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Studies of NK cell receptors and other proteins using recombinant expression and mass spectrometry
Kavan, Daniel ; Bezouška, Karel (advisor) ; Tučková, Ludmila (referee) ; Řehulka, Pavel (referee)
Charles University in Prague Faculty of Science Department of Biochemistry Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry Summary of Ph. D. Thesis Daniel Kavan Supervisor: Prof. RNDr. Karel Bezouška, DSc. Prague 2010 Daniel Kavan Introduction Introduction NK cells and CD69 as one of their surface receptors Natural killer cells (NK cells) are the subpopulation of large granular lymfocytes, which lacks the surface receptors typical for B cells or T cells. They are characterized by the presence of NKp46 and NKp30 [Moretta L. et. al. 2002], however. They were named natural killers according to their function in the organism, as they do not need any activation and nevertheless they are able to eliminate abnormal (i. e. infected or transformed) cells from the tissue [Kiessling R. et. al. 1975]. This function is dependent on scanning the major histocompatibility complex (MHC) class I molecules of ambient cells. The resulting action (killing or not killing the target cell) is dependent on the balance of activating and inhibiting signals mediated by the NK cell surface receptors and forwarded to the specific signaling pathway [Raulet D. H. et. al. 2001]. Specificity of NK cells is not based only on one type of antigen receptor as it is in case of T and B cells,...
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Similarity Search in Protein Structure Databases
Galgonek, Jakub ; Skopal, Tomáš (advisor) ; Porto, Markus (referee) ; Svozil, Daniel (referee)
Proteins are one of the most important biopolymers having a wide range of functions in living organisms. Their huge functional diversity is achieved by their ability to fold into various 3D structures. Moreover, it has been shown that proteins sharing similar structure often share also other properties (e.g, a biological function, an evolutionary origin, etc.). Therefore, protein structures and methods to identify their similarities are so widely studied. In this thesis, we introduce a system allowing similarity search in pro- tein structure databases. The system retrieves, given a query structure, all database structures being similar to the query structure. It employs several key components. We have introduced a novel similarity measure assigning similarity scores to pairs of protein structures. We have designed specific access method based on LAESA metric indexing and using the proposed measure. The access method allows to search similar structures more effi- ciently than when a sequential scan of a database is employed. To achieve further speedup, the measure and the access method have been parallelized, resulting in almost linear speedup with the respect to the number of available cores. The last component is a web user interface that allows to accept a query structure and to present a list of...
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The effect of prenatal MA exposure and crossfostering on functional changes in laboratory rat
Hrubá, Lenka ; Šlamberová, Romana (advisor) ; Tejkalová, Hana Barbora (referee) ; Šulcová, Alexandra (referee)
The aim of our study was to investigate the impact of prenatal methamphetamine (MA) exposure and cross-fostering on development of rat pups during preweaning period and on behavior, anxiety, cognitive function and nociception in adulthood. Mothers were daily exposed to injection of MA (5 mg/kg) or saline (S) approximately for 9 weeks: three weeks prior to impregnation, throughout the entire gestation period and for 23 days of lactation. Control females (C) did not receive any injection. On postnatal day (PD) 1, pups were cross-fostered so that each mother received some of her own and some of the pups of mother with the other two treatments. We obtained 9 experimental groups (C/C, C/S, C/MA, S/C, S/S, S/MA, MA/C, MA/S, MA/MA). Pups were tested during postnatal development by means of following behavioral tests: negative geotaxis, tail pull, righting reflex on surface and in mid-air, rotarod and bar-holding and were examined for physiological maturation. In adulthood, males and females rats were tested in the Open field (OF) and in the Elevated plus maze (EPM) for behavior and anxiety and in the Plantar test for thermal nociception. Adult male rats were tested on cognitive function in the Morris water maze (MWM). In adult female rats, phases of the estrous cycle were recognized and compared. Our...
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The expression and function of nucleophosmin (B23) during bovine preimplantation development
Benešová, Veronika ; Kaňka, Jiří (advisor) ; Krylov, Vladimír (referee)
Identification of genes that start their expression in different time of embryonal development is crucial for proper understanding of early mammalian embryogenesis. Genes that are differently expressed during this period are supposed to be important for normal preimplantation development. One of these genes is nucleophosmin, the multifunctional protein, which functions among others as a transporting protein between cytoplasm and nucleoli. It is involved in duplication of centrosomes, formation of ribosomes and functions as a chaperoning protein. Nucleophosmin shuttles rapidly between the nucleolus and cytoplasm. Its localization is cell cycle dependent and reflects the development of the nucleoli. For studying the effect of nucleophosmin on the bovine preimplantation development we used RNA interference that causes degradation of the target mRNA after injection of nucleophosmin double-stranded RNA. The average decrease of nucleophosmin mRNA abundance is 86.3 % between nucleophosmin dsRNA injected embryos and control group. We detected no differences in protein expression and localization of nucleophosmin between the groups by immunofluorescence method. Embryos in all groups were able to develop until the blastocyst stage, although there were significant difference between uninjected control group and...
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Measurement of the membrane potential using ion-selective electrode sensitive to tetraphenylphosphonium
Lábajová, Anna ; Kofránek, Jiří (advisor) ; Sigler, Karel (referee) ; Plášek, Jaromír (referee)
103 ABSTRACT The computerized device for membrane potential measurement using the tetraphenylphosphonium-selective electrode was constructed. Signal acquisition, processing and data storage were realized by MATLAB/Simulink software. The selective membrane was optimized incorporating TPP+ TPB- precipitate into the membrane. The electrode had a Nernstian response from 3.10-6 M TPP+ . The TPP+ TPB- precipitate was later replaced by sodium tetrakis[3,5-bis(1,1,1,3,3,3-hexafluoro-2- methoxy-2-propyl)phenyl]borate (NaHFPB). The electrode with incorporated NaHFPB had a Nernstian response from 1.10-6 M TPP+ and had better sensitivity than commercially available electrodes. The values of selectivity coefficients for K+ , Na+ , Ca2+ and Mg2+ were calculated. The device was used for mitochondrial membrane potential measurement of isolated mitochondria and for evaluating the respiratory chain function of digitonin- permeabilized cells (hepatocytes, HeLa G, BSC-40 and control transmitochondrial cybrids). This method was used also for monitoring the mitochondrial permeability transition pore (MPTP) function of isolated mitochondria and permeabilized hepatocytes. MPTP opening was induced by high calcium concentration and the action of calcium was enhanced by pro-oxidant tert-butyl hydroperoxide (t-BHP). This...
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra ; Chaloupka, Roman (advisor) ; Krůšek, Jan (referee) ; Kubala, Martin (referee)
Three synthetic peptides corresponding to transmembrane segments TMS1, TMS3 and TMS6 of secondary-active transporter MntH from Escherichia coli were used as a suitable alternative model enabling to study TMS structure, TMS interaction with membranes, TMS mutual interaction and also function of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformation in lipid membranes. Electrophysiological experiments demonstrated that individual TMS were able under certain conditions to form ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS1 and TMS6 channels, influenced the transition between closed and open states and affected the conformation of all studied peptides. For TMS3 Mn2+ was crucial for formation of ion channels. It was shown that a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings can contribute to understanding of structure-function relationship at the molecular level. However, it remains unclear to what extent...
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Tvorba konstruktů pro studium funkce DRM1 u Arabidopsis thaliana
Veselá, Petra
The aim of the study called Creating of constructs for study of function DRM1 in Arabidopsis thaliana was to create a construct harbouring the gene of interest DRM1 (dormancy-associated protein), and other components necessary for successful transgenosis, by which will be transformed Arabidopsis thaliana plants in order to study the function of this gene in the plant organism. DRM1 function has not been identified yet, but the gene was annotated as putative dormancy-associated protein. The final gene cassette, containing DRM1 gene under promoter for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was inserted into the vector pGreen II giving the recombinant plasmid pWell17A whose completeness was verified by restriction analysis. The Rubisco promoter ensure DRM1 gene overexpression in transgenic plants and on the basis of this overexpression is determined DRM1 gene function.
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Analysis of animal mitochondrial genes for DNA barcoding
Brabencová, Klára ; Škutková, Helena (referee) ; Maděránková, Denisa (advisor)
The aim of this work is a literature review on the topic of the mitochondrial genome and DNA barcoding, building a dataset of mitochondrial sequences from GenBank database and creatione of a software function for extraction of individual genes that are present in the mitochondrial genome. This function was developed in Matlab. DNA barcoding is a method that uses short DNA sequence of mitochondrial genome for identification of species. There is no comprehensive work examining the appropriateness of different mitochondrial genes. This aim investigates the potential of other mitochondrial genes and evaluate their effectiveness for DNA barcoding and calculation of intra-and interspecific variability.
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Computational Investigations of Biomolecular Systems and Comparison with Experiments in Various Environmental Conditions
KHABIRI, Morteza
Computational methods were used to study two different types of biological systems. The first study is related to the effect of three different organic solvents (formamide, acetone and isopropanol) on the structure and behavior of three globular proteins. These enzymes belong to the haloalkane dehalogenase family: DhaA, LinB, and DbjA. Moreover, the effect of mutation in the presence of DMSO was also investigated in two variants of DhaA; DhaA57 (L95V+A172V) and DhaA80 (Thr148Leu+Gly171Gln+Ala172Val+Cys176Phe). The simulation results showed that except for DhaA80, organic solvents entered the active site and influence its hydration. Not only the active site but also the enzyme?s hydration shell is influenced by organic molecules. The results showed that the water molecules are stripped out from the enzyme surfaces. It seems that the dual nature of organic molecules makes them favorable to solvate the enzymes. Radial Distribution Function (RDF) of the different parts of each organic molecule reveals that the behavior of each organic solvent in the vicinity of hydrophobic surfaces is similar to their behavior at the air-water interface. Structural analysis of root-mean-square-deviations (RMSD) and B-factors reveals that the flexibility of the enzymes decreased in the presence of most organic solvents, mainly in the CAP domain. Changes of other structural properties like radius of gyration and total solvent accessible surface areas are minimal. DbjA exists as a dimer and is more influenced by organic molecules. They penetrate to the amino acid network between monomers and influence their motion. The second study is the interaction of voltage-gated potassium channel Kv1.3 wild type and its mutants (Kv1.3_V388C, Kv1.3_V388C_H399T) with scorpion toxin (ChTX). Since there is no structure for Kv1.3, 94% sequence identity with Kv1.2 structure was used to make a homology model based on the Kv1.2 structure. MD structural analyses reveal that mutation of V388C changes the stability of selectivity filter by interrupting the amino acid network interactions behind the selectivity filter. The interaction of ChTX is also affected by the single mutant. ChTX is able to block wild type and double mutant channels but cannot occlude the pore entirely. Introducing the second point mutation H399T in the pore region reverts the structural changes back to the wild type. These results are entirely consistent with experimental results. Additionally, the binding energy of ChTX with the wild type mKv1.3 was investigated by the potential of mean force method, in the presence and absence of KCl solution. The results both in experiment and simulation show that, even though the unbinding process and dissociation rate is changing in the present of K+ ions, the binding energy is independent of K+ concentration. All together, the combination of computer simulations together with experiments provides new knowledge about channel-toxin interactions which could be helpful for drug design.
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