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Surface functionalization of the biological gold nanoparticles for micro-rna targeting
Pourali, Parastoo ; Benada, Oldřich ; Benson, Veronika
Among non-viral gene carriers with low toxicity and high transfection efficiency, the use of gold nanoparticles (AuNPs) is of particular interest due to their biocompatibility and special properties. This is the first time we attempted to functionalize the surface of the biological AuNPs in order to conjugate them with antimiR-135b through electrostatic interactions and knockdown the microRNA-135b gene expression inside the cells. A fungal strain, Fusarium oxysporum, was cultured in Sabouraud Dextrose Broth (SDB), centrifuged, and the mycelium-free supernatant was challenged with 1 mmol final concentration of HAuCl4.3H2O and incubated for 24 h at 37°C in a shake flask. AuNPs were characterized by visible spectrophotometry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDS), and a zetasizer. The washed and sterilized AuNPs were used for cytotoxicity and conjugation assays. First transferrin (Tf) and then polyethylenimine (PEI) were used to functionalize and change the surface charge of the AuNPs and then antimiR-135b was conjugated to the AuNPs trough electrostatic interactions. Their association was confirmed by visible spectrophotometry and electrophoresis. Confocal microscopy was used to investigate the internalization of the AuNPs-antimiR-135b complex. The results proved the formation of AuNPs with a maximum absorption peak at 528 nm, round and oval shapes (15-20 nm), and average zeta potential of -21.02 mV. The AuNPs-antimiR-135b showed delayed electrophoresis unlike antimiR-135b or AuNPs alone. Functionalized AuNPs did not cause any toxicity in cell culture and confocal microscopy showed successful transfection of AuNPs-antimiR-135b into the vast majority of 4T1 cells. We concluded that the biological AuNPs were non-toxic and they could carry antimiR-135b to enable gene silencing
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Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
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Analysis of pulmonary samples infected with Aspergillus fumigatus and Pseudomonas aeruginosa by scanning electron microscopy
Juříková, Tereza ; Benada, Oldřich (advisor) ; Nebesářová, Jana (referee)
Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....
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Electron cryo-microscopy techniques in biological research and nanotechnologies
Mistríková, Veronika ; Bednár, Jan (advisor) ; Nebesářová, Jana (referee) ; Benada, Oldřich (referee)
Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...
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Analysis of pulmonary samples infected with Aspergillus fumigatus and Pseudomonas aeruginosa by scanning electron microscopy
Juříková, Tereza ; Benada, Oldřich (advisor) ; Nebesářová, Jana (referee)
Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....
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Electron cryo-microscopy techniques in biological research and nanotechnologies
Mistríková, Veronika ; Bednár, Jan (advisor) ; Nebesářová, Jana (referee) ; Benada, Oldřich (referee)
Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...
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Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
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Gold chemical vapor generation by tetrahydroborate reduction for AAS: Radiotracer efficiency study and characterization of gold species
Musil, Stanislav ; Arslan, Y. ; Kratzer, Jan ; Vobecký, Miloslav ; Benada, Oldřich ; Matoušek, Tomáš ; Rychlovský, P. ; Ataman, O. Y. ; Dědina, Jiří
The main objective was to assess the generation efficiency in the optimized method of chemical vapor generation of gold by means of 198,199Au radioindicator. We also intended to characterize the volatile Au species formed by tetrahydroborate reduction.
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Příprava TiO2 pro fotokatalytické aplikace
Matějová, Lenka ; Cajthaml, Tomáš ; Matěj, Z. ; Strýhal, Z. ; Benada, Oldřich ; Šolcová, Olga
Powder nanoporous TiO2 with narrow monodisperse pore-size distribution in the range from 2 to 30 nm were prepared by self-assemble micelle system in nonpolar and polar solvents at laboratory temperature and pressure. Nonionic surfactants Triton X-102, 100, 114, 45, 15 were used for preparation of micelle systems. Various approaches were applied for removing of residual organic matter from prepared titania gels: a) calcination, b) improved extraction by supercritical (CO2) and subcritical fluids (H2O, CH3OH) and c) the combination of both forenamed approaches.
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