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Study of the key points of lincomycin and celesticetin biosynthesis
Vobruba, Šimon ; Janata, Jiří (advisor) ; Bobek, Jan (referee) ; Kutejová, Eva (referee)
Lincosamides form a small but important group of specialized microbial metabolites with antibiotic activity. The most important members of this group are celesticetin and clinically used lincomycin. Structurally, lincosamides are composed of an amino sugar and an amino acid connected by an amide bond. The amino acid precursors of both lincosamides remarkably differ. Proteinogenic L-proline is the precursor of celesticetin, while an unusual amino acid (2S,4R)-4-propyl- L-proline (PPL) is incorporated in the more efficient compound lincomycin. Surprisingly, both these precursors are recognized and activated for further biosynthetic steps by homologous adenylation domains CcbC and LmbC, respectively. The detailed description of this amino acid recognition and activation step, which is critical for the biological activity of the resulting compound, was the aim of the first part of this thesis. The site-directed mutagenesis of the LmbC substrate binding pocket and biochemical characterization of resulting mutants were employed to identify the residues crucial for the activation of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket (like in CcbC) to the PPL-specific enzyme (LmbC). The substitution of only three amino acid...
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Drones (UAVs) in the area of emergency services (Integrated Rescue System)
Bobek, Jan ; Baumgartnerová, Alena (referee) ; Rujbrová, Šárka (advisor)
Drony jsou v oblasti integrovaného záchranného systému používány stále častěji, díky jejich lepší dostupnosti a ceně. V policejní oblasti jsou drony využívány k monitorování různých akcí nebo hlídání podezřelých oblastí. Policie drony také využívá k rekonstrukci dopravních nehod. V hasičské oblasti se drony používají hlavně k monitorování požárů a předávání informací pilotovi dronu, ale jsou vyvíjeny i drony určené k hašení požárů. V urgentní medicíně jsou drony využívány k přepravě vybavení a krevních vzorků v místech, kde to pozemními prostředky není možné nebo to je obtížné. Cílem této bakalářské práce je poskytnout přehled dronů využívaných v oblasti integrovaného záchranného systému a popsat technologie související s těmito drony.
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Regulation of transcription in Gram-positive bacteria
Rabatinová, Alžběta ; Krásný, Libor (advisor) ; Bobek, Jan (referee) ; Valášek, Leoš (referee)
Bacteria are the most abundant organisms on the planet. They live almost in all environments, including those that are most extreme. All land and water ecosystems depend heavily upon their activity. Bacteria play essential roles in cycling of nutrients such as carbon, nitrogen, and sulphur. Due to their short cell cycle, they must be able to swiftly adapt to the conditions of their habitat to survive. Microbial growth itself is an autocatalytic process. There are three distinct phases of the growth curve: lag, exponential (log), and stationary. Bacterial cells must change their gene expression between these phases in order to adapt to the new conditions. The first stage of gene expression is transcription. The key enzyme of this stage is RNA polymerase (RNAP) that transcribes DNA into RNA. RNAP is regulated by a number of accessory proteins and also small molecule effectors. Understanding how RNAP functions is essential for understanding how bacteria cope with changing environments. This Thesis presents studies of selected aspects of bacterial gene expression regulation at the level of transcription, using Bacillus subtilis as the model organism. The first part of this Thesis focuses on protein determinants of the ability of RNAP to be regulated by the concentration of the initiating nucleoside...
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Analysis and mapping of binding sites of gene expression regulators in the genus of Streptomyces.
Šmídová, Klára ; Bobek, Jan (advisor) ; Krásný, Libor (referee) ; Kopecký, Jan (referee)
Streptomyces are medically important soil-living bacteria that undergo morphological changes from spores to aerial hyphae and are important producers of bioactive compounds including antibiotics. Their gene expression is tightly regulated at the early level of transcription and translation. In the transcriptional control, sigma factors play a central role; the model organism Streptomyces coelicolor possesses astonishing 65 sigma factors. The expression of sigma factors themselves is controlled on the post-transcriptional level through the action of sRNAs that modify their mRNA level. However, only several sigma factors in Streptomyces have known regulons and also their sRNAs-mediated regulation has not been studied so far. According to previously measured gene expression data, we selected several highly expressed sigma factors. Using mutant strains with HA-tagged sigma factors, regulons of two important sigma factors, SigQ and HrdB, were analyzed by ChIP-seq procedure. Other sigma factors were further studied to see if they possess asRNAs, using 5' and 3' RACE method and northern blotting. Our data confirm the essentiality of HrdB sigma factor during the vegetative phase of growth. The other sigma factor, SigQ, has been revealed to be an important regulator of nitrogen metabolism and osmotic...
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Function of stress sigma factors of RNA polymerase SigD, SigE, SigH and SigM in transcription regulation network of Corynebacterium glutamicum
Dostálová, Hana ; Pátek, Miroslav (advisor) ; Bobek, Jan (referee) ; Halgašová, Nora (referee)
Grampositive bacterium Corynebacterium glutamicum is an important industrial producer of amido acids and other metabolites. Its genome encodes 7 sigma (σ) subunits of RNA polymerase: primary factor σA , primary-like σB and five alternative sigma factors, σC , σD , σE , σH and σM (sigma factors with extracytoplasmic function). This study is focused on revealing so far unknown regulons of stress sigma factors or closer description of regulons whose genes are controled by σD , σE , σH and σM . These factors were partially described for their activity during surface (σD and σE ), heat (σE , σH and σM ) and oxidative (σH and σM ) stress response. We assumed that the genes of each regulon are transcribed from promoters of a single class. For the purpose of detailed promoter analysis, it was necessary to develop methods which can quickly and reliably assign sigma factor to particular promoters and, thus, respective genes. For this purpose, a combination of in vivo (two-plasmid system) and in vitro (in vitro transcription) techniques was developed that allow to specify this assignment. We identified 9 σH /σE - promiscuous promoters (PamtR, Pcg0378, Pcg1121, Pcg3309, Pcg3344, PclgR, PdnaJ, PdnaK and PsigB), 7 σD /σH - promiscuous promoters (Pcg0607, Pcg2047, Pcmt2, PfadD2, Plpd, PlppS and PrsdA) a 9 σH /σM...
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Preparation and testing of SNOM probes
Bobek, Juraj ; Neuman, Jan (referee) ; Spousta, Jiří (advisor)
Fotonická krystalická vlákna (PCF) představují slibný nástroj pro spojení technik známých z mikroskopie rastrovací sondou, elektronové mikroskopie a systémů pro vstřikování plynu. Přivedení světla a přenos pracovního plynu současně do blízkosti vzorku umístěného uvnitř elektronového mikroskopu přináší nové možnosti experimentů. PCF by mohly být použity nejen k charakterizaci nebo modifikaci struktur na mikroskopické úrovni, ale také k jejich výrobě. Tato práce se zabývá výzkumem literárních zdrojů s tématikou fotonických krystalů s důrazem na PCF. Leptání PCF pomocí kyseliny fluorovodíkové bez poškození jejich vnitřní struktury je experimentálně studováno s velkou precizností. Optické vlastnosti závislé na geometrii PCF jsou testovány pro různé modifikace PCF. Dále se práce zabývá spojením PCF s mikroskopií atomárních sil a následnou integrací do elektronového mikroskopu.
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The effect of trabectedin on the cells derived from colorectal carcinoma
Bobek, Jaroslav ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jaroslav Bobek Supervisor: Doc. PharmDr. Iva Boušová, Ph.D. Supervisor specialist: RNDr. Veronika Skarková, Ph.D. Title od diploma thesis: The effect of trabectedin on the cells derivated from colorectal carcinoma Trabectedin is alkaloid, which occurs naturally in sea tunicate Ecteinascidia turbinata. Its mechanism of action consist of creation complexes with DNA, transcription factors and nuclears proteins. This cause blocade of transcription and defect of repair mechanism of DNA in cell. We can use it in therapy of some types of cancer, in this particular work we was researching influence on colorectal carcinoma. We tested this substance on two cancerous cell lines SW 480 - derivated from primary deposit of adenokarcinoma and SW 620 - metastatic line from lymph nodes from same patient. Prime cell cultures marked as 36B and 39B were secured from patients in University Hospital in Hradec Králové and their following cultivation. Both samples 36B and 39B were derived from the primary colon tumor deposit. On all cell lines was performed cytotoxicity assay and then was individualy calculated IC50. These values were used in migration assay for cells 36B and 39B. After treatment with trabectedin both...
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The effect of 6S-like RNAs on physiological differentiation of Streptomyces coelicolor
Burýšková, Barbora ; Bobek, Jan (advisor) ; Branny, Pavel (referee)
The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...
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Preparation and testing of SNOM probes
Bobek, Juraj ; Pavera, Michal (referee) ; Spousta, Jiří (advisor)
The area of research that deals with surface modification and preparation of nanostructures is still very unexplored. And only a little contribution to this field is discussed in this bachelor thesis. Its goal is to manufacture and to test a probe made off hollow optical fibre that is used in Scanning Probe Microscopy. Optical fibre parameters in combination with proper and unique techniques allow the breakthrough off very interesting applications. It's worth mentioning the gas injection in the proximity of the surface of a sample (GIS) and thus its modification by use of electrons (FEBID), ions (FIBID) or laser beams.
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Multimedia module for radio controlled models
Bobek, Josef ; Hrubý, Ivan (referee) ; Musil, Vladislav (advisor)
The thesis deals with the development and realization of a multimedia module with a microcontroller for radio controlled models of ships, cars, and fighting techniques. The module uses commands from the RC transmitter to control the model illumination and accompanying sounds. The thesis summarizes principles generally used in RC models. Based on these general principles, a new control element for the RC transmitter is designed. A relevant multimedia module is designed and implemented. The last part of the thesis is devoted to the description of the basic parts of microcontroller firmware.
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