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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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Human lymphopoiesis and its examination via single-cell analysis
Novák, David ; Kalina, Tomáš (advisor) ; Drbal, Karel (referee)
Development of human B-lymphocytes is a convoluted process. A self-renewing stem cell progenitor in a primary lymphoid tissue commits to the lymphoid lineage. Subsequent B-lineage commitment entails somatic gene recombination processes which lead to the eventual expression of a surface antigen receptor. Functionality of the B-cell receptor, as well as successful testing for autoreactivity by the cell, are preconditions for the differenti- ation of a mature B-lymphocyte. Processes within this development are often investigated using single-cell analysis via flow cytometry, fluorescence-activated cell sorting and mass cytometry. Coupling these high-throughput methods with modern approaches to data analysis carries enormous potential in revealing rare cell populations and aberrant events in haematopoiesis. Keywords: B-lymphocyte, lymphopoiesis, flow cytometry, FACS, mass cytometry, clus- ter analysis, FlowSOM, PCA, t-SNE, Wanderlust.
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Immunogenic cell death in tumor specimens in the clinics
Fejfarová, Adéla ; Drbal, Karel (advisor) ; Büchler, Tomáš (referee)
Tumor development and growth are under the control of the immune system in the human body. Danger-associated molecular pattern (DAMP) molecules trigger the anti-tumor response by binding to pattern recognition receptor (PRR) on myeloid cells which in turn activate an adaptive immune system. DAMP molecules are released from cancer cells during a process of immunogenic cell death (ICD) which is a form of regulated cell death (RCD). ICD is induced by a variety of treatments in experimental settings as well as by therapeutic modalities commonly used in medicine. A typical DAMP marker of ICD is calreticulin which is translocated from the endoplasmatic reticulum to the plasma membrane attached to the CD91 receptor. Another marker is the nuclear protein HMGB1 which is released into the tumor environment at the later stage of ICD. This bachelor thesis describes a variety of detection methods and the results of DAMP externalization after ICD induction in vitro in cancer cell lines and in tumor specimens from cancer patiens. Moreover, the link between DAMP molecules and cancer patient survival is discussed. Last, it also summarizes the current status of clinical trials concerning ICD. Keywords tumor, antitumor immunity, cell death, adjuvans, DAMP, chemotherapeutics, immunogenic cell death, clinical trials
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Generation and analysis of mutant mouse model to study roles of KLKs in cutaneous inflammation
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Drbal, Karel (referee)
Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases of undisputable importance for a variety of functions, whose dysregulation has been linked to several pathological phenotypes. Among those pathologies, the Netherton syndrome stands out, since it is one of the very few that has its mechanism directly linked to KLK proteases as the main culprit of the disease, namely KLK5, KLK7 and to a lesser degree, KLK14. In this case, a mutation in the SPINK5 gene leads to uncontrolled hyperactivity of those proteases, which results in epidermal barrier breach due to excessive epidermal desquamation and severe inflammation of the skin. Inflammation mechanisms of NS are still relatively poorly understood, with important roles being attributed to the activities of KLKs in the processing of immune system molecules and also to the dysregulation of the cutaneous microbiome. TNFα signalling plays a key role in the homeostasis and immune response in the skin. Chronic skin infections may lead to deleterious effects with strong participation of TNFα signalling. To address the degree of its effects on the pathogenesis of NS, we have created a mouse model where the TNFR1 is disrupted by knockout of the Tnfr1 gene on the background of a previously established mouse model of the Netherton syndrome. We...
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Activation and regulation of cell death in senescent cancer cells.
Holíček, Peter ; Anděra, Ladislav (advisor) ; Drbal, Karel (referee)
Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...
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Immunogenicity of induced pluripotent stem cells (iPSC)
Tejklová, Tereza ; Drbal, Karel (advisor) ; Hájková, Michaela (referee)
Ectopic expression of several transcription factors into the somatic cells allows us to artificially dedifferentiate them into induced pluripotent stem cells (iPSC), which show great promise in regenerative medicine and personalized disease modelling, as well as diagnostic tools. Unique attribute of iPSC is the possibility of creating autologous cells for each patient, which could be used for transplantation without fear of immune rejection. However, cells differentiated from iPSC generally display decreased expression of MHC I glycoproteins, which leads to the activation of NK cells of innate immunity. T cells, the part of adaptive immunity, are activated after recognition of antigen peptide or foreign MHC I glycoproteins only in co-operation with costimulatory molecules, which are not usually expressed on iPSC. During dedifferentiation, cells keep the epigenetic profile of the source cell, which can result in the abnormal expression of genes within derived cell lines. Overall immunogenicity depends on the method of iPSC preparation, with respect to genomic stability. Another important factor is the immune environment of transplantation site as well as the tissue damage caused during transplantation. This results in the presentation of danger signals (DAMPs), which are then recognized by pattern...
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Immunogenic cell death
Šímová, Michaela ; Drbal, Karel (advisor) ; Javorková, Eliška (referee)
According to the danger model, the immune system is activated by endogenous molecules known as danger-associated molecular patterns (DAMP) that are externalized from the interior of a dying cell to the cell surface or released into the extracellular space. Due to the loss of plasma membrane integrity a necrotic cell death as well as several types of proinflammatory programmed cell death are considered to be immunogenic, whereas apoptosis, on contrary, has been initially defined as a tolerogenic type of cell death. However, under certain circumstances, the immune response can be initiated by an apoptotic cell after exnternalization of DAMP molecules by newly described secretory pathways. This phenomenon was observed on tumor cells as a result of some widely used therapeutic modalities and is known as immunogenic cell death (ICD). Nomenclature of selected types of cell death is part of this thesis. The aim of this bachelor thesis is to provide an evidence of the experimental support for ICD theory during in vivo initiation of the immune response. I will evaluate the correlation between ICD and the induced exposure of DAMP molecules on the surface of tumor cells or their secretion to the extracellular space.
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Detection of surface phenotype and chemosensitivity in bladder carcinoma cells in vitro
Šímová, Michaela ; Drbal, Karel (advisor) ; Vondálová Blanářová, Olga (referee)
Tumor malignancies are the second leading cause of death worldwide. One of the reasons for the failure of oncological treatment are the uniformly set clinical guidelines, which neglect the effect of high intertumoral heterogeneity. The in vitro chemosensitivity and resistance (CSRA) assays allow for the stratification of patients prior to therapy. Therefore, the CSRA are a long-considered method for personalization of components of chemotherapy regime. Nevertheless, none of them is being routinely used in clinical practice. Certain chemotherapeutics used for their cytotoxic and cytostatic effect are also able to induce so-called immunogenic cell death (ICD) of tumor cells and activate an anti-tumor immune response. Monitoring of changes in the expression of molecules associated with the regulation of the innate immune system on the surface of dying tumor cells would enable to predict the patient's ability to respond to treatment involving modern immunotherapeutics. The feasibility of CSRA using flow cytometry and microscopy is critically evaluated in this thesis on a model of bladder cancer. Simultaneously, the correlation of the immunogenic phenotype of tumor cells and their sensitivity to selected chemotherapeutics is discussed.
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Fibroblast activation protein and local immunosuppression in glioblastoma
Ternerová, Nikola ; Stollinová Šromová, Lucie (advisor) ; Drbal, Karel (referee)
Glioblastomas (GBMs) are one of the most common malignant tumors in the central nervous system. The tumor microenvironment of GBMs contains malignant and non-malignant stromal cells, whose interactions contribute to several GBMs characteristics, including aberrant angiogenesis, high proliferation rate, and systemic and local immunosuppression. Fibroblast activation protein α (FAP) is a membrane serine protease that is sparsely expressed in healthy tissues but is upregulated in solid tumors, including GBMs. FAP can be expressed by both malignant and non- malignant stromal cells in the tumor microenvironment, and its expression in stromal cells has frequently been linked to impaired anti-tumor immune response. The role of FAP and FAP expressing stromal cells in the infiltration of immune subpopulations into the GBM microenvironment is still unclear. This diploma thesis aimed to develop a flow cytometry protocol for the analysis of immune cell subpopulations present within the tumor microenvironment in wild-type and FAP knockout mouse syngeneic glioblastoma model. Four methods combining mechanical and enzymatic dissociation were evaluated for their ability to preserve cell viability and expression of studied surface molecules using mouse non-tumorous and GBM tissue. The most suitable method for mouse...
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