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Yeast as the model for for neurodegenerative diseases
Motyčková, Věra ; Schierová, Michaela (advisor) ; Zikánová, Blanka (referee)
Yeasts are traditional model organisms, which can be, surprisingly, used for studying neurodegenerative diseases such as Parkinson, Alzheimer and Huntington diseases. These diseases affect mostly elder population. It's because of decrease of cell control. Common denominator of these diseases are impaired vesicular transport, protein synthesis and degradation, increasing oxidative stress and ROS. The main cause of neurodegenerative diseases is protein misfolding, which provokes cytotoxic protein aggregation. Modified yeast strains can be used to identify genes and proteins that affect cytotoxicity of these key proteins. If these genes have human orthologues, they could be therapeutic targets. The aim of this work is to summarize methods that study causes of neurodegeneration in yeast and to describe the main results discovered in yeast models. Key words: yeast, model system, Parkinson disease, Alzheimer disease, Huntington disease
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Protein synthesis in cellular stress
Cienciala, Martin ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
Environmental stress is a daily bread for organisms across many different branches of life. Very complex response mechanisms have evolved to tackle such insults. Yeast Saccharomyces cerevisiae is adapted especially well for counteracting oxidative and osmotic stress. These unfavorable conditions usually lead to inhibition of protein synthesis. The GCN2 kinase is thought to be responsible for this phenomenon. General inhibition of protein synthesis is accompanied by an increase in expression of proteins engaging in stress response. Production of these proteins is often preceded by specialized regulatory processes, that operate on various stages of expression. This thesis will try to present the diversity and complexity of the individual regulatory layers.
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The analysis of membrane potential recovery in yeast under CCCP-induced stress
Babuka, David ; Plášek, Jaromír (advisor) ; Sigler, Karel (referee)
The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.
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Deconvolution fluorescence microscopy of yeast cells
Štec, Tomáš ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
Title: Deconvolution fluorescence microscopy of yeast cells Author: Tomáš Štec Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jarmoír Plášek, CSc., Institute of Physics of Charles Uni- versity Abstract: Fluorescence microscopy presents an fast and cheap alternative to more advanced imaging methods like confocal and electron microscopy, even though it is subject to heavy image distortion. It is possible to recover most of the original distortion-free image using deconvolution in computer image processing. This al- lows reconstruction of 3D structure of studied objects. Deconvolution procedure of NIS Elements AR program undergoes an thorough inspection in this diploma the- sis. It is then applied on restoration of 3D structure of calcofluor stained cell wall of budding yeast Saccharomyces cerevisiae. Changes of the structure of the cell wall during cell ageing are being examined. Cell wall of aged cells shows increased surface roughness and even ruptures at the end of cell life. Keywords: fluorescence, microscopy, deconvolution, NIS Elements AR, calcofluor, yeast, cell wall, ageing
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Extracellular matrix in yeast populations
Novotná, Pavla ; Kuthan, Martin (advisor) ; Dvořáček, Lukáš (referee)
The microorganisms in a natural environment are frequently found in multicellular forms, most commonly in biofilms. Biofilm is characterized as a community of cells living at the interface of two environments, embedded in the extracellular matrix. ECM is a significant component of biofilms in yeast populations. Extracellular matrix acts as a protective barrier and allows cells to survive under adverse conditions and better compete with other microorganisms. It also forms an effective barrier against antibiotics and other harmful substances, what makes biofilms a serious problem in medicine and industry. Formation of the matrix may be influenced by the morphological forms of colonies. Increased formation of ECM is commonly connected in phenotypic switching in response to changes of their living conditions. The compositeion of the ECM is a genus-and species-specific. The main component of the matrix consists of polysaccharides and proteins.
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GAL4/UAS binary system as a toll for the study of gene function
Soukup, Tomáš ; Krylov, Vladimír (advisor) ; Půta, František (referee)
GAL4/UAS system is a bipartite gene engineering tool, enabling ectopic expression in temporal and tissue-specific manner in vivo. Design of this technique is based on a mechanism of gene transcription, which was elucidated of large portion by an experimental study of Saccharomyces cerevisiae regulation of metabolic control circuit for processing galactose. It is possible to generate hundreds of stable transgenic lines by independent incorporation of the gene for the transcription factor Gal4p and its binding sequence (UAS), respectively, by using transposible or specific-sequence integration techniques. An individual organism, manifesting ectopic expression in suitable, adjustable conditions, can be obtained by cross breeding individual of GAL4 lines with individual from UAS line. This thesis represents a synthesis of the basic principles of GAL4/UAS system and its variants, particularly adapted to the needs of genetic manipulation of model organisms Drosophila melanogaster and Danio rerio. Additionally, this text summarizes the connection GAL4/UAS system with other techniques and briefly highlights the potential for practical applications mainly in research area of neurology. Powered by TCPDF (www.tcpdf.org)
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Changes of intracellular pH in yeast cells under stress conditions
Divín, Radek ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...
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Sch9p and Rim15p signaling pathways in yeast colony development
Mikešová, Jana ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
On solid surfaces, laboratory strain Saccharomyces cerevisiae BY4742 forms horizontally and vertically stratified colonies. Central part of the colony differentiates into U and L cells occupying upper and lower colony regions, respectively. There are many morphological and physiological differences between these two cell types. However, molecular mechanisms of the vertical stratification are still largely unknown. In this study, I analyzed effects of the deletion and overexpression of Sch9p a Rim15p kinases with crucial role in nutrient sensing pathways, of transcription activators Msn2p and Msn4p involved i stress response and of post-diauxic shift transcription factor Gis1p in yeast colony differentiation and U and L cell survival. Microscopic analyses showed changes in morphology of cells in colonies of these strains and decreased ability of vertical stratification in colonies of the strain rim15Δ and partially in colonies of the strain PTEF-SCH9. Moreover, changed level of these proteins significantly affected long-term survival of U and L cells. Obtained data suggest an involvement of nutrient sensing pathways in colony formation and development.
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Use of antibodies for the characterization of chromatin modifications in Saccharomyces cerevisiae
Kovaľová, Libuša ; Hodek, Petr (advisor) ; Malík, Radek (referee)
Transcription of precursor mRNA (pre-mRNA) and its splicing were originally conceived as two separate processes. Using Saccharomyces cerevisiae as a model, it was shown that the assembly of the complex catalyzing pre-mRNA splicing (spliceosome) can occur cotranscriptionally, i. e., during the time before the termination of transcription by RNA polymerase II. Research on cotranscriptional splicing revealed that proteins involved in transcription and specific chromatin modifications may affect pre-mRNA splicing and its regulation. It is also possible that spliceosome assembly and chromatin modifications can affect each other. Prp45, the yeast ortholog of the human transcription coregulator SKIP/SNW1, has been previously associated only with splicing. The results obtained in our laboratory suggest that Prp45 could be used as a regulator coupling the processes of transcription and splicing. We have shown that PRP45 has genetic interactions with factors important for transcription elongation, as well as chromatin modifications, and that it affects early stage of spliceosome assembly. The aim of this bachelor project was document the relationship between the physiological role of Prp45 and H3K4 trimethylation using chromatin immunoprecipitation. It was found that prp45(1-169) mutation does not markedly...
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Physiological role of Na+/H+ antiporters in yeast cells
Zahrádka, Jaromír ; Sychrová, Hana (advisor) ; Obšilová, Veronika (referee) ; Pichová, Iva (referee)
3 Abstract Yeast Saccharomyces cerevisiae belongs to important models for alkali-metal-cation homeostasis research. As other cells, certain intracellular content of K+ is necessary for S. cerevisiae, but Na+ or other alkali metal cations (Li+ , Rb+ ) are toxic for yeast cells. Uniporters Trk1 and Trk2 are responsible for K+ accumulation, while efflux of Na+ , Li+ , Rb+ and K+ is ensured by Ena ATPases, Na+ (K+ )/H+ antiporter Nha1 and K+ specific channel Tok1. Several regulators of K+ (Na+ ) transporters are already known, but reciprocal regulation between transporters and overall picture of the maintenance of alkali-metal-cation homeostasis is still unclear. In this work, K+ circulation (simultaneous uptake and export of K+ ) was shown to be important in alkali-metal-cation homeostasis maintenance. K+ circulation is maintained using reciprocal regulation and interactions between K+ exporters and importers. Though obtained results showed that the alkali-metal-cation homeostasis and associated physiological parameters (e.g. membrane potential, cell size, salt sensitivity) are strain specific, Nha1p was verified to be important for cell survival in ever-changing natural environment. Furthermore, two novel positive regulators of Nha1p activity were found, 14-3-3 proteins and Cka1 kinase. 14-3-3 proteins...
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