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Nuclear dynamics and interactions of myosin 1c
Dzijak, Rastislav ; Hozák, Pavel (advisor) ; Hašek, Jiří (referee) ; Kaňka, Jiří (referee)
1. ABSTRACT Myosins are proteins that convert chemical energy stored in ATP into mechanical force that is applied on an actin filament. Nuclear myosin 1 (NM1) was the first myosin detected in the cell nucleus. Together with nuclear actin they were shown to play important roles in DNA transcription and chromatin remodeling. However, the molecular details of the NM1 functions are largely unknown. To expand our knowledge about this molecular motor we studied tissue expression, mechanism of nuclear localization and molecular interactions of this myosin motor. In the first part we examined the expression pattern of NM1 in various mouse tissues. We demonstrated that NM1 is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermatogenesis. Quantitative PCR and western blots demonstrated that the expression of NM1 in tissues varies, with the highest levels in the lungs. NM1 is a nuclear isoform of earlier identified myosin 1c (Myo1c), which was described initially as a cytosolic, and plasma membrane associated protein. The only known difference between these two proteins was the presence of additional 16 amino acids at the N-terminus of NM1. Next we focused on the influence of NM1 domains, including the N-terminus, on the subcellular localization of this protein. We found...
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Identifikace genů nezbytně nutných pro normální průběh oplození a preimplantačního vývoje skotu v podmínkách in vitro
Toralová, Tereza ; Kaňka, Jiří (advisor) ; Pěknicová, Jana (referee) ; Ješeta, Michal (referee)
The aim of this work was to find and characterize genes that seem to be important for normal preimplantation development. We characterized three genes in more detail - centromeric protein F (CENPF; mitosin), ubiquitin C-terminal hydrolase-L1 (UCHL1) and nucleophosmin (NPM1; B23; numatrin; NO38). CENPF and nucleophosmin were shown to start their expression at late 8-cell stage, i.e. at major embryonic genome activation (EGA), and were hence supposed to be important during bovine preimplantation development. CENPF plays crucial role during cell division, especially by mediating the interaction of kinetochores and microtubules. Nucleophosmin is a multifunctional nucleolar phosphoprotein, whose most important roles are rRNA processing, chaperoning, ribosome biogenesis and centriole duplication control. Further, we characterized the role of UCHL1 during fertilization of bovine oocytes. UCHL1 is a deubiquitinating enzyme that controls cytoplasmic protein degradation, recycling of free ubiquitin from proteasome products and is involved in regulation of physiological apoptosis. We studied the function of CENPF and nucleophosmin using RNA interference approach. Since UCHL1 protein is very stable, this method is not suitable for studying the UCHL1 function. We thus used two UCHL1 inhibitors that block its...
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Transcriptomics of bovine preimplantation embryo genome activation in vivo and in in vitro culture conditions
Vodičková Kepková, Kateřina ; Kaňka, Jiří (advisor) ; Petr, Jaroslav (referee) ; Krylov, Vladimír (referee)
The goal of the thesis was to characterize transcriptional profiles of in vivo and in vitro derived embryos during bovine minor and major embryonic genome activation and to identify mRNA transcripts newly synthesized during these stages. In our first work we have concentrated on the study of minor genome activation at the 4-cell stage of embryo. Using SSH, we have identified 31 amplicons homologous with already identified genes. We have selected 5 of these for detailed study of their expression during the whole period of preimplantation development: centromere protein, 350/400 kDa (CENPF, mitosin), splicing factor arginine/serine-rich 3 (SRFS3), high mobility group nucleosomal binding domain 2 (HMGN2) protein and eukaryotic translation initiation factors EIF4A2 a EIF4E. All these genes play an important role in the early embryo development. SRFS3 is the first described gene with an important function in preimplantation development, which is expressed already during bovine minor genome activation, and its transcription is α-amanitin sensitive during this period. We have selected CENPF gene for a more thorough study. By silencing its expression by the injection of CENPF dsRNA into the zygote, we have studied its function throughout the whole preimplantation development of bovine embryo....
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The expression and function of nucleophosmin (B23) during bovine preimplantation development
Benešová, Veronika ; Kaňka, Jiří (advisor) ; Krylov, Vladimír (referee)
Identification of genes that start their expression in different time of embryonal development is crucial for proper understanding of early mammalian embryogenesis. Genes that are differently expressed during this period are supposed to be important for normal preimplantation development. One of these genes is nucleophosmin, the multifunctional protein, which functions among others as a transporting protein between cytoplasm and nucleoli. It is involved in duplication of centrosomes, formation of ribosomes and functions as a chaperoning protein. Nucleophosmin shuttles rapidly between the nucleolus and cytoplasm. Its localization is cell cycle dependent and reflects the development of the nucleoli. For studying the effect of nucleophosmin on the bovine preimplantation development we used RNA interference that causes degradation of the target mRNA after injection of nucleophosmin double-stranded RNA. The average decrease of nucleophosmin mRNA abundance is 86.3 % between nucleophosmin dsRNA injected embryos and control group. We detected no differences in protein expression and localization of nucleophosmin between the groups by immunofluorescence method. Embryos in all groups were able to develop until the blastocyst stage, although there were significant difference between uninjected control group and...
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Measurement of amplitude and phase of ultrashort optical pulses by using nonlinear processes in optical fibres
Vraný, Boleslav ; Kaňka, Jiří (advisor) ; Karásek, Miroslav (referee) ; Boháč, Leoš (referee)
I confirm that I worked out this doctoral thesis independently and that I quoted all the literature used. Prohlašuji, že jsem tuto dizertační práci vypracoval samostatně a že jsem uvedl veš- kerou použitou literaturu. V Praze dne 21. 6. 2010 Abstract The thesis deals with the Frequency-Resolved Optical Gating (FROG) method of mea- suring ultrashort optical pulses. As a nonlinear process involved in the method we used cross-phase modulation in a microstructured optical fiber. The FROG device made of fi- ber pigtailed components fitted well the needs of our fiber optical laboratory. The output of the measurement differs significantly from FROG methods that use other nonlinear processes like second harmonic generation. For our FROG, the standard algorithms for reconstruction of the complex envelope from measured spectrogram failed. We therefore used a more robust genetic algorithm (GA) for the reconstruction. GA has several pa- rameters that affect its runs and we explored the influence of these parameters on the success of reconstruction of a number of test pulses and found the optimal set of these parameters. In this work, we present a general discussion of the FROG method and the standard reconstruction algorithms, the experimental setup we used, a discussion of the genetic algorithm, the technical details of our...
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Gene expression during oocyte-cumulus expansion and preimplantation embryo development in mammals
Němcová, Lucie ; Kaňka, Jiří (advisor) ; Petr, Jaroslav (referee) ; Lopatářová, Miloslava (referee)
CONCLUSIONS Major resultsof this PhD thesiscan be summarizedas follows: The real-time RT-PCR has become a tool for detection and quantifrcation of RNA targetsandis being utilized in geneexpressionstudyin our laboratory. The gene expression proÍile of GDF9, during porcine oocytes, cumulus andgranulosacells fu vitro cultivation and in vivo cumulus oocyte expansion was assessed.The obtained partial sequence was up|oaded in EMBL/GenBanlďDDBJ databaseand gene was localized on pig chromosome2. Conhary to mouse,GDFS mRNA was detectedfor the ťrrsttime also in porcine somaticfollicular cells. During cultivation,thetranscriptgraduallydecreasedin all the samples. we studied signalling pathways involved in IGFI promotion of FSH-induced oocyte-cumulusexpansion.we found that IGFI in combinationwith FSH increased the production of HA. The expression of Has2 mRNA in cumulus cells gradually decreasedduring cultivation' our results suggest that the PIK3/AKT dépendent pathwayis involved in the promotionof FSH stimulatedsynthesisof HA in porcine OCCs. Bovine blastocystsdeveloped from fertilized oocýes under different in vitro or in vivo conditions were comparedin the expressionof threegenes(Bax, L37 and S3o/.The embryosdiffer in the abundanceof Bax transcripl the elevatedlevels were detected in blastocyst completely cultivated...
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Tapered optical fibers
Martan, Tomáš ; Kaňka, Jiří ; Podrazký, Ondřej ; Kašík, Ivan ; Matějec, Vlastimil
A paper is focused on a design and manufacturing microstructure optical fibers with a high nonlinear coefficient, which was reached by using a nonlinear glass and tapering a microstructure optical fiber section. In designed highly nonlinear fiber taper, supercontinum generation was simulated. Designed tapered microstructure optical fiber was manufactured with using constructed tapering apparatus. The tapering apparatus was also employed for producing fiber tips, as probes for pH measurement.
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