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The effects of hypolipidemic therapy on endothelial dysfunction and early atherogenesis in the vessel wall in experimental models of atherosclerosis II.
Steňková, Miroslava ; Nachtigal, Petr (advisor) ; Semecký, Vladimír (referee)
Arteriosclerosis is a degenerative disease of blood vessels. It's often being reffered as "20th century disease". Double knockouted mice (apoE/LDL-receptor-double-knockout) represent a new model, which is able to evolve a serious grade of hyperlipidemia and arteriosclerosis. It was discovered, that in case of apoE/LDLr-DKO mice even ordinary nourishment led to a stronger progression of arteriosclerosis when compared to apoE apoprotein - only - defective mice. Statins (3-hydroxy-3-methyl-glutaryl-coenzyme A-reductase inhibitors) form currently most effective and most widely used group of hypolipidemics with positive effects on serum lipid levels and even on total and cardiovascular mortality in general. Statins decrease mainly levels of total cholesterol and LDL cholesterol. The aim of this study was to verify whether atorvastatin posses similar antiatherogenic effects in apoE/LDL-receptor-deficient-mice as were shown in humans. To that we analyzed blood lipid spectrum, expression of inflammatory markers in atherosclerotic plaques and the size of atherosclerotic lesions. All mice were fed with experimental diet from the age of 8 weeks. The animals were randomly divided in two groups. The control group of the animals was fed with the atherogenic diet only and in the atorvastatin group, the diet was...
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The effects of hypolipidemic therapy on endothelial dysfunction and early atherogenesis in the vessel wall in experimental models of atherosclerosis I.
Tlapalová, Barbora ; Nachtigal, Petr (advisor) ; Kopecký, Martin (referee)
Atherosclerosis, or sclerosis of arteries, is a degenerative disease of arteries. Sometimes it is called "the disease of the 20th century". Double knockout mice (apoE/LDL - receptor double knockout) represent a new model for a study of atherogenesis which is capable of developing severe hyperlipidaemia and atherosclerosis. Statins (or so-called competitive inhibitors of 3-hydroxyl-3-methyl-glutaryl-coenzym A-reductase) currently belong to the most efficient hypolipidemic drugs that are used all over the world. Statins have propitious effects on lipid plasma levels and cardiovascular mortality. They mainly decrease the levels of total cholesterol and LDL cholesterol. The main aim of this diploma thesis was to evaluate whether atorvastatin possess the same antiatherogenic effects in apo/LDL-receptor deficient mice as were demonstrated in humans. Therefore we analyzed parameters of lipid spectrum in blood, the expression of inflammatory markers in atherosclerotic plaques and the size of atherosclerotic lesions. All mice were fed with experimental diets in 8 weeks of age. Animals were randomly divided into 2 groups (a control and an atorvastatin group). The mice in the control group were fed only with an atherogenic diet, and the mice in the atorvastatin group had an atherogenic diet which was enriched...
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Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry
Kavan, Daniel
CD69 is considered as the marker receptor of activated lymphocytes and is expressed at sites of active immune response. Physiologically it appears in the form of covalently bound homodimer, however after examining its three- dimensional structure we suggested Q93 on one subunit and D88 with E87 on the other one to participate on the inter-subunit interactions. Even more profound intertwining was observed in case of R134 of one subunit with A136 and Y135 on the other one. Therefore Q93, R134 or both were mutated into alanines and showed the monomeric form just in case of double-mutant. This fact influenced significantly also the binding of ligands. While the Kd values for binding of GlcNAc was approximately 10-5 M in case of monomeric form, in case of dimeric form it was 10 times lower and even 100 times lower in case of the longest covalently bound dimers. Although the gel filtration retention time decrease was observed, which could indicate a change in molecular fold, the value of experimentally determined sedimentation coefficient was identical. Moreover neither the comparison of HSQC NMR spectra before and after ligand saturation revealed any significant shifts. Hydrogen deuterium exchange is a chemical process in which a covalently bonded hydrogen atom is replaced by deuterium or vice versa. As...
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Expression and function of molecules of innate system in embryonal phagocytes
Vavrochová, Tereza ; Filipp, Dominik (advisor) ; Černý, Jan (referee)
Phagocytes of an early embryo represent a mixture of myeloid lineages that differ from adult macrophages phenotypically, biochemically and by their origin. Recent studies suggested that there are at least three waves of macrophages populating the early embryo: a maternally-derived one and two waves of extraembryonic, YS-derived origin. In addition, the occurence of early embryonic phagocytes of undetermined origin in developing anterior head mesoderm in evolutionary distinct species is well documented. This origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of specific markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. The aim of this study is to identify and characterize a set of novel markers suitable for identification of embryonic phagocytes. Here, using qRT-PCR approach, we have established the kinetics of expression of Toll like receptors (TLRs) and their TIR-domain containing adaptors during early embryogenesis (E7.5-E12.5) and demonstrate that their major cellular source are indeed phagocytes. Using whole-mount embryo immunohistochemistry we also show that negative regulator of TLR signaling Sigirr is expressed during very early stages of mouse...
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Immunogenic cancer cell death triggered by free and polymer bound doxorubicin
Kabešová, Martina ; Kovář, Lubomír (advisor) ; Anděra, Ladislav (referee)
Immunogenic cancer cell death triggered by free and polymer-bound doxorubicin Water-soluble polymeric drug carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) have been developed to avoid undesirable side-effects of systemically active cytostatic drugs. Conjugates based on HPMA copolymer passively accumulate in tumor tissue of solid tumors due to the effect of enhanced vascular permeability and retention effect (EPR effect). Active accumulation can be achieved by binding of targeting structure recognising tumor-specific receptors to the polymeric carrier. Conjugation of free doxorubicin to HPMA polymeric carrier significantly reduces its nonspecific side effects in vivo by maintaining effective antitumor activity in vitro and in vivo. Besides direct cytotoxic effect on tumor cells, HPMA conjugates with bound doxorubicin possess immunostimulatory properties and induce long-lasting anti-tumor immunity in cured mice. Recent studies have shown that free doxorubicin induce immunogenic apoptosis in tumor cells. Those cells are dying by cell death which possesses characteristic markers of apoptosis but these cells are able to activate the immune system and thus induce effective anti-tumor immune response. This phenomenon has been described as crucial for a development of treatment-induced...
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The role of immune system factors in IVF/ET success
Růžičková, Žaneta ; Madar, Jindřich (advisor) ; Pěknicová, Jana (referee)
5 Abstract The aim of this study was to evaluate the presence of receptors (CD16, CD161, NKG2D, KIR2DL4 and CD85j) on the surface of immunocompetent cells and whether their presence is involved in the in vitro fertilization (IVF) and following embryotransfer (ET) success. We observed significant difference in the age of successful versus unsuccessful patients, which is confirmed by former studies on age-dependence of IVF success. The result from cytotoxic assay confirmed, that neither the stimulation protocol, nor the PBMC source have any effect on effector function. Afterwards, we performed a large-scale analysis of surface markers, starting with healthy donors, compared to patients. Both groups of patients have lower counts of HLA-G binding KIR2DL4 or CD85j receptors bearing cells. Next we observed the expression of these two receptors on various subpopulations of cells (CD56+dim , CD56+bright , T helpers and cytotoxic T cells) and their counts. Here we observed differences in CD56 surface expression on CD56+ bright NK cells, the levels of CD56+dim NK cells in PBMC (CD16 expression on this population), levels of CD56+dim Next step was to compare the levels of cell populations between the successful and unsuccessful patients. We observed an increased surface expression of KIR2DL4 on lymphocytary PBMC of...
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Implication of the Gabaergic System in Altered Responses to Stress Associated to Maternal Separation
Martišová, Eva ; Pávek, Petr (advisor) ; Štaud, František (referee)
in English language - diploma thesis Implication of the Gabaergic System in Altered Responses to Stress Associated to Maternal Separation Eva Martišová This diploma thesis studied whether the GABA system alters responses to stress induced by maternal separation in rats. The aim was to examine the influence of maternal separation on GABA neurotransmission, in particularly, the biochemical markers of the system (levels of the neurotransmitter GABA, GABA A receptor expression), and to study of gender-related differences in behavioral and neurochemical processes in rats induced by maternal separation. In order to pursue the objectives, it was necessary to develop a model of maternal separation in rats. Rat pups were separated from their mother for three hours a day from postnatal day 2 to postnatal day 21 (in the postnatal period, when the reduced ability to respond to stressful stimuli). At postnatal day 23 pups were removed from mothers, divided into groups of the same sex. Then pups were without significant manipulation left to grow into adulthood. After reaching adulthood (> 60 days) rats were divided into two groups. The first group was killed by decapitation and different brain regions (anterior and temporal cortex, corpus striatum and hippocampus) were used to determine the levels of GABA using...
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P-selectin - a novel protein in hemostasis
Hrachovinová, Ingrid ; Brdička, Radim (advisor) ; Kvasnička, Jan (referee) ; Penka, Miroslav (referee)
Originally, P-selectin played an important role in the vascular response to inflammation. In the first half of the nineties there were published papers demonstrated that P-selectin also played a significant role in blood coagulation and thrombosis. In these experiments anti-P-selectin antibodies blocked fibrin formation and suppressed the developing of thrombus. Shortly thereafter, P-selectin was shown to upregulate tissue factor (TF) generation on monocytes. The active role of P-selectin in hemostasis was supported also with our findings that overexpression of soluble P-selectin (sP-sel) can induce a procoagulant state in plasma/blood. I have focused in my postgraduate study on pathophysiology of sP-sel , its role in formation of microparicles (MPs) bearing TF. In the detail study of sP-sel we found, that procoagulant state in plasma is due to procoagulant MPs, part of them contained TF. We confirmed that recently characterized "blood-borne" TF could be induced by sP-sel. Moreover, because the production of microparticles was suppressed by inhibiting antibody against PSGL-1 (receptor of P-selectin on leukocytes), we proposed the origin of MPs from leukocytes. Recruitment of TF-bearing MPs from monocytes I demonstrated later with FACS screening of presence of specific CD-markers. I realized that...
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The Use of Multichannel Flow Cytometry in Biomedicine and Experimental Biology
Kideryová, Linda ; Smetana, Karel (advisor) ; Elleder, Milan (referee) ; Klíma, Jiří (referee)
Flow-cytometry is a process on which large numbers of single cells are quantitatively and qualitatively analyzed. This method gives information about size, granularity surface or intracellular markers of every single cell in suspension. In modern biology is worthy to perform quick, objective multiparametric analyses of cell phenotype. This project was focused on cells, which analyses are complicated by extreme rareness or lack of clearly identifying specific markers. Analysis of stromal cells of the investigated tumors (histiocytoma and tumor fibroblasts originating in squamous epithelium: basalioma (BCCF) and spinalioma (SCCF) elucidated alteration of gene expression induced by tumor cells. Tumor-derived stromal fibroblasts acquire distinct properties to shape a microenvironment conducive to altering the phenotypic characteristics of normal epithelial cells in vitro. Reproducible, quick and highly sensitive method of detection extremely rare non-haematopoetic cells (EPC, CEC) was established. Numbers of CFU-En correlate neither with circulating endothelial progenitors nor with matured endothelial cells detected by flowcytometry. These colonies are formed in cooperation of CD14 + and CD4+ cells. Numbers of endothelial progenitors and matured endothelial cells are closely related with vessel endothelium...
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The use of multichannel flow cytometry in biomedicine and experimental biology
Kideryová, Linda
Linda Kideryová USE OF MULTICHANNEL FLOW-CYTOMETRY IN BIOMEDICINE AND EXPERIMENTAL BIOLOGY SUMMARY Flow-cytometry is a process on which large numbers of single cells are quantitatively and qualitatively analyzed. This method gives information about size, granularity surface or intracellular markers of every single cell in suspension. In modern biology is worthy to perform quick, objective multiparametric analyses of cell phenotype. This project was focused on cells, which analyses are complicated by extreme rareness or lack of clearly identifying specific markers. Analysis of stromal cells of the investigated tumors (histiocytoma and tumor fibroblasts originating in squamous epithelium: basalioma (BCCF) and spinalioma (SCCF) elucidated alteration of gene expression induced by tumor cells. Tumor-derived stromal fibroblasts acquire distinct properties to shape a microenvironment conducive to altering the phenotypic characteristics of normal epithelial cells in vitro. Reproducible, quick and highly sensitive method of detection extremely rare non-haematopoetic cells (EPC, CEC) was established. Numbers of CFU-En correlate neither with circulating endothelial progenitors nor with matured endothelial cells detected by flow-cytometry. These colonies are formed in cooperation of CD14 + and CD4+ cells. Numbers of...
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