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T cells labelling by bimodal contrast agent for in vivo studies of stroke
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Kříž, Jan (referee)
Stroke is a serious brain injury, which causes sudden death or terminates in permanent neurological disability. Nowadays, tissue plasminogen activator (tPA) is used as the only effective treatment of stroke. One of the potential targets for novel therapy are T cells. Even though the explicit role of T cells in the pathogenesis of brain injury, amounts and timing of all T cell subtypes infiltrating into brain during the stroke still needs further investigation. The research in this field is complicated by the lack of efficient methods for in vivo cell tracking. Therefore the aim of this thesis was to develop a method of T cells labelling by MRI contrast agent in order to investigate T cells distribution in ischemic mice model using in vivo MR imaging. T cells were isolated from C57/BL6 mice in two step isolation protocol using gradient centrifugation and magnetic separation with the efficiency of 97 %. The isolated cells were labelled with 100 μg Fe/mL of Molday ION Rhodamine B contrast agent. The labelling efficiency after 17 hours of cells incubation was higher than 99 %. The labelled cells were cultured with CD3 and CD28 antibodies resulting into the 74 % viability of labelled T cells compared to 83 % viability of non labelled T cells. The labelled T cells were visualized by fluorescent...
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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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The role of exosomes in chronic myeloid leukemia
Březinová, Lenka ; Krijt, Matyáš (advisor) ; Holada, Karel (referee)
Exosomes are extracellular vesicles of a size range 30-150 nm whose function has been explored in chronic myeloid leukemia (CML) due to their role in proliferation of CML cells, remodelling the bone marrow niche, angiogenesis and resistance to treatment with tyrosin- kinase inhibitors (TKIs). Although BCR-ABL kinase is effectively targeted by TKIs, 20-30 % of patients remain resistant to treatment. Resistance of CML cells to TKIs treatment is supported by exosomes. Exosomes transport proteins, nucleic acids, chemokines and small molecules that stimulate anti-apoptotic or suppress pro-apoptotic processes in leukemic cells. Anti-apoptotic processes are especially enhanced by upregulated protein levels: TGF- β1, USP6 and FGF2 and various types of RNA: miR-365, miR-21, Hsa_circ_0058493 and mRNA for BCR-ABL. In contrast leukemic cells tend to reduce the number of pro-apoptotic molecules, including miR-320, miR-328 and miR-146a-5p. Leukemic cells modify the bone marrow microenvironment through exosomes in the way to support their survival and also in order to adjust expression of adhesion and pro- angiogenic molecules. An important role in those processes play miR-126, miR-210 and miR- 92a. Neither the number of processes affected by CML exosomes nor their potential use in the treatment of CML is...
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Pathology and physiology of de novo purine synthesis
Krijt, Matyáš
Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...
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Pathology and physiology of de novo purine synthesis.
Krijt, Matyáš ; Zikánová, Marie (advisor) ; Šebesta, Ivan (referee) ; Čajka, Tomáš (referee)
Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...
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Iron metabolism in Naegleria gruberi
Arbon, Dominik ; Šuťák, Róbert (advisor) ; Krijt, Matyáš (referee)
The metabolism of iron ions is a crucial process in all living organisms and its correct regulation is essential for basic life functions. Homeostasis of iron ions is closely regulated, it usually appears as a component of various proteins and plays role in many oxidation-reduction reactions. Naegleria gruberi is a non-pathogenic, free living protozoon, that serves as a laboratory model for closely related pathogenic Naegleria fowleri. This work focuses on the study of selected metabolites of N. gruberi, that were possible to detect and quantify by the means of modern metabolomic methods, and the influence on culture cultivated in environment with lack of iron ions was shown. The discovery of effect of this condition on the energetic metabolism of this protozoan is an important aspect of understanding the biological processes on cellular level. This method proved a significant influence on certain metabolites and modification of certain metabolic pathways as a direct effect of decreased availability of iron ions. Second part of this work was focused on the enzyme alcohol dehydrogenase, that was found in the genome of this protozoon. Unusual aspects of this enzyme include a N-terminal mitochondrial presequence, prompting about mitochondrial localization, and utilization of iron ion as a prosthetic...
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T cells labelling by bimodal contrast agent for in vivo studies of stroke
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Kříž, Jan (referee)
Stroke is a serious brain injury, which causes sudden death or terminates in permanent neurological disability. Nowadays, tissue plasminogen activator (tPA) is used as the only effective treatment of stroke. One of the potential targets for novel therapy are T cells. Even though the explicit role of T cells in the pathogenesis of brain injury, amounts and timing of all T cell subtypes infiltrating into brain during the stroke still needs further investigation. The research in this field is complicated by the lack of efficient methods for in vivo cell tracking. Therefore the aim of this thesis was to develop a method of T cells labelling by MRI contrast agent in order to investigate T cells distribution in ischemic mice model using in vivo MR imaging. T cells were isolated from C57/BL6 mice in two step isolation protocol using gradient centrifugation and magnetic separation with the efficiency of 97 %. The isolated cells were labelled with 100 μg Fe/mL of Molday ION Rhodamine B contrast agent. The labelling efficiency after 17 hours of cells incubation was higher than 99 %. The labelled cells were cultured with CD3 and CD28 antibodies resulting into the 74 % viability of labelled T cells compared to 83 % viability of non labelled T cells. The labelled T cells were visualized by fluorescent...
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Preparation of analogues of MRI contrast agents and study of their internalization on cell cultures
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Martínková, Eva (referee)
Magnetic resonance imaging (MRI) is a commonly used non-invasive method to examine internal organs and soft tissues. In order to increase signal intensity and improve specificity contrast agents (CAs) have been developed, which affect T1 or T2 relaxation times. T1-contrast agents are mostly based on Gd(III) complexes. The aim of this thesis was to prepare analogues to commercial Gd(III)-CAs by changing the central atom for europium and use europium fluorescence properties for observing CAs' cell-distribution. However, due to strong cell auto-fluoresce the fluorescence of complexes did not provide convincing images reporting their biochemical fate. The toxicity of prepared complexes was tested on adherent cell cultures and evaluated by Fluorescence-Activated Cell Sorter (FACS). Internalization of the CAs' analogues was confirmed also by elemental analysis (ICP-MS). (In Czech)
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