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Analysis of adherent cells confluency in 2D culture
Bracková, Michaela ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
First part of this semester work dealing with the theoretical description of adherens cells, particularly structure and functions of this cells. Subsequently work is devoted to description of cell culturing, with mention of conditions which are necessary for cell culturing, following by substances which promote cell growth. Last part of theoretical research is concern with microscopy technique that is suitable for studying of adherent cells. Subsequently it is about fluorescence and confocal microscopes. Practical part dealing with cell culturing of adherent cells and the evaluation of realized experiments.
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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry
Soukup, Jakub
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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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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Characterization of pore opening relevant residues in TM3 and TM4 domains of Orai1
ANDOVA, Ana-Marija
Calcium (Ca2+) ions play a crucial role in almost every aspect of cellular life. The most prominent calcium entry pathway into the cell is the calcium release-activated calcium (CRAC) channel, composed of the Orai1 protein, and the stromal interaction molecule STIM1. The channel is activated through conformational changes upon STIM1 coupling to the C-terminus of Orai1 protein following store depletion, which in turn allows Ca2+ influx into the cell. The abnormal function of the CRAC channel caused by mutations gives rise to distinct pathologies. Since it has not yet been elucidated how the signal propagation moves to the pore upon coupling, this thesis dives into its investigation by focusing on characterizing the TM3 and TM4 domains and their importance in leading to an open permissive conformation of the channel. The pivotal foundation for the creation of novel strategies in the modulation of the Orai1 function lies with the understanding of the dynamics of the Orai1 pore opening.
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Study of the influence of natural drugs on estrogen receptor and confirmation of their effect in cell cultures
Elbelová, Jana ; Carazo Fernández, Alejandro (advisor) ; Pourová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Jana Elbelová Supervisor: PharmDr. Alejandro Carazo, Ph.D. Title of the diploma thesis: Study of the influence of natural drugs on estrogen receptor and confirmation of their influence in cell culture Estrogens are one of the main reproductive hormones in women. Their function in the human organism is mediated by the estrogen receptor (ER). Estrogens are involved in important physiological processes such as reproduction, bone metabolism and cardiovascular function. Long term elevated levels of these hormones may also lead to breast cancer development. Phytoestrogens are natural ligands of estrogen receptor widely found in dietary products. These compounds have beneficial properties in treatment of menopausal symptoms thank to their estrogenic activities but are able to function as antiestrogenic agents too. In addition, they may play a role in the development of other illnesses (breast cancer etc.). Therefore, they can be considered as a potential pharmacological tool for the treatment of estrogen-related conditions. Within this master thesis, a total of six compounds, two phytoestrogens and four of their more relevant metabolites, were chosen (genistein, daidzein, S equol, O...
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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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Experimental model systems to study small DNA viral infection
Bučková, Alžbeta ; Saláková, Martina (advisor) ; Horníková, Lenka (referee)
Merkel cell polyomavirus (MCPyV) and Human papillomavirus 16 (HPV 16) are members of small tumour DNA viruses Polyomaviridae and Papillomaviridae, which represent increasing risk for humans resulting from their oncogenic potential. After the acquisition HPV 16 and MCPyV are able to persist for long term in a form of asymptomatic infection, while the aggressive disease is mostly being cleared by the host immune system. Integration of viral genome into the host DNA causes cell transformation resulting in rare but fatal skin carcinomas and epithelial lesions of anogenital tract, head and oropharynx, that may progress into malignant tumours. Their mechanisms of immune system evasion and complete life cycles are not fully understood to this day which highlights some of the reasons why continuing research in this field is of importance. The aim of this thesis is to review model systems used to study infection of MCPyV and HPV 16 in vitro and in vivo. Key words: Papillomaviruses, polyomaviruses, virus-like particles, pseudoparticles, animal models, cell culture, human papillomavirus 16, Merkel cell polyomavirus, HPV 16, MCPyV
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Microglia control adenosine A2A-receptor mediated astrogliosis
Svobodová, Magdaléna ; Mladěnka, Přemysl (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Magdaléna Svobodová Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Assoc. Prof. Maria da Glória Correia da Silva Queiroz, Ph.D. Title of diploma thesis: Microglia control adenosine A2A-receptor mediated astrogliosis In the central nervous system, astrocytes and microglia are the main cells coordinating the inflammatory response. During inflammation, dying or temporarily damaged cells release ATP, as a danger-associated signal molecule, that contributes to the induction of astrogliosis and promotes clearance of the debris by immune cells such as microglia. Adenosine that results from ATP metabolism also stimulates astrogliosis. However, the effects of adenosine on astrogliosis may be more complex, since it also modulates microglia phenotype and microglia have been shown to prevent excessive astroglial proliferation mediated by nucleotides. In this context, ATP and adenosine are assumed as relevant signalling molecules in the control of astrogliosis and its modulation by microglia. However, it is still unknown whether and how microglia modulate adenosine-mediated astrogliosis. The present study aims to clarify the impact of microglia in the control of adenosine-induced astrogliosis. Two...
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Microglia control adenosine A2A-receptor mediated astrogliosis
Svobodová, Magdaléna ; Mladěnka, Přemysl (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Magdaléna Svobodová Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Assoc. Prof. Maria da Glória Correia da Silva Queiroz, Ph.D. Title of diploma thesis: Microglia control adenosine A2A-receptor mediated astrogliosis In the central nervous system, astrocytes and microglia are the main cells coordinating the inflammatory response. During inflammation, dying or temporarily damaged cells release ATP, as a danger-associated signal molecule, that contributes to the induction of astrogliosis and promotes clearance of the debris by immune cells such as microglia. Adenosine that results from ATP metabolism also stimulates astrogliosis. However, the effects of adenosine on astrogliosis may be more complex, since it also modulates microglia phenotype and microglia have been shown to prevent excessive astroglial proliferation mediated by nucleotides. In this context, ATP and adenosine are assumed as relevant signalling molecules in the control of astrogliosis and its modulation by microglia. However, it is still unknown whether and how microglia modulate adenosine-mediated astrogliosis. The present study aims to clarify the impact of microglia in the control of adenosine-induced astrogliosis. Two...
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