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Analysis of coronavirus by QRT-PCR and possible therapy by nanoliposomal carriers of recombinant antigens
Krchová, Lenka ; doc. RNDr. Milan Bartoš. Ph.D. (referee) ; Turánek, Jaroslav (advisor)
This thesis deals with the study of coronaviruses, their diagnostics and subsequently the possible construction of a recombinant vaccine based on liposomal carriers of recombinant antigens. The experimental part of the thesis touches upon two levels. First, the diagnosis of SARS-CoV-2 virus was measured and investigated by real-time PCR. Biological material of a large number of patients was worked with. Together with the basic determination of the presence of the virus, the stability of the viral particle over time, its resistance to temperature changes, the effect of dilution of the carrier medium on the Ct value and other sub-experiments were investigated. From all the metadata, the most useful variant for pathogen diagnosis is finally evaluated. The experimental part is then followed by a possible therapy with recombinant vaccines. This issue is very topical and moves the whole professional community. Liposomes have been prepared which serve as carriers of bioactive substances in recombinant vaccines. The liposomes were homogenized by extrusion to the desired size and variously surface modified. Antigen binding, stability over time and possible degradation were investigated. Liposomes were characterized by DLS, nanoflow cytometry and electron microscopy.
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Verification of practical possibilities of strengthening the immune system in men
BOHÁČOVÁ, Kateřina
This thesis focuses on the immune systém, its function and the factors that influence it. The theoretical part defines the concept of the immune systém and its functioning. Firstly, it presents the biological perspective, including a description of its basic cells, primary organs and division. The thesis then addresses important factors that have a significant impact on its function. These factors are divided into positively influencing and negatively influencing factors with each chapter discussing their effects onhuman health in case of excess or deficiency. The practical part aims to apply the knowledge in practice, specifically through immune training designesd by Dr. Hermann Geesing. The immune training is conducted with eight men over a period of five months. The participants subjectively evaluate the results of their training based on predetermined questions. The output is presented through tables and graphs along with an interview. The evaluation includes the participantts mental and physical state at the beginning and end of the immune training, along with a description of activities that were either easy or difficult for the participants to accomplish.
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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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Diagnostic of immunohistochemistry in pathology
HRDINOVÁ, Andrea
The bachelor thesis deals with the use of immunohistochemical methods in the diagnosis of neoplasms in pathology. Describes the use of antibody spectrum at the Department of Pathology, Český Krumlov Hospital, a.s. Immunohistochemical methods are nowadays an important method used in pathology for tumor typing and metastasis for the determination of primary tumors. They complement the standard staining methods for basic diagnostics such as Hematoxylin-Eosin and Van-Gieson, which are less specific, these methods mainly serve to differentiate the basic cellular components and tissue components of the preparation. Immunohistochemistry methods are based on the special antibody affinity principle and are divided into direct and indirect methods. For a direct method, an antibody labeled with, for example, an enzyme is used to detect the antigen and, upon binding, induces a color change observed under the microscope. Indirect method (two-stage, three-stage) is a more sensitive method, based on the principle of an unlabeled primary antibody to which a conjugated secondary antibody binds to an enzyme or biotin. In the three-stage, we use the affinity of avidin and biotin to form the avidin-biotin-enzyme complex (eg. peroxidase), which is shown and highlighted. In more detail, the thesis deals with the diagnosis of breast and sentinel lymph node tumors, both in the theoretical and practical part, and the incidence of these tumors since the introduction of the immunohistochemical method at the Department of Pathology of the Český Krumlov Hospital. Last but not least, a case report of individual interesting cases is described.
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Preparation of antibodies to determine the association of mitoribosomal complexes with mitochondrial membrane
ZELLNER, Laura
The assembly pathways of mitoribosomes, descendants of bacterial ancestors producing proteins encoded by vestigial mitochondrial genomes, remains largely unknown. To shed light on structural features of a precursor of the highly divergent small mitoribosomal subunits (mtSSU) from Trypanosoma brucei, called mtSSU assemblosome, recently characterized by cryoEM, we raised antibodies against two assembly factors present in the complex and against two subunits of the mature mtSSU. We used the antibodies in pilot experiments to determine whether the assemblosome associates with the inner mitochondrial membrane.
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Study of chicken antibodies against viral S protein of SARS-CoV-2
Křížová, Tereza ; Hodek, Petr (advisor) ; Svášková, Dagmar (referee)
In the beginning of the year 2020, the WHO declared a pandemic in relation with spreading of a new infection. The Covid-19 disease is mainly manifested by respiratory problems caused by the SARS-CoV-2 virus. It enters the host cell through the ACE2 receptor, to which it is bound by the RBD domain of the surface "spike" protein. A possible way of protection against viral infection is vaccination, e.g. with an mRNA vaccine, which induces the production of a "spike" protein to invoke an immune response and antibody production. Another possible alternative for protection is passive immunotherapy. In this thesis, antibodies from chicken eggs were chosen, which are extensively used in the prevention of various viral and bacterial diseases. They are characterized by high binding affinity to antigens, they are economically inexpensive and are considered a rapid, simple and safe method of passive protection. Antibodies IgY were prepared from egg yolks of chickens vaccinated with an mRNA vaccine encoding a "spike" protein. The reactivity of the prepared antibodies with the whole "spike" protein (expressed in insect cells) was tested by ELISA, demonstrating the ability of the antibodies to bind to the antigen with high affinity. Subsequently, their ability to interact with the RBD domain itself (expressed in...
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Analysis of coronavirus by QRT-PCR and possible therapy by nanoliposomal carriers of recombinant antigens
Krchová, Lenka ; doc. RNDr. Milan Bartoš. Ph.D. (referee) ; Turánek, Jaroslav (advisor)
This thesis deals with the study of coronaviruses, their diagnostics and subsequently the possible construction of a recombinant vaccine based on liposomal carriers of recombinant antigens. The experimental part of the thesis touches upon two levels. First, the diagnosis of SARS-CoV-2 virus was measured and investigated by real-time PCR. Biological material of a large number of patients was worked with. Together with the basic determination of the presence of the virus, the stability of the viral particle over time, its resistance to temperature changes, the effect of dilution of the carrier medium on the Ct value and other sub-experiments were investigated. From all the metadata, the most useful variant for pathogen diagnosis is finally evaluated. The experimental part is then followed by a possible therapy with recombinant vaccines. This issue is very topical and moves the whole professional community. Liposomes have been prepared which serve as carriers of bioactive substances in recombinant vaccines. The liposomes were homogenized by extrusion to the desired size and variously surface modified. Antigen binding, stability over time and possible degradation were investigated. Liposomes were characterized by DLS, nanoflow cytometry and electron microscopy.
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Diagnostics and management of aloimunization in pregnancy
Kučerová, Rebecca ; Hornová, Markéta (advisor) ; Žižka, Zdeněk (referee)
The bachelor thesis focuses on the issue of maternal alloimmunization in pregnancy and deals with diagnostic and management of the alloimmunization. This complication would occur in approximately 2000 women in the Czech Republic per a year without a treatment. A pregnant woman is at risk if she is Rh(D) negative and the fetus inherits Rh(D) positivity from father. The development of alloimmunization can be prevented by early preventive dosage of anti-D globulin. Despite a systematically organized system of prophylaxis, a small number of pregnant women still experience immunization. Fetuses are at risk of developing anemia in the uterus and than after birth by developing hyperbilirubinemia. In the most severe cases of intrauterine anemia, the solution is intraumbilical transfusion of erythrocytes. The aim of this study was to summarize the outcomes of IUT in retrospect. The quantitative practical part is completed with 2 model study cases of hydrops fetalis. The quantitative part of the research analyzes the results of all IUTs performed in the period from 1991 to 2021 in the Center of Fetal Medicine of the Gynecology and Obstetrics Clinic of the General Hospital and the First Medical Faculty of Charles University in Prague. Using tables and graphs, the data were then analyzed and described in...
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The Role of Lck Kinase in T-cell Antigen Receptor Signaling
Němec, Dušan ; Štěpánek, Ondřej (advisor) ; Rösel, Daniel (referee)
LCK activity is crucial for the triggering of the entire T cell activation process. The primary function of LCK is to convert the signal of TCR:pMHC ligation into the intracellular environment. The outcome of the LCK-triggered pathway is T cell activation, cytokine production, differentiation, and clonal expansion. This thesis provides a summary of recent knowledge about the unique position of LCK in the T cell signaling machinery as well as an overview of molecules and interacting partners that regulate LCK activity. It describes the importance of the LCK-coreceptor association for optimal TCR signaling and physiological thymocyte development and mentions discussed adaptor role of LCK in the T cells. Keywords: LCK, T-cell, antigen, kinase, enzyme
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Characterization of T-cell clones from naïve and virtual memory compartment
Přibíková, Michaela ; Štěpánek, Ondřej (advisor) ; Drbal, Karel (referee)
Virtual memory (VM) CD8+ T cells represent a population of antigen-inexperienced T cells with an apparent memory phenotype. In lymphoreplete germ-free mice VM CD8+ T cells represent 10-20% of all peripheral CD8+ T cells. Their origin correlates with the levels of self-reactivity where the main factor that determinates the T-cell fate decision is the strength of homeostatic signals. In the first part of this thesis, we demonstrated that VM CD8+ T cells and naïve CD8+ T cells had distinct TCR repertoire and T-cell subsets contained different clonotypes. Moreover, 'VM clones' were enriched among VM T cells and were also present in naïve T cells. In contrast, 'naïve clones' were almost exclusively detected in naïve T cells. Next, we characterized the signaling of particular OVA-reactive TCRs from both naïve and VM subsets. We confirmed that 6 out of 8 tested TCRs were responsive to Kb-OVA. In the last part of the thesis, we developed and optimized a qPCR-based method for the relative quantification of specific T-cell clonotypes prior to and during the immune response. This method will serve as a tool for studying the biology of particular VM and naïve T-cell subsets and their role during the immune response. Keywords: T-cell receptor, homeostatic signaling, self-reactivity, virtual memory cells, T cells
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