National Repository of Grey Literature 61 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Tumor in vitro chemosensitivity and resistance assays (CSRA) using flow cytometry
Drozdová, Tereza ; Drbal, Karel (advisor) ; Balounová, Jana (referee)
In vitro chemosensitivity and resistance assay determine the sensitivity of a specific tumor after a specific treatment administration in an experimental setup. A heterogeneous population of cancer cells is exposed to various approved anticancer drugs in short-term ex vivo and their combination thereof. The effect of each drug is then determined based on the viability of specific tumor cells allowing for individual patient treatment using a precise combination of drugs. This approach is an example of the personalized medicine principle, which is focusing on the adjustment of diagnostic procedures and treatment of a specific patient. Therefore, its goal is to avoid treatment failure in patients with poor response to the statistically most effective treatments based on randomized clinical trials. The number of viable cells determined by the flow cytometry provides very accurate statistics for multiparametric analysis. A necessary prerequisite is the presence of dissociated cancer cells in a single cell suspension. This is different from cloning methods, where tumor colonies grow on agar media, or from histocultures, which are specific with its three-dimensional tissue cultivation. We can also sort cells from suspension based on their pre-defined attributes for their subsequent functional testing. The...
Effects of the Interferon regulatory factor 3 on immune responses to vaccinia virus in the atopic organism
Pilná, Hana ; Mělková, Zora (advisor) ; Drbal, Karel (referee)
Vaccinia virus (VACV) is an enveloped DNA virus, member of the Orthopoxviridae genus. VACV genome size is about 200 kbp. This huge genome capacity allows VACV to encode a set of factors that are non-essential for virus replication and spread in vitro. While these factors are needed for interfering with host immune responses, VACV remains strongly immunogenic. Cell-mediated and humoral immune responses in atopic disorders are deregulated to a certain extent, leading to complications in case of infection or vaccination with vaccines based on replicating viruses, such as eczema vaccinatum caused by VACV. VACV effects on immune responses consist among others in the inhibition of expression of type I interferon (IFN) at various levels - for example in a specific inhibition of phosphorylation of the interferon regulatory factor-3 (IRF-3) via inhibition of the activity of TANK-binding kinase 1 (TBK 1) that normally phosphorylates IRF-3. Phosphorylation allows IRF-3 to translocate into the nucleus where it initiates transcription of IFNβ followed by induction of expression of IFN and interferon stimulated genes. Expression of these genes is shut down when IRF-3 activity is inhibited. To overcome this block, a recombinant VACV expressing murine IRF-3 under VACV p7.5 promotor (WR-IRF3) was generated....
Cytometric assay of antigen-specific T cell response in monitoring of BCG vaccine therapy
Hadlová, Petra ; Drbal, Karel (advisor) ; Kalina, Tomáš (referee)
Bladder carcinoma (BCa) is among the most common carcinomas in the Western world. Despite the availability of effective therapies, there is currently an urgent need to develop a stratification method, which would enable the accurate identification of patients responsive to therapy. In the theoretical part of my diploma project I describe the heterogeneity of BCa and the currently applied immunotherapeutic approaches. I specifically focused on the Bacillus Calmette-Guérin (BCG) vaccine instillation. For decades another use of BCG has been a prophylactic vaccination against tuberculosis (TB) infection. BCG serves as a model treatment because it is highly efficient when prescribed to the responsive patient. However, an effective stratification is yet to be developed for BCa and latent tuberculosis infection (LTBI) diagnosis and/or monitoring. In the experimental part of my project, I developed and tested a 10-parameter panel for T cell- specific activation test (TAT) applicable for a stratification of BCa patients as well as for the detection of LTBI. I tested the panel on positive controls using flow cytometry (FCM) method because it allows for detection and measurement of dozens of markers at a single cell level. It is easily applicable to available urine and blood samples obtained from BCa...
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...
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...
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.
Microvesicle and exosome detection in immune-related diseases
Šťastná, Evelína ; Drbal, Karel (advisor) ; Fabišik, Matej (referee)
Exosomes (ES) and microvesicles (MV), collectively called extracellular vesicles (EV), are submicroscopic vesicles encapsulated by a phospholipid bilayer. Smaller ES (40 - 100 nm) originate in endosomal compartment, while larger MV (50 - 1000 nm) shed from cell plasma membrane. EV are secreted by all types of cells. They consist of lipids and proteins, but their composition varies according to the cell they originate from. In addition, they differ in the cargo they transport (DNA, RNA and proteins). They occur in every bodily fluid in much higher amounts compared to the original cells themselves, what makes them an attractive and accessible biomarker of autoimmunity diseases, cardiovascular diseases or tumours. For detection of EV, sensitive flow cytometry (FCM) is used, which I am going to compare to alternative methodologies. Part of this work will be description of EV biogenesis and then I will focus on the role of EV in coagulation and inflammation related to autoimmune diseases, more specifically in rheumatoid arthritis (RA).
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.
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...
Immunopathology aspects and new treatment options for rheumatoid arthritis
Bartoňová, Michaela ; Šenolt, Ladislav (advisor) ; Drbal, Karel (referee)
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease, which affects joints. It significantly affects the quality of life and is associated with increased mortality. Although its etiology is not fully known, it is believed that both genetic and environmental risk factors are involved in its outbreak. Knowledge of these risk factors helps us to determine the risk of RA outbreaks in vulnerable populations and provides us with knowledge about possible prevention. Pathogenesis of RA is caused by activated mesenchymal cells and cells of the innate and adaptive immune system, such as endothelial cells, synovial fibroblasts, monocytes, macrophages, dendritic cells, T and B lymphocytes. It is an incurable disease, but there are several medications, which improve disease activity or can even cause remission. Great success has been observed in biological treatment and inhibitors of Janus kinases. In some cases, the response to this relatively new therapy is not sufficient, and new drugs based on other mechanism should be developed. Examples include inhibition of granulocyte and macrophage colony stimulating factor, inhibition of interleukin 6 or interleukin 17 and bispecific antibodies. There is a growing importance of biosimilar drugs, which would make treatment, thanks to a lower price,...

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