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Comparative study of Trichomonas vaginalis strains with various level of metronidazole resistance
Havelka, Michal ; Tachezy, Jan (advisor) ; Kopáček, Petr (referee)
Trichomonas vaginalis is an anaerobic protozoan that causes the human disease trichomoniasis. The most commonly used drug against trichomoniasis is metronidazole. Metronidazole is highly effective, yet in some cases T. vaginalis is resistant to this drug. In T. vaginalis, we distinguish resistance to metronidazole into two types based on the conditions in which it occurs, aerobic and anaerobic resistance. Both types of resistance are associated with changes in the physiology of the parasite. The aim of this study was to characterize the changes in hydrogenosome and cytosol associated with metronidazole resistance at the proteome level in aerobically resistant clinical isolates, the aerobically resistant line MR3 and the anaerobically resistant line MR100. A further aim of this work was to characterize the function of hybrid- cluster protein and to clarify its role in resistance to metronidazole since its production is more abundant in both aerobically and anaerobically resistant T. vaginalis. This work contributed to identification of several potential proteins that could play a role in the development of metronidazole resistance and established a foothold for further metabolomic and biochemical studies of metronidazole resistant T.vaginalis.
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Metronidazole resistence in clinical cases of giardiasis.
Perglerová, Aneta ; Tůmová, Pavla (advisor) ; Uzlíková, Magdalena (referee)
Metronidazole (MTZ) targets a spectrum of anaerobic pathogens ranging from bacteria to eukaryotic parasites, including the protist Giardia intestinalis (Metamonada). Despite the long time and widespread use of MTZ, understanding giardiasis treatment failure (G-TF) is incomplete. Most of the knowledge was gained on laboratory-generated resistant lines or on clinical lines without data on the treatment outcome. In cooperation with Bulovka University Hospital in Prague, 70 giardiasis cases were followed between 2020-2023, of which 25.7% exhibited the G-TF. Giardia genotyping revealed that G-TF cases belonged to the same genetic group (assemblage B, mainly BIII); and G-TF cases were all imported, out of which 60% were from returning travellers from India. To test the hypothesis that resistance is genetically determined/associated with a transmissible genotype, genetic polymorphisms shared in Giardia G-TF samples in a set of genes involved in MTZ metabolism and detoxifications (NR1, NR2, prx1, hmp) were examined. The analysis revealed amino acid substitutions (K42E, K139R) in NR2 isolates of G-TF patients, which might affect protein structure. Also, a gene dosage effect as drug resistance co-variant/cause due to gene duplications or deletions of the hmp gene was tested. Results from dPCR revealed that...
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Use of a Solid Bismuth Drop Electrode in the Determination of the Drug Metronidazole
Jarošová, Johanka ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
The subject of the present Bachelor Thesis was the study of a new commercial solid bismuth drop electrode (SBiDE) and its use to find optimal conditions for the voltammetric determination of the drug metronidazole using the differential pulse voltammetry technique (DPV). The voltammetric behavior of the selected drug was studied as a function of pH of aqueous Britton-Robinson buffer (BR buffer) solution. BR buffer at pH 12 was chosen as the optimal medium for the determination of the drug metronidazole on SBiDE, in which the concentration dependence of metronidazole was measured in the range of 1×10−6 - 6×10−4 mol/L. The limit of quantification (LQ) was 1.38×10−6 mol/L and the limit of detection (LD) was 4.41×10−7 mol/L. The newly developed voltammetric method was used for the determination of metronidazole in the pharmaceutical forms Efloran infusion solution 500 mg/100 mL, Entizol 250 mg tablet, Entizol 500 mg vaginal tablet and Noridem infusion solution 500 mg/100 mL. The selected voltammetric method was compared with the analytical method by UV-VIS absorption spectrometry in the environment of aqueous BR buffer solution at pH 12 in the concentration range of metronidazole 2×10−5 - 8×10−5 mol/L.
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Voltammetric Determination of Metronidazol at a Polished Silver Solid Amalgam Composite Electrode
Škvorová, Lucie ; Barek, Jiří (advisor) ; Zima, Jiří (referee)
The aim of this work was to find out the optimal conditions for voltammetric determination of metronidazole, which is used as an anti-tumor drug. The stability of stock solution of metronidazole in deionized water was monitored using UV-VIS absorption spectrophotometry. Optimum conditions for voltammetric determination of metronidazole at a polished silver solid amalgam composite electrode (p-AgSA-CE) have been investigated. The dependence of voltammetric behavior of metronidazole (1∙10-4 mol/L) on pH of analyzed solution has been investigated using DC voltammetry (DCV) and differential pulse voltammetry (DPV) in the pH range 2,1 - 12,1 in aqueous media of Britton- Robinson (BR) buffer. For electrochemically reducible nitro group of metronidazole, the mechanism of voltammetric reduction at p-AgSA-CE was proposed. The optimum medium of aqueous BR buffer pH 4.0 was chosen for measuring the concentration dependences for both voltammetric techniques. The concentration range 2∙10-6 - 1∙10-4 mol/L of metronidazole was measured using DCV and DPV at p-AgSA-CE with reached limits of quantification (LQs) 2∙10-6 mol/L and 4∙10-6 mol/L for DCV and DPV, respectively. For comparison of used voltammetric techniques with supplemental analytical method, the concentration range 2∙10-6 - 1∙10-4 mol/L of metronidazole...
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Resistance of Trichomonas vaginalis and other anaerobic pathogens to metronidazole
Havelka, Michal ; Tachezy, Jan (advisor) ; Rada, Petr (referee)
The aim of this bachelor thesis is to desbribe mechanisms of resistance to metronidazole in three anaerobic eukaryotic pathogens (T. vaginalis, G. intestinalis, E. histolytica). Diseases caused by these pathogens belong to the list of important but currently neglected diseases. Metronidazole acts only on microbes with an anaerobic metabolism. The drug enters the cell by passive diffusion and needs to be activated by reduction of the nitro group. Mechanisms of activation are different for every pathogen. Enzymes bound with energetic metabolism in hydrogenosomes, cytosolic thioredoxin reductase and nitroreductases play a major role in the activation. The drug damages cells in three ways - DNA damage, formation of covalent bonds with proteins, and covalent bonds with thiols. Pathogens have specific mechanisms to defend themselves against the drug. They can either down-regulate enzymes that activate metronidazole, reduce it to non-reactive aminoimidazole or they can increase the intracellular concentration of oxygen which leads to deactivation of the drug by futile cycling. These mechanisms are bound with physiological changes and subsequently with lowered viability of these pathogens. Furazolidone and benzimidazole derivatives are the best candidates to become an alternative to metronidazole for the...
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Utilization of Working Silver Solid Electrode for Voltammetric Determination of Drug Metronidazole
Mráček, Ondřej ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
This Bachelor thesis discusses the electrochemical behavior of metronidazole and seeks to find optimal conditions for the drug's voltammetric determination at a working silver solid electrode (AgSE) using differential pulse voltammetry (DPV). The voltammetric behavior of the drug was investigated, where the dependent variable was the pH of a water medium Britton-Robinson buffer (BRB). The optimum medium for voltammetric determination of metronidazole at a AgSE is a region of cathodic potential chosen to be the Britton-Robinson buffer, where the sodium hydroxide alkaline component (Na-BRB) has a pH of 10.0, and then measured further at voltammetric cell (volume of 10 ml) at concentration dependencies in the range of (1-1000)×10−6 mol/l. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 1.8×10−6 mol/l and 5.5×10−7 mol/l, respectively. In addition, the metronidazole was studied for the effect of electrochemical regeneration at the AgSE, where the parameters were: Nr = 150, Ein_r = 0 mV, Tin_r = 0.1 s, Efin_r = −1500 mV, Tfin_r = 0.1 s. The next section studies the electrochemical activity of the AgSE, which is not entirely ideal. The newly developed method might be applicable in the future for determining metronidazole through differential pulse voltammetry (DPV) to...
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Study of immunopathological mechanisms of autoimmune uveitis and the determination of new therapeutical options.
Seidler Štangová, Petra ; Svozílková, Petra (advisor) ; Rozsíval, Pavel (referee) ; Farghali, Hassan (referee)
The aim of this work was to gain new knowledge about mechanisms of autoimmune uveitis and to test new therapeutic possibilities that have not yet been studied in uveitis or whose effect is questionable. The main emphasis was placed on the role of microorganisms in the process of uveitis. A mouse model of experimental autoimmune uveitis including a germ-free model was used to achieve the aims and samples of patients' intraocular fluids were analyzed. In the experimental model, the intensity of inflammation was evaluated in vivo clinically and post mortem histologically. The effect of immunomodulatory treatment was evaluated. The intensity of inflammation was compared between groups of germ-free and conventional mice. The therapeutic effect of antibiotics administered to affect microbiome was investigated in conventional mice. In intraocular fluid samples of patients with autoimmune uveitis signs of infection were monitored and levels of cytokines and other factors were evaluated. Evaluation of the effect of immunomodulatory therapy has demonstrated the efficacy of mycophenolate mofetil, which supports its wider use in the treatment of autoimmune posterior uveitis in human medicine. The decrease in bacterial load has led to a decrease in the intensity of inflammation, thereby confirming the importance of...
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Maintenance of chromosomes integrity in Giardia intestinails as a model organism.
Uzlíková, Magdalena ; Nohýnková, Eva (advisor) ; Lalle, Marco (referee) ; Stejskal, František (referee)
Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...
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Voltammetric Determination of Selected Nitroimidazole Drugs
Škvorová, Lucie ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
The aim of presented Diploma Thesis was to study an electrochemical behavior of nitroimidazole drugs metronidazole and ornidazole and to find optimal conditions for their voltammetric determination at a mercury meniscus modified silver solid amalgam electrode using DC voltammetry (DCV) and differential pulse voltammetry (DPV). Voltammetric behavior of selected drugs was investigated in dependence on the pH of the medium used (realized using a Britton-Robinson buffer (BR buffer)) and a mechanism of the reduction of both drugs was investigated using cyclic voltammetry (CV). The optimum medium for voltammetric determination of studied nitroimidazole drugs at the m-AgSAE in a region of cathodic potentials was found to be the BR buffer of pH 8.0. Then, the concentration dependences were measured in this optimum medium in the concentration range from 2·10-7 mol/L to 1·10-4 mol/L. The limits of quantification (LQs) for both metronidazole and ornidazole were found in the concentration order of 10-7 mol/L by using DCV and DPV at the m-AgSAE. The applicability of the newly developed voltammetric methods of the determination of nitroimidazole drugs was verified on the model samples of drinking and river water, with LQ ≈ 2·10-7 mol/L for both DC voltammetry and differential pulse voltammetry at the m-AgSAE....
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Resistance to antimicrobial therapy of Helicobacter pylori strains
Moravcová, Monika ; Keil, Radan (advisor) ; Nyč, Otakar (referee)
Helicobacter pylori (hereinafter referred to as H. pylori) is a gram-negative bacteria which colonises the human stomach mucosa. Its role in the aethiopathogenesis of chronic gastritis, ulcer disorders of the gastroduodenum and MALT-lymphoma has been clearly demonstrated, and in connection with the occurrence of stomach cancer it has been indicated by the World Health Organisation (WHO) as a class I carcinogen. H. pylori infection can be detected from samples of stomach mucosa taken in an endoscopic examination (rapid urease test, microscopic examination, culture), or the non-invasive method can be used (13 C-Urea Breath Test or H. Pylori stool antigen test - HpSA). Effective therapy of H. pylori infection resides in the administration of a combination of antibiotics and a proton pump inhibitor. In recent years the resistance of bacterial strains to used antibiotics has been increasing on a worldwide scale, and we can also observe this trend in the case of H. pylori. If the level of resistance exceeds 20 % for clarithromycin and 40 % for metronidazole, these antibiotics are not recommended for the treatment of an infection caused by this bacteria. In a group of 61 patients at the Department of Internal Medicine at the University Hospital Motol who had undergone an endoscopic examination of the...
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