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Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

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

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

Because of the profound effects of signal transduction on cell behaviour, the activity of signalling pathways must be carefully regulated. Otherwise, dysregulation of signalling might harm the organism by not responding to danger or by an excessively strong reaction. Therefore, various regulatory mechanisms became essential parts of signal transduction pathways. They affect these pathways at all levels, including ligands, receptors, signalling enzymes, adaptor proteins and other signalling mediators, as well as transcription factors further downstream. In this thesis, I present the results of the research on the role of transmembrane and membrane associated adaptor proteins LST1, SCIMP, PSTPIP2 and WBP1L in the regulation of leukocyte signalling and homeostasis. Transmembrane adaptor protein LST1 is a short protein expressed in the cells of the myeloid lineage. Observation of LST1-/- mice revealed that these animals are overall healthy without visible phenotype, with the exception of mild reductions in myeloid, NK and NKT cell populations at the steady state. On the other hand, LST1 deficiency had significant protective effect during acute colitis induced by dextran sodium sulphate, suggesting the role of LST1 in the regulation of gut inflammation. Studies on PSTPIP2 and SCIMP presented in this...

The development of autoinflammatory diseases is caused by the dysregulation of innate immune mechanisms. This leads to the development of spontaneous inflammation. Mice lacking adaptor protein PSTPIP2 develop chronic autoinflammatory osteomyelitis due to higher activity of neutrophil granulocytes and their increased production of IL-1β. .β. PSTPIP2 interacts with PEST phosphatases and kinase CSK. These proteins are impor- tant negative regulators of Src family kinases. In this diploma thesis, the role of Src family kinases and the role of their positive regulator phosphatase CD45 in the development of chronic autoinflammatory osteomyelitis was studied. For this purpose, a mouse model of chronic autoinflammatory osteomyelitis (CMO) lacking CD45 was used. These mice deve- lop the disease with delayed kinetics. Bone marrow cells isolated from these mice produce less IL-1β. upon silica activation and have lower phosphorylation of ERK MAP kinase. It isβ. probably caused by higher phosphorylation of the inhibitory tyrosine of Src family kinases resulting in their lower activity. The presence of different immune cell populations in the bone marrow, spleen and blood of these mice was also monitored in these mice. The re- sults of this work contribute to a better understanding of the role of Src family...

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...

Signaling pathways must be finely tuned to assign a signal of appropriate strength and duration to the receptor stimulation. Their dysregulation can be very harmful. The consequences of dysregulated signaling pathways vary from autoimmunity, immunodeficiency, and autoinflammation to abnormal proliferation and cancer. In my thesis I aimed to characterize the roles of kinases and membrane associated or transmembrane adaptor proteins in signaling pathways downstream of different receptors. First, I was comparing the roles of SRC family kinases (SFK) in the initiation of antigen receptor signaling in B cells and in T cells. This effort resulted in the manuscript where we re-evaluated current data, which suggested that SYK can initiate BCR signaling independently of SFK. We show that much lower SFK activity is required for the initiation of BCR signaling than for TCR signaling, but we did not find any evidence for SFK-independent signal transduction. We also found that multiple factors are responsible for setting the higher threshold for SFK activity required to initiate signaling by TCR, including differences between SYK and ZAP-70, structure of the antigen receptor itself and separation of the receptor from transmembrane adaptor LAT, which is a major hub coordinating the formation of TCR signalosome....

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

Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases

Acute myeloid leukemia (AML) is a cancer disorder of hematopoiesis, characterised by production of dysfunctional progenitor cells of myeloid cell lineage. Mutations provide malignant cells with the ability to proliferate independently of growth factors and to resist to cell death induction. In addition, transformed cells are often capable of escaping the immune system. T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was originally discovered on the surface of immune cells, where it acts as an inhibitory receptor. Later, Tim- 3 was also found on leukemia cells, but its function on this cell type is much less clear. Upon ligation with galectin-9 (Gal-9), Tim-3 acts on AML blasts as an activating receptor. The Tim- 3/Gal-9 complex signalization stimulates NF-κB, β-catenin, HIF-1, PKC and mTOR pathways, which substitute to some extent the function of growth factors. They support continuous regeneration of leukemic cells, provide stimulatory signals, and further increase the production and secretion of Tim-3 and Gal-9 in a positive feedback loop. An important function of Tim-3 is the transport and localization of Gal-9, which blocks the activation of T lymphocytes by interacting with Tim-3 on their surface, inhibits formation of immunological synapse and execution of effector...

T lymphocytes specifically recognize and promote immune response against pathogenic agents, which endanger health of host organism. However, this ability must be tightly controlled to prevent response against innocuous or self-tissue antigens, which would unnecessarily damage an organism. For a maintenance of this tolerance, there is a number of molecular mechanisms regulating T lymphocytes. The protein NDFIP1 is important component of these mechanisms. This transmembrane adaptor protein binds to E3 ubiquitin ligases from the NEDD4 family, which are under normal circumstances in inactive state. Binding of NDFIP1 leads to their activation and facilitates regulation of T cell behaviour by these ligases. NDFIP1 deficiency in mice causes the development of inflammatory disease affecting the skin, lungs and gastrointestinal tract. It is characterized by increased numbers of activated CD4+ T lymphocytes and eosinophils, which infiltrate the affected tissues. The disease is ultimately fatal. The aim of this work is to describe the function of the NDFIP1 protein in T lymphocytes, which are essential for the development of this pathology. The absence of NDFIP1 in T cells disrupts several molecular mechanisms, including the degradation of the transcription factors JUNB and RORγT and the regulation of mTOR...

Major histocompatibility complex class II (MHC-II) is a group of glycoproteins responsible for the presentation of exogenous antigens to T-lymphocytes. Besides the "classical" antigen presenting cells (APCs), numerous cell types were proven to be able to express MHC-II molecules either constitutively or under specific conditions. Often, the stimulus for MHC-II expression is interferon g, a pro- inflammatory cytokine typically activating promoter IV of the Class II Transactivator. Many of the non- classical MHC-II-expressing cells can serve as APCs, activating or attenuating T-cell proliferation depending on the expression of costimulatory molecules. Additional research identified some unusual functions of MHC-II molecules on non-classical cell types, including a role in prenatal development or mating. Modulation of the MHC-II expression could potentially serve many promising therapeutic purposes and new research can lead to deeper understanding of the topic. Keywords: MHC-II, ILC, basophils, TEC, antigen presentation, CIITA, IFN-gamma

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...

Strict regulation of the immune response is critical for appropriate protection against in- fection, preventing tissue damage, and maintaining homeostasis. A significant part of this regulation is mediated at the level of signaling pathways in which tyrosine phosphorylati- on plays a key role. It is regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTP). An important PTP expressed on all nucleated hematopoie- tic cells is the CD45. Its role has been studied primarily in T- and B-lymphocytes. There CD45 plays an important role in antigen-induced signaling and signaling triggered by other stimuli. It becomes apparent that also in neutrophils CD45 plays an importat role in many mechanisms that contribute to appropriate protection against infection. These include, for example, adhesion, extravasation, chemotaxis, phagocytosis, production of cytokines and oxidative burst. In many cases, CD45 affects these processes by regulating Src family kinases. Other means of CD45 participation in specific pathways are often not clear. This thesis summarizes our current understanding of role of CD45 in neutrophil granulocytes and its effects on the function of these cells. 1

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...

Membrane adaptor proteins are characterized by the lack of enzymatic activity and the presence of various interaction sites for other proteins and cellular membranes. They typically function as scaffolds connecting receptors or other adaptors with proximal signaling molecules at cellular membranes. Their overall effects on signaling can be activating or inhibiting depending on the nature of the effector molecules they recruit. SCIMP is one of the membrane adaptors discussed in this thesis. It is expressed in antigen- presenting cells and it has been previously shown to enhance MHCII signaling in B cells. This thesis covers the analysis of SCIMP functions beyond B cells and describes the first analysis of SCIMP deficient mice. Although the results of this analysis did not show any alterations in immune cell populations, the novel function of SCIMP in dendritic cell signaling downstream of DECTIN- 1 was uncovered. DECTIN-1 is a pattern recognition receptor involved in antifungal immunity. The data presented in this thesis describe the role of SCIMP in sustaining DECTIN-1 signaling over relatively long periods of time and the contribution of SCIMP signaling to maintaining prolonged production of pro-inflammatory cytokines. PSTPIP2 is another interesting adaptor discussed in this thesis. It is...

Tertiary lymphoid organs (TLOs) are associated with inflammatory reactions in autoimmune diseases or chronic inflammation. These organized T and B cell aggregates are structurally and functionally similar to secondary lymphoid organs (SLOs). TLOs, due to their location in peripheral tissues, enable the immune system to respond more quickly and effectively to antigens and pathogens occurring in the body and to develop adaptive immune response. It is currently debatable whether TLOs have pathogenic or protective functions. This work focuses on the postnatal emergence of these new structures, the so-called neogenesis and interaction of cells, cytokines and chemokines occurring in TLOs. And last but not least, the impact of TLOs on the transplant response. Keywords: tertiary lymphoid organs, immune system, inflammatory reaction, lymphoid neogenesis