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Identification of new regulators of proinflammatory signaling pathways
Dráberová, Helena ; Štěpánek, Ondřej (advisor) ; Krulová, Magdaléna (referee) ; Funda, David (referee)
Identification of new regulators of proinflammatory signaling pathways Helena Dráberová Protein 4.1R has been described in immune system as regulator of migration and cell adhesion, but was also shown to play a role in activation of T lymphocytes. Polymorphism in gene ORMDL-3 is associated with asthma risk in children and correlates with increased ORMDL-3 expression. This disertation thesis describes the function of proteins 4.1R and ORMDL-3 in activation of mast cells after stimulation of FcεRI receptor. IL-17 is a proinflammatory cytokine that plays a role in immune response against fungal and yeast infections. IL-17 however also plays a role in the pathology of autoimmune diseases such as reumatoid arthritis, psoriasis and multiple sclerosis. IL-17 signaling is tightly regulated, however the exact mechanism has not been described. This disertation thesis describes the IL-17R complex by mass spectrometry and analyze the function of its known and newly discovered components in cells deficient in individual proteins by method CRISPR-Cas9. Last part focuses on the discovery of new subunit of IL-17RC protein CMTM4, which role in IL-17 signaling has not been described so far. CMTM4 stabilizes IL-17RC and is required for its surface expression. In vitro data are supported by data from autoimmune model of...
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The role of mTOR complexes in immunophenotype of leukemia cells
Kořánová, Tereza ; Kuželová, Kateřina (advisor) ; Krulová, Magdaléna (referee)
Acute myeloid leukemia (AML) is a cancerous disease of hematopoiesis characterised by accumulation of immature cells (blasts) of the myeloid lineage. AML blasts utilise a range of mechanisms to escape the immune system including alteration of their metabolism or expression of inhibitory molecules. Activation of these mechanisms is not yet fully understood. One of the pathways used to regulate a great number of cellular processes is the mammalian target of rapamycin (mTOR) pathway. mTOR kinase forms two complexes, mTORC1 and mTORC2, each regulating different substrates and cellular functions. The aim of this thesis was to analyze the influence of inhibition of each of the mTOR complexes on the metabolism (oxidative phosphorylation and glycolysis) and expression of immune escape markers (HLA-I, HLA-DR, CLIP, PD-L1, TIM-3) was analysed. The inhibitor JR-AB2-011 (an mTORC2 inhibitor) reduced the mitochondrial respiration rate in the majority of the cell lines, but its impact on the cell immunophenotype was only weak. Importantly, we found that the effect on the cell metabolism did not stem from the inhibition of mTORC2. Rapamycin (an mTORC1 inhibitor) decreased both metabolic rates, as well as glucose uptake. At the same time, CLIP, PD-L1, and TIM-3 expression was reduced in all the studied cell lines,...
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Combined immunotherapy of tumors with different expression of MHC class I molecules
Piataková, Adrianna Julia ; Šmahel, Michal (advisor) ; Krulová, Magdaléna (referee) ; Reiniš, Milan (referee)
Immunotherapy experienced ups and downs before being recognized as a paramount therapy for cancer. Evidence from the latest studies revealed that the tumour microenvironment (TME) plays a decisive role in the outcome of immunotherapeutic treatment. In addition, one of the mechanisms used by cancer cells to evade immunosurveillance is reduction of the expression of major histocompatibility complex class I (MHC-I), by which cancer cells become invisible to cytotoxic T lymphocytes (CTLs). Therefore, cancer immunotherapy should involve combined strategies to target both tumour cells and TME from different sites by activating other immune cells in addition to CTLs, such as tumour-associated macrophages (TAMs). This Ph.D. thesis aimed to investigate combined immunotherapy, composed of DNA immunization, immunostimulatory compounds, and an immune checkpoint inhibitor to activate adaptive and innate immunity and inhibit immunosuppression, respectively. For this purpose, murine models related to HPV-16-induced tumours with either reversibly (TC-1/A9 cell line) or irreversibly (TC-1/dB2m) reduced MHC-I expression were used. The development of the TC-1/dB2m clone was a part of this project and this clone was obtained by deactivating the B2m gene. An important focus of the research was the analysis of TAMs isolated from...
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Genetic regulation of Leishmania infection
Sohrabi, Yahya ; Lipoldová, Marie (advisor) ; Krulová, Magdaléna (referee) ; Kolářová, Iva (referee)
6 Abstract Leishmaniasis is a neglected tropical disease, which belongs to the top health problems because it is endemic in 98 countries in Asia, Africa, the Americas and the Mediterranean region, and is gradually expanding to new areas, including Central Europe and USA. Clinical manifestations of leishmaniasis include a diverse range of forms, ranging from non-lethal cutaneous leishmaniasis to potentially lethal visceral leishmaniasis. Asymptomatic cases are known to exist in endemic areas. Different species of Leishmania induce distinct symptoms, but even the patients infected by the same species develop different symptoms and may respond differently to the treatment. Thus, one of the challenges is to explain the observed variability of leishmaniasis that cannot be attributed to the currently known factors. To find novel regulatory factors of the disease we tested molecules that were shown to play role in other infections and mapped loci controlling parasite load after L. major infection. We also determined genetic control of survival after infection with tick-borne encephalitis virus (TBEV) in order to establish whether there are common elements in response to L. major and TBEV. Interferon-induced GTPases (guanylate-binding proteins, GBPs) play an important role in inflammasome activation and mediate...
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Detection and characterization of macrophages in the tumors of viral and non-viral etiology
Dalewská, Natálie ; Tachezy, Ruth (advisor) ; Krulová, Magdaléna (referee)
Head and neck cancers are etiologically associated with smoking and alcohol consumption. Part of these tumors is induced by HPV and their incidence is increasing in the last decade. Patients with virally induced tumors have better prognosis even though they are usually diagnosed with tumors in advanced stage. One of the possible explanations may be better stimulation of the immune system by viral antigens. Macrophages are cells of the innate immune system which belong to professional phagocytes. They are called TAM upon infiltration to the tumor where they represent heterogeneous group of cells. Two main phenotypes are antitumor M1 and protumor M2 macrophages. TAMs are a major component of tumor microenvironment of many types of tumors, one of them are also head and neck cancers. In my thesis I focused on the immunohistochemical detection of M1 and M2 macrophages in the head and neck tumors of viral and non-viral etiology and at the same time RT-qPCR analyses of gene expression of macrophage-associated and/or immunosuppressive genes IDO1, ARG1, CD163, NOS2 a PTGS2 was performed. My data showed that HPV- negative tumors had higher number of M2 macrophages with typical markers CD163, ARG1 and PTGS2. It is known that patients with these tumors have worse prognosis of the disease. Due to high...
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Strong Epistasis in Genetics of Leishmaniasis - Identification of Genes and Mechanisms
Krayem, Imtissal ; Lipoldová, Marie (advisor) ; Černá, Marie (referee) ; Krulová, Magdaléna (referee)
Leishmaniasis, a disease caused by Leishmania parasites, ranks as the leading neglected tropical disease in terms of morbidity and mortality. Genotype of the infected organism is an important factor that influences susceptibility to and manifestations of this disease. To study human disease using mouse models, several strains are required, which could collectively exhibit different human pathophysiology. In the current thesis, we investigated the genetic influence on resting levels of immune cells in mice, because these resting levels could influence susceptibility to many of clinical disorders, including infectious diseases; and we performed a systematic review of the role of host genetics and cytokines in Leishmania infections. Moreover, we employed systems genetics to map genes causing susceptibility to leishmaniasis, and to identify additional mechanisms controlling response to Leishmania parasites. Also, we fine mapped the locus Leishmania major response 15 (Lmr15) in order to functionally characterize its role and to identify novel potential candidate genes regulating the response to L. major. Finally, we show a novel role of guanylate binding proteins 2b and 5 in the resistance to L. major. Strain B10.O20 carrying 3.6% of O20-derived genes on the C57BL/10 genetic background, on chromosomes...
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Effect of statin treatment on macrophage polarisation in vitro
Muffová, Barbora ; Kauerová, Soňa (advisor) ; Krulová, Magdaléna (referee)
Statins are widely used for their eminent hypolipidemic effect as anti-atherosclerotic drugs from the 90's of the 20th century. Even though there are new approaches, statins are still the first choice in the prevention of cardiovascular diseases. At the beginning of the 21st century, the anti- inflammatory effect independent of lipid-lowering properties was discovered. This diploma thesis deals with the effect of statin treatment on macrophage polarisation in vitro. Macrophages differentiated from blood monocytes were used in this thesis. The effect of statin treatment on the expression of surface markers (CD16, CD15, CD36, CD163, CD206, ABCA-1 and Trem-2) was evaluated by flow cytometry. The qPCR method was used to quantify the effect of statin treatment on the gene expression of inflammatory genes (NFκB, IL-1β, IL-6, TNFα and iNOS), anti- inflammatory genes (Arg-1, TGFβ) and genes which play a role in the adhesion and migration of monocytes and macrophages to vessel intima (VCAM-1 and MCP-1). Griess method was used to evaluate the effect of statin treatment on the inducible NO-synthase activity. Last, but not least, the effect of statin treatment on proteosynthesis of inflammatory cytokines (IL-1β, IL-6 a TNFα) and anti-inflammatory cytokine IL-10 was measured. Flow cytometry results show that...
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Mitochondrial transfer-mediated modulatory action of stem cells on immune cells
Somova, Veronika ; Krulová, Magdaléna (advisor) ; Balounová, Jana (referee)
Stem cells use different mechanisms of intercellular communication to modulate an immune response. Mitochondrial transfer is one of the mechanisms which induce metabolic changes, support cell survival, and change the phenotype of immune cells. Nevertheless, little is known about the mechanism used for transfer of mitochondria between different cell populations and the faith of mitochondria inside the acceptor cell. This thesis aims to describe the mechanism of transfer and the provided modulation. Factors that could affect mitochondrial transfer including reactive oxygen species production, apoptosis and mitochondria function were analyzed. And the impact of mitochondrial transfer on cell survival and mitophagy was described. The next aim was to compare the ability of mesenchymal stem cells (MSC) and Sertoli cells (SC) to transfer mitochondria, with MSC being more productive in the transfer of mitochondria than SC. Significant differences in the presence of mitochondria from donor MSC or SC in individual populations of immune cells were also detected. To explain these findings, the impact of reactive oxygen species on the transfer of mitochondria was analyzed in detail, although it wasn't confirmed. However, it needs to be highlighted that mitophagy plays an important role before and after...
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Importance of glycolysis and oxidative phosphorylation in the metabolism of mesenchymal stem cells
Fráňová, Markéta ; Krulová, Magdaléna (advisor) ; Rohlenová, Kateřina (referee)
Mesenchymal stem cells (MSCs) are classified as multipotent stem cells. They possess the ability to differentiate into many cell types, promote angiogenesis, increase cell survival in damaged tissue and modulate the immune response. These functions of MSCs are used in the treatment of various injuries and some diseases. This work characterizes MSCs, with a focus on their energy metabolism, specifically on the switch in their metabolic phenotype between glycolysis and oxidative phosphorylation in different states of MSCs, during cell culture and after transplantation. Finally, two modulations of MSC metabolism are presented, including cultivation in a hypoxic environment and quiescence induced by serum deprivation, which increase cell survival under the ischemic conditions that MSCs enter after transplantation. Key words: mesenchymal stem cells, metabolism, glycolysis, oxidative phosphorylation
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The role of IL-17 in kidney transplantation
Menšíková, Markéta ; Stříž, Ilja (advisor) ; Krulová, Magdaléna (referee)
The role of IL-17 in kidney transplantation - abstract Naive CD4+ T-lymphocytes (Thp) can develop into Th17 line in the presence of TGF- and IL-6. Th17 cells are characterized by expression of Ror- t and by production of interleukin-17 (IL-17). It is secreted as a glycoprotein homodimer. Binding to IL-17 receptor (IL-17R), which is present in all cell types, stimulates the production of proinflammatory cytokines and chemokines. The ratio of Th17: Treg in the graft showing signs of rejection is higher than in the graft without rejection. The presence of IL-17 in a culture of proximal tubular epithelial cells (PTEC) stimulates the production of IL-6, IL-8, MCP-1 and C3 complement component. Simultaneous action of IL-17 and CD40L synergistically increases the production of IL-6, IL-8 and RANTES. Signaling from the receptor on the surface of PTEC associated with its increased expression is effected via the src kinase and MAP kinase, and probably leads to the transcription factor NF- B. In rat models of transplantation, the IL-17 appears in allografts on the second day after surgery, the level rises until the fifth day, then decreases and disappears before the death of the animal. IL-17 is not detectable in isografts and negative controls. It appears before the IFN- , which had been considered a trigger of...
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