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Molecular mechanisms of checkpoint signalling and termination
Benada, Jan ; Macůrek, Libor (advisor) ; Brábek, Jan (referee) ; Truksa, Jaroslav (referee)
Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...
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Effect of cancer-associated fibroblasts on the survival, proliferation and invasiveness of cancer cells.
Nováková, Gita ; Anděra, Ladislav (advisor) ; Brábek, Jan (referee)
Tumour microenvironment, in addition to cancer cells themselves, represents important structural and functional part of the tumour. Similarly to the normal organs tumour microenvironment comprises several cell types (fibroblasts, immune cells, endothelial cells etc.) and non-cellular components, particularly extracellular matrix. All of them form favourable conditions for the growth, proliferation, protection from the immune system- mediated destruction and nutrition of cancer cells. Cancer associated fibroblasts (CAFs) represent the most abundant cell type of tumour microenvironment. Their origin can be traced to local normal fibroblasts, endothelial cells or epithelial cells and the transition into the CAFs phenotype is influenced with several factors secreted by cancer cells (particularly TGF-β). In contrast to fibroblasts activated during wound healing newly formed cancer associated fibroblasts expressing α-SMA are not subsequently eliminated from the respektive tissue. They persist and produce a number of pro-tumorigenic factors - SDF-1, HGF, IGF-1, IL-6, VEGF, PDGF-C, TGF-β, MMPs etc. CAFs and their secreted factors target several signalling pathways enhancing basic characteristics of the tumour, so called Hallmarks of Cancer. Cancer associated fibroblasts promote proliferation and invasiveness of...
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Adhesion structures of leukemia cells and their regulation by Src family kinases
Obr, Adam ; Kuželová, Kateřina (advisor) ; Brdička, Tomáš (referee) ; Brábek, Jan (referee)
Adhesion signaling is a field of cell biology studied mostly on adherent cell types. However, hematopoietic cells grow in suspension, and use adhesion to the extracellular matrix (ECM) only in their early development, or - in case of differentiated cells - to perform the tasks they are specialized for. Peripheral leukemic cells are derived from more or less immature hematopoietic precursors that have, among other alterations, defects in adhesion to the bone marrow microenvironment. On the other hand, leukemic stem cells (LSC) use adhesion to the bone marrow ECM as a mean to evade chemotherapy, and are a source of the minimal residual disease, and of the disease relapses. Kinases of the Src family (SFK) are known regulators of adhesion signaling in adherent cell types, and their overexpression and/or hyperactivation is often seen in malignant diseases. They are also involved in hematooncologic disease progression and resistance to therapy, particularly in several types of leukemias. In the present work, we used a variety of methods including microimpedance measurement, fluorimetric measurement of adhered cell fraction, immunoblotting, confocal microscopy, and interference reflection microscopy. Our results indicate that active Lyn kinase, a hematopoietic SFK, is present in adhesion structures of...
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Proximity proteome of intramembrane serine protease RHBDL4
Boháčová, Šárka ; Stříšovský, Kvido (advisor) ; Brábek, Jan (referee)
Regulated intramembrane proteolysis is an interesting process involved in a multitude of cellular pathways. Enzymes which catalyse this are termed intramembrane proteases (IMPRs), cleaving proteins passing through the membrane within their transmembrane domain. Rhomboid proteases are serine IMPRs. They are widely distributed among organisms and evolutionarily conserved, but despite many efforts, their physiological roles are largely unexplored. RHBDL4 is a mammalian rhomboid protease localised to the endoplasmic reticulum. It is involved in the development of colorectal cancer, which makes it an important focus of research, but its physiological function is not well understood. In order to explore it, I established and employed a proximity proteomics approach, termed APEX2. It is based on biotinylation of proteins in the spatial proximity of the target in the physiological environment of intact living cells. Labelled proteins are subsequently purified, identified and quantified by mass spectrometry. Exploring the physiological vicinity of RHBDL4, its interaction partners and substrates can be revealed and the detailed subcellular compartment, where RHBDL4 resides, can thus be inferred. During three independent experiments in HCT116 cell line, three proteins emerged repeatedly in the RHBDL4...
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Clonning and characterization of the membrane microdomain targeted componets
Falteisek, Lukáš ; Černý, Jan (advisor) ; Brábek, Jan (referee)
3 Abstract Lipid rafts are defined as islets in the membranes of eucaryotic cells with different composition from the rest of the membrane. They appear spontaneously due to phase separation of different membrane components and are proposed to serve as platforms for concentration of selected signaling proteins. However, evidence for their existence is still indirect, despite more than decade of intense research. Some new approaches show that the fluctuations of membrane composition are more diverse and are caused more likely by presence of proteins than by lipid phase separation. We performed bioinformatical search looking for new signaling proteins targeted to putative rafts. We have identified several proteins and out of them phosphodiesterase 8a (PDE 8a) was salected for further research. To prepare the mouse monoclonal antibody we expressed and purified fragment of PDE 8a fused with GST. After immunisation we obtained one clone producing antibodies tentatively positive on western blot and by indirect immunofluorescence. We predicted that PDE 8a is targeted to putative rafts by N-terminal myristoylation and palmitoylation. To clarify, whether these modifications are present in the strusture of PDE 8a we prepared mutants of the PDE 8a N-terminal region lacking myristoylation, palmitoylation or both. These...
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Gold nanoparticles as a tool of targeted therapy of cancer
Knoblochová, Lucie ; Hodný, Zdeněk (advisor) ; Brábek, Jan (referee)
Nanomaterials have caught the interest of biomedical science because of their size (which enables them to interact with cellular structures), high surface area, and unique physical properties. Gold nanoparticles (GNPs) can be synthesised in various shapes. Their common property is surface plasmon resonance, which makes it possible to detect these particles with high resolution using dark field microscopy. GNPs can be efficiently modified with various ligands such as drugs, antibodies, or aptamers; this can be utilized to selectively bind GNPs to tissues, e.g. for drug delivery. Conjugated GNPs can also be used in diagnostics of tumor cells as well. Photothermal therapy consists of GNPs selectively binding to the tumor tissue, where they transform light into heat upon irradiation by near-infrared (NIR) light, thereby damaging nearby cells. The toxicity of GNPs is currently unclear. Research into modified gold nanoparticles is of great interest for targeted tumor therapy, as it may yield a tool for the selective destruction of tumor cells.
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Role of STAT3 signalling in oncogenesis and cancer therapy
Machalová, Veronika ; Hodný, Zdeněk (advisor) ; Brábek, Jan (referee)
STAT3 (Signal Transducer and Activator of Transcription 3) is considered to be one of the possible targets of cancer treatment. The ability of STAT3 constitutive activation to form tumors is a foundation of such theories. Additionally, constitutively activated STAT3 is present in many types of cancer with high occurrence, such as breast and prostate carcinoma. This protein is required in normal body cells as well. STAT3 is a transcription factor targeting many genes that are essential for the cell. STAT3 is activated by phosphorylation of its tyrosine residue and homodimerization. Proteins transcribed with help of STAT3 function in cell cycle progression, cell growth, replication, negative regulation of apoptosis, and other roles, typical for cancer. Moreover, STAT3 is modulating mitochondrial function and maintaining ROS production in mitochondria, but in form of transcriptionally inactive monomers. The purpose of this Thesis is to review known data about STAT3 in oncogenesis and by that, to show STAT3 has great potential to become the target of cancer treatment. This Thesis contains a short overview of known STAT3 inhibitors as well. Key words: Signal Transducer and Activator of Transcription 3 (STAT3), JAK/STAT3 pathway, constitutive activation, cancer, tumor, inhibitor, mitochondria, apoptosis
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