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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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Vliv strigolaktonu na větvení kořenů u rostlin huseníčku rolního
Macoszek, Ondřej
In this bachelor study the influence of the concentration of GR24, the synthetic analogue of strigolactone, on the length of the primary root of different ecotypes of Arabidopsis plants was studied. Six ecotypes of A. thaliana were chosen and the experiment was performed at the Gregor Mendel Institute in Vienna in the laboratory of Dr. Wolfgang Busch. The used GR24 concentrations ranged from 12.5 nM to 50 microM. It was known from previous work (Ruyter and Spira 2010, Kol-tai 2011, Vacková 2013) that GR24 influences the root architecture of Arabidopsis thaliana, but the results differed. From the experimental results it is clear that the influence of GR24 on the primary root length depends on the individual plant genotype, but generally it is possible to suggest that low strigolactone concentrations cause inhibition, subsequently higher stimulation of the primary root length and finally concentration above 10 microM are highly inhibitory in all genotypes. Based on the results the concentration of 100 nM was chosen for the subsequent Genome wide association study (GWAS).
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Influence of environmental factors on stomatal development in epidermis of Arabidopsis thaliana - signal trasduction.
HOŠTIČKOVÁ, Irena
The stomatal function and genetically based regulation of their number are interesting and important subjects of scientific research. Stomatal density on plant leaf surface is affected also by environmental factors, e.g. atmospheric CO2 concentration and irradiance (PAR). These two factors have common affect ? leaf internal CO2 concentration (Ci) in the leaf can be changed. The question might be if the Ci is the signal for expression of genes controlling stomatal development. According to the hypothesis which this thesis is based on I proposed an increase of expression of positive regulator STOMAGEN in the case when Ci is reduced. The expression of STOMAGEN was increased in plants with Ci decreased by higher PAR in contrast with plants where the Ci was decreased by adding of abscisic acid into nutrient solution. These results show no dependence of STOMAGEN expression on inner CO2 concentration.
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Elicitins impact on the proteome of tobacco
Dokládal, Ladislav
Title in English: Elicitins impact on the proteome of tobacco Abstract in English: Cryptogein is a proteinaceous elicitor secreted by an oomycete Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modeling, a series of cryptogein mutants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded to expectations based on the structural data reported previously. Induction of synthesis of reactive oxygen species (ROS) in tobacco cells suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant elicitins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defense genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the -loop, might not be principal factors in either ROS production or resistance induction. Nevertheless results support importance of...
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Funkce genů rodiny TTL a jejich efekt na tvorbu postranních kořenů u Arabidopsis
Hilgert Delgado, Alois Antonín ; Soukup, Aleš (advisor) ; Cvrčková, Fatima (referee)
The work is focused on the lateral roots, which are an indispensable part of the root system and play a key role in communication with rhizosphere and the efficient exploitation of natural resources. Tetratricopeptide-repeat Thioredoxin-like 3 gene (TTL3) was identified by forward screening based on its gene expression pattern in the search for genes associated with the development of lateral roots in Arabidopsis thaliana. TTL3 also known as VIT (VH1 interacting TPR containing protein), belongs to a family of four paralogues in Arabidopsis showing a very similar arrangement of domains. It seems that this type of proteins is widespread in plants and provides protein-protein signaling as an important component of phytohormone response pathways. The presented diploma thesis is focused on characterization and comparison of expression patterns of the four members of TTL family, through creation of pTTL::uidA (GUS) transcriptional fusions. The promoter area was estimated to be contained in about 2000bp upwards the TTLs coding sequences and transcriptional fusions with uidA gene were individually constructed and transformed into Arabidopsis via Agrobacterium tumefaciens. The expression patterns of analyzed genes were microscopically confirmed and allowed comparison among members of TTL gene family with regard...
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Iron-sulfur proteins and the role of iron in the gene expression of Trichomonas vaginalis
Horváthová, Lenka ; Tachezy, Jan (advisor) ; Rasoloson, Dominique (referee) ; Vyoral, Daniel (referee)
Iron is an essential nutrient for the parasitic protist Trichomonas vaginalis as a component of iron-sulfur (FeS) proteins that are indispensable for energy metabolism of the parasite. The FeS clusters are formed by FeS cluster (ISC) assembly machinery that resides, together with a number of FeS proteins, in Trichomonas hydrogenosomes. These double-membrane bound organelles, which are related to mitochondria, metabolize malate and pyruvate and produce ATP and molecular hydrogen. To obtain more complete information about hydrogenosomal pathways with particular focus on FeS proteins and ISC machinery, we participated on analysis of T. vaginalis genome sequence. To predict hydrogenosomal localization of putative gene products, we developed an application "Hunter" for the in silico searching for N-terminal presequences that are required for protein targeting into the hydrogenosomes. This approach substantially contributed to annotate genes coding for hydrogenosomal proteins that provided base for construction of novel map of hydrogenosomal metabolism as well as for following proteomic studies. Investigation of hydrogenosomal proteins led to identification of three members of Hyd machinery that is required for the maturation of the specific FeS cluster of hydrogenases named H cluster. T. vaginalis is,...
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Růst Mycobacterium smegmatis na agarovém médiu a agarovém médiu pokrytém celofánovou folií - morfologická a proteomová studie
Ramaniuk, Volha ; Weiser, Jaroslav (advisor) ; Beranová, Jana (referee)
Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...
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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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