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Study of the Influence of Faradaic Processes on the Efficiency of Yeast Electroporation
Martinů, Dominik ; Krčma, František (referee) ; Ehlich, Jiří (advisor)
Electroporation is a widely used method in biotechnology and healthcare. It involves the application of short, intense electric pulses, which create pores in the cell membrane. These pores allow the exchange of molecules between the electrolyte and the intracellular environment. Although electroporation is a standard technique, its exact mechanism remains unknown and is the subject of current research. It is assumed that the primary mechanism of electroporation is the effect of the electric field itself. Recent studies, however, suggest that one of the accompanying mechanisms may also be the effect of reactive oxygen species (ROS) and generally the products of faradaic processes. These highly oxidizing molecules can influence both the efficiency of electroporation and cell viability. The hypothesis was that by anodizing electroporation electrodes, i.e., creating a thin dielectric layer on their surface, the production of ROS can be prevented. By subsequently comparing the efficiency of electroporation using prepared capacitive electrodes and classic faradaic electrodes, it would be possible to reveal the effect of ROS on electroporation. The production of H2O2, as a representative of ROS, was mapped using standard electroporation buffers, protocols, and various electrode materials. The concentration of H2O2 was determined spectrophotometrically by its reaction with 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by horseradish peroxidase (HRP). Electrodes were selected to achieve low, medium, and high production of H2O2. These selected electrodes were used for in vitro electroporation of Saccharomyces cerevisiae yeast, with the efficiency of permeabilization determined by electrochemical impedance spectroscopy (EIS) and fluorescence measurement of propidium iodide (PI) intake. It was found that the action of ROS is not the primary mechanism of pore formation in the cell membrane during electroporation and does not significantly affect the efficiency of this method. However, the results do not exclude their role in secondary processes that affect the kinetics of pore closure and cell viability after electroporation.
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Characterization and application of microwave plasma on wound healing
Truchlá, Darina ; Němcová, Andrea (referee) ; Krčma, František (advisor)
Non-thermal plasma has a lot of ways for using in nowadays medicine. It presents many useful actions like charged particles, UV light, electric field, radicals, excited atoms and molecules. That complicated chemistry directs to uncountable synergistic interaction between cold plasma and biological systems, involve cells and tissues. This thesis is about effects of cold plasma to wound healing. Two different microwave plasma systems were used for the presented study. The first one was argon plasma torch generated by surface wave using the quartz capillary, the second one was plasma torch with reverse vortex argon flow. Diagnostics of plasma jet by optical emission spectroscopy shown the presence of active particles, which are responsible for a lot of impact of plasma treatment. Concentrations of active particles generated by plasma are dependent on conditions of plasma generation like power of generator and gas flow. For visual evidence of effects on skin caused by active particles was created simulation of skin tissue. Interaction between plasma jet and artificial skin tissue shown that UV light and temperature are not responsible for all observed effects which are noticed after plasma treatment. Some part of experiments was realized in collaboration with Medical University of Sofia in Bulgaria. The theory of positive effect to wound healing was supported by experiments based on treating artificially created wounds on laboratory mice by cold plasma. It was proved, that process of wound healing is significantly shorter after using plasma treatment in comparison with normal wound healing. Plasma treating of wound for 10 seconds in two consequent days seems like more effectively than application of plasma only one day. This Thesis was carried out as a part of international project PLASMABORDER that was supported by European commission under cohesion funds; programme INTEREG SK-CZ under contract No. 304011P709.
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Influence of electrical pulses on human blood phagocytes
Chorvátová, Michaela ; Číž,, Milan (referee) ; Kubala,, Lukáš (advisor)
The phagocytic cells circulating in the bloodstream play a key role in both the defense of the body and the pathology of inflammatory diseases. Thus, targeting their functions has potential to modulate an immune response, especially during the inflammatory phase. This master's thesis was focused on the influence of electric pulses on the most abundant phagocyte population in human peripheral blood, namely neutrophils. The theoretical part describes the role of neutrophils in the development of the immune response and the effects of the electric field on various cells. Consequent part of the thesis was the optimization of the electrical stimulation of neutrophils using a unique platform with a network of gold electrodes. In stimulated cells by electrical pulses, activation of selected signaling pathways, degranulation, ROS production, citrullination of histone H3 and expression of surface markers were monitored. Overall, electrical stimulation was observed to induce neutrophil activation but only electrical pulses of size 1 V were found to be statistically significant in the case of ROS production and 10 mV and 100 mV electrical pulses in the case of metalloproteinase MMP8 degranulation. The absence of significant effects in the most observed parameters was probably due to unwanted activation of neutrophils in control samples.
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Influence of oxidative pressure on bacterial cells
Dugová, Hana ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the impact of oxidative pressure on the bacterial strain Cupriavidus necator in its two forms: Cupriavidus necator H16 producing PHB granules and Cupriavidus necator PHB-4 as a mutant that is not capable of producing granules. The thesis compares different influences causing oxidative stress that is demonstrated by the occurrence of ROS in the cell. Furthermore, the cells were analysed with a flow cytometer and a fluorescence microscope. During the analysis it was necessary to use different types of fluorescence probes. The oxidative stress was created by means of hydrogen peroxide at various concentrations. Further tests focused on Fenton’s reaction including ammonium iron sulfate and cobalt chloride hexahydrate. Propidium iodide as a fluorescence probe was used to determine the viability of the bacterial cells, and BODIPY was tested as a lipophilic dye. Finally, the ROS in the cell was detected by H2DCFDA and CM–H2DCFDA, fluorescence probes that had to be optimised for the respective technique. The result of this bachelor thesis is the confirmation of the hypothesis that PHA granules production increase the resistance of Cupriavidus necator against oxidative stress.
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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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Redox signaling to chromatin
Plšková, Zuzana
Chromatin is a highly dynamic structure, which is constantly subjected to regulation in response to environmental conditions. This response is mediated by chromatin modifiers, such as histone acetyltransferases, which deposit histone post-translational modifications, resulting in changes in gene expression. One of such modifiers in plants is GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5). Despite its importance in plant growth and development, it remains poorly understood how GCN5 is regulated. Emergine evidence points towards redox regulation of chromatin remodeling that can be executed via redox modifications of chromatin modifiers. To investigate possible redox regulation of GCN5, we used redox insensitive lines, carrying GCN5 with mutated cysteine to serine. Even though gcn5 mutants displayed enhanced susceptibility to paraquat-induced oxidative stress, the mutated lines phenocopied the wild type. We further probed the interactome of GCN5 to identify putative functional partners, whose association with GCN5 could be altered under oxidative stress conditions, or affected by its redox status. Mutating of a single cysteine residue in GCN5 did not result in significant changes of its interactome, suggesting that additional single and higher order mutants need to be explored.
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Dissecting the effects of salicylic acid on redox balance in plant cells using biochemical and fluorescent imaging techniques
Růžičková, Gabriela ; Burketová, Lenka (advisor) ; Janda, Martin (referee)
Topic of bachelor thesis is plant immunity, specifically it focuses on salicylic acid, reactive oxygen species, it also analyses hox salicylic acid effects formation of reactive oxygen species and which proteins are involved in this biosynthesis. Also in this thesis is described basic methods for measuring of reactive oxygen species, what are advantages, disadvantages, how they work and what they can be used for. Salicylic acid is one of phytohormones involved in immune reaction in plant defence, this thesis is describing salicylic acid signalling, associated proteins and overall effect on plant. Reactive oxygen species have wide framework of action in organisms, they can be called double-edged sword, they can help plant, but also, they can harm depending on their concentration and regulation of their forming, the problem of their quenching is also described in this thesis - their formation, antioxidants, effect on plant immunity. Chapter about measuring methods is divide into three parts - fluorescence methods, chemiluminescence methods and spectrophotometric (histochemical) methods. Key words: salicylic acid, reactive oxygen species, plant immunity, plant stress, phytohormones
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Comprehensive analysis of putrescine metabolism in A thaliana using GWAS, genetic risk score, metabolic modelling and data mining
Schwarzerová, Jana ; Bartoň, Vojtěch ; Walther, Dirk ; Weckwerth, Wolfram
Polyamines are known to be functionally involvedin plant responses to stress conditions. Despite a large amount ofphysiological and genetic data, the mode of action of polyamines,especially putrescine, at the molecular level still remains unclear.An increasing number of studies point to a role of putrescine instress defense of plants due to influencing the formation of reactiveoxygen species (ROS) in cellular stress conditions. Moreover,putrescine has been found to modulate abscisic acid (ABA)biosynthesis at the transcriptional level in response to lowtemperature, revealing a novel mode of action of polyamines asregulators of hormone biosynthesis. This study presents a newholistic approach towards the analysis of putrescine metabolismusing genome-wide association studies and the calculation ofgenetic risk scores extended by Boolean analysis in metabolitesnetwork as well as data mining from available databases andliterature. It can lead to a better understanding of biologicalprocesses involved in adaptation of plants to environmentalchanges.
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Influence of plasma activated water on fungal diseases
Moskvina, Anastasia ; Krčma, František (referee) ; Kozáková, Zdenka (advisor)
This bachelor thesis focuses on influence of plasma treated water on fungal diseases. The theoretical part is dedicated to plasma-liquid interactions, properties and application of plasma activated water and its influence on different microorganisms. Plasma activated water contains reactive oxygen species which cause inactivation of living cells, making it a potential sterilizer. The experimental part of this work compares the effectiveness of three plasma treated water preparation techniques. Dielectric barrier discharge system was used to activate water above its surface. For the under the surface activation, a two-electrode system of both alternating and direct current was used. Plasma treated water was then used to prepare a suspended mixture with Aspergillus niger spores. The latter was then cultivated on agar plates for 72 hours. The overall effect was evaluated in amounts of colony forming units. The experimentally obtained data was processed and discussed in the results and discussion section of the thesis. It was found that the underwater surface activation had more significant effect on mold deactivation, which corresponds with the information contained in the theoretical part. Although the effect of dielectric barrier discharge was not as promising, all three methods used led to a decrease of colony forming units in comparison to the non-treated control sample.
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Role of oxidative stress in male infertility.
Dolečková, Barbora ; Tlapáková, Tereza (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is a phenomenon caused by an excess of reactive oxygen species (ROS), or by insufficient activity of antioxidants, that reduces these ROS levels and thereby protect the organism from oxidative damage. ROS have two types of origin: endogenous, which includes leukocytes and immature sperm, and exogenous, which includes factors such as air pollution caused by heavy metals, smoking tobacco products, obesity and others. Low levels of ROS have a positive effect on the physiological functions of the organism, including the process of spermatogenesis, where ROS participates in the course of hyperactivation and capacitation. However, increased levels of ROS trigger a number of cellular pathologies, whether the loss of fluidity of biological membranes due to lipid peroxidation, deformation of enzymatic proteins or DNA fragmentation, which negatively affects individuals' infertility. Due to the significant positive correlation of ROS scavenging by antioxidants with improving sperm parameters of an infertile individual, antioxidant therapy has recently begun to be used as a possible successful component of male idiopathic infertility treatment.
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