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Bioenergetic studies in the parasitic protist Trypanosoma brucei: functional characterisation of the mitochondrial FoF1-ATP synthase/ATPase complex
HIERRO YAP, Carolina
This Ph.D. thesis focuses on the functional characterisation of the mitochondrial FoF1-ATP synthase/ATPase complex in the parasitic protist Trypanosoma brucei, a eukaryotic system that enables the study of both operation modes of the complex in physiological settings. The results presented in this thesis are divided in four topics: (i) bioenergetics consequences of mitochondrial FoF1-ATP synthase deficiency in two life cycle stages of the parasite, with emphasis on the roles of subunits ATPaseTb1 and OSCP, (ii) changes in energy metabolism of bloodstream form parasites depleted of cardiolipin, (iii) role of the FoF1-ATP synthase dimerisation in mitochondrial biogenesis and bioenergetics, with focus on the functional characterisation of the dimer-specific subunit g in the insect stage of the parasite, and (iv) role of subunit OSCP in the structural and functional integrity of the FoF1-ATPase in trypanosomes lacking mitochondrial DNA (akinetoplastic).
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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Biogenesis of mitochondria in parasitic protist Trypanosoma brucei
Mach, Jan
In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...
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Iron metabolism of parasitic protist Trypanosoma brucei
Krejbichová, Lucie ; Mach, Jan (advisor) ; Pyrih, Jan (referee)
Trypanosoma brucei is a parasite most frequently occurring in Sub-Saharan Africa that causes sleeping sickness in humans and various similar illnesses in animals. The bloodsucking tsetse flies (Glossina) transfer the parasite to humans, their final hosts. Throughout its complex life cycle, Trypanosoma occurs in different environments and undergoes various morphological and metabolic changes. Iron is an important element for all living organisms, including Trypanosoma. The metal plays a crucial role in the host-parasite interaction since trypanosomes are dependent on the iron they acquire from the host or vector. Trypanosomes use iron in metabolic reactions, such as energy metabolism, respiration, nucleic acid synthesis, detoxification, and cellular homeostasis. It is an important element in the synthesis of iron-sulfur clusters which function as cofactors during the above-mentioned reactions. The understanding of iron metabolism in the cell can facilitate the development of new medicaments, an example being iron chelators.
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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The role of mitochondrial SCoAS substrate-level phosphorylation in the bloodstream form \kur{T. brucei}
HUSOVÁ, Michaela
Mitochondrial metabolism of Trypanosoma brucei is considered highly reduced because of its dysfunctional electron transport chain and tricarboxylic acid cycle. But previously published paper suggests significant mitochondrial ATP pool created by substrate phosphorylation via succinyl coenzyme A synthetase, which plays crucial role in T. brucei survival. This thesis is therefore focused on substrate phosphorylation and on influence of succinyl coenzyme A synthetase heterodimer RNAi on T. brucei cells.
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Mitochondrial gene expression in trypanosomatids
PROCHÁZKOVÁ, Michaela
This thesis comprises of diverse projects all focused towards analysis of mitochondrial translation in unicellular parasites. As only two mitochondrially encoded genes are required during the life cycle stage when Trypanosoma brucei resides in the bloodstream of a mammalian host, this protist provides a simplified background in which to study mitochondrial translation termination phase. The leading project utilizes T. brucei to examine mitochondrial translation termination factor TbMrf1 by gene knockout. Subsequently, it is suggested that the peptidyl-tRNA hydrolase TbPth4 is able to abate the TbMrf1 knockout phenotype by its ability to rescue mitoribosomes that become stalled when TbMrf1 is absent. Additionally, modifying methyltransferase of TbMrf1, the TbMTQ1, was characterized. And finally, this work contributed to the development of the protein expression regulation method in Leishmania parasites, a protocol for measurement of proton pumping activity of FoF1 ATPase complex in native mitochondria, and optimization of purification protocol for hydrophobic recombinant proteins.
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Biogenesis of mitochondria in parasitic protist Trypanosoma brucei
Mach, Jan ; Tachezy, Jan (advisor) ; Yurchenko, Vyacheslav (referee) ; Hashimi, Hassan (referee)
In last decade, investigations of mitochondria including their various reduced forms such as hydrogenosomes and mitosomes revealed unexpected diversity of this indispensable organelle. Interestingly, the single mitochondrion of parasitic protist Trypanosoma brucei is able to undergo remarkable functional and structural changes reflecting available carbon sources. Moreover, it was proposed that trypanosomes belong among the most ancient eukaryotes and as such, their mitochondria raised high attention of biologists. To contribute to the knowledge of mitochondrial biogenesis and function, we focused on studies of two key mitochondrial processes, the processing of preproteins that are imported to the mitochondria, and mechanism of pyruvate transport to these organelles. Moreover, we also investigated uptake of iron by T. brucei. This metal is essential for function of numerous proteins, particularly for iron-sulfur proteins in mitochondria. Evolutionary history of trypanosomes and their mitochondrion is a question of debates. According to some reports, mitochondrion of trypanosomes represent an ancient form of this organelle, which is supported by identification of putative "archaic" translocase of the outer mitochondrial membrane (ATOM) and finding of only a single type of translocation pore in...
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The effect of iron on the metabolism of the protozoan Trypanosoma brucei
Arbon, Dominik ; Konvalinka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Iron ions are essential components of numerous cell processes. Their utilisation is strictly regulated, since any impairment can have devastating effect on the cell. In living organisms, iron ions are bound to proteins, for storage, transportation, or as a vital part of catalytic centers of enzymes. Transportation of iron ions between different compartments is important for the correct function of the cell. It was recently shown on yeast, how a mitochondrial transporter of iron ions is essential for the synthesis of iron-sulfur clusters of enzymes. This work aims to describe the localization and function of a homologous protein of a parasitical organism Trypanosoma brucei, which causes African trypanosomiasis, also known as sleeping sickness. This parasite is entirely dependent on uptake of iron ions from its host and therefore the utilization of iron ions is studied as a potential therapeutic target. This work is focused on the characterization of protein Mcp17, which is assumed to function as a transporter of iron ions into the mitochondria of T. brucei. Utilizing expression of marked Mcp17, the transporter was confirmed to be localized on the mitochondrial membrane of the cell. Measuring of enzyme activity of selected enzymes indicated that cells with inhibited expression of the gene mcp17...
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