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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 ; 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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Is Ceanorhabditis Elegans a Suitable Model Organism for the Study of Human Lysosomal Enzymopathies? A Study of Fabry, Schindler, Pompe and Mucopolysaccharidosis IIIC Diseases in C. Elegans and Humans, Respectively
Uřinovská, Jana ; Elleder, Milan (advisor) ; Smetana, Karel (referee) ; Raška, Ivan (referee)
The Ph.D. thesis is focused on lysosomal storage disorders (LSD) with impaired lysosomal metabolism of glycans, namely on Fabry, Schindler, Pompe diseases and muccopolysaccharidosis (MPS) type IIIC. We hypothesized that the worm, which has active glycan metabolism, will have orthologs corresponding to human enzymes deficient in the above disorders and could be a suitable model for study of cellular pathology of these LSDs. The protein families of glycosidases which are deficient in the first three disorders are well characterised while the protein deficient in MPS IIIC was unknown and its enzymatic action was only partially characterized. The gene deficient in MPS IIIC was recently identified in our lab (publication 2) and, as is shown below, it belongs to the lysosomal proteins that have no apparent ortholog in the worm. The main task of the thesis was to find and characterize the C. elegans orthologs of human α-galactosidase (α-GAL), α-N-acetylgalactosaminidase (α-NAGA), acid α- glucosidase (GAA) and heparin acetyl-coenzyme A:α-glucosaminide N-acetyltransferase (HGSNAT) whose deficit leads to Fabry, Schindler, Pompe and MPS IIIC diseases, respectively, and to study their biological functions in C. elegans by RNA-mediated interference (RNAi). Additional aims were to perform basic bioinformatic analysis and...
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A contribution to analysis of molecular basis of selected lysosomal disorders
Hřebíček, Martin ; Elleder, Milan (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
4 Conclusion As discussed before, lysosomď disorders can provide clues about the function of a deÍicient protein and about important cellular mechanisms. Because of that it is worthwhile to study patients with these rare disorders and learn from complex disease manifestations at biochemicď, morphologicď and molecular levels. This thesis is accompanied with papers on sphingolipidoses, the most frequent group of lysosomď disorders, and a mucopolysaccharidosis. The core of the thesis is anďysis of the molecular causes of diseases in relation to clinical manifestations. There is a practicď application to that, because enzyrne replacement therapy, an effective, but extremely expensive treatment, is avďlable for severď of lysosomď storage disorders. Prediction of the future severity of the disease can identiý patients, who can benefit most from the treatment. There is a trend to preventive treatment which can stop development of irreversible changes in the patients tissues. Because of excessive costs of the treatment even the richest countries do not treat ďl patients with these diseases. In Fabry disease we have shown for the first time that X-inactivation influences the severity of the disease in heterozygous femďes. Analysis of irractivation has a potentiď to become a predictive test in Fabry femďes - this,...
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