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Mechanisms of iron-sulfur cluster biogenesis in eukaryotes
Temešinko, Tomáš ; Doležal, Pavel (advisor) ; Malych, Ronald (referee)
Many essential cellular proteins use iron-sulfur (Fe-S) clusters as cofactors. These proteins often serve as enzymes, components of the electron-transport chain or as intracellular sensors. Prior to the use of the cluster in a protein, it needs to be formed or created de novo. In total, four different mechanisms of Fe-S cluster biogenesis can be used by the eukaryotic cell - ISC, CIA, SUF and NIF. All of these pathways include a specific targeting system for delivering the cluster to its acceptor protein. Errors in biosynthesis ofFe-Sclustersaremostlylethalandcanleadtofailureindevelopmentofmulticellularorganisms.Despite this a better characterization of these mechanisms is needed as research is currently still in progress. This bachelor's thesis provides current information regarding the mechanisms of Fe-S clusters biogenesis in eukaryotes acquired mostly from mammalian cells, including humans, and from well-known model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana, and parasitic protist Giardia intestinalis.
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Molecular consequences of electron transport chain deficiency in proliferating and quiescent cells
Magalhães Novais, Silvia Carina ; Rohlena, Jakub (advisor) ; Panicucci Zíková, Alena (referee) ; Eelen, Guy (referee)
Oxidative phosphorylation (OXPHOS) system is best known for producing ATP in mitochondria. As a major metabolic hub, OXPHOS also supports other biological functions that are unrelated to ATP synthesis. Are these additional roles of OXPHOS unique to different cell states, such as to proliferation and quiescence, two of the most contrasting states of a mammalian cell? In this thesis we used genetic models of OXPHOS deficiency to show that in proliferating cells and in tumors OXPHOS primarily supports biosynthesis of nucleotides, whereas in quiescent cells OXPHOS provides oxidative stress resistance. Mechanistically, in growing tumors OXPHOS drives dihydroorotate dehydrogenase (DHODH), an enzyme crucial for de novo synthesis of pyrimidines. Complex II of OXPHOS also acts as a sensor of mitochondrial dysfunction and regulates pyrimidine synthesis for energy conservation. In contrast, in quiescent cells OXPHOS maintains autophagy by continuous low level ROS generation and together with the antioxidant defense provides constitutive oxidative stress protection. In summary, beyond ATP production OXPHOS has additional vital roles tailored to the particular requirements of quiescence and proliferation, with relevance for pathophysiology in cancer and vascular biology.
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Mutations in brassinosteroid biosynthetic genes and their effects on plant phenotype
Frimlová, Klára ; Rothová, Olga (advisor) ; Mašková, Petra (referee)
Brassinosteroids are important group of phytohormones, whose presence affects anatomy, morphology and development of plants. They occur in all vegetative and mainly generative organs. Brassinosteroids are biosynthesized via two main paths named early C-6 oxidation pathway and late C- 6 oxidation pathway. Brassinosteroid biosynthesis is catalyzed by enzymes from the group of cytochrome P450. Gene mutations - which affect brassinosteroid biosynthesis - have been studied especially in Arabidopsis thaliana L., Pisum sativum L., Solanum lycopersicum L. and Oryza sativa L. Mutations lead to changes in the composition and amount of brassinosteroids in plants. The reduced content of brassinosteroids is manifested by typical phenotype that includes reduced growth, reduced size of plant organs, and dark green leaves in mutant plants. The mutant phenotype can be reversed by exogenous application of brassinosteroids, which is nevertheless dependent on the concentration of the brassinosteroids used. Higher concentrations of brassinosteroids cause mostly inhibition of root elongation. Keywords: brassinosteroids, biosynthesis, mutant, phenotype
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Analysis of the biosynthetic gene cluster encoding biosynthesis of the manumycin antibiotic U-62162, and the ways of its modification.
Urbanová, Daniela ; Petříček, Miroslav (advisor) ; Schierová, Michaela (referee)
Streptomyces is the largest antibiotic-producing genus in the microbial world. Manumycin-type antibiotics are a small group of its metabolites. Their antibiotic activities are not very important but they show biological properties which can be potencially used e. g. to treat inflammation, cancer or Alzheimer's disease. The structure of manumycin compounds is formed by a central unit with connected upper and lower polyketide chain. The lower chain is mostly terminated by so called C5N unit. The substance U-62162 produced by the strain Streptomyces verdensis differs significantly from the other members of the manumycin-type metabolites in the structure of the lower chain which is fully saturated and lacking the C5N unit. The U-62162 biosynthetic gene cluster was sequenced and functions of identified open reading frames were deduced. Heterologous expressions of the cluster showed some genes reguired for the biosynthesis of the upper chain to be encoded on a different part of the chromosome. The insertional inactivation of the vrdER gene confirmed the enoylreductase to be responsible for the saturation of the lower chain. DSBA oxidoreductase, which gene is located at the edge of the cluster, is probably not involved in the biosynthesis. The insertion of genes for the biosynthesis of the C5N unit did...
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Plant cuticle
Voloshina, Mariia ; Schwarzerová, Kateřina (advisor) ; Synek, Lukáš (referee)
The cuticle is a lipidic structure covering plant aerial organs, providing mechanical rigidity and acting as a protective barrier. It contains the cutin polyester and waxes, which are derived from very-long-chain fatty acids. These compounds are synthesised in two separate pathways. The cuticular biosynthetic machinery is incredibly complex and employs a multitude of enzymes, some of which are functionally redundant, are present in different tissues or catalyse reactions with substrates of various chain lengths. The mechanisms of how these compounds are transported and how the cuticle is assembled rely on ABC transporters, LTP lipid carrier proteins, cutin synthases, and cutinsomes. Knowledge of these highly dynamic processes is very fragmented and the integrated model of cutin synthesis is yet to be elucidated. A tight connection between the cuticle and the cell wall, conventionally seen as two separate entities, has also been implied. The complexity of these mechanisms is also reflected in their transcriptional and post-transcriptional regulation. While SHINE and MIXTA-like transcriptional factors and the WW-domain protein CFL1 regulate the cuticle's synthesis throughout a plant's development, ABA-dependent MYB transcriptional factors are important during abiotic stress. Recent research also...
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Konverze biologicky aktivních látek při přípravě zelených a černých čajů
Procházková, Anna
This graduation theses deals with the cultivation, processing, preparation of tea and the chemical composition of tea leaves in the theoretical part. Furthermore deals with the synthesis of biologically active substances in plant materials and their conversion from the preparation of tea to the transformation in the human organism after its consumption. It also briefly summarizes and defines the issues of free radicals and antioxidants as part of their functioning in the human body, and then attempts to objectively summarize their main pharmacological effects. The practical part, focused on the determination of phenolic substances and the total antioxidant capacity of selected green and black teas, is supplemented by their sensory analysis.
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Studium vzniku a vlastností fluorescenčně aktivních nanočástic
Prášková, Markéta
Nanoparticles belong to the future of mankind, both in the scientific world and objects of everyday life. Their potential is apparent not only in the diagnostics and treatment of various diseases, but also in engineering or informatics. Nanoparticles with fluorescent properties can be used for the fluorescent labelling of molecules for the monitoring of various biological processes, tracking of the fate of administered drug in cells or whole organisms, or for the construction of biosensors. Presented work deals with two types of nanoparticles - liposomes and quantum dots (QDs). In this work, the properties of liposomes with encapsulated fluorescent active drug doxorubicin were evaluated. Moreover, QDs of various sizes were characterized, synthesized by microwave synthesis and biosynthesis. The nanoparticles were analysed by their optical properties, zeta-potential and toxicity propertis. Other methods, such as gel electrophoresis, electrochemistry, liquid chromatography, mass spectrometry or Raman spectroscopy were also used in this work.
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Ergothioneine and mycothiol in the biosynthesis of lincosamides
Seidlová, Bára ; Kameník, Zdeněk (advisor) ; Kopecký, Jan (referee)
Specialized microbial metabolites are described as low-molecular-weight bioactive compounds, which are dispensable for the growth, evolution, or reproduction of its producer. This group of substances includes the lincosamides, which are produced mainly by the bacteria of the Streptomyces genera. Apart from other precursors, two low-molecular-weight thiols, ergothioneine and mycothiol, are essential participants of the lincosamide biosynthesis. Mycothiol (MSH) serves in this pathway as a source of sulphur, on the other hand, ergothioneine (ESH) constitutes a conjugate with the aminosugar moiety of lincosamide structure. The conjugate is condensed with an activated amino acid, which is catalyzed by an unusual enzyme to form a core of the lincosamide molecule. The objective of this diploma thesis is to isolate the conjugate of ESH and aminooctose, which serves as a substrate of the LmbD biosynthetic protein. Another aim is to study the links between the thiol metabolism and the biosynthesis of three lincosamides, lincomycin, celesticetin, and intervencin, which are produced by different bacterial strains. Bacterial strains were cultivated under laboratory conditions and methods of liquid chromatography with UV and MS detection were used for the analysis. The parameters of the methods were developed...
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Mutations in brassinosteroid biosynthetic genes and their effects on plant phenotype
Frimlová, Klára ; Rothová, Olga (advisor) ; Mašková, Petra (referee)
Brassinosteroids are important group of phytohormones, whose presence affects anatomy, morphology and development of plants. They occur in all vegetative and mainly generative organs. Brassinosteroids are biosynthesized via two main paths named early C-6 oxidation pathway and late C- 6 oxidation pathway. Brassinosteroid biosynthesis is catalyzed by enzymes from the group of cytochrome P450. Gene mutations - which affect brassinosteroid biosynthesis - have been studied especially in Arabidopsis thaliana L., Pisum sativum L., Solanum lycopersicum L. and Oryza sativa L. Mutations lead to changes in the composition and amount of brassinosteroids in plants. The reduced content of brassinosteroids is manifested by typical phenotype that includes reduced growth, reduced size of plant organs, and dark green leaves in mutant plants. The mutant phenotype can be reversed by exogenous application of brassinosteroids, which is nevertheless dependent on the concentration of the brassinosteroids used. Higher concentrations of brassinosteroids cause mostly inhibition of root elongation. Keywords: brassinosteroids, biosynthesis, mutant, phenotype
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