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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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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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