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The Role of T-cell Receptor in Lymphoma
Kulinich, Viktoriia ; Havránek, Ondřej (advisor) ; Alberich Jorda, Meritxell (referee)
T lymphomas are malignant tumors that could arise from the T cell of any type and developmental stage. Their clinical presentation could largely vary from indolent to very aggressive form. Its exact pathophysiology is still not completely understood; therefore, it is critical to uncover the main mechanism underlying T-lymphoma development and growth to guide rational treatment. T- cell receptor (TCR) is a critical sensor and major determinant of T-cell fate. Therefore, we aimed to assess the function of TCR in T-cell lymphomas. We hypothesized that TCR might provide tumor cells with proliferative signals even in the absence of antigen stimulation. Using model human T-cell lymphoma derived cell lines expressing TCR, we investigated consequences following the knockout (KO) of TCR and CD3. We showed that TCR KO was associated with decreased cellular growth and related changes in cell cycle, however other proliferative functions seem to be uncompromised. TCR KO was also associated with a decrease of AKT kinase activity. Our RNA sequencing-based comparison of unmodified and TCR KO cells uncovered alterations in several signaling pathways important for cell survival. Among the altered were WNT, NF-κB, Jak/STAT and others. Additionally, TCR loss was associated with defects in antigen presentation....
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Vliv expozice těžkých kovů na expresi mRNA isoforem metalothioneinu u adherentních buněčných linií
Jordanová, Lucie
Metallothionein is a small cysteine-rich protein that plays an important role in metal homeostasis and protection against heavy metal toxicity, DNA damage and oxidative stress. This bachelor thesis was conducted to investigate the effect of heavy metal exposure on the mRNA expression of metallothionein isoforms in adherent cell lines. The effect of zinc exposure in the form of ZnSO4 on the mRNA expression of MT-1A, MT-1X and MT-2A isoforms in adherent breast cancer cell lines MCF-7, MDA-MB-231 and T-47D was investigated. Using MTT viability assay, the cell viability changes at different ZnSO4 concentrations and RNA integrity is verified using denaturing agarose gel. Polymerase chain reaction combined with reverse transcription qRT-PCR is used to study mRNA levels.
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Comparison of in vitro methods for the study of cytotoxicity
Eliášová, Pavla ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavla Eliášová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Comparison of in vitro methods for the study of cytotoxicity People are exposed to a growing number of toxic substances from the environment. Endocrine disrupting chemicals (EDCs) are a broad category of molecules that are thought to cause adverse effects on the endocrine system by interfering with the synthesis, transport, degradation or action of endogenous ligands. One of the aims of this thesis was to determine the in vitro toxicity of 17 selected endocrine disruptors on the human hepatocellular carcinoma HepG2 cell line. Cell viability was determined using the CellTiter96® AQueous One Solution Cell Proliferation Assay colorimetric method, the principle of which is the reduction of MTS to the colored product formazan by mitochondria in viable cells. The cytotoxic potential of the compounds was expressed by using the toxicological parameter IC50, which was measured in three time intervals (6, 12 and 24 hours). For 14 substances: Atrazine, DHEP, Bisphenol A, Carbofuran, 3-hydroxycarbofuran, Cypermethrin, DDE, DES, MEHP, PCB 118, PCB 153, PFOA, PFOS, Propiconazole, IC50 > 100 µM (respectively > 250 µM) was...
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Determination of renal toxicity of antineoplastics in vitro
Zádrapová, Marie ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Marie Zádrapová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of renal toxicity of antineoplastics in vitro BRAF inhibitors are important antineoplastics. They work on the principle of inhibition of certain types of protein kinases and turned out to be very efficient for the treatment of melanoma. One of their disadvantages is relatively early onset of resistance; thus, it is important to look for new combinations of drugs that are already in use or work on the development of new structures with similar inhibition efficacy on melanoma cells. Encorafenib and its combination with binimetinib have been shown to be very promising drugs from the group of BRAF inhibitors, however, potential renal toxicity may be a therapeutic limitation. This thesis was focused on the determination of in vitro cytotoxicity of encorafenib on different types of renal cells in three time intervals and on its comparison with two drug standards - amphotericin B and paracetamol. Three types of morphologically and functionally different kidney cells (PODO / TERT256, HK-2 and HEK293) were used for this purpose. The cytotoxic potential was measured by colorimetric method CellTiter 96®...
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In vitro models for studying Syncytin-1-induced fusion of trophoblast cells
Jech, Lukáš ; Trejbalová, Kateřina (advisor) ; Zíková, Martina (referee)
Trophoblast cell fusion is essential for human placenta development. Apart from initiating blastocyst implantation, syncytialization is critical for optimal nutrient and gas exchange between mother and fo- etus. Multicellular syncytia called syncytiotrophoblast covers the surface of the branched structure of chorionic villi, which is in direct contact with maternal blood. Impairment of the syncytialization process leads to insufficient fetal nutrition and severe pregnancy complications. Syncytia formation is induced by the interaction of the surface glycoprotein of retrovital origin, Syncytin-1, with its receptor. Despite the significance of these processes, the details of cell fusion and trophoblast differentiation remain unk- nown. Furthermore, because of its uniqueness, the human placenta cannot be covered by animal models. As a result, research into human placental development, especially Syncytin-1-induced trophoblast cell fusion, is limited to in vitro trophoblast models. These models include primary trophoblast cell cultures and trophoblast cell lines, which can be obtained by immortalizing cell cultures or extracted from trophoblast tumours. Dedifferentiated trophoblast stem cell cultures were also established. The most recent approach, however, involves the direct reprogramming of dermal...
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Determination of toxicity using a cell model.
Fojkesová, Karolína ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Karolína Fojkesová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of toxicity using a cell model The subject of the diploma thesis was measure and evaluate the cytotoxicity in vitro of 22 newly synthesized substances with potential use for their antifungal, antibacterial and antimycobacterial activity on the standard HepG2 cell line. The tested substances were synthetized at the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University and their chemical structure is mafenide derivative. The compounds are MAF, MAF-4, MAF-8, MAF-9, MAF-10, MAF-11, MAF-12, MAF-13, MAF- 14, MAF-16, A-5-F, A-3,5-F2, A-3-Br-5-Cl, A-3,5-Cl2, A-3,5-Br2, A-5-Br, A-THIOF, A-SA, A-3,5-I2, A-5-Cl, A-3-I-5-Cl, A-5-I. Cytotoxicity has been measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay, which is a colorimetric method based on the reduction of the tetrazolium salt of MTS to the colored soluble formazan, and substances have been compared and evaluated according to the IC50 parameter. In case of 9 substances, specifically MAF, MAF-8, MAF-11, MAF-12, MAF-16, A-5- F, A-3,5-F2, A-SA, A-5-Cl, the IC50 was greater than...
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Determination of potential cytotoxic effect of experimental substances in vitro.
Osinková, Barbora ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Barbora Osinková Supervisor: RNDr. Jana Maixnerová Ph.D. Title of diploma thesis: Determination of potential cytotoxic effect of experimental substances in vitro The subject of this master's thesis was to evaluate the cytotoxic potential of newly synthesized substances in vitro in the cell model HepG2, which is cell line obtained from a human liver carcinoma. These substances were synthesized within the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University (GUAM, GUAM-NH2, THIOSEMIK, GUAM-2, GUAM-6, GUAM- 11, GUAM-12, GUAM-18, GUAM-19, GUAM-2-1, GUAM-6-3). These substances are being investigated for assumed antimicrobial activity against G+ bacteria and fungi or for their ability to inhibit cholinesterases. The commercial colorimetric method CellTiter 96® AQueous One Solution Cell Proliferation Assay was used for in vitro determination of cytotoxicity, the principle of which is to measure the metabolic activity of cells based on the reduction of the tetrazolium salt of MTS to coloured formazan. The half-maximal inhibitory concentration IC50 was used to determine the number of viable cells and for evaluation of cytotoxic potential of...
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Study of inflammation using epithelial cells
Majerová, Barbora ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The human respiratory system is in constant contact with heterogeneous agents from the environment. There must be effective mechanical lung barriers and sufficient immune protection due to continuous deposition of various substances in the respiratory system. The mutual balance between the mechanisms of natural and adaptive immunity of the lungs is essential for destruction of infectious agents without initiation of inflammatory response. Overreaction of the immune system of the lungs may lead to the production of various inflammatory mediators and cytokines such as interleukins IL-1, IL-6 and IL-18. When determining the immunogenicity of a substance, it has to be exposed to lung epithelial cells, and then the concentration of cytokines produced is measured. To determine the immunogenicity of mammalian immunoglobulin G and chicken immunoglobulin Y the subsequent twenty-four hour exposure to A549 lung cancer cell line was made. Concentration measurement of cytokines IL-1 and IL-6 was performed using Luminex method, which pointed out the immunogenicity of goat immunoglobulin G and certain chicken immunoglobulins Y.
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Interaction of bacterial lectins with human lung epithelium
Vyhnalová, Kateřina ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Recessive autosomal disease cystic fibrosis (CF) is caused by a mutation in the CFTR gene ("regulator of cystic fibrosis transmembrane conductance"), which encodes the same named chloride channel. This mutation leads to incorrect ion transport, which causes the formation of an excessively viscous mucus on the surface of the airways and subsequently to the susceptibility to bacterial diseases. This disease mainly affects the respiratory system, where infections are associated with various causes of death in patients with CF. The most common pathogen causing infections is Pseudomonas aeruginosa (PA), which uses many virulence factors, such as pili or adhesins. Lectin PA-IIL, from the group of PA adhesins, is characterized by a high affinity for L-fucose, so it contributes to the adhesion of PA to the low sialylated epithelium of CF patients. In this work the interactions between PA-IIL and lung epithelium were investigated. The cell lines CuFi-1 (CF patient) and NuLi-1 (healthy individual), which were examined ex vivo, were used. A part of these cell lines were exposed to neuraminidase. The PA-IIL lectin was isolated from the E. coli cell line pET25_PAIIL and subsequently fluorescently labeled with DyLight 488. The activity of mentioned lectin was verified by red blood cell agglutination. The...
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Determination of renal toxicity of antineoplastics in vitro
Zádrapová, Marie ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Marie Zádrapová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of renal toxicity of antineoplastics in vitro BRAF inhibitors are important antineoplastics. They work on the principle of inhibition of certain types of protein kinases and turned out to be very efficient for the treatment of melanoma. One of their disadvantages is relatively early onset of resistance; thus, it is important to look for new combinations of drugs that are already in use or work on the development of new structures with similar inhibition efficacy on melanoma cells. Encorafenib and its combination with binimetinib have been shown to be very promising drugs from the group of BRAF inhibitors, however, potential renal toxicity may be a therapeutic limitation. This thesis was focused on the determination of in vitro cytotoxicity of encorafenib on different types of renal cells in three time intervals and on its comparison with two drug standards - amphotericin B and paracetamol. Three types of morphologically and functionally different kidney cells (PODO / TERT256, HK-2 and HEK293) were used for this purpose. The cytotoxic potential was measured by colorimetric method CellTiter 96®...
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