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Preparation of Monoclonal Antibodies Against VP2 Protein of Human Polyomaviruses
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Růžičková, Šárka (referee)
Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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Selected localities of Prague for teaching biology and geology
Cibíková, Markéta ; Rajsiglová, Jiřina (advisor) ; Drda Morávková, Alena (referee)
This bachelor thesis is focused on the most important Prague locations that are suitable for biological and geological excursion. Very important is practical teaching biology and geology. It is desirable that students understand the processes of ancient geological history, that they have seen with their own eyes preserved fossils and that they know how plants and animals which are integral part of teaching biology look like. In this point of view the theoretical teaching in the classrooms is insufficient. In the work is included biological and geological characteristics of selected sites aimed at their geologicla formation, rock, fossils, plants and animals that are found there. Itinerary for each excursion should be designed. There are links to worksheets made by methodical portal RVP which are the part of my work. Short introduction of teaching biology and geology in foreign countries and their access to excursion is presented as well. This thesis was designed for teachers of biology and geology to make preparations of their own excursions easier.
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Use of model ecosystems near Chomutov in basic education
Řeháková, Tereza ; Havlová, Michaela (advisor) ; Drda Morávková, Alena (referee)
This paper focuses on the teaching of eco-tour by cyclo around Chomutov in basic education. The main objective of my DP is a cycling tour with a focus on the comparison of selected characteristics of ecosystems occurring in the area. As I chose the selected ecosystems - meadows and pastures, forests and water bodies. Selected location near Chomutov offers direct observation of these ecosystems provide a number of paths for nontraditional ways of teaching, using bicycle wheels. Developed materials include design incentive hours for after-school teaching materials in the field and the draft final hours. For after-school education in the field are handled by individual worksheets ecosystems - meadows and pastures, forests and water bodies. Besides these main worksheets, I created a set of other activities in the outdoors. They are practicing first aid package of games and competitions designed for environments and other attractions that can fill the time spent in nature. Next, I created a complete methodological guide for teachers, which are summarized in detail all the information for planning, progress and completion of the excursion. Excursions and its content complies with the current general education program for primary education in the country. Keywords: tour, cycling tours, ecosystems, Chomutov
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Interaction of polyomaviruses with proteasomal system of host cell
Verdánová, Martina ; Drda Morávková, Alena (advisor) ; Horák, Martin (referee)
Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...
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Construction of mouse polyomavirus chimeric VLP bearing melanoma epitopes
Kojzarová, Martina ; Drda Morávková, Alena (advisor) ; Tachezy, Ruth (referee)
Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...
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Research of protective immunity on patients after transplantation.
Pelák, Ondřej ; Kalina, Tomáš (advisor) ; Drda Morávková, Alena (referee)
Depletion of immune functions in patients after hematopoetic stem cell transplantation (HSCT) results in higher frekvencies of several types of oportunistic infections. Especially the reactivation of human cytomegalovirus (CMV) may cause life-threating infections and require antiviral treatment. Within this diploma thesis we compare the function signatures of CD4+ and CD8+ T-cells stimulated by CMV antigens, which were obtained from 63 pacients after HSCT. Polychromatic flow cytometry measurements of CD154 (CD40L), intracellular cytokines (INF-γ and IL-2) and marker of degranulation (CD107a) allowed us to determine the function status of various T-cells simultaneously. We have discovered, that dual production of INF-γ and IL-2 on CD8+ T-cells was present only in pacients controling their CMV reactivations, but was absent in noncontrolers. Single production of INF-γ on CD8+ T-cells was the most abundat subtype, but they most probably represent non-protective memory cells. We have further created two test of NK-cells functionality, one that aims on the detection of their degranulation ability and second, that detects cytotoxic capability of NK-cells. Key words: transplantation, hematopoetic stem cells, T-lymfocytes, immune system recovery, cytomegalovirus, INF-γ, IL-2
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Analysis of BK virus life cycle
Bakardjieva - Mihaylova, Violeta ; Drda Morávková, Alena (advisor) ; Mindlová, Martina (referee)
Polyomaviruses are small unenvelope DNA viruses, whose replication take place in cell nucleus. Despite its small genome size, these viruses can cause significant changes in the host cell, one of the most significant is cell transformation. Most studies of human pathogens from this family is the focus of clinical research, but do not provide enough information about the individual events of the life cycle of viruses. This thesis mainly aims to determining the exact time when the creation of the individual viral products and generate a timeline of events during natural infection in cells that are targets for BKV in the human body. It was found that the time course of the life cycle of BKV is very similar to those for model polyomaviruses MPyV and SV40 and in permissive cells takes about 40 - 50 hours.
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Implementation of new findings to biology classes - the example of stem cells
Petrová, Ivana ; Švecová, Milada (advisor) ; Drda Morávková, Alena (referee)
The aim of this study is to collect the recent data about the establishing the lines of pluripotent stem cell and the usage of these cells for biomedical purposes. On this example, we integrated the education in life science and social science. Pluripotent stem cells were used as a key topic. During our project, we completed the PowerPoint presentation which provides the elementary knowledge on the method for establishing of pluripotent cell lines and the usage of these cells for biomedicine. We created the Czech version of didactic game PlayDecide based on the European Union project. Based on the experiences from this didactic game, the students will learn basic information on pluripotent stem cells and their usage in biomedicine. Students will establish their own opinion on ethical aspects of this topic. Students could be able to defend their opinion and to acknowledge all problems which stem from the suggested solutions. We tested this didactic game in schools.
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Expression analysis of new follicullar B cell populations characterized by absence of CD27 molecule and down-modulation of CD38 molecule.
Kerdíková, Zuzana ; Růžičková, Šárka (advisor) ; Drda Morávková, Alena (referee)
Two novel B cell populations were characterized in peripheral blood of patients with common variable immunodeficiency and healthy controls were observed using flow cytometry in the study supported by the grant IGA MZ ČR NKT11414-3. These B cell populations were defined as CD19+ CD27- CD21+ CD38low CD24+ IgM+ FO I and CD19+ CD27- CD21+ CD38low CD24++ IgM++ cells. Since none of found populations has ever been described, the aim of this thesis was to characterize these populations with focus on analysis of variable regions of the heavy chains of immunoglobulins and genes coding proteins participating in the process of VHDHJH formation (Rag 1, Rag 2, and TdT) produced by cells of these populations. Flow cytometry, single cell sorting, single-cell RT-PCR, IgVH, Rag 1, Rag 2, and TdT specific PCR amplification and cycle sequencing were employed to perform the molecular analysis in individual B lymphocytes. Both populations in two patients with common variable immunodeficiency, two healthy controls, and in two patients with autoimmune diseases - rheumatoid arthritis and systemic lupus erythematosus (as the disease control) - were examined. Finally, the statistical analysis was used to evaluate the differences in expression of variable regions of the heavy chains of immunoglobulins and in Rag1 and 2, and...
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GM crops for new food, pharmacological and technical use: inovation of secondary school study
Koblihová, Kateřina ; Opatrný, Zdeněk (advisor) ; Drda Morávková, Alena (referee)
The demand for plant and other production is increasing with the growth of human population when the territory where plants can be grown is continually decreasing. The present agricultural production of food, industrial materials and drugs of plant origin is not enough for people, as for the effort to achieve higher environmental comfort, especially in developed countries. We are trying to find a simpler, cleaner and cheaper ways of crop production. The genetic modification is one of the modern ways how to achieve this goal. Students get a lot of information about this topic from various sources, but mainly just from the internet and television. It is recommended that students should be able to compare information received from the media with school knowledge as for avoiding opinion pressure and manipulations.
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