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Regulation of intracellular calcium levels as tool to control NK cell cytotoxicity.
Graman, Vojtěch ; Frič, Jan (advisor) ; Šimíček, Michal (referee)
NK cells, a subset of innate lymphoid cells, play a crucial role in recognizing and eliminating virally infected or cancerous cells, making them a promising cell-based immunotherapy for AML patients. However, NK cell-based immunotherapies face unforeseen efficacy problems. Intracellular Ca2+ signalling was shown to play a crucial part in NK cell cytotoxicity. Maintaining the intricate balance of intracellular Ca2+ signalling is vital for NK cell-mediated target cell killing. In the complex microenvironment of the patient's body, NK cells encounter various stimuli, which can potentially disrupt the balance of intracellular Ca2+ signalling. Stimulation of PRRs was shown to affect intracellular Ca2+ , further influencing overall NK cell cytotoxicity. This study investigated the impact of TLR stimulation on Ca2+ signalling and NK cell functions. The effect of TLR stimulation was assessed using Ca2+ influx measurement, functional cytotoxicity, and degranulation assay, as well as gene expression analysis. Exposure to TLR ligands resulted in elevation of intracellular Ca2+ levels, accompanied by a reduction of cytotoxic activity at low effector-to-target ratios. An increasing trend in degranulation was observed. Furthermore, gene expression analysis unveiled upregulation of NFAT and Orai1 in NK cells...
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Alkaloid isolation from Vinca minor and their biological activity II.
Míšek, Vít ; Hošťálková, Anna (advisor) ; Chlebek, Jakub (referee)
Míšek, V.: Alkaloid isolation from Vinca minor and their biological activity II. Diploma thesis, Faculty of Pharmacy in Hradec Králové, Charles University, Department of Pharmacognosy and Pharmaceutical Botany, Hradec Králové, 2023 Supervisor: PharmDr. Anna Hošťálková, Ph.D. Key words: secondary metabolites, Apocynaceae, Vinca minor, cholinesterases, cytotoxicity, alkaloids, isolation, biological activity Alzheimer's disease (AD) is a neurodegenerative disease characterized by the progressive development of cognitive dysfunction, impact on normal daily functions and neuropsychiatric symptoms. There is a limited number of compounds for pharmacotherapy AD, moreover, AD cannot be treated causally. Therefore, research focused on the discovery of new active substances, including natural origin ones, could be one of the possible options for widening the pool of substances used in AD treatment. An extract was prepared by extracting the aerial parts of Vinca minor L. with ethanol and subsequent liquid-luquid extractin at different pH. Using column and preparative thin layer chromatography, this extract was further processed to isolate individual alkaloids. The structure of isolated alkaloids was determined by spectrometric methods (GC/MS and NMR). After comparing the obtained structures with the literature...
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Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus
Faschingbauer, Jakub ; Chlebek, Jakub (advisor) ; Šafratová, Marcela (referee)
Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....
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Study of the effect of zinc oxide nanoparticles on cell migration
Kutějová, Monika ; Zumberg, Inna (referee) ; Chmelíková, Larisa (advisor)
Zinc oxide nanoparticles (ZnO NPs) are widely used across various industries due to their properties, and with the development of nanomaterials, they are also finding applications in medicine. This bachelor’s thesis investigates the cytotoxic effects of these nanoparticles on cultured cell lines. The study focused on the human osteosarcoma cell line Saos-2 and the fibroblast cell line NIH-3T3. During in vitro experiments, the cells were exposed to different concentrations of ZnO NPs. The cytotoxicity of each concentration was assessed using the MTT assay, which provided information on cell viability. Based on the MTT assay results, appropriate concentrations of ZnO NPs were selected for conducting a Scratch assay, with the highest concentration corresponding to the determined inhibitory concentration for NIH-3T3. The cytotoxic effects were thus evaluated by studying cell migration. Subsequent statistical analysis determined the extent of cytotoxic effects of ZnO NPs depending on the concentration and cell type.
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Interaction of yeast and microalgae extracts with cell cultures
Serikbay, Adilkhan ; Holub, Jiří (referee) ; Márová, Ivana (advisor)
The objective of this bachelor thesis was to optimize the extracting of bioactive compounds from microorganisms for the purpose of characterizing and evaluating their cytotoxicity on human cell line HaCaT. During the characterization phase, extract samples were analyzed using spectrophotometric techniques. After exposing the cell line to selected extracts, a cytotoxicity test was performed to assess cell viability using MTT analysis. The theoretical part encompassed an extensive literature review focused on cultivation techniques, human cell lines, cytotoxicity analysis, and viability assessments. The practical section provided a detailed exploration of contemporary extraction techniques utilizing a wide range of both polar and non-polar solvents to enhance the efficiency of extracting active compounds from the yeast strain Rhodotorula toruloides and microalgae Chlorella vulgaris. Active compounds such as ubiquinone, ergosterol, lycopene, and torularhodin were identified in the yeast, while the microalgae were found to contain lutein, chlorophylls, as confirmed by HPLC analysis. The biocompatibility of the optimized extracts from selected strains was verified using the MTT assay on HaCaT cell lines. The aqueous extract from microalgae Chlorella vulgaris exhibited lower toxicity to HaCat cell line, with viability rates exceeding 85%. Yeast extracts from Rhodotorula toruloides initially showed higher viability rates, which, however, decreased with increasing extract concentrations.
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Characterization of cytotoxic effect of combined antimicrobial nanomaterials
Kozlíčková, Hana ; Fialová, Lenka (referee) ; Márová, Ivana (advisor)
This thesis deals with the study of the effects of combined nanomaterials on human skin cells. Pure antimicrobial substances, two types of liposomes enriched with antimicrobial substances, nanofibers with antimicrobial substances and, finally, four types of combined nanomaterials were analyzed from the point of view of cytotoxicity. The analysed active substances were eugenol, thymol, cavarcrol, curcumin, vitamin E and the antibiotics streptomycin and ampicillin. In the theoretical part of the work, the cell line of human keratinocytes, used in the experimental part of the work for cell tests, was characterized. Furthermore, individual active substances with an antimicrobial effect were described and the principles of biological effects were described, which include antimicrobial, antioxidant, cytotoxic and synergistic effects. Additionally, the theoretical part described individual nanomaterials, their preparation and usage in cosmetics and medicine. The experimental part was based on the characterization of prepared nanomaterials and on testing the influence of individual antimicrobial substances on the proliferation and viability of human HaCaT cells. Using the DLS method, the size of the prepared liposomes was measured and the effect of PHB and the type of active substance on their size was studied. MTT and LDH tests were chosen to test the cytotoxicity of individual substances. Furthermore, a scratch test was performed to monitor the effect of the investigated substances on proliferation and the rate of wound healing by cells. The last performed tests were immune response assays, in which were tested the samples for production of the human anti-inflammatory cytokines IL-6 and IL-8. In experimental part of this thesis, it was found that the prepared nanofibrous materials are safe for use in healthcare or cosmetics and, in the future, suitable to produce nanofibrous wound coverings enriched with antimicrobial substances, which can give them exceptional properties.
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Preparation and characterization of nanoparticles with encapsulated antimicrobial component
Dohnalová, Marie ; Strečanská, Paulína (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on the preparation and characterization of particles with encapsulated natural substances with antioxidant and antimicrobial properties. Selected antioxidants were anthocyanins, eugenol and vitamin E, as one of the most powerful antioxidants. At the same time, antimicrobial agents were chosen, in this case eugenol. In the theoretical part, individual active substances, materials for the preparation of particles, methods of their preparation were characterized and the principle of determining the safety of used materials. In the experimental part, selected active substances were characterized and the preparation of particles was optimized. Two types of liposomes were prepared, for which the encapsulation efficiency, size and stability were determined. Also prepared aqueous extracts of blueberries and blackberries were prepared, for which total anthocyanin content has been determined. The extracted substances from the plant material were encapsulated into alginate particles. Furthermore, the gradual release of active substances from particles into model environments using a spectrophotometer was determined. During the gradual release of active substances from the particles, the antioxidant effect was also monitored, in which it was possible to observe a decrease with the passage of time. The antimicrobial effect of active substances and liposomes against model microorganisms Escherichia coli and Micrococcus luteus was also tested. Cytotoxicity tests on human keratinocytes were performed on selected active substances, extracts and prepared nanoparticles, where it was proven that the selected materials are non-cytotoxic. Finally, a proposal for a possible application form in the form of an alginate hydrogel was created.
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Natural drugs in cancer treatment and their cytotoxicity
Hájková, Tereza ; Raudenská, Martina (referee) ; Provazník, Ivo (advisor)
The thesis deals with the natural substances in context with the cancer disease. The natural substances have a positive effect on the human organism and they are able to influence the viability and the growth of the cancer cells. The main mechanical device is to influence the mechanisms needed to start the apoptosis of the cancer cells and stopping further proliferation. The cancer cell lines utilization in the cancer disease is discussed in the thesis too. The thesis states common methods of determining the natural substances cytotoxicity. For the experimental part of the thesis it was chosen the MTT test method and the xCELLigence system for monitoring in real time. The mechanical device of the tested substance capsaicin in application on the prostate cell lines, tumorous PC3 and nontumorous PNT1A influence will be observed within the experimental part of the thesis.
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Ruthenium-based nanoparticles and testing of their anticancer activity
Žáková, Eliška ; Fohlerová, Zdenka (referee) ; Heger,, Zbyněk (advisor)
Neoplastic diseases hold the second place of the most common causes of death worldwide. Available treatments include various combinations of surgery, chemotherapy, radiation, hormone therapy, immune therapy and targeted therapy. The emphasis is currently laid on nanomedicine, where new nanosized complexes are developed and applied for the targeted treatment and chemotherapy. The aim is to significantly improve the anticancer effect and decrease the damage to organism. In this thesis, ruthenium nanoparticles with a size of 12–14 nm were synthesized and their surface modified with polyvinylpyrrolidone. Furthermore these were subsequently modified with polyoxyethylene(40)stearate for binding of doxorubicin. These nanoparticles were tested on breast carcinoma cells (MDA-MB-231), ovarian carcinoma cells (A2780) and neuroblastoma cells (UKF-NB-4). Apoptosis and necrosis testing showed 60—64 % increase in apoptosis when comparing ruthenium nanoparticles modified with doxorubicin to nonmodified ruthenium nanoparticles. The modification increased level of oxidative stress in tumorous cells and slightly a genotoxicity to non-tumorous cells, nevertheless the hemocompatibility was significantly improved. Testing has proven with IC50 0.98 g/ml, 3.91 g/ml and 1.95 g/ml higher sensitivity to these cells and confirmed expected anticancer activity. Compared to one of the most common chemotherapeutic agents cisplatin the modified ruthenium nanoparticles are significantly more toxic to cell lines A2780 (IC50=21 µg/ml), MDA-MB-231 (IC50=9 µg/ml) and UKF-NB-4 (IC50=4 µg/ml).
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Use of some microbial oils in foods and cosmetics
Požgayová, Viktória ; Szotkowski, Martin (referee) ; Márová, Ivana (advisor)
This bachelor thesis focuses on preparation of microbial oils from oleaginous yeasts and the subsequent characterization of their properties and biological effects on organisms. The thesis is divided into two main parts, theoretical and experimental part. The theoretical part provides an overview of information about microbial oils and the possibilities of their current or future applications in food and cosmetic industry. It also describes other lipophilic metabolites synthesized by carotenogenic yeasts and briefly characterizes the carotenogenic yeast strains as well as analytical methods used in the experimental part. Experimental part was focused on the process of optimization of the extraction of lipophilic active compounds from yeast biomass. The best option proved to be the combination of three solvents such as dimethyl sulfoxide, hexane, and ethanol. Using this approach, the extracts were prepared from Rhodotorula kratochvilovae, Rhodotorula mucilaginosa, Rhodosporidium toruloides, Cystofilobasidium macerans and Sporobolomyces pararoseus biomass. All of the prepared extracts were characterised based on the content of lipophilic metabolites by UV-VIS spectrophotometry and by analysis on HPLC/PDA and GC/FID. Furthermore, the antioxidant activity, SPF, antimicrobial effect, and short-term stability of these extracts were determined. The best temperature to preserve these extracts proved to be 7 °C. Three extracts with the highest SPF values were chosen and added to the prepared cosmetic emulsion which was characterized and the sensory analysis was carried out. The MTT cytotoxicity assay on human HaCaT keratinocytes was realized for the three extracts that were added to the prepared cosmetic product as well as for their combination.
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