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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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