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Smart hydrogen sensor
Kašpárek, Petr ; Pavlík, Michal (referee) ; Boušek, Jaroslav (advisor)
The main goal of this diploma thesis is to get familiar with organic sensors developed at the Faculty of Chemistry at Brno University of Technology. The outcome is the implementation of smart hydrogen detector which is based on use of these sensors. This device will allow to test and use relevant sensors outside the test apparatus placed in the laboratory of the Faculty of Chemistry at Brno University of technology.
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Development and optimization of system for measuring of watering
Kašpárek, Petr ; Macháň, Ladislav (referee) ; Žák, Jaromír (advisor)
The main goal of this thesis is to become acquainted with questions of sensors and the problems of measuring the plant watering. The result is creating an intelligent prototype of a module that should measure temperature, conductivity and pH of the watering. Automatic measuring of these properties makes the parameter evaluation easier and quicker. This measuring module extends contemporary device with the possibility to measure properties of the plant watering.
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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Generation and analysis of mutant mouse models to study pathophysiological roles of KLK5 and KLK7 in epidermis
Kašpárek, Petr ; Sedláček, Radislav (advisor) ; Stopka, Pavel (referee) ; Machoň, Ondřej (referee)
Kallikrein-related peptidases (KLKs) constitute a family of closely related serine proteases encoded by genes clustered in one chromosomal locus. KLKs are widely expressed in a variety of tissues and numerous in vitro experiments suggest their important roles in many physiological and pathological processes. However, the biological roles of KLKs in vivo are often obscured mainly due to unavailability of suitable animal models. Although gene deficient mouse models were generated for several KLK genes, they had limited use for understanding the roles of individual proteases in the complex environment in vivo. One of the main obstacles which hampers in vivo analysis is partial functional overlap between some KLKs. This makes traditional single-gene deficient animal models an inadequate tool to address the biological impact of the gene deficiency as compensatory mechanisms often result in a lack of phenotype. In this work, we used the transcription activator-like effector nuclease (TALEN) technology to generate several novel mutant mouse models to study the complex KLK proteolytic pathways and their roles in healthy organism and in disease. We prepared a novel mouse model for Netherton syndrome (NS), an autosomal recessive skin disorder caused by mutation in the gene SPINK5, which encodes the KLK-inhibitor...
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Generation and analysis of mutant mouse model to study roles of KLKs in cutaneous inflammation
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Drbal, Karel (referee)
Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases of undisputable importance for a variety of functions, whose dysregulation has been linked to several pathological phenotypes. Among those pathologies, the Netherton syndrome stands out, since it is one of the very few that has its mechanism directly linked to KLK proteases as the main culprit of the disease, namely KLK5, KLK7 and to a lesser degree, KLK14. In this case, a mutation in the SPINK5 gene leads to uncontrolled hyperactivity of those proteases, which results in epidermal barrier breach due to excessive epidermal desquamation and severe inflammation of the skin. Inflammation mechanisms of NS are still relatively poorly understood, with important roles being attributed to the activities of KLKs in the processing of immune system molecules and also to the dysregulation of the cutaneous microbiome. TNFα signalling plays a key role in the homeostasis and immune response in the skin. Chronic skin infections may lead to deleterious effects with strong participation of TNFα signalling. To address the degree of its effects on the pathogenesis of NS, we have created a mouse model where the TNFR1 is disrupted by knockout of the Tnfr1 gene on the background of a previously established mouse model of the Netherton syndrome. We...
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The role of mitochondrial complex II in cancer cell biology
Kraus, Michal ; Neužil, Jiří (advisor) ; Kašpárek, Petr (referee)
Mitochondria are essential organelles for most eukaryotic cells, containing intricate networks of numerous proteins. These include, among others, complexes I-IV of the electron transport chain. Being at the crossroads of the tricarboxylic acid cycle and the respiratory chain, mitochondrial complex II plays a key role in cellular metabolism. The protein complex, also known as succinate dehydrogenase, is capable of not only succinate oxidation and electron transfer but also contributes to the production of reactive oxygen species. Mitochondrial complex II consists of four subunits, SDHA-D, and four dedicated protein assembly factors SDHAF1-4 that participate in complex II biogenesis. Mutations and epigenetic modulations of genes coding for succinate dehydrogenase subunits or assembly factors are associated with pathological conditions such as neurodegenerative diseases, or may result in tumor formation. However, inborn complex-II-linked mitochondrial pathologies are rather understudied, compared to diseases with causative errors of other mitochondrial complexes, presumably due to the fact that none of complex II subunits is encoded in the mitochondrial genome. Recent studies have shown that impairment of mitochondrial complex II function or assembly leads to accumulation of alternative assembly forms...
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The tumor immune microenvironment and its crosstalk with kallikrein-related peptidases in mammary carcinoma of a mouse model
Šlaufová, Marta ; Kašpárek, Petr (advisor) ; Brábek, Jan (referee)
Breast cancer is the most common cancer type with a high annual death rate. Finding meaningful tissue-related or body-fluid-accessible biomarkers is necessary to characterize cancer subtype, predict tumor behavior, choose the most effective therapy, predict severe treatment-related toxicities, and also the opportunity to personalize treatments for each patient. There is increasing evidence that various kallikrein-related peptidases (Klk) gene family members can modulate the immune response and are differentially regulated in breast cancer, and therefore are proposed to be potential prognostic biomarkers. This work established and validated an experimental setup to study the roles of selected kallikrein-related peptidases (KLK5, KLK7, KLK14) in breast cancer in vivo using gene-deficient mouse models previously generated in our laboratory. We used the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) editing system to generate several E0771 cell line-based reporter and gene-deficient cell lines. These allowed enhanced monitoring of cancer progression in vivo and studying KLKs roles in tumor immune microenvironment of C57Bl/6N mice. Finally, we present the analysis of the initial in vivo experiments using these tools combined with established Klk-deficient mouse models. Our...
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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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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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Generation of large animal models using genome editing
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Kašpárek, Petr (referee)
The principle of gene engineering is the intervention to the DNA of the studied organism. After the discovery of the programmed endonucleases, there has been a great expansion of this technique and it also accelerated the possibilities to create large animal models. Until recently, large animal models were very difficult to be generated. These endonucleases include zinc finger nuclease (ZFN), transcription activator like effector nuclease (TALEN) and CRISPR/Cas9. All endonucleases produce locally specific splicing in the targeted segment of the genome. This splicing is most easily corrected by the non-homologous ends joining (NHEJ), so then it is possible to create a so -called knock-out (KO) model. The second type of repair is homologous recombination (HR) using a DNA template with homologous arms. This makes it possible to create a knock-in (KI) model that cannot be created without specific endonucleases in large animal models due to the low natural HR. This work summarizes the history, technique and the use of programmed endonucleases for the creation of large animal models. These models have a great use in biomedicine, mostly in preclinical research, they are also significant in agriculture and even in the environment protection. Key words: large animal model, transgenesis, genome editing,...
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