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Průnik obsahu mykotoxinů ze zrna obilovin do hotového výrobku a dopad na zdraví člověka
Machálková, Monika
This thesis deals with the issue of mycotoxins, which are important pathogens of many agricultural crops and may pose a high health risk to humans and livestock. It contains an overview of the most important mycotoxins their producers and mycotoxicoses. The main focus of this thesis is the occurrence of mycotoxins in cereals and the effect of processing it on their content. The experimental part focuses on monitoring the content and the penetration of deoxynivalenol and zearalenone in winter wheat from grain to bread. We examined a total of 10 samples of treated and 10 samples of untreated variants of winter wheat. For each sample grain, flour and bread were examined for DON and ZEA content and all tested samples were positive for DON and ZEA. None of the examined samples exceeded or even approached the legislative limits.
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Molekulárně-biologická diagnostika lidských polyomavirů
Ryšavá, Markéta
Most of the human population encounters human polyomaviruses during childhood, when the first infection is asymptomatic or with mild symptoms. However, these viruses persist in the human body and most often, they reactivate during immunosuppression. Together with JC virus reactivation, the progressive multifocal encephalopathy is associated. Hemorrhagic cystitis is associated with BK virus, resulting in the loss of allograft during kidney transplantation. Accurate diagnostics can detect viruses in a timely manner and mitigate tissue damage. This diploma thesis deals with biotechnologies, which can be used in virus detection with a focus on real time PCR, which is the gold standard in virus diagnostics. The literary research summarizes basic information about polyomaviruses with a focus on human BKV and JCV polyomaviruses. It summarizes the biotechnological methods used for detection of polyomaviruses, both in routine diagnosis and in alternative approaches involving biosensors and CRISPR. The experimental part of the work includes the design of a detection system for polyomaviruses from in silico genome analysis, through the design of potential primers, to theoretical specificity analysis. The practical part of the work compares the efficiency and sensitivity of amplification of two reaction mixtures, where one is intended for simple systems and the other for multiplexes. It also includes testing of selected additives of PCR and determining the sensitivity and validity parameters of the reaction mixture test selected for PCR system development. The results showed that detection using a multiplex reaction mixture is sufficiently sensitive, as it meets the conditions and requi-rements of clinical recommendations and at the same time shows very good values of sensitivity and specificity of the test comparable to published technologies. This reaction mixture appears to be relevant to the use of the development and optimization of a PCR system for the detection of polyomaviruses.
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Analysis of serological cross-reactivity between antibodies against BK polyomavirus variants
Caisová, Helena ; Horníková, Lenka (advisor) ; Roubalová, Kateřina (referee)
BK polyomavirus (BKPyV), which asymptomatically infects about 80 % of the human population, can reactivate in immunosuppressed patients after kidney transplantation and cause nephropathy. In the European population, there are predominantly two BKPyV sub- types, BKPyV-I and BKPyV-IV, which behave as different serotypes. Recipients who receive a kidney from a donor positive for a different subtype are at a greater risk of graft rejection. So far, it is impossible to distinguish between these two subtypes using a serological ELISA test as serum antibodies against these two subtypes cross-react. This work aimed to iden- tify epitopes on the major antigenic protein VP1, which are responsible for the binding of cross-reacting and/or subtype-specific antibodies. The identification of such epitopes could further lead to improvement in pre-transplant diagnostics and better management of pati- ents after transplantation. Therefore, two types of antigens composed of the VP1 protein of the BKPyV-IV subtype with introduced mutations characteristic of BKPyV-I in the DE and EF loops of VP1 were prepared and tested using an antigen competition assay with a panel of human sera. The results showed that the DE loop region is an immunogenic epitope that binds specific antibodies for BKPyV subtype I. Conversely, a mutation in...
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Biomarkers of neurodegeneration in serum and cerebrospinal fluid in patients with selected neurological diseases
Nosková, Libuše ; Fialová, Lenka (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Švarcová, Jana (referee)
Neurofilaments are the key structural component of the cytoskeleton of neurons, where they are essential for many functions. They consist of 3 subunits: light chain (NFL); medium chain (NfM) and heavy chain (NfH). Except neurofilament proteins there is also α-internexin in the central nervous system (CNS) or peripherin in the peripheral NS. Due to various pathophysiological processes, neurofilament proteins are released into the extracellular space, where they can interact with the components of the immune system. While the involvement of the immune system in the pathogenesis of neurodegenerative diseases is obvious, less knowledge about the antibody response to the neurofilament proteins is available. It is eligible to expand our knowledge in this area. Determination of free antibodies against neurofilaments together with their immune complexes with corresponding antigen provides us more detailed insight into the antibody immune response against neurofilaments. We have optimized the ELISA methods to determine free antibodies against light and heavy chain of neurofilaments together with their corresponding immunocomplexes in both serum and cerebrospinal fluid. Implementation of these methods is precondition for analysis of those parameters in serum and cerebrospinal fluid of patients with...
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Cathepsin L of Sphaerospora molnari - localisation, function and diagnostic tools
NECHVILE, Rudolf Lukas
The aim of this thesis was to develop diagnostic and quantitative assays/tools based on western blotting, confocal microscopy and immunogold electron microscopy, as well as flow cytometry, to determine the localisation and potential function of the highly expressed cathepsin L of the myxozoan parasite Sphaerospora molnari (SmolCL) in its fish host, the common carp. Furthermore, a recombinant SmolCL was used in a vaccine trial to estimate its potential for raising antibodies in carp and test their immunoprotective potential towards S. molnari. This thesis provides new methodological tools for research and allows a greater understanding of myxozoan parasite-fish host interactions based on proteolytic enzymes.
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Comparison confirmation ELISA to recomLine test for the determination of hepatitis C
BAČKOVÁ, Marcela
My bachelor thesis deals with hepatitis C disease and its laboratory diagnosis, especially confirmatory tests. The theoretical part focuses on the disease in general, describes the hepatitis C virus that causes the disease. It also discusses the acute and chronic phases, virus transmission and treatment options. In the practical part I describe the examination of anti-HCV screening antibodies and how to proceed in case of a positive finding. The main task of my practical part is to compare two confirmatory tests - ELISA and immunoblot recomLine. At the end of the work I evaluate both tests and compare the final results.
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Implementation and optimization of ELISA method
KOPAČKOVÁ, Tereza
Enzyme-Linked ImmunoSorbent Assay or ELISA is a method that has a wide application, especially in immunology. Antigens and antibodies can be detected by the method. The basic principle of the method is the reaction of antigen and antibody and the subsequent formation of an immunocomplex. The immunocomplex is detected by a conjugate that causes a color change of the substrate in the presence of antigen or antibody in the test sample. The ELISA test is a simple method important fot the diagnosis of various diseases or laboratory research. This method is a special type of enzyme immunoassay. The bachelor thesis is focused on the general implementation and optimization of the ELISA method. The aim of the theoretical part of the work is a general introduction to immunological methods. The division of individual types of ELISA tests, including the principles and use in practice, is elaborated in detail. In the practical part, the ELISA method is introduced and optimized using a DYNAREAD photometer for reading 96well microtiter plates and AlaDYN software. In this way, a total of 8 test samples of sera are examined and subsequently the obtained data are processed. The test measurement was performed using the QuatiVac ELISA test kit and human IgG antibodies against SARS-CoV-2 in serum were determined. The peaks of SARS-CoV-2 spike proteins expresses recombinantly in the human cell line HEK 293 were used as antigens. The measurement was performed using a DYNAREAD instrument and data processing was performer using AlaDYN software. 6 calibrators, a positive control, a negative control and then individual serum samples were measured. The measurement results are 5 serum samples in which IgG class anti-SARS-CoV-2 antibodies were detected and 3 samples in which no antibodies were detected.
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Vývoj protilátkové imunitní odpovědi proti kryptosporidiovým infekcím
FENCLOVÁ, Jana
This bachelor thesis is focused on the development of humoral immune response against cryptosporidial infections in BALB/c mice. Mice were infected with various Cryptosporidium spp., namely C. parvum, C. proliferans, C. baileyi and C. andersoni. Cryptosporidium-specific IgG antibodies were detected by ELISA. In this work, cross-reactivity of antibodies with different Cryptosporidium species was also monitored.
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Serological overviews- history, present, future
ŠOJSLOVÁ, Denisa
In the theoretical part, I dealt with the meaning of serological surveys, their history, the last survey from the year 2013, and the future of surveys. Then I described the advantages of vaccination and different vaccination types. Last but not least, I talked about tick-borne encephalitis. After that, I am dealing with the history of this illness, its occurrence and spreading, the clinical process, and diagnosis of the illness. The purpose of my bachelor thesis is to prove antidotes against the tick-borne encephalitis, as with people that were vaccinated, as well as with those that had already suffered this illness. The practical part turns to the diagnosis of antidotes against tick-borne encephalitis virus, using the ELISA method. Laboratory results were statistically processed into graphs. Blood samples were being tested from April to October 2019. From these samples, we learned that more women were tested than men in the age group of 30 to 39. Results of my work confirm the general fact that the tick-borne encephalitis is a seasonal illness. Most samples are being tested during summer months, which is related to a higher activity of ticks during this season. Two hypotheses were suggested. The first hypothesis related to the impact of age on the amount of antidotes after having suffered tick-borne encephalitis. The second hypothesis related to the impact of age on the amount of antidotes after vaccination.
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Problems of laboratory examination in Lyme disease
HAVLEOVÁ, Lucie
Lyme borreliosis is a multisystem infectious disease most often transmitted by ticks of the genus Ixodes. In the Czech Republic, specifically by ticks Ixodes ricinus. The initiator of this disease is a complex of spirochetal bacteria Borrelia burgdorferi sensu lato. The incidence of the disease is relatively often as several thousand cases are reported in the Czech Republic every year. However the knowledge about Lyme borreliosis is relatively extensive and simultaneously an intensive research is constantly ongoing, the opinions of experts on this issue are still unfolding accordingly. So far, it is still rather difficult to diagnose Lyme disease, especially because of the various and sometimes non-specific clinical symptoms that might often lead to failure in diagnosis. As no effective vaccine has yet been developed, the only way to reduce the risk of infection is prevention and protection against the ticks. This work investigates and summarizes current knowledge about the history, etiology, epidemiology, pathogenesis, clinical course, prevention, therapy, and diagnosis of Lyme disease. Patients suspected of having contracted Lyme disease were tested using a two-level screening, firstly by applying ELISA method in order to select either positive patients or patients with uncertain results so that a confirmation Western blot method could follow subsequently. The outcome of my work confirmed the well-known fact that Lyme disease is seasonal. The higher number of positive samples in the summer months is related to the increased activity of ticks during this period. However, the results of positive patients who are divided into groups by age or gender represent very interesting findings. Particularly the higher incidence of the positive samples can be found among women.
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