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Analysis of chemotherapeutics in extracellular vesicles
Kožnarová, Simona ; Hložková, Michaela (referee) ; Vašinová Galiová, Michaela (advisor)
Extracellular vesicles are a newly discovered way of cell-to-cell communication. The issue is still significantly unexplored, especially in the case of cancer. The goal of this pilot study was to attempt to detect platinum contained in extracellular vesicles by ICP-MS. In this study, samples of ovarian cancer cell lines treated with platinum derivatives, a type of chemotherapeutic drug, were used. This method was proved as able to detect platinum, even as able to determine its concentration reliably. The quantity of platinum in vesicles was around 1–2 % of the total platinum added to the system. The results varied according to the used platinum derivative, the cell line and the number of cells releasing the vesicles. Most platinum was determined in vesicles of the SK-OV-3 line, which is naturally resistant to this treatment, for all platinum derivatives. From these results it can be concluded that the use of ICP-MS is also advisable for additional research on this issue.
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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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Extracellular vesicles and middle T antigen of mouse polyomavirus
Kropáček, Václav ; Šroller, Vojtěch (advisor) ; Brázdová, Andrea (referee)
This study is focused on middle tumor antigen (MT Ag) of mouse polyomavirus (MPyV), potential consequences of it's secretion via extracellular vesicles (EVs) and it's effect on cellular signaling. MT Ag is membrane bound protein able to induce cellular transformation thanks to it's ability to interfere with cellular signal transduction. Mainly due to aberrant activation of MAP kinase pathway. Firstly we followed up previous observations of our group concerning ability of MT Ag to be secreted from cells via extracellular vesicles. We were interested if MT Ag could contribute to malignant transformation in recipient cells. We performed 2 types of EVs isolation from cell lines stably expressing middle T antigen (3T6MT). We confirmed presence of MT Ag in isolated EVs. Then we characterized isolated EVs by detection of exosomal markers and cryo-electron microscopy. In next step we exposed recipient cell line (3T6) to isolated EVs and with use of flow cytometry tried to detect internalization of MT Ag. Simultaneously we tried asses levels of Erk phosphorylation in 3T6 cells exposed to EVs. Secondly we tried to confirm and analyse previous unpublished observations of elevated levels of NF-kB phosphorylation in cells stably expressing MT Ag. We used western blot and detection of NF-kB dependent secreted...
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Diagnostic potential of extracellular vesicles
Klementová, Helena ; Holada, Karel (advisor) ; Živný, Jan (referee)
Extracellular vesicles (EV) are membrane particles produced by cells. Many published papers describe changes in the level of EV in body fluids under various pathological conditions. In our study, we analyzed the numbers of EV in the peripheral blood of patients with multiple sclerosis (MS) and in the umbilical cord blood of preterm newborns with inflammation using flow cytometry. We extensively optimized the detection method and verified the presence of EV in the samples using electron microscopy. We found a reduced number of T-cell and phosphatidylserine-positive EV in patients with newly diagnosed MS compared to healthy blood donors. In MS patients requiring a change in the treatment, the number of B-cell EV was lower compared to neurological controls. Similarly, the level of endothelial EV was lower in MS patients receiving Natalizumab treatment. In the umbilical cord blood of preterm newborns with inflammation, we observed significantly increased numbers of placental, endothelial and platelet EV compared to preterm newborns without inflammation. Compared to the full-term controls, the changes in EV numbers were not significant. The number of endothelial EV in umbilical cord blood correlated with the concentration of IL-6 in the venous blood of newborns. Likewise, the number of platelet and...
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Analysis of chemotherapeutics in extracellular vesicles
Kožnarová, Simona ; Hložková, Michaela (referee) ; Vašinová Galiová, Michaela (advisor)
Extracellular vesicles are a newly discovered way of cell-to-cell communication. The issue is still significantly unexplored, especially in the case of cancer. The goal of this pilot study was to attempt to detect platinum contained in extracellular vesicles by ICP-MS. In this study, samples of ovarian cancer cell lines treated with platinum derivatives, a type of chemotherapeutic drug, were used. This method was proved as able to detect platinum, even as able to determine its concentration reliably. The quantity of platinum in vesicles was around 1–2 % of the total platinum added to the system. The results varied according to the used platinum derivative, the cell line and the number of cells releasing the vesicles. Most platinum was determined in vesicles of the SK-OV-3 line, which is naturally resistant to this treatment, for all platinum derivatives. From these results it can be concluded that the use of ICP-MS is also advisable for additional research on this issue.
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Analysis of protein cargo of extracellualr vesicles isolated from the yeast Candida albicans
Hlubučková, Lucie ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Candidate: Lucie Hlubučková Consultant: RNDr. Klára Konečná, Ph.D. Title of thesis: Analysis of protein cargo of extracellular vesicles isolated from the yeast Candida albicans Backgroung: The aim of this diploma thesis was to analyze the protein cargo carried in extracellular vesicles released from the yeast Candida albicans (C. albicans), which is one of the most important mycotic agens. Extracellular vesicles (EVs) are utilized as "transport vehicles", for the delivery of effector molecules into extracellular milieu. These molecules and primarilly proteins can play different roles in host-pathogen interactions. Proteins isolated from EVs and identified by proteomic approach were sorted into categories according to their molecular function and localization for the purpose of finding out, which proteins are predominantly distributed via extracellular vesicles into extracellular space after induction of nutrition starvation. Analysis of EVs protein cargo with focus on virulence factors could extend the knowledge about extracellular vesicles and their potential role in pathogenesis. Methods: Chosen C. albicans yeast strain was a clinical isolate strain isolated from a premenopausal women suffering from recurrent...
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Analysis of protein cargo of extracellualr vesicles isolated from the yeast Candida albicans
Hlubučková, Lucie ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Candidate: Lucie Hlubučková Consultant: RNDr. Klára Konečná, Ph.D. Title of thesis: Analysis of protein cargo of extracellular vesicles isolated from the yeast Candida albicans Backgroung: The aim of this diploma thesis was to analyze the protein cargo carried in extracellular vesicles released from the yeast Candida albicans (C. albicans), which is one of the most important mycotic agens. Extracellular vesicles (EVs) are utilized as "transport vehicles", for the delivery of effector molecules into extracellular milieu. These molecules and primarilly proteins can play different roles in host-pathogen interactions. Proteins isolated from EVs and identified by proteomic approach were sorted into categories according to their molecular function and localization for the purpose of finding out, which proteins are predominantly distributed via extracellular vesicles into extracellular space after induction of nutrition starvation. Analysis of EVs protein cargo with focus on virulence factors could extend the knowledge about extracellular vesicles and their potential role in pathogenesis. Methods: Chosen C. albicans yeast strain was a clinical isolate strain isolated from a premenopausal women suffering from recurrent...
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Extracellular vesicles derived from mesenchymal stem cells and their use in the treatment of neurological disorders
Kratochvílová, Anna ; Hájková, Michaela (advisor) ; Holada, Karel (referee)
Extracellular vesicles (EVs) are cell-derived membrane vesicles, which represent an important part of intercellular communication by transferring cytosolic proteins, lipids, and RNAs between cells. It has been proved that mesenchymal stem cells (MSCs) are potent producers of EVs with a therapeutic effect comparable to their parental cells. Moreover, recent findings suggest that EVs may strongly contribute to their physiological function. The aim of this thesis is to introduce extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) as a novel cell-free alternative to the cell-based therapy and focus on their neuroprotective properties studied in various neurological diseases.
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