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Metabolic setup of Drosophila macrophages during the immune response
KREJČOVÁ, Gabriela
Adjustment of cellular metabolism is a key function that allows macrophages to fulfill their roles in the body. While the pro-inflammatory polarization of macrophages has been extensively studied in mammalian models, it has not yet been satisfactorily investigated in insects. The study presented in this thesis therefore attempts to elucidate the metabolic setup of macrophages during the immune response in Drosophila melanogaster.
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The role of macrophages in the regulation of systemic metabolism in Drosophila
KREJČOVÁ, Gabriela
Macrophages are immensely versatile cells in the mammalian body, fulfilling roles ranging from protection against pathogenic intruders and engulfing apoptotic cells to morphogenesis and maintenance of tissue homeostasis. This impressive functional versatility may be achieved due to plasticity of macrophage cellular metabolism called metabolic polarization. The adoption of different polarization phenotypes by macrophages determines their function and is essential for the health of the organism. Nonetheless, if the cells lose their metabolic plasticity or polarize inadequately to a particular situation, it can lead to the development of chronic pathological states such as metabolic syndrome. Metabolic polarization of immune cells is thus a key factor in determining whether macrophage function within the organism will be adaptive or pathological. Despite Drosophila melanogaster represents a major model organism for immunological studies, the metabolic setup of activated immune cells has not been addressed up to now. The results of this thesis document that Drosophila immune cells undergo metabolic polarization toward aerobic glycolysis when challenged by extracellular bacteria. Mammals alike, this cellular metabolic switch is regulated by the transcription factor HIF1, thus documenting the conservation of this process between insects and vertebrates. Furthermore, we show that the adoption of aerobic glycolysis is directly linked to the production of the signaling factor IMPL2, which induces the mobilization of lipid stores from the fat body via the silencing of insulin signaling. By this mechanism, immune cells secure sufficient nutrients for successful elimination of the pathogen. Moreover, the mammalian ImpL2 homolog IGFBP7 appears to act analogously in the mammalian liver not only during severe infectious states but also in the liver of obese individuals. While such macrophage activity in regulating systemic metabolism is beneficial to the host during bacterial infection, it becomes maladaptive when chronically activated. Further evidence for a metabolism-regulatory role of immune cells has been found during insect metamorphosis and early post-metamorphic development. This thesis documents that during this period, macrophages infiltrate and engulf the histolyzing larval fat body and convert nutrients into storage peptides and lipoproteins. Subsequently, these nutrients are exploited by the maturing adult structures.
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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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Apoptóza mononukleárnych buniek a makrofágov mliečnej žľázy hovädzieho dobytka
Bátik, Andrej
This bachelor thesis is aimed on apoptosis of mononuclear cells and macrophages in bovine mammary gland. The first part is about mononuclear cells. Monocytes can quickly migrate from blood to tissue and differentiate to macrophages. Macrophages are professional phagocytes. They have significant role in immunity respond. These cells react on every antigen and they remove source of this antigen and also remove old and unuseful cells. Next part is about apoptosis. Apoptosis as known as programed cell death. In this part is described morphology, biochemistry and genetics of programed cell death. Apoptosis is completely natural way of dying, without it live cannot exist. Bovine mammary gland is often attacked by staphylococcus and other bacteria. Result of these attacks is in most cases mastitis, inflammatory disease. Mastitis causes serious health danger for cows also causes economical and finance lost in dairy farms. Thesis describes understanding of these processes in order to improve knowledge and battle against inflammatory diseases.
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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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Aplikace L-carnitinu do peritoneální dutiny potkana
Drozd, Eliška
This thesis (Application of L-carnitine to the peritoneal cavity of rat) deals with the effect of L-carnitine on the inflammatory response of the organism. L-carnitine is a common part of the body's physiological functions. It is part of the lipid metabolism in the mammalian organism. Its effect helps to transfer fatty acids from the cytosol to the mitochondria. The literary part deals with the current state of L-carnitine research and its effects in the body. It also deals with methods of applying active substances to or-ganisms and blood elements, namely leukocytes and their functions associated with inflammatory reaction. The experimental section deals with the influence of L-carnitine on the course of inflammatory reaction in the peritoneal cavity. It also deals with the determination of the dynamics of the inflammatory response, which is evalu-ated using absolute and differential leukocyte counts at two time points.
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