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Cell death as a result of iron-induced cellular damage
Běhounek, Matěj ; Balušíková, Kamila (advisor) ; Truksa, Jaroslav (referee)
Iron is an essential trace element for almost all living organisms. Iron overload in cells and tissues, however, leads to their disruption. Most oftenly damaged are parenchymatic organs such as the liver, pancreas and heart. The aim of this thesis was to create cellular in vitro models for the investigation of effects of excess iron on hepatocytes and pancreatic beta cells and on these models to investigate cellular processes which lead to cellular damage during iron overload. We focused on examining the presence of oxidative and endoplasmic reticulum stress and the activation of apoptotic cell death. For our experiments, we used HEP-G2 cell line which represents human hepatocytes and NES2Y cell line which represents human pancreatic beta cells. To study the mechanisms of cellular damage during iron overload, we used two approaches by which we observed both acute and long-term effects of high levels of iron on damage of the tested cell lines. When studying the acute effect of excess iron on the cells, we applied high doses of iron (using 15 mM ferric citrate in medium) that led to the activation of cell death in hours. Long-term effects of iron overload were tested on cells regularly cultivated in the presence of 50 μM and 100 μM ferric citrate over a period of several months. Iron concentrations...
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Wnt signaling in intestinal homeostasis and tumorigenesis
Janečková, Lucie ; Kořínek, Vladimír (advisor) ; Macůrek, Libor (referee) ; Truksa, Jaroslav (referee)
The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....
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Polymeric conjugates for deliveryof cytostatic drugs and ABC transporter inhibitors in multi drug resistant tumors.
Sivák, Ladislav ; Kovář, Marek (advisor) ; Truksa, Jaroslav (referee)
Significant problem and important cause of failure of the cancer chemotherapy is that even originally very sensitive tumors become resistant to effects of cytostatic drugs. Loss of sensitivity to specific chemotherapeutics agent may not directly cause the loss of sensitivity to other chemotherapeutics. However, it have been described that tumors resistant to one type of chemotherapeutics were found to be resistant to several other anticancer drugs that are different in both structure and mode of action. This phenomenon has been described as multidrug resistance (MDR). MDR can develop in several ways, with the predominant mechanism being the overexpression of ATP- binding cassette (ABC) transporters, such as P-glycoprotein. These transporters acts as energy driven pumps and which, maintain intracellular drug concentration below toxic levels. The aim of this study was to examine the potential of novel polymeric therapeutics based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing either anticancer drug, inhibitor of ABC transporters or both, for overcoming MDR mediated by P-glycoprotein in doxorubicin (Dox) resistant murine monocytic leukemia cell line P388/MDR. Series of low-molecular weight inhibitors reversin 121 (R121), reversin 205 (R205) and ritonavir (RIT) and their derivatives...
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Charakterizace vlivu senescence na indukci a regulaci smrti nádorových buněk
Nováková, Gita ; Anděra, Ladislav (advisor) ; Truksa, Jaroslav (referee)
4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....
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Expression and regulation of the ABC transporters in tumour cells
Tomková, Veronika ; Truksa, Jaroslav (advisor) ; Němcová, Vlasta (referee)
Estrogen signalling pathway plays crucial role in carcinogenesis of breast cancer. Estrogen receptor (ER) is a prototypical hormone receptor that upon binding its ligand, estradiol, translocates into the nucleus and turns on target genes related to cellular proliferation and survival. Although estrogen signalling physiologically supports normal breast tissue development, deregulations of this pathway contribute to development of breast tumours that are estrogen receptor dependent. One of the main obstacles in breast cancer treatment is acquired resistance to common anticancer drugs also known as multidrug resistance (MDR). The switch between chemotherapy responsive to chemotherapy resistant cell phenotype is usually accompanied by increased expression of ABC transporters, special membrane proteins responsible for export of various kinds of commonly used anticancer drugs from the intracellular to extracellular space and is also linked to the existence of cancer stem cells (CSCs). ABC transporters can not only export chemotherapeutic drugs but may modulate tumour microenvironment through the transport of endogenous intracellular substrates such as leukotrienes (LTs), sphingolipids and prostaglandins PGs). This function may also play important role in carcinogenesis. The aim of the thesis was to...
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The role of autophagy in apoptosis induction by fatty acids in pancreatic beta cells.
Žigová, Ivana ; Němcová, Vlasta (advisor) ; Truksa, Jaroslav (referee)
Type 2 diabetes mellitus represents a metabolic disease reaching epidemic dimensions in the 21st century. Fatty acid-induced apoptosis of pancreatic β-cells significantly contributes to its pathogenesis. Saturated fatty acids (FAs) are strongly cytotoxic for β-cells, whereas unsaturated FAs are well tolerable by β-cells, they are even able to inhibit proapoptotic effects of saturated FAs when co-incubated. According to recent studies, FAs-induced apoptosis in pancreatic β-cells is partly regulated by autophagy, a catabolic process involved in the degradation and recyclation of cell components in lysosomes. The aim of this diploma thesis was to contribute to the clarification of the role of autophagy in FAs-induced apoptosis regulation. We induced apoptosis in human pancreatic β- cell line NES2Y by 1 mM stearic acid (SA) and inhibited it with 0.2 mM oleic acid (OA) co- incubated with SA. We revealed, that the saturated SA used in apoptosis-inducing concentration simultaneously inhibits the autophagic flux in pancreatic NES2Y cell line. When SA is co- incubated with unsaturated OA in concentration sufficient for inhibition of proapoptotic effect of SA, OA is also able to inhibit the block of autophagy induced by the effect of SA. Application of unsaturated OA alone in this concentration did not...
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Mechanism, regulation and use of TRAIL-induced apoptosis in cancer cells
Horová, Vladimíra ; Anděra, Ladislav (advisor) ; Truksa, Jaroslav (referee) ; Živný, Jan (referee)
Tumour necrosis factor-Related Apoptosis Inducing Ligand (TRAIL), a membrane- bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors (DRs). However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. In the first paper we focused on the influence of endosomal acidification. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A (CCA) we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pre- treated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL-receptor complexes were temporary attenuated. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. NF-κB or MAP kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization, mitochondrial amplification loop...
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Determining the expression of iron transport and metabolism molecules in chosen chronic diseases.
Chmelíková, Jitka ; Kovář, Jan (advisor) ; Truksa, Jaroslav (referee)
Iron is an essential element for human organism, because it cooperates as a cofactor of enzymes in many metabolic pathways. Iron is a component of hemoglobin, and thus it is indispensable for the oxygen transport to tissues. It can exist as a ferrous or ferric form. However, ferrous iron paticipates in reactions in which highly reactive hydroxyl group can be formed. This product is harmful for the organism. Non-heme iron is taken up to the circulation through duodenal enterocyte. Iron excretion is carried out only by desquamation of the enterocytes or by bleeding. Therefore, iron intake must be strictly regulated. Iron overloading is observed in some chronic diseases (hereditary hemochromatosis, alcohol liver disease). In contrary, iron depletion can be a case of iron deficiency anemia. The aim of this master thesis is to determine the expression of iron transport molecules in duodenum in chronic diseases which originate due to disturbances of iron intake regulation. We determine the expression of molecules of iron transport (DMT1, Dcytb, ferroportin, hephaestin) on mRNA level by qPCR and on protein level by western blot. The level of serum hepcidin was determined by ELISA. Our results show an increased expression of mRNA of transporters DMT1 and ferroportin as well as ferrireductase Dcytb and ferroxidase...
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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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