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Identification and characterisation of selected properties of some strains of lactic acid bacteria
Sásková, Denisa ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Nanotechnology is currently one of the fastest growing scientific disciplines. An interesting area of research is the biosynthesis of metal nanoparticles using microorganisms including lactic acid bacteria. In the first part this diploma thesis focuses on verification of identity of bacterial species Lactobacillus casei and Lactobacillus paracasei by genus- and species-specific PCR. In the next part of experimental work the capability of six Lactobacillus strains to produce silver nanoparticles is tested. Synthesis of nanoparticles was confirmed for all the strains depending on the amount of added AgNO3 and on time of cultivation. Differences between the strains were detected.
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Low power module with thermocamera
Horák, Daniel ; Najman, Jan (referee) ; Chalupa, Jan (advisor)
This bachelor thesis is focused on contactless temperature measuring with given thermographic camera and lossless image compression obtained by this camera. The theoretical part describes principles for better understanding of the issue and the practical part contains the solution itself, prepared for above mentioned thermographic camera.
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Izolation and identification of DNA from probiotic bacteria in complex matrices
Balogová, Petra ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Nowadays, probiotic lactic acid bacteria (LAB) and bifidobacteria are plentifully exploited in food processing industry. LAB and bifidobacteria are important part of microflora of gastro intestinal tract (GIT). Probiotics (most often just lactobacilli and bifidobakteria) can be supplied to the GIT of the organism like food complements. Species identification is therefore very important. New methods of identification of LAB and bifidobacteria are based on analysis of DNA. Mostly exploited method is polymerase chain reaction (PCR). In my diploma work, genus and species specific PCRs were used for identification of different species of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of six food supplements (Zenflo, Linex Forte, Probian, Nutra Bona, GS lactobacillus Forte, Pangamin Bifi plus). Total DNA was isolated from crude lysates of cells present in tablets by magnetic particles coated by carboxyl groups . The preparation of cell lysates was optimalised. Different amounts of lysozyme (3 mg/ml, 10 mg/ml), time of incubation at laboratory temperature (1,5 hour, 3 hour) and time of incubation with SDS and proteinase K at 55 °C (1 hour, 3 hour, over all night) were tested. Isolated DNA was quantified and checked in PCR. Primers specific for genus Lactobacillus and Bifidobacterium and for species Lb. acidophilus, Lb. casei, Lb. rhamnosus and Lb. plantarum and B. animalis, B. bifidum, B. infantis, B. longum were used, respectively. All identified bacteria were in accord with the data declared by producer in 3 food supplements (Zenflo, Linex Forte and Pangamin Bifi Plus). The genus indentification was in accord with declaration of producer in other food products only (Probian, Nutra Bona, GS Laktobacily Forte).
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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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Software Design of Single Purpose Machine for Visual Inspection
Horák, Daniel ; Krejsa, Jiří (referee) ; Štarha, Pavel (advisor)
This master’s thesis deals with the fundamentals of machine vision application and its practical implementation. The research part is focused on the basic possibilities of image acquisition and image processing in different dimensions. The practical part describes the design of the dimension control algorithm using a 3D camera. This algorithm is then implemented in a single-purpose machine for optical dimension control.
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Photon-upconversion nanoparticles for single-molecule immunosensing of cancer biomarkers and bacteria
Farka, Z. ; Mickert, M. J. ; Hlaváček, Antonín ; Poláchová, V. ; Kostiv, Uliana ; Pastucha, M. ; Horák, Daniel ; Gorris, H. H. ; Skládal, P.
The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg·mL−1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU·mL−1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.
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Low power module with thermocamera
Horák, Daniel ; Najman, Jan (referee) ; Chalupa, Jan (advisor)
This bachelor thesis is focused on contactless temperature measuring with given thermographic camera and lossless image compression obtained by this camera. The theoretical part describes principles for better understanding of the issue and the practical part contains the solution itself, prepared for above mentioned thermographic camera.
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Effect of iron oxide nanoparticles with ascorbic acid on neural stem cells
Jiráková, Klára ; Moskvin, Maksym ; Horák, Daniel ; Jendelová, Pavla
Cells labelled with iron oxide nanoparticles (ION) can be tracked by magnetic resonance imaging (MRI) in several applications. However, various studies demonstrated toxicity and oxidative stress induction associated with nanoparticles exposure. We analysed biologic effects after the exposure of two types of iron oxide nanoparticles (with and without an antioxidative agent, an ascorbic acid) on human neural stem cells. The labelled cells in gel phantoms were detected in MRI and they showed decreased relaxation rates in comparison with control. ION slightly decreased cell proliferation in comparison with unlabelled cells, which was dependent on concentration and presence of ascorbic acid. None of the nanoparticle type showed negative effect on cell viability and both demonstrated minor effect on reactive oxygen species (ROS) formation. Unfortunately, ascorbic acid bound to nanoparticles did not show any effect on ROS attenuation. Cells exposed to both types of nanoparticles showed increased positivity for a phosphorylated form of H2AX a marker of double strand breaks. We showed that ION in low concentrations do not affect cell viability, but have negative effect on cells on DNA level. Their potential use for oxidative stress reduction is dependent on the concentration of ascorbic acid bound to the nanoparticles and this should be further increased.
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Design and characterization of advanced polymer-coated upconversion nanoparticles
Kostiv, Uliana ; Horák, Daniel (advisor) ; Kaman, Ondřej (referee) ; Brožek, Jiří (referee)
Lanthanide-based upconversion nanoparticles are able to absorb low-energy near-infrared (NIR) photons and emit visible or ultraviolet light. This unique optical property enables luminescent detection without autofluorescence from biological samples that is crucial for bioimaging and diagnostics. Moreover, NIR irradiation allows to deliver light deep into the tissue, which can be used for drug or gene delivery and NIR-induced photodynamic therapy (PDT) of tumors. Despite huge effort during the last 10 years devoted to the upconversion nanoparticle synthesis and their surface engineering, preparation of the particles suitable for biomedical applications still remains a big challenge. The current work is focused on synthesis of the upconversion nanoparticles with controllable morphology, size, distribution, crystallinity, and high upconversion efficiency, as well as on desirable surface modification by biocompatible inorganic or organic polymers. Neat and functionalized homogeneous silica shell was introduced on the NaYF4:Yb3+ /Er3+ and NaGdF4:Yb3+ /Er3+ nanoparticles to enhance their biocompatibility and colloidal stability in water. To render the nanoparticles with targeting moieties, their surface was decorated with cell-adhesive RGD or cell-penetrating TAT peptides. The RGD- and TAT- conjugated...
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