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Are currently used chelators of heavy metals also chelators of calcium and magnesium?
Skoupilová, Karolína ; Mladěnka, Přemysl (advisor) ; Šuťák, Róbert (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Karolína Skoupilová Supervisor: prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of diploma thesis: Are currently used chelators of heavy metals also chelators of calcium and magnesium? Calcium and magnesium are important elements for the human body. Calcium plays an important role in blood clotting and intracellular signalling, thus participating in muscle contraction and platelet aggregation, among other processes. Magnesium is a cofactor for many enzymes. A significant deficiency of both ions can manifest as disorders of heart rhythm and increased neuromuscular excitability. The aim of this study was to determine the chelating activity of currently used heavy metal chelators and to determine the stability of the indicator. First, the ideal pH for the experiment was determined. In the next step, the chelation rate of 21 known metal chelators was measured using an in vitro spectrophotometric method. Finally, the chelation of the most active calcium chelators was verified on platelet aggregation in a human blood sample. At the same time, the long-term stability of the indicator o- cresolphthalein complexone (CC) was determined. In the first step, pH 7.5 was chosen, which was the only one...
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From iron metabolism in parasitic protists to drug discovery
Arbon, Dominik ; Šuťák, Róbert (advisor) ; De Koning, Harry P. (referee) ; Petrák, Jiří (referee)
Iron is essential for the correct function of various biochemical processes. Most living organisms developed elaborate strategies for homeostasis of this metal, including specific approaches for its acquisition and trafficking, to incorporate it into different pathways in various cellular compartments. Across the domain of parasitic protists, iron plays a crucial role in the interaction between pathogen and its host, where the struggle for scavenging available iron is a basis for nutritional immunity. This work summarizes the current knowledge about iron acquisition and trafficking in a spectrum of facultative and obligative unicellular parasites, with experimental discoveries of the iron acquisition strategies and response to iron-deficient conditions in parasitic protists Naegleria fowleri and Acanthamoeba castellanii. Based on this, the validity of exploiting iron chelation therapy is theoretically and experimentally assessed. Further work focuses on the utilization of mitochondrial targeting as a method of improving the potential of therapeutic compounds, including iron chelators, and describing their action in vitro and in vivo against a range of parasites.
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Copper metabolism in Acanthamoeba castellanii
Doležalová, Taťána ; Šuťák, Róbert (advisor) ; Mladěnka, Přemysl (referee)
Copper is an essential element that, due to its redox properties, is involved as a cofactor in many enzymes, the most well-known example is the cytochrome c oxidase. At the same time, in higher concentrations, this element shows toxic effects on several levels, it can disrupt iron-sulfur clusters, damage proteins and lead to cell death. This is the reason why this thesis studies copper metabolism in the parasitic amoeba Acanthamoeba castellanii, which causes serious diseases such as acanthamoeba keratitis or granulomatous amoebic encephalitis. Understanding how copper homeostasis is maintained in this organism could lead to targeted disruption of this balance and the exploitation of the toxic effects of copper. In this work, the function of the P-type ATPase homologous to CCC2 from Saccharomyces cerevisiae, responsible for the transfer of copper ions from the cytoplasm to the Golgi apparatus, was characterized by functional complementation. Furthermore, a carrier from the Ctr family has been described that transfers copper ions presumably across the cytoplasmic membrane. The expression of these carriers was monitored in copper excess and deficiency. To get a better picture of how A. castellanii maintains copper homeostasis, the cellular accumulation of copper and the ability of this amoeba to deal...
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The effect of amoeba predation on the evolution of virulence in human pathogenic microorganisms
Drncová, Eliška ; Šuťák, Róbert (advisor) ; Konupková, Anežka (referee)
Amoebae act as one of the main regulators of microbial communities, where, as a result of their predation, selection pressure is exerted for the emergence of defence mechanisms to achieve resistance. This adaptation allows microorganisms to randomly infect the human body and successfully defend against components of innate immunity, especially macrophages, which, like amoebae, are phagocytic cells. The manifestation of virulence in opportunistic pathogens is due to conserved macrophage pathways used for degradation of ingested material, which the microorganism has already encountered in amoebae. Because of this similarity, amoebae can be used to investigate the interaction between a pathogen and its host, which includes research on the virulence mechanisms of many human microbial infections. Among the most extensively studied organisms whose pathogenicity results from long-term interaction with amoebae are the bacterium Legionella pneumophila and the microscopic fungus Cryptococcus neoformans, with very different virulence strategies and manifestations. Understanding the evolutionary context and the advantages that microorganisms gain during interaction with amoebae informs us about the origins of virulence of opportunistic human pathogens.
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Effect of various stress factors on mitochondrial processes of pathogenic protists
Ženíšková, Kateřina ; Šuťák, Róbert (advisor) ; van Hellemond, Jaap (referee) ; Leitsch, David (referee)
Mitochondria perform various important functions in cells. They are the main site of energy metabolism, biosynthetic and regulatory processes, and the center of iron metabolism. Additionally, mitochondria are also the central organelle responsible for the production of potentially dangerous reactive oxygen species and possess a self-destructive arsenal capable of inducing whole-cell apoptosis. This single organelle thus controls the fate of the entire cell. Given these facts, this organelle has become the focus of interest for many scientists and pharmaceutical companies developing drugs targeting mitochondria. During evolution, unicellular parasites have evolved several mechanisms to survive, defend themselves and reproduce in the hostile environment of their host. One of these mechanisms is the ability to adapt its mitochondrial metabolism to maintain the viability of the whole cell. This work focuses on mitochondria from two different perspectives: First, concerning the phenomenon of nutritional immunity, we studied the effect of iron and copper deprivation on the mitochondria of the "brain-eating" amoeba Naegleria fowleri. Proteomic analysis of cells pre-incubated with specific chelators, together with the determination of several enzyme activities and measurements of oxygen consumption,...
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Iron and copper transporters in amphizoic amoebae "Naegleria fowleri" and "Acanthamoeba castellanii"
Grechnikova, Mariia ; Šuťák, Róbert (advisor) ; Lorenzo Morales, Jacob (referee) ; Maciver, Sutherland (referee)
Amphizoic amoebae Naegleria fowleri and Acanthamoeba castellanii are distributed worldwide in diverse natural and anthropogenic environments. N. fowleri can infect healthy individuals, causing a rare but deadly brain disease called primary amoebic meningoencephalitis with a mortality rate of over 95%. Infection develops if amebae from contaminated water get into the nose. A. castellanii can infect the central nervous system of immunocompromised patients, causing granulomatous amoebic encephalitis with a survival rate of 2-3%, and the eyes of healthy people, leading to a severe sight-threatening infection called Acanthamoeba keratitis. N. fowleri and A. castellanii are aerobic organisms and require iron and copper cofactors. Both metals are crucial for almost all known organisms and toxic in excess, so the pathogens tightly control the homeostasis of iron and copper, which is critical for their virulence. However, the information about metal homeostasis in amphizoic amoeba is scarce. This work aimed to characterize some of the mechanisms employed by N. fowleri and A. castellanii for iron and copper acquisition and detoxification. Despite the similar morphology and lifestyle of studied amoebae, their mechanisms of iron homeostasis under iron-limiting conditions are entirely different. Both amoebae...
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The role of iron in the virulence of Acanthamoeba castellanii
Šimáčková, Aneta ; Šuťák, Róbert (advisor) ; Krijt, Matyáš (referee)
Iron is an essential nutrient for all living organisms. It plays a crucial role in the virulence of pathogenic bacteria as well as parasitic eukaryotes. Although iron is an abundant element in the host organism, it cannot be easily obtained by the parasite. The ability of parasites to induce disease depends largely on how efficient the mechanisms for iron acquisition have evolved. This thesis deals with the effect of iron on the virulence of Acanthamoeba castellanii. It is a free-living amoeba that, in exceptional situations, can cause fatal inflammatory brain disease or damage a person's vision. Culturing with mammalian cells allowed us to demonstrate the effect of iron on amoeba virulence using flow cytometry. Based on fluorescent labeling, we localized the protein AC_IDIP (Iron-Deprivation Induced Protein) in the cytosol. This protein was also isolated and a polyclonal antibody was raised against it and used to monitor the effect of iron availability on the AC_IDIP level. Using comparative proteomic analysis, it was possible to monitor changes in the A. castellanii proteome depending on the nutrient source. Altered levels of many proteins were detected under conditions where mammalian cells were the nutrient source. These were mainly cytoskeletal proteins. In contrast, the proportion of proteins...
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FeS cluster assembly in cytosol of Trichomonas vaginalis
Stojanovová, Darja ; Tachezy, Jan (advisor) ; Šuťák, Róbert (referee)
Iron-sulfur (FeS) cluster assembly is extensively studied in model organisms, e.g. Saccharomyces cerevisiae, Homo sapiens, and more recently in Trypanosoma brucei. However, little is known about FeS assembly in divergent anaerobic organisms such as Trichomonas vaginalis, which parasites in the human urogenital tract. This parasitic protist possesses anaerobic form of mitochondria, the hydrogenosome, in which some component of FeS cluster assembly machinery (ISC) has been identified, whereas the cytosolic CIA pathway has not been studied so far. Our work deals mainly with TvIscU, a component of ISC pathway, and T. vaginalis CIA pathway. We suggest that both hydrogenosomal and cytosolic FeS cluster assembly pathways of this parasite differ from typical models. We examined possible ISC-CIA relationship. Next, we found homologues for several key components involved CIA machinery, namely Nbp35, Cfd1, Nar1, Cia1 and Cia2. However, we did not identify any homologous proteins to Tah18, Dre2 and Mms19. We expressed identified proteins with HA-tag and localized them by cell fractionation and immunofluorescence microscopy in T. vaginalis. Finally, we immunoprecipitated two Cfd1 paralogues, TvCfd1A and TvCfd1B to search for their interacting partners. The results suggest that these two paralogues interact with...
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Metabolism of iron in amoebas
Glavanakovová, Marie ; Šuťák, Róbert (advisor) ; Pyrih, Jan (referee)
Ferritins are complex protein structures for iron storage and detoxification. They are typically found in the cytoplasm of the cell; however, they were also identified in mitochondria and chloroplasts. They are distributed among all living organisms but uncommon in non-photosynthetic protists. Surprisingly, a ferritin gene was found in the genome of free-living amoeba Naegleria gruberi. Bioinformatic analysis confirms that it belongs to a group of eukaryotic ferritins. Upon creating antibodies in a laboratory rat, we localized the protein in mitochondria of N. gruberi. The native recombinant protein without the mitochondrial presequence was used for further in vitro experiments. Using growth experiments, we established that the expression of ferritin is dependent on a concentration of iron in cultivation media; however, we were not able to directly prove its ability to bind iron in vitro. We concluded that the function of the ferritin of N. gruberi is most likely related to metabolism of iron. Side experiments were focused on a different protein from the ferritin family - Dps protein in Acanthamoeba castellanii. Despite of a successful creation of antibodies, we were not able to detect the expression of protein neither in total cell lysates nor in subcellular fractions of acanthamoebas under...
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Role of sulfhydryl oxidase 1 in cancerogenesis
Beranová, Lea Marie ; Truksa, Jaroslav (advisor) ; Šuťák, Róbert (referee)
Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...
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