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Differences in immunophenotype of CD27+ B lymphocyte subpopulations in healthy controls and patients with various immunopathologies
Valatová, Pavla ; Růžičková, Šárka (advisor) ; Petanová, Jitka (referee)
The immune system will maintain the integrity of the organism from harmful non-malicious recognizes and protects the body against exo- and endogenous toxic substances and together with the nervous and endocrine system are among regulatory systems of the organism. Recently the evidence has supported the emerging concept of different B cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B cell homeostasis. This work is focused on the study of peripheral CD27+ B lymphocytes (one of the least explored human B lymphocytes) in healthy controls and patients with various immunopathologies, in this case we present representative results for patients with inflammatory bowel disease. Using polychromatic flow cytometry we examined 31 of peripheral blood samples, including 14 controls, 7 patients with Crohn's disease (CD) and 5 with ulcerative colitis (UC). In 6 patients with CD, we were able to perform immunophenotyping also 2 hours after intravenous administration of infliximab, and in one patient 14 days after drug...
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Rare B lymphocyte populations in the context of peripheral B lymphopoiesis in humans
Grígelová, Andrea ; Růžičková, Šárka (advisor) ; Hrušák, Ondřej (referee)
B-cells of peripheral blood in humans represent a heterogeneous cellular environment displaying many important functions in the immune system. Recently, there is an increasing amount of evidence that B cell subpopulations are involved in the pathogenesis of many different diseases. However, there is little or no knowledge on how the individual differentiation stages of B lymphocytes are involved in pathological processes, and how they are distributed and represented under the physiological state and under pathological conditions. There is a reasonable assumption that, as with dendritic cells, NK / NKT cells and T lymphocytes, also B cell populations will contain minor and/or rare subpopulations reaching relative frequencies in the range of 0.01% to 0, 1 ‰. The primary aim of this thesis was to investigate the extent of phylogenetic and ontogenetic heterogeneity of the peripheral B cell population and lymphopoietic tissues on the basis of a comparative study across different vertebrate species. Another goal of the work was to use polychromatic flow cytometry with 183 individuals, out of them 50 controls and 133 patients with different (immune) pathologies or tumors in order to identify an optimal combination of surface features, and to use it to detect and demonstrate the existence of minor/rare...
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Cryptic Rearrangements of Human Chromosomes Associated with Autism Spectrum Disorders
Křivánková, Anna ; Šolc, Roman (advisor) ; Růžičková, Šárka (referee)
Autism spectrum disorders (ASD) are heterogeneous group of neurodevelopmental disabilities characterized by antisociality and atypical behavioral patterns. Its etiology is very complex, autism is usually formed by combining many factors. One of the causes may be genetic (gene mutation). It is known about 450 candidate genes for ASD so far. Minority of these genes occur in loci which are affected by cryptic rearrangements. These rearrangements significantly contribute to manifestation of this disorder. Patologies they cause, lead to syndromes with high penetrance for ASD such as Angelman/Prader-Willi or DiGeorge syndrome. Other loci are found on chromosome 1, 2 or 16. Due to short time of studies of cryptic rearrangements, phenotypic variability and number of patients we can expect more researches in the future. These researches are expected not to overlook the impact of the aberrations on formation of autism spectrum disorders.
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Changes in distribution of B lymphocyte subpopulations in patients with Crohn disease before and after biological therapy
Suchá, Renata ; Růžičková, Šárka (advisor) ; Krulová, Magdaléna (referee)
B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...
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Immunophenotype differences in non-memory B lymphocyte populations in healthy controls and patients with immunopathologies
Polák, Milan ; Růžičková, Šárka (advisor) ; Šplíchal, Igor (referee)
B-lymphocytes are a subset of immune cells that can be distinguished mainly by carrying clonally diversified membrane-bound immunoglobulin specialized to specific antigen recognition. Together with other immunocytes B-lymphocytes play a central role in adaptive immune system which takes part in defense of the host against wide variety of pathogens. Recently the evidence has supported the emerging concept of different B-cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B-lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B-cell homeostasis. That is the reason for the thesis to be focused on an analysis of B-cell population profile changes in disease, identification of any association present among specific B-cell subpopulations, as well as association between these subpopulations and clinical parameters. Using polychromatic flow cytometry we analyzed frequencies of 11 B-cell subpopulations including stages of transient B-lymphocytes through effector antibody-producing plasma cells. We examined 81 individuals including 22 healthy controls and 59 patients with...
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Possibilities of Cognitive Lingvistic Concept Metaphor
Růžičková, Šárka ; Hník, Ondřej (advisor) ; Peterka, Josef (referee)
Résumé This diploma thesis under the title Posibilities of Cognitive Lingvistic Concept Methaphor has two goals. The first is describe teachers attitudes towards methaphor in literary education. The second is show a posibilities of new concept of methaphor. There are two parts of thesis: theoretical and practical. The first part defines methapor as linguistic, literary and cognitive phenomenon. I also deal with a way how cognitive lingvistic concept of methaphor improves a process of education and how it interconnects the lessons of literature and Czech language. The second part of thesis is based on a research of results. I describe a general approaches of teachers toward methaphor during an interpretation of poetic text. I compared these approaches with an inovative education model. Finally I made a few suggestions of new approach to educational process in literary education
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DNA damage and signalling pathways in cellular senescence
Hubáčková, Soňa ; Hodný, Zdeněk (advisor) ; Dvořák, Michal (referee) ; Růžičková, Šárka (referee)
Organisms such as mammals need tissue renewal as an important process for maintenance of their viability. Because proliferation is essential also for tumourigenesis, cells need tumour-suppressor mechanisms to protect organism against cancer. Cellular senescence, the permanent state of cell-cycle arrest, features one of these intrinsic barriers against tumourigenesis after DNA damage and understanding of this process may lead to finding of novel therapeutic targets and to optimization of chemotherapy for patients with cancer. In the first part of the PhD thesis, we investigated activation of JAK/STAT signalling pathway in drug-induced senescence. We used genotoxic drugs like aphidicolin, camptothecine, 5-bromo- 2'-doexyuridin, etoposide or thymidine to induce premature senescence in normal and cancer cells. All this chemicals were able to persistently activate JAK/STAT signalling in monitored cells. Activation of STATs was accompanied with up-regulation of expression of interferon-stimulated genes (ISGs), such as MX1, IRF1, IRF7 and PML. Since IRF1 and IRF7 can be directly involved in stimulation of the IFN genes, we show activated expression as well as secretion of IFNbeta and IFNgamma, but not IFNalpha in drug-induced senescent cells. Furthermore, an inhibition of JAK1 as a major kinase of STAT...
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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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Heat shock proteins - - their role in diagnosis and prognosis of pregnancy related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Růžičková, Šárka (referee)
Heat shock proteins increase their gene expression after exposure of cells or organisms to some forms of stress, which may be high temperature, infection, inflammation, hypoxia, lack of nutrients and water. Stressful situations for the body are also pregnancy-related complications associated with placental insufficiency - preeclampsia and IUGR, as well as other pregnancy-related complications such as fetal growth restriction and gestational hypertension. Therefore, I examined whether the occurrence of pregnancy-related complications (preeclampsia, fetal growth retardation, gestational hypertension) affect the gene expression of heat shock proteins. Five hsp systems was detected, namely Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 in placental tissue samples and whole maternal peripheral blood. Samples came from women with physiological pregnancy and from women with certain pregnancy-related complications (PE, FGR, GH). RNA was isolated from the samples. Detection of hsp expression was performed by using real-time RT-PCR and the comparative Ct method. Changes in gene expression between the test samples and reference sample were examined. To assess the difference between physiological pregnancies and pregnancies with selected pregnancy- related complications, an analysis of variance (ANOVA) was used....
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