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MicroRNA expression in glucocorticoid-treated patients with systemic autoimmune
Uher, Martin ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...
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Radiolabeling of ramucirumab followed with the study of its internalization in vitro
Gajdoš, Jakub ; Bárta, Pavel (advisor) ; Kuchařová, Monika (referee)
v anglickém jazyce Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Bc. Jakub Gajdoš Supervisor: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: Radiolabeling of ramucirumab followed with the study of its internalization in vitro. The process of angiogenesis ensures the formation of the bloodstream at the site of its increased need. Therefore, it is not surprising that angiogenesis is often included in the tumor production process, because it provides the tumor cells nutrition supply and metabolite removal. The targeting of angiogenesis has become a key topic of some scientific research. The process of tumor blood supply formation provides a family of vascular endothelial factors (VEGFs) and their respective receptors, which have become the target of the angiogenesis attenuation in a cancer treatment. One of many therapeutics is the monoclonal antibody ramucirumab targeted against VEGF receptor type 2 (VEGFR-2). Radioactive labeling of ramucirumab with a suitable radionuclide could bring benefits in either radiotherapy or radiodiagnostics. The aim of this diploma thesis was the indirect radioactive labeling of monoclonal antibody ramucirumab using 99m Tc as radiodiagnostic nuclide via the chelation agent succinimidyl-6-...
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The influence of spinal anesthesia on the degree of DNA damage
Koščáková, Mária ; Kuchařová, Monika (advisor) ; Vokřál, Ivan (referee)
Background: The human organism is exposed daily to many endogenous and exogenous substances that are the source of oxidative damage. Cell structures, including DNA (deoxyribonucleic acid) in the nucleus are damaged due to high concentrations of these substances and accumulation of oxidative stress in cells. The predominance of these damaging processes may later be responsible for human diseases such as cancer, neurodegenerative diseases or heart failure. In our study, we observed oxidative damage at the DNA level due to spinal anesthesia. Methods: Sample processing was performed by comet analysis. The principle consists in fixation of cells (lymphocytes) in the agarose gel, lysis of cell structures for nucleotide release, incubation with specific enzymes and exposure to electrophoresis. Damaged, negatively charged parts of the DNA in the electric field are directed to the positive charged anode, creating a typical comet shape. For visualization, the gels were stained with ethidium bromide (DNA intercalating dye). Results: We have quantified single-strand breaks, oxidized purines and pyrimidines (use of enzymes to detect specific damages). The results are reported in percentage of DNA in the comet's tail. The principle is to compare the intensity of the comet's tail with the total comet intensity....
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Comparison of the effect of sphingoid bases on cell lines derived from fibroblasts and keratinocytes
Šulcová, Kristýna ; Macháček, Miloslav (advisor) ; Kuchařová, Monika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kristýna Šulcová Supervisor: RNDr. Miloslav Macháček, Ph.D. Title of diploma thesis: Comparison of the effect of sphingoid bases on cell lines derived from fibroblasts and keratinocytes The skin is the largest organ of the human body which fulfills several tasks during the whole human lifespan. Apart from the esthetic function it creates a organism cover which functions as a barrier between the inner and outer environment. In this way it contributes to homeostasis and defense of the whole organism. The skin diseases are widespread and might occur in all ages. They can affect any part of the human body. The consequence of this is a great variety of those disorders. The seriousness can range from a rash to the life-threatening conditions. Atopic dermatitis, psoriasis, scabies and acne ranks among the most common and widespread illnesses of this kind. These are mostly non-infectious illnesses which can develop even into local inflammation as affected skin is often a place of the input of various pathogens. Affected people often suffer from dry skin lesions, itched rashes, cracking crusts, blisters and peeling skin. The skin of the patients is more sensitive to outer conditions which makes their...
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Miniaturized and fast method for solubility and level of supersaturation determinations of drug nanocrystals
Paľová, Romana ; Nováková, Veronika (advisor) ; Kuchařová, Monika (referee)
Title of thesis: Miniaturized and fast method for solubility and level of supersaturation determinations of drug nanocrystals Author: Romana Paľová Department: Biophysics and Physical Chemistry Supervisor: Doc. Veronika Nováková, Ph.D. Specialized supervisor: Assoc. Prof. Leena Peltonen, Ph.D. As a formulation strategy for improving the bioavailability of poorly water-soluble drugs, drug nanocrystals can be employed. Several studies have shown that drug nanocrystals exhibit higher dissolution rate compared to conventional drug formulations. However, when orally administered, various conditions in gastrointestinal tract, such as changes in pH may result in problematic absorption. In this work, the preparation of drug nanosuspension of itraconazole (ITZ), as model poorly water-soluble compound, was carried out. The nanosuspension was prepared by using wet milling method of preparation, with suitable stabilizers added (Poloxamer 407 and Hydroxypropyl methylcellulose). Hydrochloric acid buffer pH 1.2 was chosen to create an environment similar to gastric fluids. In order to investigate changes in solubility of ITZ nanosuspension, UV spectrophotometer was used. Measurement was performed at the wavelength 551 nm, where dissolved ITZ does not absorb light and no change in absorbance occurred....
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Determination of fatty acids in human tissues
Turňová, Ivana ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Ivana Turňová Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Consultant: prof. MUDr. Zdeněk Zadák, CSc. Title of Diploma Thesis: Determination of fatty acids in human tissues The fatty acids are non-negligible component of lipids as one of the basic nutrients. This thesis in its theoretical part presents above all the group of polyunsaturated fatty acids (PUFA), which are important structural units of the cell membranes, they are also the precursors to several significant biologically active substances. In the human organism PUFA participate in many physiological and pathological processes this way, where they cause the large spectrum of actions. Onwards the thesis describes the gas chromatography method that is used in chemical analysis of lipids for the fatty acid determination as the gold standard. Experimentally, the fatty acid representation was determined in the blood, muscle, heart, liver and kidney among 26 cadavers divided into two groups according to the presence/absence of inflammatory process at the time of death. The data obtained were statistically analysed. The results were descriptively evaluated and there were indicated possible explanations of differences...
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The influence of anesthesia on the degree of DNA oxidative damage
Zubáňová, Veronika ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Background: Oxidative damage is one of the most frequent types of cell components damage leading to oxidation of lipids, proteins and the molecule of DNA. As a consequence, there is a higher occurrence of several pathologies such as atherosclerosis, neurodegenerative diseases, cancer; or diabetes. In our study, influence of whole body anesthesia during minor surgery on the level of DNA damage was examined using comet assay technique. Methods: The basic principle of this method is fixing the cells (lymphocytes) in agarose, their lysis for the removal of membranes, incubation with the specific enzymes and electrophoresis of the released cell nuclei. During the electrophoresis, free low-molecular weight and negatively charged fragments of DNA move towards anode which causes the formation of the typical comet cell shape. Finally, the gels are stained by ethidium bromide (DNA intercalating dye) and visualized. Results: We have observed single strand breakages (SSBs) and, with the use of modified assay using specific enzymes for detection of specific lesions, also oxidized purines and pyrimidines. The extent of DNA damage as determined by the intensity of the tail of the comet was quantified using LUCIA Comet Assay (Laboratory Imaging, Czech Republic) software for image analysis. The results were used...
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