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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Jablonská, Dominika ; Čmiel, Vratislav (referee) ; Svoboda, Ondřej (advisor)
This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.
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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Sanetrníková, Dominika ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.
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Monitoring the success of transfection of cell line 293 HEK
Dvořák, Tomáš ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.
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Visualization of Marked Cells of a Model Organism
Kubíček, Radek ; Kršek, Přemysl (referee) ; Herout, Adam (advisor)
This master thesis is focused on volumetric data rendering and on highlighting and visualization of the selected cells of the model organisms. These data are captured by a confocal deconvolution microscope. Input data form one large volumetric block containing separate slices. This data block is rendered by an applicable method and then are identified and visualized the cells marked by the GFP (Green Fluorescent Protein) process or by chlorophyle fluorescency. The principal aim of this work is to find out the preferably optimal effective method enabling this highlighting, most preferably working without a manual check. Due to the data structure, this ambition seems hardly realizable, so it suffices to find out a manual working method. The last step is to embed the results of this work into FluorCam application, the confocal deconvolution microscope data visualizer.
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Preparation of organelle markers in the yeast Saccharomyces cerevisiae
Dekha, Daniella ; Vopálenská, Irena (advisor) ; Papoušková, Klára (referee)
The aim of the work was to prepare a set of strains with fluorescently labelled organelles. The organism used is a haploid strain with a high frequency of homologous recombination Saccharomyces cerevisiae BY4742. The yeast S. cerevisiae is a unicellular organism and is one of the most studied experimental living systems. Its generation time tends to be 1.5-2 hours, which allows rapid monitoring of culture development on both solid and liquid media. For each organelle, specific proteins were selected, namely proteins localized on the vacuole membrane (Vph1, Vtc3), peroxisomes (Pex3), nucleus (Nvj1), endoplasmic reticulum (Sec63), mitochondria (Cox4) and in the vacuole lumen (Prc1). Selected proteins were labeled by attaching fluorescent proteins (yEGFP, yomCherry or yomRuby2) to their C-termini. The prepared strains were analyzed in liquid and solid media. Growth curves were constructed, fluorescence at different growth stages was compared and the effect of two different carbon sources on the expression of selected genes was determined. A total of 12 strains with fluorescent organelle markers for the vacuolar membrane, peroxisomes, nucleus, endoplasmic reticulum, mitochondria, and vacuolar lumen and 5 strains with a pair of fluorescent markers for vacuolar membrane and selected other organelle...
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Nuclear Transport Signals in the proteins of the ARP2/3 complex
Němcová, Barbora ; Bellinvia, Erica (advisor) ; Cvrčková, Fatima (referee)
The intracellular movement of macromolecules such as proteins and RNA is essential for maintaining cellular homeostasis and coordinating various cellular processes. In eukaryotic cells, the transport of these molecules between the cytoplasm and the nucleus is carefully regulated. Nuclear transport signals (NTS) play a key role in facilitating the import and export of proteins across the nuclear envelope. The ARP2/3 complex, which is an important regulator of activity dynamics, has been studied mainly for its functions in the cytoplasm, such as cell movement and cell division. However, new findings suggest that the ARP2/3 complex might also have nuclear functions in plants. Plants are unique multicellular organisms that rely on precisely coordinated cellular activities for growth, development, and response to stimuli. Understanding the molecular mechanisms behind nuclear processes in plants has recently become a focus of research. The ARP2/3 complex, which consists of seven subunits, is known for its ability to branch actin filaments and thereby control cellular processes requiring actin remodeling. However, recent studies have revealed a potential link between the ARP2/3 complex and nuclear functions in plants. Proteins associated with the ARP2/3 complex have been found to localize within the plant...
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Characteristics and gene expression levels of plant transformants modified by one related constructs shearing a gene for proteinase inhibitor.
BEZUNK, František
Tobacco plants were transformed by bacteria Agrobacterium tumefaciens bearing a plasmid construct with a fusion gene SPI2:GFP and selectable marker NPTII coding for resistance of transformants to antibiotic kanamycin. SPI2:GFP gene was created by a fusion of SPI2- (for serine protease inhibitor) and GFP gene (coding for a green fluorescent protein - GFP) sequences. Transformed tobacco plants were selected on a medium with kanamycin. Positively selected plants were selfed and segregation ratios for NPTII determined for each of genotypes. The presence and function of transgenic DNA was verified by methods of PCR, RT-PCR, Western blot and using the stereomicroscopic fluorescence study of transformants of T0 and T1 generations. This Transgenic genotypes of tobacco Nicotiana tabacum, cv. Petit Havana, SR1 WT showing the expression of the fused gene SPI2:GFP on a level of mRNA and GFP protein were obtained. Thesis was perfomed with a support of the project No. M106030 of Ministry of Education, Youth and Sports of the Czech Republic.
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Sex in Trypanosomatids
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Čepička, Ivan (referee)
3 Abstrakt Rody Leishmania a Trypanosoma jsou původci vážných lidských onemocnění: leishmaniózy a trypanosomózy. Dlouhá léta nebyly u těchto parazitů nalezeny přesvědčivé důkazy o genetické výměně, a proto byly rody Trypanosoma a Leishmania považovány za klonálně se rozmnožující, a to binárním štěpením jako většina prvoků. Výzkum ztěžovaly i skutečnosti, že pohlavní dimorfismus není patrný a chromosomy nekondenzují, tudíž nejsou viditelné. Nicméně klonální model začaly zpochybňovat pozorování přirozeně se vyskytujících hybridních druhů. Nejdříve byla existence sexu popsána u trypanosom a to prvním přímým důkazem hybridů T. brucei, získaných po společném přenosu rodičů mouchou tsetse. U leishmanii byl důkaz poskytnut na základě dvojitě rezistentních hybridů a sexuální výměna podstupovala stejný meiotický proces jako T. brucei. Byli pozorovaní přirozeně se vyskytující hybridi Nového i Starého světa jak u rodu Viannia, tak i u rodu Leishmania. Otázkou dalších výzkumů bylo, jaký je mechanismus genetické výměny, ale odpověď dodnes není jasná. Klíčová slova: genetická výměna, Trypanosoma, Leishmania, klonalita, meióza, GFP, přenašeč Abstract Genera Leishmania and Trypanosoma are agents of serious human diseases: leishmaniasis and trypanosomózy. For many years these parasites were considered clone-replicating by...
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Formation of blastema during limb regeneration in Amphibia
Paušlyová, Lucia ; Tlapáková, Tereza (advisor) ; Paňková, Daniela (referee)
Total limb regeneration among vertebrates is basically restricted to some amphibians. Urodeles have the ability to regenerate amputated limbs through their life span. Anurans have the ability of complete regeneration of amputated limbs only in their larval stage. The key process of the limb regeneration is the formation of undifferentiated cell group which is called blastema. There are many cell types that contribute to formation of the blastema while the most important part in this process belongs to the skeleton muscle tissue and dermal fibroblasts. Another critical factor in formation of the blastema and its growth are the nerves in the area of wound and neurotrophic factors produced by them. In the last 20 years it has been great improvement in using different markers for tracking the fate of blastema cells.
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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