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Pseudotyping in baculoviruses
Zobalová, Eliška ; Španielová, Hana (advisor) ; Fraiberk, Martin (referee)
The baculoviruses are a group of enveloped DNA viruses that infect the larval stage of arthropods, mainly insects. They are widely used in biotechnology and well known for their utility as biological pesticides and gene expression vectors for the production of proteins in insect cells and larvae. However, they are also able to enter in mammalian cells and deliver-genes for expression under the control of mammalian cell-active promoters. Recombination or pseudotyping can result in formation of baculoviruses that provide a higher transduction frequency or are able to recognize specific mammalian cells. This bachelor thesis describes the phenomenon of viral pseudotyping and summarizes published information about the use of pseudotyped baculoviruses for gene therapy and vaccination.
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Programmable nucleases in human therapy
Šlaufová, Marta ; Kašpárek, Petr (advisor) ; Černý, Jan (referee)
Most genome disorders cause severe symptoms and are usually incurable. Recent, rapid development of programmable nucleases (PNs) brought new possibilities for the treatment of many diseases, such as genetic disorders, infectious diseases or cancer. PNs are enzymes, which enable site specific DNA cleavage that can lead to targeted modification of desired genomic loci. They are composed of separable non-specific cleavage domain and DNA- binding domain. The DNA binding domain is in the form of modular DNA-binding proteins or complementarity-based pairing of the oligonucleotide. The non-specific cleavage domain mediates DSB stimulation, which is necessary for further genome editing. Development of zinc finger nucleases (ZFNs) followed by transcription activator-like effector nucleases (TALENs) enabled the first therapeutic approaches based on targeted manipulation of human genome. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology brought further simplification to the method and broadened the availability of PN-based toolkits. This thesis will provide a summary of the recent developments, application of PNs in the therapy of human patients and potential obstacles preventing their implementation in clinics.
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Possibel approaches to gene therapy of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Vaněk, Ondřej (referee)
1 Abstract Cystic fibrosis is an autosomal recesive disorder caused by a mutation in the CFTR gene, which leads to inefficiency or absence of CFTR chlorid channel. One way to induce production of CFTR protein in target cells, is to use gene therapy. The principle od gene therapy is to transfer DNA or mRNA molecules inside malfunctioning cells. The aim of this study was to optimise the detection of CFTR protein using the Western blot analysis. Then, using this method the effectiveness of CFTR-mRNA transfection was studied. To study the CFTR protein, a number of cell lines was used: a healthy human ephitelial cell line (NuLi-1), an ephitelial cell line with ΔF508 mutation (CuFi-1), and a human lung carcinoma cell line (A549). This study compared four different ways of cell lysis - lysis by sonication and lysis by three distinct lysis buffers. Lysis by RIPA buffer with protease inhibitors was determined for the detection of CFTR protein. Moreover, three different primary monoclonal antibodies were also tested. The CF3 antibody, which is specific to an extracellular epitope of CFTR protein, was found able to detect CFTR protein specifically. A couple of different glycosylated forms of CFTR protein was detected. The highest amount of CFTR protein was determined in the NuLi-1 cell line. CFTR protein was also...
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Polymer systems for siRNA delivery
Blažková, Jana ; Pechar, Michal (advisor) ; Šťovíček, Vratislav (referee)
The process of RNA interference (RNAi) is a natural phenomenon posttranscriptionally controlling gene expression by means of small double-stranded RNA molecules (dsRNA). Small interfering RNA (siRNA) is a small dsRNA that can be used for targeted gene silencing as an alternative therapeutic treatment of genetic diseases. For in vivo administration, siRNA must be protected against degradation to ensure its efficient delivery to target cells using sophisticated vectors. This work is focused on description of non-viral vectors based on cationic polymers, forming polyelectrolyte complexes with siRNA (polyplexes), and surface-modifying hydrophilic polymers enabling protection of the vector during its transport in the bloodstream.
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Biocompatibility and immunocompatibility of polymers for gene therapy
Matyášová, Veronika ; Šírová, Milada (advisor) ; Tučková, Ludmila (referee)
Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...
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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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