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Study of exosomes in polyomavirus infection
Hyka, Lukáš ; Šroller, Vojtěch (advisor) ; Saláková, Martina (referee)
Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...
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Comparison of DNA isolation methods
ŠESTÁKOVÁ, Kristýna
Isolating DNA is crucial in genetic laboratories. I tis the first method of further steps to obtain DNA information. The isolation method requirements are high, Due to the similarity of DNA to other biopolymers, the method must exhibit high selectivity and also sensitivity due to the low DNA content of the biological material. The input sample from clinical practice is usually human blood and nuclear cells contained in it. This thesis describes why blood is most commonly used. From the volunteer blood obtained, DNA was isolated by four methods: salting out, based on a change in DNA solubility depending on the change in ion concentration in the solution; phenol-chloroform extraction, which is one of the cheapest and oldest methods; in addition, using a commercially available kit, a fast and simple method using DNA binding to a silicate surface; and isolating with an automated magnetic particle separator (Anzenbacher and Kovář, 1986). Each method has its advantages and disadvantages and its choice depends on the further handling of DNA. The most common criteria for choosing an isolation method is its time-consuming , the cost of the method, the yield and the purity of the DNA. In this thesis these requirements are compared amongst methods.
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