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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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Noncanonical functions of IL-1α
Novák, Josef ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Brdička, Tomáš (referee)
1α (IL 1α) is a multifunctional cytokine 1α is 1α independent on the receptor sig 1α is responsible for 1α to the plasma membrane. 1α activates express κB, binds to 1α 1α 1α to the plasma membrane 1α to signal 1α is required for membrane 1α exter 1α anchoring 1α 1α 1α with tumor suppressor p53 following genotoxic stress is further described in human cell 1α coloca
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Role of IL-34 in the pathogenesis of autoimmune rheumatic diseases
Reichlová, Petra ; Andrés Cerezo, Lucie (advisor) ; Brdička, Tomáš (referee)
Interleukin-34 (IL-34) is a cytokine described in 2008 using proteomic analysis. As IL-34 does not share any sequence similarity with other interleukins, it fell into the group so-called "orphan" cytokines. IL-34 binds to three receptors: CSF-1R, syndecan-1 and PTP-ζ. These interactions influence signal pathways which lead to proliferation, differenciation of monocytes or to an increased expression of other cytokines. Up-regulated expression of IL-34 has been observed in many autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögrenʼs syndrome or systemic scleroderma. These findings indicate that IL- 34 may be involved in the pathogenesis of these diseases and may become one of the diagnostic markers or even the target of medical therapy in the future. Key words: interleukin-34, cytokine, autoimmune rheumatic diseases, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, Sjögrenʼs syndrome
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Targeting IRAK4 kinase in autoimmune diseases and cancer
Synáčková, Alžběta ; Dráber, Peter (advisor) ; Brdička, Tomáš (referee)
Immune system provides host protection against invading pathogens. However, aberrant activation can lead to development of autoimmune diseases or cancer. Understanding the mechanisms of inflammation and immune responses is crucial for treatment of such conditions and reestablishing immune balance. Toll-like receptors and interleukin-1 family receptors are a key component of the innate immune system. Their downstream molecules, MyD88 and IRAK4, are essential for receptor signaling as their deficiency causes host susceptibility to infection. On the other hand, overactivation of this pathway was shown to be able to promote autoimmunity and cancer. The main focus of this text will be to summarize current knowledge about the mechanism of IRAK4 signaling and how it can be exploited in the development of therapeutics. Keywords IRAK4, MyD88, Toll-like receptors, IL-1 receptor, cytokines, autoimmunity, cancer
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Immunomodulation of dendritic cells by adenylate cyclase toxin from B. pertussis
Jáňová, Hana ; Adkins, Irena (advisor) ; Brdička, Tomáš (referee)
Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...
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Biochemická a funkční charakterizace nového transmembránového adaptorového proteinu NVL
Vonková, Ivana ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
In T cells, signalling events initiated at the immunological synapse by T cell receptor (TCR) have been already thoroughly studied. In contrast, signalling pathways involved in MHCII-mediated signal transduction on the APC side of this structure are still rather poorly understood. Transmembrane adaptor proteins (TRAPs) are a class of proteins with an essential role in signalling triggered by TCR and other immunoreceptors. So far no TRAPs involved in MHCII signalling have been identified. In this work a new TRAP, highly enriched in the immunological synapse in APCs is described and termed Nvl. Nvl is expressed exclusively in professional antigen presenting cells - B cells, monocytes/macrophages and dendritic cells, and the level of its expression positively correlates with the expression of MHCII. Nvl is present in tetraspanin microdomains and together with tetraspanins it is localized to the polarized structures of the cell, including uropod, cleavage furrow, and, most interestingly, the immunological synapse. The presence in the immunological synapse indicates possible role for Nvl in MHCII- mediated signal transduction. Indeed, Nvl becomes tyrosine-phosphorylated after stimulation via MHCII, although strong phosphorylation has also been observed after FcγR stimulation or treatment with...
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Mechanisms of MHCII signaling in B lymphocytes
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
During the initiation of an antigen-specific immune response, peptide fragments originating from the antigen are presented in complex with MHC class II glycoproteins (MHCgpII) on the surface of the antigen presenting cells (APC). Antigen recognition by T lymphocyte is accompanied by the formation of the molecular structure at the interface with APC called immunological synapse (IS). During this contact, signal transduction is initiated at both, T lymphocyte and APC, sides of the IS. For a long time it was thought that the only function of MHCgpII is presentation of antigen. However, later it was found that stimulation of MHCgpII led to triggering of signals contributing to decision about the further fate of APC. MHCgpII do not have any signaling motifs in their cytoplasmatic domains, and so associated molecules are necessary for the transduction of the signals. This work focuses on B lymphocytes in which the associated molecules are Ig alfa/beta, MPYS, CD19 and CD20. After the stimulation of MHCgpII these proteins mediate signaling events including activation of several families of protein kinases, phospholipase C, mobilization of calcium and activation of transcriptional factors NFAT and AP-1. In B lymphocytes, activities of these pathways may result in proliferation and differentiation but also in the...
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Study of the effect of mesenchymal stem cells in combination with immunosuppressive therapy on inflammatory response in in vivo model
Jabůrek, Filip ; Krulová, Magdaléna (advisor) ; Brdička, Tomáš (referee)
Immunosuppressive drugs have been used for many years for the treatment of autoimmune diseases and post-transplantation treatment. While these drugs have a lot of advantages, they also show several undesirable side effects. The most common side effects are higher blood pressure, lowered renal function and susceptibility to infections. Therefore, in recent years there has been a demand for other medical approaches that do not exhibit the above-mentioned adverse effects. Among one of the newly tested approaches is the application of mesenchymal stem cells (MSCs), which possess several advantages such as immunomodulatory abilities, safety and relatively easy isolation, however, stem cell use alone has not yet provided sufficiently strong immunomodulation. Only a small part of research of MSCs is focused on their use in the combination with immunosuppressive therapy. Therefore, in my thesis I focused on the model which allows to reduce the dose of immunosuppressive drugs in the combination with MSCs. Combined therapy is more advantageous than both monotherapies thanks to lower dosages of these drugs used. It enables to decrease negative side effects of immunosuppressive drugs, when combined with MSCs to provide sufficient immunomodulation in comparison to classical therapy. The aim of my work was to...
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Adhesion structures of leukemia cells and their regulation by Src family kinases
Obr, Adam ; Kuželová, Kateřina (advisor) ; Brdička, Tomáš (referee) ; Brábek, Jan (referee)
Adhesion signaling is a field of cell biology studied mostly on adherent cell types. However, hematopoietic cells grow in suspension, and use adhesion to the extracellular matrix (ECM) only in their early development, or - in case of differentiated cells - to perform the tasks they are specialized for. Peripheral leukemic cells are derived from more or less immature hematopoietic precursors that have, among other alterations, defects in adhesion to the bone marrow microenvironment. On the other hand, leukemic stem cells (LSC) use adhesion to the bone marrow ECM as a mean to evade chemotherapy, and are a source of the minimal residual disease, and of the disease relapses. Kinases of the Src family (SFK) are known regulators of adhesion signaling in adherent cell types, and their overexpression and/or hyperactivation is often seen in malignant diseases. They are also involved in hematooncologic disease progression and resistance to therapy, particularly in several types of leukemias. In the present work, we used a variety of methods including microimpedance measurement, fluorimetric measurement of adhered cell fraction, immunoblotting, confocal microscopy, and interference reflection microscopy. Our results indicate that active Lyn kinase, a hematopoietic SFK, is present in adhesion structures of...
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Studies on interactions between NKR-P1D and Clrb membrane receptors
Hanč, Pavel ; Novák, Petr (advisor) ; Brdička, Tomáš (referee)
Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...
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