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Rozdílná jaderná lokalizace aberantních a normálních homologů chromosomu 8 v intaktních nebo NaBt diferencovaných buňkách HT-29
Harničarová, Andrea ; Bártová, Eva ; Kozubek, Stanislav
An accumulation of numerous chromosomal structural aberrations is a typical feature of the human colon adenocarcinoma cell line HT-29. In these cells the chromosome 8 territories were visualized using fluorescence in situ hybridisation (FISH), which enabled to distinguish the aberrant chromosome 8 homologue from the normal one. Analyses of nuclear parameters revealed differences in radial positioning between chromosome homologues analysed. In comparison with an normal chromosome 8, the aberrant chromosome 8 territory was positioned closer to the nuclear periphery. Under standard conditions HT-29 cells displayed relatively undifferentiated phenotype but incubation of these cells in the presence of sodium butyrate (5mM) induced biochemical and morphological changes that are typical of well-differentiated phenotype of enterocytes.
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Cytologická studie modelů DNA metylace a metylace histonů u lidských buněčných linií
Skalníková, M. ; Bártová, Eva ; Kozubek, Stanislav ; Kozubek, Michal
Epigenetic processes are defined as heritable changes in genome function that occur without a change in DNA sequence. Gene expression, chromosome segregation, DNA replication, repair, and recombination all act, not on DNA alone, but on the chromatin template. DNA methylation, along with histone lysine methylation, establishes the framework for long-term epigenetic maintenance. The discovery that enzymes can (re)organise chromatin into accessible and inaccessible configurations revealed epigenetic mechanisms that considerably extend the information potential of the genetic code. In mammals, heterochromatin is characterised by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone 3 (H3-K9), whereas euchromatin is associated with methylation at lysine 4 of histone 3 (H3-K4).
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Analýza genové exprese kolorektálního karcinomu detekované cDNA mikročipy pomocí kombinace různých vyhodnocovacích přístupů
Jansová, E. ; Krontorád, P. ; Svoboda, Z. ; Koutná, I. ; Pavlík, T. ; Kozubek, Michal ; Jarošová, M. ; Žaloudík, J. ; Kozubek, Stanislav
We have compared colorectal carcinoma tissues with parallel samples of epithelial tissue and identified, by means of Human 1.7K cDNA microarrays, a set of genes with significantly altered expression in 12 patients. We used a combination of several approaches of microarray data analysis to be sure that the results were reliable. Although we have found differences between the results obtained by various image analysis software packages and using different statistical tools, we have identified the group of 22 genes with significantly altered expression in colorectal carcinoma tissue as compared with epithelial tissue using all evaluation approaches. Hierarchical clustering showed the possibility of dividing the patients into two groups according to the presence of metastases in regional lymph nodes. We have proposed 6 genes as potential markers for the detection of the presence of regional metastases in patients.
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Dynamika pohybu a umlčování transgenních lokusů HP1 v živých buňkách
Ondřej, Vladan ; Kozubek, Stanislav ; Lukášová, Emilie ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
Intranuclear localization and mobility of Cy3 labelled transgene loci were studied in living cells. Observations showed occurrence of several transgene copies in each cell nucleus. The majority of copies were localized in the centromeric heterochromatin defined by HPlbeta-GFP fusion protein, the minority of copies in euchromatin. The tracking of loci showed restricted diffusive motion of these in the short-time range. During long-time observation of HPlbeta domains and transgene loci, we have found that in most cases the proximity of these objects decreased within time. The changes of positions of HPl domains were very small but transgene loci displayed directional movement towards the relevant HPl domain. Our data records support the idea that the cell nucleus consists of several separated higher-order compartments. The genes relocate within these compartments, which is finally connected with changes of their expression status.
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Obrazová analýza pohybu struktur uvnitř buněk
Matula, Pe. ; Ondřej, Vladan ; Kozubek, Stanislav
Analysis of time-lapse series of images of living cells requires automation. This contribution concerns the main features of the software package developed for this purpose in our laboratory. Some improvements of our recently published method for tracking of sub-cellular structures based on graph theory are presented. The behaviour of the method is evaluated on images of centromeric heterochromatin defined by HPlbeta-GFP fusion protein. The results show that the method is very well applicable for this type of data.
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Rychlé snímání obrazu živých buněk a jeho limity
Matula, Pa. ; Kozubek, Michal ; Kozubek, Stanislav ; Ondřej, Vladan
Technique of high-resolution cytometry (HRCM) developed in our laboratory is capable of automated (2D as well as 3D) acquisition and analysis of fluorescent stained cells. The cytometer can process large number of cells with high resolution. The acquisition can be repeated in time and that enables live cell studies. If very short events in living cells are studied then the sampling frequency must be high. The highest sampling frequency depends on the technical parameters of the motorized parts of cytometer (especially on the camera frame rate, the speed of filter exchange and the speed of axial movement) and on the control software, which must be appropriately optimized. The limits of the current setup are discussed and available sampling frequencies for different acquisition modes are listed.
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