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The role of iron in the regulation of proteins connected with tamoxifen-resistance
Potomová, Petra ; Truksa, Jaroslav (advisor) ; Balušíková, Kamila (referee)
Cancer cells are highly dependent on nutrient uptake to sustain their increased proliferation, one of these nutrients being iron. In recent years, a heightened dependency on iron was observed in cancer cells, allowing for the proper function of numerous enzymes, DNA synthesis and mitochondrial respiration. Here, we further delve into the iron metabolism of malignant cells, attempting to understand the differences between tamoxifen-sensitive and resistant (Tam5R) ones using two breast cancer cell lines of luminal A origin, MCF7 and T47D. These cells show numerous changes in iron homeostasis and iron-dependent mechanisms. Based on alterations in proteomes of Tam5R cell lines, we focused on iron regulation of proteins that are deregulated in tamoxifen resistance - assessing their regulation on transcriptional (mRNA) and post-transcriptional level (protein) as well as comparing their responsiveness to their sensitive parental cell line. We assessed two main types of regulation - iron-responsive element interaction with iron-regulatory proteins (IRE-IRP pathway) and tristetraprolin (TTP) driven mRNA degradation via AU-rich elements (ARE). Using iron loading and chelation, we challenged the cells - confirming the known IRE-IRP regulation of ferritin heavy chain (FTH), transferrin receptor 1 (TfR1),...
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Iron metabolism of parasitic amoeba Acanthamoeba castellanii
Krejbichová, Lucie ; Mach, Jan (advisor) ; Horáková, Eva (referee)
Acanthamoeba castellanii, an opportunistic parasitic amoeba, rarely causes infection in humans. Yet, the treatment and diagnosis is often complicated. It causes two diseases: granulomatous encephalitis and acanthamoeba keratitis (inflammation of the cornea of the eye). There is a modern way to deal with parasitic protists and other infectious diseases - interference with iron metabolism. Iron is a crucial element for living organisms; it is involved in many metabolic processes. However, it is also toxic for the organisms and therefore, its uptake, storage and homeostasis in the cell is a strictly controlled process. Ferric reductase takes part in iron homeostasis inside the acanthamoeba cell. Fluorescent microscopy has shown that ferric reductase is located in the vacuole membranes and its activity increases when iron availability decreases. This protein probably mediates the reduction of iron ions that are used in other cellular processes, especially for the transport of iron across membranes. Interestingly, the reduced availability of iron doesn't affect the mitochondrial Fe-S cluster pathway as we proved by fluorescent microscopy with an antibody against one of the proteins of this pathway (IscU). Iron deficiency doesn't affect respiration but alternative oxidase activity is reduced. Moreover, deprived...
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Proteomic Analysis of Trichomonas vaginalis hydrogenosone
Campo Beltran, Neritza ; Tachezy, Jan (advisor) ; Nohýnková, Eva (referee) ; Yarlett, Nigel (referee)
Trichomonas vaginalis is a human pathogen that affects annually approximately 258 million people worldwide. This parasite possesses organelles of mitochondrial origin called hydrogenosomes, which generate ATP under anaerobic conditions. The identification of the protein content at the subcellular level may provide new targets for antiparasitic drugs developments as well as it contributes for our understanding of the organelles function and evolution. The availability of protocols for organelles purification and the complete genome sequence allow the study of the organellar proteomes using mass spectrometry and bioinformatics, providing a powerful strategy that combine cell biology and proteomics. In our research, we used several approaches to identify the protein composition in hydrogenosomes and mitosomes. We performed transcriptomic and proteomic analysis to investigate the molecular responses of Trichomonas vaginalis upon iron availability. Furthermore, the changes in the proteome during the development of metronidazole resistance were also studied. The organelles separated by differential and Optiprep-sucrose gradient centrifugation were analyzed with nano- RP-HPLC/MALDI-TOF/TOF. We also used Triton X-114 phase partitioning to separate membrane proteins and iTRAQ technique to label the peptides...
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Chelating Polymers for the Haemochromatosis Treatment
Groborz, Ondřej ; Hrubý, Martin (advisor) ; Kotek, Jan (referee)
5 Chelating Polymers for the Haemochromatosis Treatment Author: Ondřej Groborz Tutor: Mgr. Martin Hrubý, Ph.D., DSc. Advisors: Ing. Kristýna Kolouchová Ing. Pavel Švec Institute of Macromolecular Chemistry, Czech Academy of Sciences Abstract Haemochromatosis is a group of hereditary diseases which are characterised by toxic accumulation of iron in parenchymal organs, leading to organ toxicity and irreversible damage. Currently, there are only a few approved medications for this disease, yet all of them possess severe side effects. Herein, we have proposed a new paradigm for treatment: insoluble polymers with negligible systemic biological availability would form stable complexes with iron ions in the gastrointestinal tract, hence decreasing biological availability of iron. The insolubility of polymers prevents them from being absorbed into the organism in the first place while having no systemic side effects or toxicity. We have prepared polymers with several covalently bound iron-chelating ligands and based on the biological data we selected the most successful chelators for possible future applications. These polymers exhibited negligible resorbability and toxicity, superior in vitro iron chelating activity and their efficacy was proven in an in vivo model. Therefore they could be used as a...
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Effect of chronic hypoxia on antioxidative capacity of rat myocardium.
Závišková, Kristýna ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia activates endogenous signaling cascades, which lead to cardiac protection against acute ischemia/reperfusion (I/R) injury. The molecular mechanism of this phenomenon has not been fully clarified yet. However, it was proved that reactive oxygen species (ROS) take part in cardioprotective signaling pathway inducted by chronic hypoxia. The high level of ROS must be precisely regulated by antioxidative system of a cell. The aim of diploma thesis was to examine the effect of intermittent hypobaric hypoxia (IHH, 7 000 m) on relative amount of antioxidative enzymes (peroxiredoxin 6 - PRX6, thioredoxin 1 and 2 - TRX1 and TRX2, thioredoxin reductase 1 - TRXR1) and also enzymes of iron metabolism (heme oxygenase 1 and 2 - HO1 and HO2, aconitase 1 and 2 - ACO1 and ACO2), which participate in regulation of cell redox state. Moreover, we studied the effect of adaptation to IHH and an antioxidant tempol on relative amount of calcium-independent phospholipase A2 (iPLA2). iPLA2 can remove peroxidized fatty acids from membrane phospholipids. On the other hand, iPLA2 can damage cell in I/R conditions. All enzymes were studied in homogenates from normoxic and IHH adapted rat left ventricular myocardium by Western blot. Adaptation to IHH caused a decrease of PRX6 and on the opposite an increase of...
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Proteomic analysis in hematology: Identification of alfa2-macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the of leukemic K562 cell differentiation induced by sodium butyrate.
Pešlová, Gabriela ; Vyoral, Daniel (advisor) ; Krijt, Jan (referee) ; Suttnar, Jiří (referee)
The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate
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Molecular mechanisms of tamoxifen resistance in breast cancer
Tomková, Veronika ; Truksa, Jaroslav (advisor) ; Brábek, Jan (referee) ; Mráček, Tomáš (referee)
The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...
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Mechanisms of resistance and iron metabolism in cancer stem cells
Lettlová, Sandra
(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....
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Molecular mechanisms of tamoxifen resistance in breast cancer
Tomková, Veronika ; Truksa, Jaroslav (advisor) ; Brábek, Jan (referee) ; Mráček, Tomáš (referee)
The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...
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Iron homeostasis in malaria
Wernerová, Klára ; Šuťák, Róbert (advisor) ; Voleman, Luboš (referee)
Although malaria is a well-studied infectious disease, we are still unable to fight it effectively, as evidenced by a large number of infected people. Many drugs are available against malaria. However, because of incessantly emerging resistances, new, more effective antimalarials need to be developed. One possibility is to target the parasite's iron metabolism, the essential element of all organisms. Iron participates in DNA synthesis, respiration, energy production. It acts as a cofactor of ribonucleotide reductase, and metalloproteins with FeS clusters or heme. During the infection, the parasite must compete with the host for nutrients, including iron. The mechanism of iron uptake or excretion in malaria parasite is not completely clear. Only two iron transporters are known, but it is already evident, that there must be more of them. The Plasmodium parasite digests a large amount of hemoglobin, which is degraded into free heme and denatured globin. Free heme is toxic to the cell though. Plasmodium defends itself from the toxicity of free heme by forming chemically inert hemozoin. This unique mechanism of protection against the free heme toxicity is very useful for Plasmodium and other blood parasites, but it also becomes an advantageous target for drugs because the mechanism is present only in...
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