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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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Role of interaction between circadian and immune system in development of obesity and type 2 diabetes mellitus
Honzlová, Petra ; Sumová, Alena (advisor) ; Okuliarová, Monika (referee) ; Vodička, Martin (referee)
Disruptions within the circadian, metabolic, and immune systems contribute to the onset of metabolic illnesses, including type 2 diabetes. This doctoral thesis aimed to examine the mutual interactions between these three systems in relation to the development of metabolic disorders. The circadian system regulates a variety of mammalian physiological processes. On the molecular level, the circadian clock is in individual cells maintained by a complex of interconnected transcription-translation feedback loops. The circadian system is hierarchically organized, consisting of the central circadian clock residing within the suprachiasmatic nuclei (SCN) and of peripheral oscillators found in various organs and tissues, including the pancreas and compartments of the innate immune system such as macrophages and microglia. The central circadian clock synchronizes the peripheral oscillators via multiple pathways, including feeding- related metabolic changes to which some of the peripheral oscillators are highly sensitive. Misaligned feeding can thus induce dyssynchrony between individual peripheral oscillators. Our results show that a reverse restricted feeding (rRF) regimen, when food is available only for a limited time during the resting period of the day, has distinct effects on the circadian clocks in...
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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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Danio rerio as a model of serious human diseases
Hason, Martina ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
(ENGLISH) Over the last five decades, zebrafish (Danio rerio) has become a useful vertebrate model organism for the field of developmental biology and disease control. Using zebrafish in xenotransplantation studies is becoming more popular and progressed towards drug screening of anti-cancer drugs. Zebrafish are particularly suitable for high-throughput pre-clinical drug screening, due to the small size of embryos and the striking evolutionary conservation of cancer- related pathways between human and zebrafish. The fast, large-scale evaluation of the cancer- drug response in vivo could facilitate progress in personalized cancer therapy. Nevertheless, there is still a lack of methods which would allow for rapid and sensitive evaluation of tumor cell growth to facilitate high-throughput screening of drugs in vivo. In our bioluminescent zebrafish transplantation model, we proposed and validated a new screening platform for pre-clinical drug discovery in zebrafish embryos. In our experiments we used the NanoLuc luciferase, which enabled us to rapidly screen inhibitors of cancer growth in a sensitive and quantitative way with very low background compared to the conventional fluorescence signal. In our screen we evaluated the in vivo drug response of 180 kinase inhibitors in zebrafish embryos...
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Macrophages and nitric oxide in leishmania - sandfly - host interactions
Kratochvílová, Tereza ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
Leishmania reside fagolysosome of macrophages immediately after their entry to host where they multiply and consequently infect other macrophages or eventually other cells. A synthesis of a reactive reactant of oxygen and nitrogen is one of the mechanisms that some mammal cells are equipped with and that also contributes to eradication of leishmania. Nitric oxide rising during a metabolic change of L-arginine under the catalysis of NO synthase is of a large importance. Beyond cytotoxic function, nitric oxide is involved in signalling pathways for a neurotransmission (nNOS) and vasorelaxation (eNOS). Not all types of macrophages have ability to produce NO (iNOS). It is a heterogeneous group differing in immunological function and also in physiology. A group of classical activated macrophages represents an effective APC capable of efficient killing of intracellular pathogens. In addition to NO, they also secrete an inflammatory cytokines, which evolve an immune reaction towards to Th1. Contrary to this, a group of alternative activated macrophages is not capable of any efficient antigen presentation and nitric oxide production but produces L-ornithine, which is a precursor of polyamines, which leishmania utilizes for its own intracellular growth. For the mouse model, status of resistance and/or...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Signaling effects of adenylate cyclase toxin action on phagocytes
Černý, Ondřej
The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...
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Přítomnost nízkomolekulárních imunomodulátorů ve slinách klíštěte \kur{Ixodes ricinus}
HAUSEROVÁ, Simona
Ticks use proteinaceous molecules contained in their saliva to suppress immune response of the host to complete their succesful feeding. In some ticks (e.g. Rhipicephalus sanguineus) presence of non-proteinaceous molecules was discovered. The aim of this work was to determine the amount of these molecules in the saliva of Ixodes ricinus tick. Namely, prostaglandin E2 and adenosine were analyzed. The second aim of this work was to evaluate the role of both low molecular weight components (or at least one of them) in the suppression of TNF-alfa cytokine production in suitable cells by ticks saliva.
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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