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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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Phenotype of melanocytes under physiological and pathological conditions
Strnadová, Karolína ; Lacina, Lukáš (advisor) ; Mokrý, Jaroslav (referee) ; Balvan, Jan (referee)
In addition to the dominant keratinocytes and fibroblasts, melanocytes are also indispensable representatives of skin cell populations. Melanocytes are pigment cells whose primary function is to produce the pigment melanin, which is important for protecting keratinocytes from harmful ultraviolet radiation. Excessive exposure to this radiation is a risk factor for the development of skin tumours, including malignant melanoma of the skin, in which pathological transformation of melanocytes into melanoma cells occurs. The presented thesis focuses on 4 thematic areas associated mainly with malignant melanoma. In the first thematic area, the increasing incidence of malignant skin melanoma is associated with the ageing of the population. One of the reasons seems to be the more frequent occurrence of proinflammatory setting in the ageing organism. It prepares a suitable environment for tumour development. The second thematic area focuses on new approaches that could expand the range of diagnostic methods for the early detection of malignant melanoma. The first approach methodically uses the detection of proinflammatory molecules in the patient's serum. Higher serum levels of IL-6 and IL-8 correlate with an unfavourable patient prognosis. The second approach is based on the possibility of detecting a...
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miRNA analysis of small extracellular vesicles isolated from plasma after diet intervention
Kratochvílová, Michala ; Koc, Michal (advisor) ; Šrámek, Jan (referee)
The aim of this thesis was to establish a protocol for the isolation of small extracellular vesicles (sEVs) from human plasma. Plasma samples were collected from 44 healthy women who underwent a 60-hour dietary intervention in the form of fasting as part of a clinical trial. These women were divided into two groups based on their BMI - lean and obese. Plasma was collected from each participant before and after the dietary intervention and sEVs were isolated using the polyethylene glycol precipitation method. Each sample was then characterized by the size of sEVs and their concentration. The amount of sEVs in plasma before the dietary intervention was the same for both groups and did not change with dietary intervention. However, the 60-hour fasting resulted in a change in sEVs size in the lean group. Another goal of this thesis was to analyze miRNA expression in sEVs. There were differences found in the expression of certain miRNA between the two groups. More specifically, differences were observed in the expression of hsa-miR-17-5p, hsa-miR-20a-5p and hsa-let-7b-5p. The 60-hour fasting period resulted in an increased expression of hsa-miR-5100 in the lean group and hsa- miR-27a-3p and hsa-miR-27b-3p in the obese group. However, these changes induced by the dietary intervention were not evaluated...
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Small extracellular vesicles as microRNA carriers and their role in neural cell regeneration
Šprincl, Vojtěch ; Romanyuk, Natalyia (advisor) ; Kriška, Ján (referee)
Acute spinal cord injury is a serious type of injury, the treatment of which still represents a challenge for contemporary medicine. Neural stem cells (NSCs) transplantation is one of many promising ways to contribute to the regeneration of damaged tissue. NSCs communicate with the rest of the tissue by means of small extracellular vesicles (sEVs), in which their regenerative potential is thus assumed. The aim of this thesis is to verify the antiapoptotic effect of sEVs isolated from the culture medium of two types of NSCs in an rat in vitro model of spinal cord injury. To meet this goal, different methods of isolating sEVs from the culture medium were tested. Subsequently, sEVs were characterized according to their size and the presence of surface markers. This thesis includes an PCR analysis of the cargo of sEVs, which showed an abundance of neuroprotective and antiapoptotic miRNAs. Fluorescent staining of sEVs proved that sEVs penetrate into the cytoplasm of stem cells. Finally, sEVs were applied to a rat in vitro model of spinal cord injury and their neuroprotective effect was demonstrated using the immunoblotting method. Keywords Small extracellular vesicles, exosomes, acute spinal cord injury, regeneration of nervous tissue, miRNA, microRNA, neural stem cells
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The role of exosomes in chronic myeloid leukemia
Březinová, Lenka ; Krijt, Matyáš (advisor) ; Holada, Karel (referee)
Exosomes are extracellular vesicles of a size range 30-150 nm whose function has been explored in chronic myeloid leukemia (CML) due to their role in proliferation of CML cells, remodelling the bone marrow niche, angiogenesis and resistance to treatment with tyrosin- kinase inhibitors (TKIs). Although BCR-ABL kinase is effectively targeted by TKIs, 20-30 % of patients remain resistant to treatment. Resistance of CML cells to TKIs treatment is supported by exosomes. Exosomes transport proteins, nucleic acids, chemokines and small molecules that stimulate anti-apoptotic or suppress pro-apoptotic processes in leukemic cells. Anti-apoptotic processes are especially enhanced by upregulated protein levels: TGF- β1, USP6 and FGF2 and various types of RNA: miR-365, miR-21, Hsa_circ_0058493 and mRNA for BCR-ABL. In contrast leukemic cells tend to reduce the number of pro-apoptotic molecules, including miR-320, miR-328 and miR-146a-5p. Leukemic cells modify the bone marrow microenvironment through exosomes in the way to support their survival and also in order to adjust expression of adhesion and pro- angiogenic molecules. An important role in those processes play miR-126, miR-210 and miR- 92a. Neither the number of processes affected by CML exosomes nor their potential use in the treatment of CML is...
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Exosomes in viral infection and cancer
Sekavová, Alžběta ; Španielová, Hana (advisor) ; Hirsch, Ivan (referee)
Exosomes facilitate intercellular communication and transport of cellular cargo. Understanding the mechanisms underlying the cargo sorting to exosomes and the transport itself is crucial for vaccine development and diagnostic research. Exosome-mediated transfer contributes to immune response as well as progression of several diseases, including cancer and viral infections. Research on exosomes and their role in life cycles of tumorigenic viruses links already known mechanisms of viral carcinogenesis to the transport mechanisms of both cellular and viral proteins and nucleic acids. Epstein-Barr virus employs exosomes for transmission of the LMP1 oncoprotein and regulatory RNAs, whereas human immunodeficiency virus exploits cellular exosomal pathway for hijacking its membrane during budding, which helps it evade the immune system. It has been discovered that hepatitis C virus transfers its infectious virions between cells in exosomes. Exosomes containing oncoproteins and viral RNAs are also released from cells infected with other human tumorigenic viruses. However, mechanisms and implications of such events remain to be discovered. Keywords: exosome, cancer, viral infection, tumorigenic viruses, immunity, in- tercellular communication, hepatitis C virus, Epstein-Barr virus, human immuno- deficiency virus
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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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