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The effect of aspartate β-hydroxylase inhibition on immune reactions
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Filipp, Dominik (referee)
Aspartate β-hydroxylase (ASPH) is an enzyme that contributes to tumor progression by enhancing the proliferation, migration and invasiveness of cancer cells. Its expression is mostly negligible in normal cells, whereas it is often overexpressed in cancer cells. An inhibitory effect of ASPH on immune cells, specifically on natural killer cells, has also been demonstrated. Thus, the ASPH enzyme could be a new target for cancer immunotherapy. The aim of this thesis was to show the effect of ASPH inhibition on the immune response. Firstly, it was found that ASPH inhibition contributes to the anti-tumor effect of DNA vaccination. The reduction of tumors induced by ASPH inhibition in combination with DNA vaccination was shown to be mainly caused by CD8+ T lymphocytes. Subsequently, the specific activation of T lymphocytes was confirmed by the ELISPOT assay. In case of non-specific activation of T lymphocytes, ASPH inhibition had no effect on the direct activation of CD8+ T lymphocytes. Finally, it was found that plasmacytoid dendritic cells did not contribute to CD8+ T lymphocyte activation after ASPH inhibition. Thus, the results demonstrate that inhibition of ASPH contributes to the activation of adaptive immunity induced by DNA vaccination, but the mechanism of this activation remains unknown.
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Novel methods of treatment of B cell malignancies based on immunotherapy with genetically modified T cells
Novotná, Natálie ; Otáhal, Pavel (advisor) ; Šmahel, Michal (referee)
CAR T cell therapy represents a promising method in treatment of hematological malignancies. Gene immunotherapy uses modified T cells that express a chimeric antigen receptor (CAR) on their surface. Modified T lymphocytes are able to recognize and destroy target cells based on specific surface markers. Although CAR T cell therapy is used in clinical practice, there is a number of limitations that reduce its effectiveness. The aim of this thesis is to explore new possibilities of making the entire therapy more efficient through endogenous secretion of interleukins (IL-7, IL-15, IL-21) under the control of inducible promoters, and thus to strengthen the persistence and expansion of CAR T cells in vivo. For this purpose, inducible expression systems containing the gene for CAR19 receptor specifically recognizing the CD19 molecule and the interleukin gene located under inducible NFAT or NR4A promoters, were constructed. The assembled vectors were electroporated into PBMC cells using the PiggyBac transposon system to achieve stable expression in T lymphocytes. After co-cultivation with RAMOS cell line, data were obtained by measurement on a flow cytometer and the ELISA method. Based on the results, it is evident that stimulated CAR T cells are able to generate higher concentrations of interleukins,...
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Metasearch for Reviews on the Czech Web
Šmahel, Michal ; Doležal, Jan (referee) ; Smrž, Pavel (advisor)
The main purpose of this work is to create a metasearch engine for review articles with built-in sentiment analysis. In addition, a complex survey of main text extraction tools and web browser automation tools for web crawling has been carried out to achieve of the best possible results. The resulting metasearch engine provides a web interface for searching relevant review articles, thus saving time spent on manual searching. Thanks to multi-level transformer-based filtering, it can return 10—15 relevant review articles on frequently reviewed topics in about 4 minutes with no effort, just by clicking on a button.
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Role of Zinc-α2-glycoprotein in tumors
Kotvalová, Hanka ; Šmahel, Michal (advisor) ; Kubů, Martin (referee)
The Zinc-α2-glycoprotein (ZAG) encoded by the AZGP1 gene particularly contributes to the regulation of lipid and glucose metabolism. In different types of tumors, its altered production was found and the association of the ZAG level with the patient's prognosis was suggested. However, the role of ZAG in tumor progression can differ in individual malignancy types and mechanisms of ZAG function are not sufficiently identified. The objectives of the bachelor's thesis are a description of the structure of the ZAG protein and the regulation of AZGP1 expression, a summary of the identified ZAG functions in nonmalignant diseases, and a focus on the role of ZAG in tumors, particularly on the mechanisms of its action and its potential use as a biomarker.
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Post-transcriptional modification of mRNA molecules in viruses of the Poxviridae family
Jakešová, Kristýna ; Vopálenský, Václav (advisor) ; Šmahel, Michal (referee)
Post-transcriptional modifications of mRNA molecules in viruses belonging to the Poxviridae family, including the vaccinia virus, play a key role in regulating viral gene expression and also influence virus-host cell interactions. These modifications include the synthesis of a 7-methylguanosine cap, 2'-O-methylation, 3' polyadenylation of the mRNA strand, hydrolysis of the 7-methylguanosine cap, and 5' polyadenylation of the mRNA strand. Studying post-transcriptional or co-transcriptional mRNA modifications in Poxviridae viruses can contribute to a better understanding of viral pathogenesis and the development of new strategies for treating infections caused by viruses in this family. The aim of this bachelor's thesis is to summarize current knowledge of co-transcriptional or post-transcriptional mRNA modifications in Poxviridae viruses, with a special focus on vaccinia virus.
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Derivation and characterization of tumor cell lines labeled with fluorescent proteins
Majerová, Miriam ; Šmahel, Michal (advisor) ; Drbal, Karel (referee)
The effective treatment of cancer is hindered by the mechanisms of tumor cells allowing them to escape from immunosurveillance. One such mechanism is the downregulation of MHC I expression by tumor cells. As a result, CD8+ T lymphocytes are not able to eliminate tumor cells. These cells are often characterized by different expression of MHC I, which leads to the heterogeneity of the tumor environment. This thesis describes a production of a model of MHC I heterogeneity in tumors. Expression plasmids carrying genes for FP were created. Tumor cell lines TC-1, TC-1/A9 and TC-1/dB2m with different expression of MHC I molecules were successfully labeled with these plasmids. When monitoring stability of FP expression by these cell lines, a decrease was observed both in vitro and in vivo. The assumption that the cytokine environment of the tumor induces FP expression could not be confirmed because of unstable FP expression. In a tumor from a mixture of TC-1+TC-1/dB2m cell lines, it was possible to distinguish between these two lines based on the expression of β2m. In a mixture of TC- 1/A9+TC-1/dB2m lines, that could not be done due to the heterogeneity of TC-1/A9 MHC I expression. The use of combined immunotherapy showed the greatest impact on immune cell infiltration in tumors from a mixture of TC-1+...
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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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Roles of cytoskeleton in mouse polyomavirus trafficking
Klímová, Lucie ; Forstová, Jitka (advisor) ; Šmahel, Michal (referee)
6 Roles of cytoskeleton in mouse polyomavirus trafficking ABSTRACT: Mouse polyomavirus (mPyV) is small non-enveloped DNA virus. Its endocytic pathway is studied for a potential utilisation of polyomaviral virus-like particles in gene therapy and/or immunotherapy. mPyV enter cells by internalisation into smooth monopinocytic vesicles. During it's journey through the cell, it pass through early endosomes, and at the time 3 hours post infection, it is localised in endoplasmic reticulum and recycling endosomes. Many aspects of mPyV trafficking and nuclear entry are not clear yet. Time-lapse live imaging fluorescence confocal microscopy was used to describe the mouse polyomavirus intracellular movements. For these studies, we utilised mPyV fluorophore-labeled virions and cells expressing GFP-tagged g-actin or alpha-tubulin. Some virion-loaded vesicles were seen to move with actin organised into dynamic structures. Some of these structures resembled actin comets created by Listeria or vaccinia virus. At the same time post infection (40-60 min post infection), movement of the virion loaded vesicles along mirotubules was observed suggesting the simultaneous involvement of actin and tubulin during mPyV trafficking. Dynamitin, a dominant negative inhibitor of dynein-dynactin function reduced mPyV infection. Taken...
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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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Characterisation of the cell line TRAMP-C2 side population, mouse model of prostate cancer
Žlabová, Anna ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
Side population is a minor subpopulation (SP) of some cell lines, exporting staining dye Hoechst 33342 out of their cytoplasm. It is discussed as a possible source of "cancer stem cells", "tumour initiating cells" or "metastasis initiating cells". However, broad literature suggest, that stemness and other privileged properties of SP are very variable between different cell types, cell lines and stage of disease. Cell lines TRAMP are the only widely available murine models for testing of prostate cancer therapy. We noticed in literature a mention about existence of 1-2% of cells constituting side population, but detailed characteristic have not been described until now. In this diploma thesis, we worked on characterisation of SP of the TRAMP-C2 cell line in comparison to other cells (nonSP). In the first part, we compared stem properties of SP and nonSP. We started with checking the existence of SP by its verapamil sensitivity. Using mRNA analysis, we showed that neither SP nor nonSP have increased c-Kit expression and that there are no differences in Bmi-1 expression. We found that SP is heterogenic mixture of CD24-CD44-, CD24-CD44+ and CD24+CD44+ cells, while nonSP is almost solely CD24-CD44+. We documented that SP and nonSP returned back to original SP ratio during cultivation. Then we showed on...
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