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Function of SWI/SNF chromatin-remodeling complex in tumor initiation and progression of melanoma cells
Ondrušová, Ľubica ; Vachtenheim, Jiří (advisor) ; Goetz, Petr (referee) ; Hejnar, Jiří (referee)
There is an increasing evidence that alterations in chromatin remodeling play an important role in tumorigenesis. The SWI/SNF chromatin remodeling complexes contribute to the regulation of gene expression by altering the local chromatin structure. Depending on the context, they can act as either transcriptional activators or repressors. All SWI/SNF subcomplexes contain one of two ATPase subunits, Brm (Brahma) or Brg1 (Brahma related gene 1), which provide the energy for remodeling. Malignant melanoma is an aggressive cancer and is known for its notorious resistance to conventional anticancer therapies. MITF (microphthalmia-associated transcription factor) plays an essential role in melanoma biology and is placed on the central crossroad in the regulation of melanocyte development, differentation, maintenance of lineage identity, and survival of both normal and malignant melanocytes. Our results show that the active SWI/SNF complex is strictly required for the expression of MITF. This complex is also required for expression of some transcriptional MITF targets. The survival of melanoma cells is absolutely dependent on functional SWI/SNF complex and all subunits of this complex are expressed at high levels in melanoma cell lines. Primarily, Brg1-containing subcomplexes are more important for MITF...
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Molecular mechanisms of tumor pathogenesis of Hedgehog signaling pathway in selected tumor types
Kreisingerová, Kateřina ; Vachtenheim, Jiří (advisor) ; Macůrek, Libor (referee) ; Uldrijan, Stjepan (referee)
The presented doctoral thesis is focused on the role of the Hedgehog (HH) signaling pathway in cancer pathogenesis. HH signaling pathway is an evolutionarily conserved signaling pathway that plays an essential role in embryonic development. Its activity is strictly limited to stem and progenitor cells for example in brain, lung, skin or prostate. HH pathway also plays a key role in tissue homeostasis and regeneration. Aberrantly activated HH pathway is essential in cancer progression. The aim of the presented thesis was to elucidate new details about the HH signaling pathway. We identified a new target gene of the HH pathway - the anti-apoptotic protein survivin. Survivin is considered to be an important tumor marker associated with a poor prognosis of patients. We showed that the inhibitor of HH pathway effectors GLI1 and GLI2 GANT61 reduced the survivin level in cancer cells. Subsequently, we used GANT61 and the inhibitor of the anti-apoptotic BCL2 protein family obatoclax to inhibit melanoma cells growth. We showed that the combination of these inhibitors was very effective in the eradication of melanoma cells in vitro. We also proved that GANT61 triggers the process of apoptosis in melanoma cells. We found out that the HH signaling pathway is canonically activated in many cell lines of various...
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Study of Epithelial Mesenchymal Interactions in Squamous Epithelium Derived Tumors
Kodet, Ondřej ; Lacina, Lukáš (advisor) ; Borovanský, Jan (referee) ; Ehrmann, Jiří (referee)
This thesis is focused on the epithelial mesenchymal interactions in tumors derived from squamous epithelium including tumors arising from minor cell population (melanocytes). This study is also reflecting aspects of epithelial glycobiology resp. the study of endogenous lectins, the galectins, in head and neck squamous carcinomas. Galectins represent, in the current concepts of cell and tumor biology molecules with a remarkable potential. Galectins participate, besides in regulation of pre- and postnatal homeostasis in normal tissues, also in many pathological processes such as autoimmune reactions or malignancies. In this thesis, we demonstrated the presence of galectin-1 and -2 and their glycoligands in interphasic and mitotic nuclei, which may contribute to regulation of the cell cycle. Furthermore, we demonstrated galectin-9 as a sensitive marker of transformation normal to the dysplastic squamous epithelium in head and neck. The epithelial mesenchymal interactions represent mechanisms, which are responsible for dynamic maintenance of the homeostasis of the organism during prenatal development, postnatal growth and during cyclic renewal of certain tissues. These interactions also participate in wound healing. On the other hand they play a crucial role in the process of tumor transformation,...
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Cytokine expression in regressive melanoma on porcine MeLiM model
Miltrová, Veronika ; Skalníková, Helena (advisor) ; Krulová, Magdaléna (referee)
Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...
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Intercellular interactions in skin tumors.
Kučera, Jan ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Kovář, Marek (referee)
The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...
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Examination of the Spatial Structure of Pigs' Melanoma in Tissue Sections Based on Histology and Mass Spectrometry
Anýz, J. ; Vysloužilová, L. ; Horák, Vratislav ; Štěpánková, O. ; Vaculovič, T. ; Adam, V.
We examine the spatial structure of the melanoma in tissue sections. The pigs melanoma was examined in 10 tissue samples obtained from animals of age between 4 and 22 weeks. The tissue sections were measured by light microscopy and Laser Ablation Ion Inductively Coupled Plasma Mass Spectrometry to obtain spatial metal (Cu, Zn) distribution. The exploratory analysis of the tissue sections indicates there is clearly a pattern in the spatial structure. Different projections of the spatial structure of the melanoma are obtained by the different measurement methods. The spectral clustering on the data was utilized to describe the structure in the data. According to the clustering results, there are distinct clusters of observations in the histological data. The spatial elemental distribution of the metals Cu and Zn cannot be clustered -the data form one compact cluster. The clustering of the histological images produces clusters which are related to the annotation of the biological samples in broader terms -the differences between fibrous and cancerous tissue.
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The diagnostics of tumorous and pigmented skin lesions
TICHÁ, Lea
The skin is the largest organ of the human body. It is a protective shield against heat, light, injury, and infection, regulates body temperature, stores water and fat and serves as a sensory organ. Above all, the human health is very often reflected on the skin. Thus, there are many types of pigmented skin lesions and skin tumors that require proper diagnosis to determine. Early detection of skin cancer allows timely treatment and improves clinical outcome. The collection of equipment and methods used for diagnosing skin cancer has been growing notably over the last decades, yet histologic examination still remains a gold standard. Histopathology is the microscopic examination of biological tissues in order to observe the appearance of diseased cells in very fine microscopic detail, therefore major types and modifications of pigmented skin lesions and skin tumours can be distinguished by these methods. The theoretical part of this theses describes the main types of pigmented skin lesions and skin tumors in terms of morphology, occurrence and other important aspects. It also describes the factors that influence the formation and development of skin cancer and types of prevention against them. The methodical part is focused on the histopathology procedures, from tissue sampling to histopathological evaluation. There is also data analysis describing the incidence of skin lesions a skin cancer at five years (2013-2017) and Euro Melanoma Day 2018 based on collected skin excisions.
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Introduction testing of mutation V600E in the BRAF gene in clinical laboratory
VÍTKOVÁ, Markéta
Nowadays, skin cancer belongs among very frequent tumor diseases. It has been proven that sun and skin cancer are maximally related, and thus it may affect everyone, however, ´light-type´ people having blonde or ginger-coloured hair, light skin and high numbers of freckles are much more likely to be affected. Skin tumors are most frequently localized in body areas that are exposed to sunshine in the long term, such as eyelids, nose, neck, shoulders and hands. The most frequent types of skin cancer are the following: Basaliom, Melanoma and Spinaliom. The aim of my bachelor thesis was to acquire theoretical knowledge about skin cancer, especially about the Malignant Melanoma. The Malignant Melanoma is, unlike the other epitelial tumors, such as Basaliom or Spinaliom, not characterized by local destructive growth, but by the danger of an early haematogenic or lymphogenic metastasis. The Malignant Melanoma often metastasizes into lymph nodes, however, distant metastases may also appear in skin, lungs, the brain and gastro-intestinal tract, mainly in the small intestine. The presence of mutations may be understood to be the biological cause of the Malignant Melanoma. One of the most significant and most described mutations (in connection to the Malignant Melanoma) is the BRAF gene V600E mutation, appearing in position No. 1799 in the area of codon No. 600. Codon GTG>GAG mutation leads to the exchange of valin for glutamic acid. Activating the mutated BRAF protein is a result of conformation changes within the protein structure. The mutated V600E BRAF cells are not subject to apoptosis and aging, the result of which is an incontrollable replicational potential of the cells. The mutated V600E BRAF Melanoma then escapes the organism´s immunity response. The aim of the practical part of my bachelor thesis was to acquire the basic molecularly biological methods in a genetic laboratory. Mainly isolating the genom DNA obtained from a buccal swab as well as peripheral blood, preparing and carriyng out a PCR reaction, preparing agarose gel, and detecting PCR products using gel electrophoresis. Mutation V600E within the BRAF gene examination was carried out via the PCR-ARMS method. The principle of this method relies in using four different primers able to detect both mutated and unmutated DNA sequence during one experiment. The methodology was taken over from Huang et al. (2013) and subsequently adapted to the needs of the laboratory.
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The Role of the Tumour Microenvironment on Melanoma Cell Invasiveness
Jobe, Njainday ; Rösel, Daniel (advisor) ; Kořínek, Vladimír (referee) ; Bušek, Petr (referee)
Cancer cell invasion and metastasis are hallmarks of cancer. It is becoming apparent that the interaction between cancer cells and the surrounding microenvironment are involved in their ability to invade and metastasise. In general, cancer cells can either migrate individually, in an amoeboid or mesenchymal manner, or collectively. The first aim of this thesis was to analyse the role of NG2 in amoeboid to mesenchymal transition (AMT) and Rho/ROCK signalling. We found that NG2 promotes an amoeboid morphology, and increased invasiveness, in a Rho-dependent manner. Secondly, we analysed the role of the major tumour microenvironment (TME) component, cancer-associated fibroblasts (CAFs), on melanoma cell invasiveness. We found the CAF interaction with melanoma cells leads to increased levels of interleukin-6 (IL-6) and IL-8, and this leads to increased invasiveness. Simultaneous blocking of IL-6 and IL-8, using neutralising antibodies, inhibits CAF-dependent invasion. Further analysis of another major component in the melanoma TME, keratinocytes, has highlighted the importance of the tumour cell niche in invasion. Our results indicate that cancer cells have the ability to change morphology, and that the TME plays an important role in melanoma cell invasiveness. Metastatic melanoma treatment has proven...
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