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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry
Soukup, Jakub ; Holada, Karel (advisor) ; Šebestová Janoušková, Olga (referee) ; Živný, Jan (referee)
Prions (PrP) are the main cause of neurodegenerative diseases such as Scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. Although the cellular PrP (PrPC ) is involved in many cellular processes, its precise function still needs to be discovered. The disease is caused by the accumulation of a pathological form of PrP (PrPTSE ), which is caused by direct contact of PrPTSE and PrPC . PrP is anchored in the membrane by GPI and can be transmitted by cell-to-cell contact, tunnelling nanotubes, or extracellular vesicles (EVs). EV factions are divided by different biogenesis into exosomes, microvesicles, and apoptotic bodies. PrPTSE was found in exosomes and microvesicles, but these fractions were never compared to each other. The first aim of the doctoral thesis is a comparison of PrP content, prion-converting activity and infectivity in these fractions on CAD5 and N2a-PK1 cellular models of infection. We isolated a fraction of large EVs (20,000× g) and small EVs (110,000× g) by centrifugation from a conditioned medium. We characterised EVs by cryo-electron microscopy and western blot with Alix, TSG-101, CD63, CD9, and HSP70 markers. The contamination from other cellular compartments was checked by calnexin. EV fractions differed...
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Low treshold working consulting
Šilarová, Vilma ; Richterová-Těmínová, Martina (advisor) ; Janoušková, Olga (referee)
The paper deals with less researched topic of job related (re)integration of unstabilized drug users. It is a comparative study of two emplyoment agencies - the Prague based PSA SANANIM and Wiena based WBB. Case studies derived from these agencies are compared through the scope of provided social services, methodology of work and other factors that are part of the functioning process of the agencies. The study takes into account different conditions in which the above mentioned agencies operate, specificaly: active policy of employment and setting the national drug policy priorities of the Czech Republic and Austria. Research data are based on research papers and specialized texts in Czech and German languages, statistical bulletins, lessons learned during internship conducted abroad, case histories and dekurses of clients PSA. Conclusions serve to improve methodology of emplyoment consultancy for unstabilized clients. It also detects factors that are so far limiting.
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Conditions of prion propagation in cell cultures
Hobzová, Kristýna ; Janoušková, Olga (advisor) ; Rusina, Robert (referee)
Prion diseases are fatal neurodegenerative diseases that affect mammals, including humans, which are characterized by accumulation of pathologi- cal prion protein isoform (PrPTSE ) in the brain. The animals were commonly used for the prion disease research in the past but in recent years, the tissue cultures are being used as well. Tissue cultures have many advantages com- pared with animals. E.g. the possibility of a detailed study of the biochemical processes associated with prion diseases, and rapid and sensitive PrPTSE de- tecting method. However no reliable in vitro model was developed for human prion diseases so far. We focused on monitoring of transmission and propagation efficiency of different prion strains and on the influence of cultivation conditions on the transfer of the neuronal cell line CAD5, which is highly sensitive to prion infection. We confirmed the sensitivity of CAD5 cells to mouse-adapted scra- pie prion strains and we presented new facts about their ability to propagate mouse adapted prions of human strains and bovine spongiform encepha- lopathy. We have used CAD5 cell sensitivity to be infected with different prion strains in other parts of this work. In the second part, we focused on the cell sensitivity to prion infection and propagation of prion strains under different culture...
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Nanostructured Modifcation of Material Surfaces for Controlled Cell Cultivation
Kotelnikov, Ilya ; Proks, Vladimír (advisor) ; Chytil, Petr (referee) ; Šebestová Janoušková, Olga (referee)
Commercial media and surfaces for cell cultivation do not promote conditions for cell cultivation and proliferation with specific interactions cell-material surface. The aim of this thesis is developing surfaces for cell cultivation which mimic conditions in living tissues. The approach introduced in this thesis is based on applying biomimetic peptide ligands on inert supporting materials with non-fouling properties. Considering that a choice of a ligand sequence and distance between peptides can dramatically influence the outcome, a few model peptides with varying parameters were synthesized and investigated. The cell adhesive peptides were synthesized by solid phase peptide synthesis and scrambled peptides were synthesized and tested as well. Spatial deposition of peptides is another highly important point of study. The 'click'- reaction was used to successfully immobilize the peptides on produced surfaces. The distance between the peptide molecules on the surfaces was controlled by their concentration in a reaction mixture. The reference samples were immobilized with radiolabeled peptides for quantitative estimation of the peptides present. Then, the materials with different types of peptides and range of concentrations were examined via cultivation of cell cultures. The experiments were focused on...
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Effects of silicon nanoparticles on human cells
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Janoušková, Olga (referee)
In past years, nanoparticles have been studied as possible platform to be used in biomedicine. In order to establish the application potential of nanoparticles, its impact to biological systems have to be determined. Herein, several silicon-based nanoparticles of different origins were studied in respect of their influence on metabolic activity of human cells, namely osteoblast cell line SAOS-2 and monocytic cell line THP-1. The obtained results proposed that the impact of nanoparticles on cells is highly dependent on cultivation conditions in which nanoparticles are administered to cells. Furthermore, microscopy experiments were implemented in order to localize the particles within cells, where conventional microscopy limitations are evident. Key words: silicon nanoparticles, quantum dots, cell-particle interaction, cytotoxicity
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Role of expression of cellular prion protein in the differentiation of neuronal cell lines
Kučerová, Johanka ; Holada, Karel (advisor) ; Janoušková, Olga (referee)
Cellular prion protein (PrPC ) is a membrane bound glycoprotein. The protein is expressed in all vertebrates, mainly in the nervous system, but it is present also in the cells of gastrointestinal tract, bone marrow, germ cells and heart. PrPC is necessary for pathogenesis of prion diseases, which are deadly and without the possibility of therapy. The pathogenic isoform of prion protein is formed by changing of secondary structure of PrPC and it's the main constituent of infectious prion particles. Pathological form of prion protein accumulates in brain of infected patients and this process is associated with neurodegradation. Physiological function of PrPC is poorly understood. Knock-out of the PrPC gene (PRNP) is not connected with any noticeable phenotype. Potential functions of PrPC are dispersed, protein may have antiapoptotic effect, it can be involved in ions metabolism or in protection against oxidative stress. Latest results show, that PrPC can play important role in cell differentiation. During the differentiation PrPC can influence the development of cells and their typing. It could affect cell cycle and have an influence on formation of nervous system. Aim of the present study was to elucidate, whether the down-regulation of PrPC or infection with prions has an impact on differentiation of...
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Low treshold working consulting
Šilarová, Vilma ; Richterová-Těmínová, Martina (advisor) ; Janoušková, Olga (referee)
The paper deals with less researched topic of job related (re)integration of unstabilized drug users. It is a comparative study of two emplyoment agencies - the Prague based PSA SANANIM and Wiena based WBB. Case studies derived from these agencies are compared through the scope of provided social services, methodology of work and other factors that are part of the functioning process of the agencies. The study takes into account different conditions in which the above mentioned agencies operate, specificaly: active policy of employment and setting the national drug policy priorities of the Czech Republic and Austria. Research data are based on research papers and specialized texts in Czech and German languages, statistical bulletins, lessons learned during internship conducted abroad, case histories and dekurses of clients PSA. Conclusions serve to improve methodology of emplyoment consultancy for unstabilized clients. It also detects factors that are so far limiting.
|
|
|
Conditions of prion propagation in cell cultures
Hobzová, Kristýna ; Janoušková, Olga (advisor) ; Rusina, Robert (referee)
Prion diseases are fatal neurodegenerative diseases that affect mammals, including humans, which are characterized by accumulation of pathologi- cal prion protein isoform (PrPTSE ) in the brain. The animals were commonly used for the prion disease research in the past but in recent years, the tissue cultures are being used as well. Tissue cultures have many advantages com- pared with animals. E.g. the possibility of a detailed study of the biochemical processes associated with prion diseases, and rapid and sensitive PrPTSE de- tecting method. However no reliable in vitro model was developed for human prion diseases so far. We focused on monitoring of transmission and propagation efficiency of different prion strains and on the influence of cultivation conditions on the transfer of the neuronal cell line CAD5, which is highly sensitive to prion infection. We confirmed the sensitivity of CAD5 cells to mouse-adapted scra- pie prion strains and we presented new facts about their ability to propagate mouse adapted prions of human strains and bovine spongiform encepha- lopathy. We have used CAD5 cell sensitivity to be infected with different prion strains in other parts of this work. In the second part, we focused on the cell sensitivity to prion infection and propagation of prion strains under different culture...
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