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Knockout isoforem metalothioneinu pomocí metody CRISPR-Cas9 u adherentních tkáňových kultur
Duda, Jakub
It is not too long ago, that genome modifications were very complex, or, in some cases, almost impossible to achieve, but with the discovery and description of the CRISPR system in prokaryotes, a lot of breakthroughs came to the human knowledge, and thanks to some of these breakthroughs the CRISPR system has been modified to be used as a genome editing tool in eukaryotic organisms. Genetic modifications could in theory be the key to cancer therapy in modern pharmacy, and this thesis focuses on Metalmetallothionein (MT) proteins, which are commonly found in organisms and are beneficial to the organism (as inhibotors of oxidative stress, or as antioxidants binding heavy metals, for example), but can also be dangerous. Because, according to the isoform, and also the type of tissue, MTs can cause the development of cancer tissue and tumour growth, sometimes because of their presence, in other cases because of their absence. This thesis was focusing on the possible knock-out of all isoforms of MT at once, using CRISPR/Cas9 method, and thereby infucencing one of the very important factors in carcinogenesis. The result of this thesis is the achievement of knock-out of the MT3 isoform.
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Neuronal cell culture in vitro
Kohoutová, Šárka ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Neuronal cell cultures are in vitro cultures of dissociated neurons that have become an essential part of many neurobiological experiments in the last century. Cultured neurons not only allow to answer questions about their physiology under complex in vivo conditions, but also can serve as a model of neurodegenerative diseases. Neuronal cells can either be isolated directly from the nervous tissue of animals at the prenatal or adult stage of development, or they can be obtained through targeted manipulations of stem cells and secondary cell lines that lead to their neuronal differentiation. Primary neurons are considered the gold standard of neurobiological research not only because of their long tradition of cultivation, but also because primary neurons retain typical neuronal properties under in vivo conditions. There are several disadvantages associated with primary neurons, including the fact that fully differentiated neurons do not proliferate and are relatively demanding in terms of culture conditions For this reason, their role is often replaced by mitotically active secondary cell lines or stem cells. This bachelor thesis summarizes the knowledge about cell cultures used to study the functions of neuronal cells and highlights the advantages and disadvantages of their use. Key words Primary...
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Effect of chromatin on the repair of double-strand DNA breaks after cleavage by CRISPR/Cas and other programmable nucleases in plants
Trojan, Jakub ; Přibylová, Adéla (advisor) ; Procházková, Klára (referee)
Plants are highly resistant to ionizing radiation, also thanks to a high-quality repair system for repairing double-stranded breaks. Double-strand breaks in plants are repaired by four repair pathways. Most often, double-strand breaks are repaired by non-homologous end joining (NHEJ), which joins the broken ends without further processing. More accurate but slower and more complex is repair through homologous recombination (HR), which repairs the break using a homologous sequence. HR repair takes place preferentially in a region with active transcription and during the S and G2 phases of the cell cycle. Alternatively, repair further proceeds through single-strand annealing (SSA) or Theta mediated end joining (TMEJ). Both pathways are based on short homology between the overlapping ends of the double-strand break. An often neglected part of repairs is the overcoming of repressive chromatin, which protects the genome from DNA damage and prevents access of nucleases but also acts as a barrier for repair proteins. This work summarizes the current knowledge about DNA repair in plants. Furthermore, describe the influence of chromatin not only on the repair but also on the activity of programmable nucleases used in genetic engineering, such as zinc finger nucleases (ZFNs), transcription activator-like...
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Molekulárně-biologická diagnostika lidských polyomavirů
Ryšavá, Markéta
Most of the human population encounters human polyomaviruses during childhood, when the first infection is asymptomatic or with mild symptoms. However, these viruses persist in the human body and most often, they reactivate during immunosuppression. Together with JC virus reactivation, the progressive multifocal encephalopathy is associated. Hemorrhagic cystitis is associated with BK virus, resulting in the loss of allograft during kidney transplantation. Accurate diagnostics can detect viruses in a timely manner and mitigate tissue damage. This diploma thesis deals with biotechnologies, which can be used in virus detection with a focus on real time PCR, which is the gold standard in virus diagnostics. The literary research summarizes basic information about polyomaviruses with a focus on human BKV and JCV polyomaviruses. It summarizes the biotechnological methods used for detection of polyomaviruses, both in routine diagnosis and in alternative approaches involving biosensors and CRISPR. The experimental part of the work includes the design of a detection system for polyomaviruses from in silico genome analysis, through the design of potential primers, to theoretical specificity analysis. The practical part of the work compares the efficiency and sensitivity of amplification of two reaction mixtures, where one is intended for simple systems and the other for multiplexes. It also includes testing of selected additives of PCR and determining the sensitivity and validity parameters of the reaction mixture test selected for PCR system development. The results showed that detection using a multiplex reaction mixture is sufficiently sensitive, as it meets the conditions and requi-rements of clinical recommendations and at the same time shows very good values of sensitivity and specificity of the test comparable to published technologies. This reaction mixture appears to be relevant to the use of the development and optimization of a PCR system for the detection of polyomaviruses.
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Optimization of CRISPR/Cas9 technique for the Ixodes ticks genome editing
KITZBERGER, Daniel
CRISPR/Cas9 technique was used for genome editing of several arthropod species, but it has not yet been used on Ixodes spp. tick species. Therefore, in my work, I performed a bioinformatic analysis of the I. ricinus transcriptome and I. scapularis genome to reveal the sequence of the target gene for the CRISPR/Cas9-mediated knock-out. We prepared single guide RNA and mixed it with the Cas9 endonuclease to produce the Ribonucleoprotein complex and cleave the target sequence of the I. ricinus gene beta-galactoside alpha-2,6-sialyltransferase.
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The struggle for human dignity in the era of modern eugenics. Molecular genome editing tool CRISPR/CAS9 and its use in human genome therapy from the perspective of theological ethics
Auxt, Miroslav ; Štica, Petr (advisor) ; Fošum, Jan (referee)
The recent breakthrough discovery of the molecular genome editing tool CRISPR/CAS9 represents a complete revolution in the field of molecular biology, biomedicine and other related fields. It is a highly effective biomolecular tool, derived from the bacterial immune system, with which it is possible to introduce precise changes in the genomes of all organisms. The thesis is limited to the ethical evaluation of the use of CRISPR/CAS9 exclusively in human gene therapy. Thanks to its efficiency, simplicity, accuracy and low financial costs, the CRISPR/CAS9 editing tool, in compliance with ethical parameters, already has a broad spectrum of use in therapeutic procedures on somatic or body cells in the treatment of human genetically determined diseases without introducing a change into the future offspring of the given individual. In addition to great therapeutic potential, the application of CRISPR/CAS9 raises many ethical questions related to the possibilities of its further use, possibly misuse. Ethically problematic genetic procedures include: human hereditary genome editing, i.e. the targeted alteration of the genome of sex cells, progenitor cells and cells of early embryonic development stages with the therapeutic goal of eliminating a genetically determined disease associated with the...
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Biogenesis, structure and physiological functions of mitochondrial ATP synthase
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Doležal, Pavel (referee)
Mammalian mitochondrial ATP synthase is an enzyme composed of 18 protein subunits, which is localised in the inner mitochondrial membrane. Its main function is to utilise proton gradient, produced by respiratory chain complexes (RCC), for the synthesis of ATP. Aside from the creation of ATP it is known that its dimers contribute to the correct mitochondrial morphology through the formation of cristae apexes. Furthermore, ATP synthase was proposed to have a role in the mitochondrial permeability transition phenomenon, which is important for regulation of programmed cell death. Over the recent years, our understanding of mammalian ATP synthase biogenesis has been tremendously improved. Its assembly process is now clarified, however the knowledge about assembly intermediates of its peripheral stalk and of subunit c are still not sufficient. We focused precisely on those unsolved questions in the fields of ATP synthase biogenesis and its secondary functions, by the production of a KO model of catalytic β subunit of mammalian ATP synthase F1 domain (βKO). This model was successfully prepared on the background of HEK293 cell line. Its characterisation revealed that disruption of the F1 structure resulted in the inability to assemble functional monomer and resulted in a decay of individual subunits. The only...
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Analysis of Gretchen Hagen 3 gene family in tobacco BY-2 cell culture
Helusová, Lenka ; Müller, Karel (advisor) ; Pěnčík, Aleš (referee)
Auxin conjugation is one of the crucial metabolic processes regulating auxin activity in plant cells. Gretchen Hagen 3 (GH3) is a family of acyl amido synthetases that conjugates auxin with amino acids and belongs amongst important enzymes involved in auxin conjugation. Due to the existence of more sensitive methods to detect auxin metabolites and the current study of abiotic stress effects, research on GH3 enzymes is intensified these days. These enzymes are best known in thale cress (Arabidopsis thaliana), soya bean (Glycine max), rice (Oryza sativa). These models don't allow to study their activities in a biochemical way. Therefore, the aim of this work was to monitor the auxin metabolism in the established model tobacco BY-2 cell lines (Nicotiana tabacum). The NtGH3.1 and NtGH3.6 genes, which were shown to have a variability in their expression regulation by auxin, were targeted and mutated using tne CRISPR/Cas9 method. Mutations in the derived lines were detected by sequencing. In the derived lines, auxin metabolic profililing was analysed by LC/MS. Metabolic profiling showed a correlation between the NtGH3.6d form and the specific production of the metabolite oxIAA- Gln (N-(2-onindole-3-acetyl)-glutamine). The study of an eventual substitution of individual GH3 gene forms in mutant lines...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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