifications in tRNA editing and in mitochondrial import of tRNAs. Finally, it provides evidence that in contrast to protein import, mitochondrial membrane potential is dispensable for import of tRNAs into the mitochondrion of T. brucei.
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