Transgenic techniques based on the cell transfer

Katedra fyziol. živočichů a vývoj. biol. (zrušena)Dep. of Physiology and Develop. Biology (obsolete)Faculty of SciencePřírodovědecká fakult

Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.

Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal..

Transfection of stem cells and other testicular cells of Xenopus tropicalis

Xenopus tropicalis represents one of the most important model organism used in developmental and cellular biology. Laboratory of Developmental Biology at the Faculty of Science, Charles University in Prague has successfully established a long-term culture of X. tropicalis juvenile testicular cells. Based on expression profiling analysis of selected specific markers (Sox9, WT1, etc) it was proven that the major cell type in this culture is pre-Sertoli cells. Furthermore these pre-Sertoli cells allow a longterm cultivation of the germinal stem cells. By performing a histochemical test for the presence of alkaline phosphatase in the colony of these cells were proven the features of stem cells. In this diploma thesis we focused on optimization of work with the mixed cell culture. In particular we define conditions of dissociation and subsequent separation of a feeder-layer formed by the pre-Sertoli cells. We also attempted to develop suitable conditions for transfection of the germinal cells. With these techniques we will to investigate the functional properties of the germinal stem cells. Moreover, it provides us a powerful tool for performing another experiments focused on transgenesis and/or different gene inactivation. Powered by TCPDF (www.tcpdf.org

Transgenic techniques based on the cell transfer

Katedra fyziol. živočichů a vývoj. biol. (zrušena)Dep. of Physiology and Develop. Biology (obsolete)Faculty of SciencePřírodovědecká fakult

Stanovení olova v kostech metodou vysokorozlišovací atomové absorpční spektrometrie s elektrotermickou atomizací a kontinuálním zdrojem záření za využití přímého dávkování suspenzí

A straightforward, quick, sensitive and reliable method is introduced for the determination of lead in bones using slurry sampling and high-resolution continuum source electrothermal atomic absorption spectrometry (HR-CS-ETAAS). The spectral interference caused by the molecular absorption of PO molecules with rotational fine structure coinciding with the analyte absorption at the most sensitive resonance line 217.001 nm in time was identified and successfully corrected by applying a mathematical correction algorithm using the spectrum obtained by vaporization of hydroxyapatite. The slurry preparation and measuring conditions were determined by means of a response surface methodology. Experiments were designed according to a 2(7-4) replicate (n = 3) fractional factorial design for seven factors (particle size, glycerol and HNO3 concentration, sonication time, concentration of the chemical modifier, pyrolysis and atomization temperature) each at three different levels, including central points. The optimized conditions were 100 mg of a ground sample with particle size < 315 mm, dilution in a liquid-phase composed of 10% w/w glycerol and 5.0% w/w nitric acid solutions, a sonication time of 2 min and the final slurry volume of 10.0 mL. A detection limit of 9.1 ug kg(-1) and characteristic mass of 7.6 pg were achieved using the suggested method under the optimized experimental conditions. Sufficient analyte stabilization was achieved by using 1 ug Pd and 50 ug citric acid. Accurate data were obtained with the use of matrix-free calibration. The accuracy of the method was established by analysing NIST SRM 1486 Bone Meal. Further, the results acquired for ten river otter samples by slurry sampling were compared with those determined after microwave-assisted digestion by inductively coupled plasma time of flight mass spectrometry (TOF-ICP-MS) to assess the accuracy of the method. The results obtained by the two methods were compared using a paired t-test (at the 95% confidence level) and showed no significant difference. The precision of the introduced method was better than 5.5%.Vyvinuta byla jednoduchá, rychlá a ekologicky šetrná metoda pro stanovení Pb v kostech metodou atomové absorpční spektrometrie s elektrotermickou atomizací s kontinuálním zdrojem záření a vysokým rozlišením (HR-CS-ETAAS). Optimalizace metody umožňující analýzu reálných vzorků formou přímého dávkování suspenzí byla provedena za využití nástrojů frakcionálního faktorového plánování. Pro korekci spektrální interference způsobené strukturovaným pozadím v důsledku přítomnosti PO molekulových absorpčních pásů bylo použito matematické korekce. Určeny byly analytické charakteristiky stanovení Pb pomocí navržené metody. Validace metody byla provedena s využitím komerčně dostupného certifikovaného materiálu NIST SRM 1486 Bone Meal a pomocí referenční metody TOF-ICP-MS

Nové postupy mikrovlnným polem asistované extrakce a mineralizace komplexních vzorků pro potřeby anorganické prvkové analýzy

A large number of case studies are presented covering new approaches and new experiments, which meet the requirement for industrial application of sustainable chemistry. Nevertheless preparations of samples with complex matrix such as ZrO2, TiO2, sludge, phosphate fertilizers, fly ash, sediments, etc. still contains energy-intensive and time consuming steps or includes chemicals such as hydrofluoric acid for sample dissolution. By replacing highly toxic reagents with the employment of microwave assisted extraction (MAE) or digestion (MWD), simple and environmentally friendly methods were developed in accordance to current green chemistry trends.V posledních letech je z hlediska požadavků oblasti udržitelné chemie kladen důraz na vývoj nových ekologicky šetrných, ekonomicky a časově úsporných postupů [1]. Nicméně i v současné době příprava celé řady komplexních vzorků vyžaduje využití časově a energeticky náročných postupů, nebo korozivních a toxických činidel, jako např. kyseliny fluorovodíkové. Za využití mikrovlnným polem asistované extrakce (MAE) či mineralizace (MWD) je ovšem možné v souladu s trendy současné zelené chemie dosáhnout požadovaných výsledků i s využitím ekologicky šetrnějších činidel, jak je demonstrováno na příkladech přípravy komplexních vzorků, jako např. ZrO2, TiO2, uhelných popílků, kalů či sedimentů

Pre-amplification in the context of high-throughput qPCR gene expression experiment

Background: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). Results: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. Conclusions: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step

Proceedings of the 29th National Conference of the Czech Association of Occupational Therapists

Proceedings of the Conferenc