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Macrophages selectively sense mechanical cell death in CLASP-depleted melanoma cells to trigger CXCL10 response
Otruba, Matúš ; Labzin, Larisa (advisor) ; Filipp, Dominik (referee)
Melanoma represents a significant and often fatal form of cancer, with metastasis being a primary cause of cancer-related deaths. Immunotherapy aims to stimulate the immune system to eliminate tumors, but even when used together with traditional chemotherapy, still only has <50% success rate. Chemotherapeutic drugs induce tumor cell death, primarily through apoptosis, which is 'silent' and does not incite inflammation or immune cell infiltration into the tumor. During metastasis, tumor cells migrate through confined spaces. CLASP proteins protect organelles and cellular integrity during tumor cell metastasis; when CLASP proteins are depleted, tumor cells migrating through tissue die through a mechanical cell death. The immunological impact of CLASP depletion-induced cell death remains unknown. This study aims to explore whether macrophages, innate immune cells that sense neighboring dying cells, trigger inflammatory cytokine responses specifically to mechanical, as opposed to chemotherapeutic, melanoma cell death. To model mechanical cell death during metastatic migration, CLASP1, a microtubule stabilizing protein, was depleted, and cells were physically compressed to simulate forces encountered during metastasis. Chemotherapy was simulated using a B-Raf inhibitor and the apoptosis inducer...
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Characterization of cytotoxic effect of combined antimicrobial nanomaterials
Kozlíčková, Hana ; Fialová, Lenka (referee) ; Márová, Ivana (advisor)
This thesis deals with the study of the effects of combined nanomaterials on human skin cells. Pure antimicrobial substances, two types of liposomes enriched with antimicrobial substances, nanofibers with antimicrobial substances and, finally, four types of combined nanomaterials were analyzed from the point of view of cytotoxicity. The analysed active substances were eugenol, thymol, cavarcrol, curcumin, vitamin E and the antibiotics streptomycin and ampicillin. In the theoretical part of the work, the cell line of human keratinocytes, used in the experimental part of the work for cell tests, was characterized. Furthermore, individual active substances with an antimicrobial effect were described and the principles of biological effects were described, which include antimicrobial, antioxidant, cytotoxic and synergistic effects. Additionally, the theoretical part described individual nanomaterials, their preparation and usage in cosmetics and medicine. The experimental part was based on the characterization of prepared nanomaterials and on testing the influence of individual antimicrobial substances on the proliferation and viability of human HaCaT cells. Using the DLS method, the size of the prepared liposomes was measured and the effect of PHB and the type of active substance on their size was studied. MTT and LDH tests were chosen to test the cytotoxicity of individual substances. Furthermore, a scratch test was performed to monitor the effect of the investigated substances on proliferation and the rate of wound healing by cells. The last performed tests were immune response assays, in which were tested the samples for production of the human anti-inflammatory cytokines IL-6 and IL-8. In experimental part of this thesis, it was found that the prepared nanofibrous materials are safe for use in healthcare or cosmetics and, in the future, suitable to produce nanofibrous wound coverings enriched with antimicrobial substances, which can give them exceptional properties.
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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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Mebrane adaptor proteins in hematopoiesis and immune response
Pavliuchenko, Nataliia ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee) ; Smrž, Daniel (referee)
Membrane adaptor proteins are proteins associated with cellular membranes that do not themselves serve as receptors. Instead, they propagate or modify the signals of these receptors by recruiting other signaling and regulatory proteins and arranging them into supramolecular complexes. In this thesis, I sought to describe selected membrane adaptor proteins and their roles in inflammation and regulation of hematopoiesis in mouse models using a reverse genetics approach. The main part of the work focused on the role of the membrane adaptor protein PSTPIP2 in suppressing inflammation. In mice, missense mutations in the Pstpip2 gene causing loss of PSTPIP2 protein lead to the development of autoinflammatory disease chronic multifocal osteomyelitis (CMO) characterized by sterile inflammatory lesions in the bones and adjacent soft tissue. These mice represent a model of the human autoinflammatory disease, chronic recurrent multifocal osteomyelitis. At the molecular level, neutrophils in the absence of PSTPIP2 exhibit pathological hyperactivity of pathways regulating IL-1β and reactive oxygen species (ROS) production, which are both implicated in the etiology of the disease. PSTPIP2 interacts with several signaling regulators, including PEST family protein tyrosine phosphatases (PEST-PTPs) and inositol...
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Host immune response in cutaneous versus visceral form of leishmaniasis
Matějková, Barbora ; Leštinová, Tereza (advisor) ; Jelínková, Kristýna (referee)
Parasitic protozoans of the genus Leishmania circulate between vectors and hosts during their life cycle, in which they come into contact with the immune system. In the host body, infection can lead to the development of a disease called leishmaniasis. This can manifest itself in a number of ways, with the best known forms being referred to as visceral, mucocutaneous and cutaneous leishmaniasis. This thesis focuses on the host immune response during the cutaneous and visceral forms of leishmaniasis. In addition, attention has been given to host, vector and parasite factors that differ between cutaneous and visceral leishmaniasis forms and that may influence the different clinical manifestations. Attention is paid not only to factors related to immunity, but also to factors such as parasite inoculum dose sizes, temperature differences between skin and internal organs, parasite genetics, and others. Keywords - Leishmania, visceral leishmaniasis, cutaneous leishmaniasis, immune response, parasite, immunity
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The Role of TRPV1 in Macrophage Activation and Polarisation
Fikarová, Natálie ; Krulová, Magdaléna (advisor) ; Frič, Jan (referee)
The ability to sense painful stimuli is essential to protect the body. Up to date, the underlying molecular mechanisms are still not completely understood; therefore, the treatment of chronic inflammatory pain remains challenging. The TRPV1 channel is one of the known nociceptors mediating the sensation of burning stimuli. Its agonist is capsaicin, the pungent compound of chilli peppers. This channel has been extensively studied in neurones; however, its function in immune cells is not well understood. Especially in macrophages, data regarding the role of TRPV1 in macrophage polarisation are often contradictory. Thus, further research in this area is desired to clarify the function of TRPV1 in immune cells. This diploma thesis aims to investigate the role of TRPV1 in macrophage polarisation during the inflammatory response. In this work, macrophages were stimulated with capsaicin prior to, after or concurrently with the application of LPS to determine the effect of TRPV1 activation on the inflammatory response. The involvement of MAP kinases in signalling after TRPV1 activation by capsaicin was addressed confirming that ERK 1/2 is part of the signalling cascade. Furthermore, this work proposes that activation of TRPV1 in the context of the LPS-induced inflammatory response could lead to the switch...
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Translation potential of current preclinical techniques for gene therapy of neurological diseases in clinic. A critical review.
Žideková, Paulína ; Novák, Ondřej (advisor) ; Jendelová, Pavla (referee)
Research in the field of gene therapy has potential to become a revolutionary way to the existing treatment for a wide spectrum of neurological diseases. To treat these disorders causally, by specific substituting, deleting, silencing or editing faulty genes could be a privilege of gene therapy. The concept of translational medicine is to facilitate the transfer of working principles in preclinical research into treatment in humans. Its key issue is to overcome limitations associated with the gap between the tremendous variety molecular biology tools of preclinical research and the lack of simple corresponding options in humans. Clinical implementation of most of the preclinical approaches is still considered to be limited. The main focus of this thesis is to summarize latest advancements of molecular and genetic engineering tools that themselves or in combination have the potential to promote most preclinical gene therapy of neurological diseases to clinical use. Based on that, this study aims to suggest perspective methods of treatment for selected neurological diseases.
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Úloha Adenylát kinázy 1 v aktivaci a metabolismu imunitních buněk larev \kur{Drosophila melanogaster}
KAISLEROVÁ, Nikola
The aim of this thesis was to study the role of Adenylate kinase 1 (Ak1) in the immune system of Drosophila melanogaster larvae upon the infection by parasitoid wasp Leptopilina boulardi. Using the immune specific Ak1 RNA interference, it was analyzed the effect of Ak1 reduction on the immune response and viability of Drosophila. The importance of Ak1 was also evaluated within the metabolism of immune cells. It has been shown that Ak1 is crucial in energy metabolism of immune cells and important for the proper functioning of immune system.
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Role makrofágy produkovaného cytokinu IMPL2 v regulaci systemického metabolismu během bakteriální infekce u \kur{Drosophila melanogaster}
VARGOVÁ, Hana
The main goal of this bachelor thesis is to verify whether ImpL2, produced by activated immune cells, is responsible for changes in systemic metabolism during the immune response during the acute phase of bacterial infection in Drosophila melanogaster. The theoretical part includes previously known information about the immune system of mammals and D. melanogaster, especially about polarization and cellular metabolism of macrophages, as well as knowledge about the roles of the ImpL2 gene and its mammalian homologue Igfbp7. The practical part of the bachelor thesis deals with the role of the ImpL2 gene in the induction of changes in systemic metabolism during the immune response. The effect of the cytokine IMPL2 on selected metabolic genes in adipose tissue after Streptococcus pneumoniae infection was tested by means of macrophage-specific RNA interference. The work also clarifies whether this signaling factor has an effect on the concentration of selected metabolites in circulation and in macrophages during the acute phase of bacterial infection.
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