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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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Role of microvesicles and exosomes in reproduction of mammals
Straková, Johana ; Frolíková, Michaela (advisor) ; Petr, Jaroslav (referee)
Microvesicles and exosomes are extracellular vesicles of nanometer size derived from cell membranes. Due to their ability to transfer proteins, lipids and RNA, microvesicles and exosomes are now considered to be a common form of communication between somatic cells. Microvesicles and exosomes have been detected in the reproductive organs of male and female mammals, where they mediate the transport of molecules between cells and thus promote their communication during physiological and pathological processes. Signalling through microvesicles and exosomes is involved in sperm maturation processes, which enrich these vesicles for molecules required for motility and fertilization capacity. Similarly, during oocyte maturation, microvesicles promote communication between follicle cells and its growth. During fertilization, the molecules transported by microvesicles promote capacitation and acrosomal reaction and, after gamete fusion, support the developing embryo and its subsequent implantation in the uterus. The main focus of this bachelor thesis is to discuss the role of microvesicles and exosomes in various processes in mammalian reproduction from sperm and egg maturation to successful fertilization and embryo implantation. A significant part of the thesis is devoted to the presentation of molecules...
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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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Molecular events associated with resistance to tyrosine kinase inhibitors in leukemia cells.
Hrdinová, Tereza ; Vyoral, Daniel (advisor) ; Klener, Pavel (referee) ; Holoubek, Aleš (referee)
Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disease characterized by the expression of BCR-ABL oncoprotein with constitutive tyrosine kinase activity. Although the development of tyrosine kinase inhibitors (TKI) such as imatinib dramatically improved the treatment of CML, a certain subset of patients develops resistance to TKI drugs. The most common cause of TKI resistance are point mutations in the BCR-ABL1 gene, followed by other mutation-independent mechanisms. Survival and proliferation of CML cells in the presence of TKI drugs are accompanied by adaptive changes in their metabolism. Drug resistance can be maintained by extrinsic signals, among which exosomes, small vesicles released by (drug-resistant) cells, have been shown to play an important role. The aim of this thesis was to characterize two CML cell lines sensitive and resistant to imatinib, as well as the exosomes derived from imatinib-resistant CML cells by proteomic approaches. Identification of metabolic vulnerabilities in drug-resistant cells enables their targeting by clinically available drugs, thus offering potential therapeutic targets for their selective elimination. Analysis of exosomes derived from imatinib-resistant cells can identify specific membrane surface proteins exploitable as clinically relevant...
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Role of Exosomes in the Progression, Diagnosis, and Treatment of Brain Tumors
Vaníková, Lucie ; Zíková, Martina (advisor) ; Lacina, Lukáš (referee)
Recent studies have confirmed the importance of extracellular vesicles, particularly exosomes, in the development of brain tumors. Considerable attention has been paid mainly to the influence of exosomes on biological processes in brain tumors. Exosomes mediate intercellular communication in the tumor microenvironment by transporting biomolecules. Most often they transmit various types of ribonucleic acids, specifically microRNAs, which affect the signalling pathways related to tumour growth in target cells. Thus, exosomes play an important role in tumor cell proliferation and differentiation, metastasis, and tumor resistance to chemotherapy or radiation. Due to their small size, exosomes can cross the blood-brain barrier and thus promote tumor progression. The topic of the bachelor thesis is a summary of the current knowledge on the role of exosomes in brain tumor progression, diagnosis and treatment.
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Study of exosomes as drug delivery system in therapy of glioblastoma
Tomášková, Lucia ; Šimůnek, Tomáš (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucia Tomášková Supervisor: prof. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of exosomes as a drug delivery system in the treatment of glioblastoma Central nervous system disorders are among the most serious diseases affecting humans. They affect not only the patient's life, but also his/her surroundings. Therefore, their therapy, whether at the level of complete cure or alleviation of accompanying symptoms, is a challenge for scientific research. In our research, we focused on glioblastoma multiforme, a brain cancer not yet treatable. The main drawback in therapy is overcoming the blood-brain barrier. Exosomes, such as the body's natural nano-vesicles, have been shown to be a suitable system for delivering drugs to brain tissue. Our research has shown that by a suitable method we are able to obtain sufficient quality exosomes from macrophage and fill them very efficiently with antitumor agents paclitaxel, doxorubicin and temozolomide, while the delivered substances show higher efficacy and fewer side effects than the free form.
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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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Rab-2A protein: its localization and function in human spermatozoa and fertilization
Sadílková, Lucie ; Postlerová, Pavla (advisor) ; Lánská, Eva (referee)
The Rab-2A protein belongs to the Rab family of monomeric G-proteins involved in the regulation of intracellular vesicular transport. Although several studies describing the role of Rab-2A protein in mammalian sperm have been published, the exact localization and function of this protein in male gametes have not been fully understood yet. In the diploma thesis, we were the first to describe the presence of Rab-2A in human sperm using 5C5, produced by the Laboratory of Reproductive Biology. Using the 5C5 antibody, we were able to identify several Rab-2A isoforms with molecular weights of 26, 24, 22, and 18 kDa in the human sperm lysate, confirmed by mass spectrometry analysis. Using the indirect immunofluorescence method, we localized Rab-2A in the acrosome area of both ejaculated and capacitated sperm. We noticed that Rab-2A leaves the sperm during the acrosomal exocytosis. We also found a reduced Rab-2A protein level in sperm of patients with various pathologies of ejaculate. Based on studies describing the role of Rab-2A in acrosome biogenesis and the presence of Rab-2A in bovine sperm perinuclear theca, we tried to detect Rab-2A in this protein layer in human sperm. However, the chosen method is apparently not applicable to human sperm and isolation has failed. Additionally, we demonstrated the...
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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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Study of exosomes as drug delivery system in therapy of glioblastoma
Tomášková, Lucia ; Šimůnek, Tomáš (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucia Tomášková Supervisor: prof. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of exosomes as a drug delivery system in the treatment of glioblastoma Central nervous system disorders are among the most serious diseases affecting humans. They affect not only the patient's life, but also his/her surroundings. Therefore, their therapy, whether at the level of complete cure or alleviation of accompanying symptoms, is a challenge for scientific research. In our research, we focused on glioblastoma multiforme, a brain cancer not yet treatable. The main drawback in therapy is overcoming the blood-brain barrier. Exosomes, such as the body's natural nano-vesicles, have been shown to be a suitable system for delivering drugs to brain tissue. Our research has shown that by a suitable method we are able to obtain sufficient quality exosomes from macrophage and fill them very efficiently with antitumor agents paclitaxel, doxorubicin and temozolomide, while the delivered substances show higher efficacy and fewer side effects than the free form.
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