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Study of Anticancer Effect of New Low-molecular-weight PIKK Inhibitors
Kulhavá, Monika ; Řezáčová, Martina (advisor) ; Pejchal, Jaroslav (referee) ; Vinklárek, Jaromír (referee)
Conventional radiotherapy and chemotherapy interfere with the basic functions of dividing cells and are very effective againts rapidly proliferating tumor cells. They are still an indispensable tool in the fight against cancer, but their interventions also affect healthy cells and cause a number of side effects. Due to the instability of the gemone of tumor cells and frequent mutations, increased aggressiveness and resistence to treatment can occur. Tumor resistence to treatment is a very serious problem. There are a number of mechanisms by which cells defend themselves. One of the fundamental causes of resistence is the natural ability of all cells to repair damaged DNA. On the one hand, this is indispensable for cell life. On the other hand, it reduces the effect of chemotherapy or radiotherapy, which are based on the principle of causing DNA lesions. Small interventions in the signaling pathways of the cell's repair system can contribute to increasing the effectiveness of cytotoxic treatment. DNA-PK is serine/threonine kinase that is activated by DNA double-strand breaks which are considered the most lethal type of damage caused by radiation and some chemotherapy drugs. Activation of this kinase triggers signaling cascades that can lead to DNA repair, cell cycle arrest, or apoptosis. By...
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Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Řezáčová, Martina (referee) ; Mosinger, Jiří (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biochemical sciences Candidate Mgr. Miloslav Macháček Supervisor doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer. Photodynamic therapy (PDT) of cancer is non-invasive treatment modality for solid tumour treatment using three basic components - molecular oxygen, light and photosensitizer (PS). These elements are essentially non-toxic on their own, but in the combination they induce reactive oxygen species (ROS; singlet oxygen mainly) production, causing damage to cellular components and subsequent cell death. Type of cell demise is dependent mainly on the type of PS, length of irradiation and subcellular localization of the drug. Singlet oxygen is highly reactive and is capable of limited diffusion in biological environment. Apart from direct cytotoxic effect, vascular shutdown (oxygen and nutrition deprivation) and activation of immune system are involved in tumour eradication. Combination of effective compound with delivery system, conjugation with targeting substances or synthesis of highly effective non-aggregating water soluble compounds are the main pathways in design of modern PSs. In our...
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Alterations in protein expression induced by valproic acid in human T-lymphocyte cell line MOLT-4
Tichá, Zuzana ; Kvasničková, Eva (advisor) ; Řezáčová, Martina (referee)
Univerzita Karlova Farmaceutická fakulta Katedra biochemických věd EFFECT OF VALPROIC ACID (VA) ON CHANGES IN EXPRESSION OF PROTEINS OF HUMAN T-LYMPHOCYTE LEUKEMIA CELL LINE MOLT-4 Valproic acid (VA) is histondeacetylase inhibitor (HDACI) and it leads to inhibition in proliferation and apoptosis induction during incubation with leukemia cells. The aim of our article was to determine the changes in expression of proteins after incubation leukemia cells with VA. After 4-hours-long incubation of MOLT-4 cells with 2 and 4 mmol/l VA, the nuclear extracts from MOLT-4 cells were used to detection of changes in levels of proteins. The nuclear extracts were separated for first dimension-IEF in immobilized pH 4-7 gradient. Immediately after being focused, the proteins were separated for electophoretic way in the second dimension, 9-16% gradient DS was used. Proteins separated by 2D electrophoresis were visualized by silver staining , and silver-stained gels were digitized using a laser personal densitometer. Protein maps were quantified using a Melanie 3 software package. Proteins were identified by MALDI mass spectrometer. The finding protein process was finished by using ProFound and PeptIdent programs. Identification process of protein spots exhibiting significant abundance alterations induced by VA help to...
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Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Řezáčová, Martina (referee) ; Mosinger, Jiří (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biochemical sciences Candidate Mgr. Miloslav Macháček Supervisor doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer. Photodynamic therapy (PDT) of cancer is non-invasive treatment modality for solid tumour treatment using three basic components - molecular oxygen, light and photosensitizer (PS). These elements are essentially non-toxic on their own, but in the combination they induce reactive oxygen species (ROS; singlet oxygen mainly) production, causing damage to cellular components and subsequent cell death. Type of cell demise is dependent mainly on the type of PS, length of irradiation and subcellular localization of the drug. Singlet oxygen is highly reactive and is capable of limited diffusion in biological environment. Apart from direct cytotoxic effect, vascular shutdown (oxygen and nutrition deprivation) and activation of immune system are involved in tumour eradication. Combination of effective compound with delivery system, conjugation with targeting substances or synthesis of highly effective non-aggregating water soluble compounds are the main pathways in design of modern PSs. In our...
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Possibilities of flow cytometry in analysis of cellular response to genotoxic stress
Havelek, Radim ; Řezáčová, Martina (advisor) ; Bílková, Zuzana (referee) ; Štětina, Rudolf (referee)
Possibilities of flow cytometry in analysis of cellular response to genotoxic stress Flow cytometry is a modern tool for interrogating the immunophenotype and functional characteristics of cells. It is a technique of quantitative single cell analysis that works by sensing optical properties of cells in a flow stream with laser beam. Analysis and differentiation of the cells is based on size, granularity, and whether the cell is carrying fluorescent molecules in the form of either conjugated antibodies or dyes. The cells may be stained with fluorescent dyes, incubated with fluorogenic substrate or labelled with fluorochrome-linked antibodies specific for molecules either on the surface or in the intracellular components of the cell. Flow cytometers are multiparameter, recording several measurements on each cell. Therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population of cells. When cells are exposed to genotoxic agent, they respond quantitatively and qualitatively according to the absorbed dose and the cell type. To cope with the resulting damage to cellular DNA, the temporary cell-cycle checkpoints and DNA damage response mechanisms are activated to allow more time for effective repair. However, if these mechanisms fail or the damage is irreparable, then...
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Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Řezáčová, Martina (referee) ; Mosinger, Jiří (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biochemical sciences Candidate Mgr. Miloslav Macháček Supervisor doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer. Photodynamic therapy (PDT) of cancer is non-invasive treatment modality for solid tumour treatment using three basic components - molecular oxygen, light and photosensitizer (PS). These elements are essentially non-toxic on their own, but in the combination they induce reactive oxygen species (ROS; singlet oxygen mainly) production, causing damage to cellular components and subsequent cell death. Type of cell demise is dependent mainly on the type of PS, length of irradiation and subcellular localization of the drug. Singlet oxygen is highly reactive and is capable of limited diffusion in biological environment. Apart from direct cytotoxic effect, vascular shutdown (oxygen and nutrition deprivation) and activation of immune system are involved in tumour eradication. Combination of effective compound with delivery system, conjugation with targeting substances or synthesis of highly effective non-aggregating water soluble compounds are the main pathways in design of modern PSs. In our...
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Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Řezáčová, Martina (referee) ; Mosinger, Jiří (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biochemical sciences Candidate Mgr. Miloslav Macháček Supervisor doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer. Photodynamic therapy (PDT) of cancer is non-invasive treatment modality for solid tumour treatment using three basic components - molecular oxygen, light and photosensitizer (PS). These elements are essentially non-toxic on their own, but in the combination they induce reactive oxygen species (ROS; singlet oxygen mainly) production, causing damage to cellular components and subsequent cell death. Type of cell demise is dependent mainly on the type of PS, length of irradiation and subcellular localization of the drug. Singlet oxygen is highly reactive and is capable of limited diffusion in biological environment. Apart from direct cytotoxic effect, vascular shutdown (oxygen and nutrition deprivation) and activation of immune system are involved in tumour eradication. Combination of effective compound with delivery system, conjugation with targeting substances or synthesis of highly effective non-aggregating water soluble compounds are the main pathways in design of modern PSs. In our...
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The molecular mechanisms of stem cells response to cytostatic treatment
Seifrtová, Martina ; Řezáčová, Martina (advisor) ; Vávrová, Jiřina (referee) ; Skálová, Lenka (referee)
The molecular mechanisms of stem cells response to cytostatic treatment Apoptosis has been suggested as a common mechanism by which the cells are eradicated during chemotherapy. Most studies have focused on the reaction to DNA damage in tumor cell lines, but it is obvious that not only the tumour cells, but also normal cells are exposed to DNA-damage inducing agents during an anticancer treatment. Within this study we focus on the reaction of stem cells which are an important component of tissues and represent promising tool for a tissue engineering. Before their use, however, their reaction to the DNA damage caused by various genotoxic stressors requires elucidation and characterization of these cells under genotoxic stress contributes to the evaluation of their safety usage. In this study we assessed the effects of the frequently used chemotherapeutic agents mitoxantrone (MTX) and cisplatin (CP) on mesenchymal stem cells isolated from a dental pulp (DPSCs) and compared it with the response of human dermal fibroblasts (HDFs). Our data indicate that both cell types react to MTX and CP by the induction of apoptosis as well as by a cell cycle arrest in a dose-dependent manner. DPSCs and HDFs treated with clinically relevant concentrations of MTX underwent a permanent cell cycle arrest called...
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Possibilities of flow cytometry in analysis of cellular response to genotoxic stress
Havelek, Radim ; Řezáčová, Martina (advisor) ; Bílková, Zuzana (referee) ; Štětina, Rudolf (referee)
Possibilities of flow cytometry in analysis of cellular response to genotoxic stress Flow cytometry is a modern tool for interrogating the immunophenotype and functional characteristics of cells. It is a technique of quantitative single cell analysis that works by sensing optical properties of cells in a flow stream with laser beam. Analysis and differentiation of the cells is based on size, granularity, and whether the cell is carrying fluorescent molecules in the form of either conjugated antibodies or dyes. The cells may be stained with fluorescent dyes, incubated with fluorogenic substrate or labelled with fluorochrome-linked antibodies specific for molecules either on the surface or in the intracellular components of the cell. Flow cytometers are multiparameter, recording several measurements on each cell. Therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population of cells. When cells are exposed to genotoxic agent, they respond quantitatively and qualitatively according to the absorbed dose and the cell type. To cope with the resulting damage to cellular DNA, the temporary cell-cycle checkpoints and DNA damage response mechanisms are activated to allow more time for effective repair. However, if these mechanisms fail or the damage is irreparable, then...
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