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Cell biology of iron transport in plants.
Batík, Adam ; Žárský, Viktor (advisor) ; Tylová, Edita (referee)
Plants use iron as a cofactor of proteins used in photosynthetic systems, electron transport chain and many more. Iron bioavailability for plants in soil is low because it tends to oxidise and create insoluable compounds.For this reasonplants haveevolvedtwo distinct iron uptake mechanisms.Because of the iron toxicitycaused by production ofreactiveoxygen species via the Fenton reaction and the unspecific transport of metals other than iron, plants have to regulate cellular iron concentrationtightly.Theyhave evolved a complex system of signalling networks that has recently begun to uncover. In additionto the regulation ofiron uptake, the plant cell combats iron toxicity by sequestering iron into storage organelles and by chelating it. Iron is essential for seed sprouting but this work is focused on transport of iron into the plant from the soil,subcellulartransportandlongdistance transport ofironin the vasculature.
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra
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...
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Biochemical and mechanical processes in synovial fluid - modeling, analysis and computational simulations
Pustějovská, Petra
vi Title: Biochemical and mechanical processes in synovial fluid - modeling, mathematical analysis and computational simulations Author: Petra Pustějovská (petra.pustejovska@karlin.mff.cuni.cz) Department: Matematický ústav UK, Univerzita Karlova v Praze Institut für Angewandte Mathematik, Universität Heidelberg Supervisors: prof. RNDr. Josef Málek CSc., DSc. (malek@karlin.mff.cuni.cz) Matematický ústav UK, Univerzita Karlova v Praze, Prof. Dr. Dr. h.c. mult. Willi Jäger (jaeger@iwr.uni-heidelberg.de) Institut für Angewandte Mathematik, Universität Heidelberg Abstract: Synovial fluid is a polymeric liquid which generally behaves as a viscoelastic fluid due to the presence of polysaccharide molecules called hyaluronan. In this thesis, we study the biological and biochemical properties of synovial fluid, its complex rheology and interaction with synovial membrane during filtration process. From the mathematical point of view, we model the synovial fluid as a viscous incompressible fluid for which we develop a novel generalized power-law fluid model wherein the power-law exponent depends on the concentration of the hyaluronan. Such a model is adequate to describe the flows of synovial fluid as long as it is not subjected to instantaneous stimuli. Moreover, we try to find a suitable linear viscoelastic model...
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Functions of RAB GTPases and SNARE proteins in post-Golgi trafficking pathways in response to abiotic stress in plants
Moulík, Michal ; Hála, Michal (advisor) ; Mašková, Petra (referee)
Plants as sessile organisms are strongly affected by abiotic and biotic stress factors. Thus, they have developed an array of morfological, biochemical and physiological adaptations to reduce the negative effects of these factors. The membrane trafficking, among others, plays very important role in adaptation to abiotic stress. In my bachelor thesis I have focused on two important protein families involved in this trafficking, namely on RAB GTPases and SNARE proteins. In the first part, the phenomenon of stress is characterized and the strategies how plants cope with the effect of stressors are described, especially the production of reactive oxygen species (ROS) and autophagy. Following chapter deals with an introduction to the membrane trafficking in plants. In the rest of the thesis, I characterize RAB GTPases and SNARE proteins and provide contemporary insight in the mechanism of their function. The aim of the key parts of these chapters is to summarize current knowledge of RAB GTPases' and SNARE proteins' functions in post-Golgi trafficking pathways during response to abiotic stressors or secondary oxidative stress. Key words: plants, abiotic stress, membrane trafficking, secretion, endocytosis, vacuole, RAB GTPases, SNARE proteins
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Determination of permeability and active transport of selected butyrylcholinesterase inhibitors in vitro
Machan, Radek ; Červený, Lukáš (advisor) ; Čečková, Martina (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Radek Machan Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Determination of permeability and active transport of selected butyrylcholinesterase inhibitors in vitro European Medicine Agency (EMA) and Food and Drug Administration agency (FDA) emphasise drug membrane permeability and drug-drug interactions on ABC transporters expressed in physiological barriers should be investigated for compounds in preclinical studies or for those already clinically used but evidence free. In this work we aimed to assess the capability of several experimental butyrylcholinesterase inhibitors that had been designed to treat dementia to permeate blood-brain barrier and to elucidate role of ATP-binding (ABC) cassette transporters in this transport. For this purpose, we employed in vitro bidirectional transport study across monolayers formed by polarized and highly differentiated Caco-2 cells. The permeability values gained from measurements were similar to values of several commonly used drugs for treatment of CNS disorders (e.g. antidepressants, antiepileptics). In addition, the compounds showed values of efflux ratio (basolateral- to-apical/apical-to-basolateral) approximately one...
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Monitoring the effect of medically important drugs on activity of human P-glycoprotein using fluorescent probes
Veľas, Lukáš ; Gášková, Dana (advisor) ; Sigler, Karel (referee)
One of the main causes of failure in cancer treatment when using chemotherapy is the phenomenon of multidrug resistance - MDR. The most important protein mediating MDR in human cells is P-glycoprotein. The main goal of this thesis was the modification of fluorescent method - developed for studying yeast MDR pumps at the Department of Biophysics at the Institute of Physics of Charles University - to study the activity of P-glycoprotein in human tumor cells. The fluorescent method is based on the use of redistribution potentiometric probe diS-C3(3) which we found to be a substrate of P-glycoprotein. The optimalization of the method (experimental window) allowed sensitive monitoring of changes in the activity of P-glycoprotein caused by the effect of its several known inhibitors/substrates: oligomycin, amiodarone, verapamil, vinblastine, ketoconazole, itraconazole and FK506. New important results regarding the effect of these medically significant drugs on human cells were obtained. The developed method will undoubtedly be of great benefit in the search for new effective inhibitors and the study of their mechanisms in the future.
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Biochemical and mechanical processes in synovial fluid - modeling, analysis and computational simulations
Pustějovská, Petra
vi Title: Biochemical and mechanical processes in synovial fluid - modeling, mathematical analysis and computational simulations Author: Petra Pustějovská (petra.pustejovska@karlin.mff.cuni.cz) Department: Matematický ústav UK, Univerzita Karlova v Praze Institut für Angewandte Mathematik, Universität Heidelberg Supervisors: prof. RNDr. Josef Málek CSc., DSc. (malek@karlin.mff.cuni.cz) Matematický ústav UK, Univerzita Karlova v Praze, Prof. Dr. Dr. h.c. mult. Willi Jäger (jaeger@iwr.uni-heidelberg.de) Institut für Angewandte Mathematik, Universität Heidelberg Abstract: Synovial fluid is a polymeric liquid which generally behaves as a viscoelastic fluid due to the presence of polysaccharide molecules called hyaluronan. In this thesis, we study the biological and biochemical properties of synovial fluid, its complex rheology and interaction with synovial membrane during filtration process. From the mathematical point of view, we model the synovial fluid as a viscous incompressible fluid for which we develop a novel generalized power-law fluid model wherein the power-law exponent depends on the concentration of the hyaluronan. Such a model is adequate to describe the flows of synovial fluid as long as it is not subjected to instantaneous stimuli. Moreover, we try to find a suitable linear viscoelastic model...
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Subcelulární lokalizace a úloha komplexu exocyst v savčích buňkách během cytokineze
Ulrychová, Lenka ; Hudeček, Jiří (advisor) ; Entlicher, Gustav (referee)
Cytokinesis is the last step of cell cycle when two individual daughter cells separate in process called abscission. This process involves various cellular membrane structures such as endoplasmic reticulum or trans-Golgi network. Moreover, recent investigation has also highlighted an important role of recycling endosomes. The membrane dynamics appear to be important during cell division especially for the formation of new plasma membrane between two daughter cells. Numerous studies suggest that cytokinesis is tightly linked with highly sophisticated transmembrane shuttle that is controlled by Ras-superfamily members such as Rab and Ral proteins. Moreover, during last years has also been revealed the involvement of tethering factors which mediate the fusion of intracellular vesicles with the target plasma membrane. The best known tethering factor is the evolutionary conserved exocyst complex found in all eukaryotic cells. This protein complex is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) and was found to interact with members of Ras- superfamily suggesting its involvement in the regulation of cytokinesis. Although the exact mechanism remains shrouded in fog this work suppose the possible interactions among Ras- like proteins and exocyst members which may...
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Biophysical studies of membrane transport proteins from Nramp/MntH family and their function
Ňuňuková, Věra
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...
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