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Use of high resolution melting analysis for the study of lactic acid bacteria
Knápková, Monika ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Using different methods of DNA isolation of lactic acid bacteria in molecular biological methods
Chvalkovská, Eva ; Skoumalová, Petra (referee) ; Brázda, Václav (advisor)
This thesis focused on the probiotic bacteria, DNA isolated from these bacteria by three different methods and the effect of isolation on DNA identification using molecular biological methods. Probiotic bacteria are an important part of human intestinal tract. They have an important role in the function of the immune system due to adhesion to the mucosa of the intestinal flora. They create a inhostile environment for pathogens. Probiotic bacteria are commonly taken in the food like dairy products or food supplements. However, overuse of antibiotics is at risk of passing on the intrinsic resistance that probiotic bacteria have to the pathogenic bacteria. The intrinsic resistence they have to maintain the natural homeostasis of the intestinal tract. It is important to effectively identify risky probiotic bacteria that have the ability to transmit resistance to eliminate their presence in food and dietary supplements. Three methods of DNA isolation like phenol extraction method, magnetic particle isolation and commercial kit isolation were used in the experimental part. DNA was isolated from three dietary supplements, namely Biopron 9 premium, Linex forte and GS Lactobacily forte 21. The purity and concentration of the isolated DNA was detected spectrophotometrically. The presence of individual DNA strains in dietary supplements was confirmed by real-time polymerase chain reaction. The best method of isolation in terms of purity and concentration of isolated DNA was evaluated by RT-PCR and spectrophotometry using a commercial kit isolation method.
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The application of magnetic particles for DNA isolation from cereal products
Starenkova, Anastasiia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.
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Imunochemická detekce proteinu TNF-α pomocí magnetických částic
Koudelková, Žaneta
This thesis is focused on development and optimization of a method for detection of the cytokine TNF-α by the sandwich ELISA using magnetic particles. These particles were functionalized with carboxylic functional groups to enable the antibody binding. Optimal pH of the MES buffer, EDC concentration, and method of activation were determined by optimizing the conditions of particle activation. In the second part of the thesis, the conditions of the magnetic particle-based sandwich ELISA were optimized. It was discovered that mixing the samples during incubation resulted in a higher number of molecules being captured by the antibodies. The optimal incubation times for the antigen and detection antibody were determined to be 3 hours and 1.5 hours, respectively. The optimized conditions for particle activation and ELISA were used to create a calibration curve of TNF-α standards. Optimized magnetic particle-based ELISA protocol can simplify and enhance the analysis of complex samples like blood serum, urine, or saliva.
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Detection of soluble endogline in Lab-in-Syringe
Ilićová, Marie ; Svobodová, Zuzana (advisor) ; Chocholouš, Petr (referee)
The main target of the thesis was to develop an automated method for the isolation of endoglin from a sample using a magnetic immunosorbent in Lab-In-Syringe with subsequent immunodetection by affiblot. The theoretical part deals with endoglin, in its membrane and soluble form, especially with their connection to endothelial dysfunction. The principles of anti-endoglin therapy using the monoclonal antibody carotuximab or TRC105 are also briefly described here. Furthermore, the theoretical part summarizes the basics of the automation in Lab-In-Syringe and of the affiblot technique, their principles, and their applications. The last chapter deals with the biofunctionalization of magnetic particles by antibodies in order to prepare a magnetic immunosorbent which is used for the isolation of proteins from a complex sample. The experimental part describes methods for testing the affiblot prototypes. The main goal was to develop a method for the preparation of anti-endoglin magnetic immunosorbent and to increase the capacity of the method by upscaling it from a 1 ml to a 5 ml syringe. Furthermore, methods for the isolation of the soluble form of human endoglin from the culture medium containing the drug TRC105 were developed for 1 ml and 5 ml syringes. Various experiments were carried out to optimize the...
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Automated preparation of anti-COVID magnetic immunosorbent
Křížová, Lucie ; Svobodová, Zuzana (advisor) ; Smělá, Denisa (referee)
The target of the diploma thesis is the automation of the manual preparation of anti-COVID-19 magnetic immunosorbent. The theoretical part of the work is focused on the research processing of information about the SARS-CoV-2 virus causing the disease COVID-19. The basic characteristics of the disease, its causative agent, clinical picture, methods for detecting the SARS-CoV-2 virus in the human body, and treatment are described here. Furthermore, the principle of the LIS method is described here and the methods of its use in other areas of analysis are described here. The experimental part describes how the manually demanding batch method for antibody immobilization on magnetic particles was converted to an automated method in the Lab-In-Syringe (LIS) system. To convert the batch method to LIS, a series of experiments, optimizations, and searches for analogies were carried out to fully automate the method with minimal operator involvement. Using the device, we prepared anti-COVID-19 immunosorbents, which were subsequently tested using the PCR method on patient samples in the laboratory of the University of Pardubice with BSL 3 protection Keywords: COVID-19, SARS-CoV-2, magnetic particles, Lab-In-Syringe, LIS
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Automated preparation of anti-COVID magnetic immunosorbent
Křížová, Lucie ; Svobodová, Zuzana (advisor) ; Smělá, Denisa (referee)
The target of the diploma thesis is the automation of the manual preparation of anti-COVID-19 magnetic immunosorbent. The theoretical part of the work is focused on the research processing of information about the SARS-CoV-2 virus causing the disease COVID-19. The basic characteristics of the disease, its causative agent, clinical picture, methods for detecting the SARS-CoV-2 virus in the human body, and treatment are described here. Furthermore, the principle of the LIS method is described here and the methods of its use in other areas of analysis are described here. The experimental part describes how the manually demanding batch method for antibody immobilization on magnetic particles was converted to an automated method in the Lab-In-Syringe (LIS) system. To convert the batch method to LIS, a series of experiments, optimizations, and searches for analogies were carried out to fully automate the method with minimal operator involvement. Using the device, we prepared anti-COVID-19 immunosorbents, which were subsequently tested using the PCR method on patient samples in the laboratory of the University of Pardubice with BSL 3 protection Keywords: COVID-19, SARS-CoV-2, magnetic particles, Lab-In-Syringe, LIS
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Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization
Hromádková, Lenka ; Bílková, Zuzana (advisor) ; Fialová, Lenka (referee) ; Krejsek, Jan (referee)
Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,...
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Immobilization of protease V8 on magnetic particles for application to proteolytic cleavage of pepsin A
Čepa, Adam ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
This thesis is part of a long-standing research in the field of diagnosis of the stomach diseases, which is based on the gastric enzyme pepsin A mapping. It was found that a phosphorylation in the primary structure of this enzyme may serve as a marker of incipient stage of carcinogenesis. This thesis is focused on the immobilization of protease V8 isolated from microorganism Stafylococcus aureus to magnetic agarose beads. Protease V8 is a promising candidate for producing peptide maps of pepsin A. The influence of pH, temperature and reaction time on the enzyme to activity has been studied and the optimal conditions for hydrolytic catalysis of formation of peptide fragments of pepsin A.
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