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The role of PKN family kinases in cancer
Novotná, Petra ; Rösel, Daniel (advisor) ; Ramaniuk, Volha (referee)
This bachelor thesis is focused on the PKN family of Ser/Thr kinases. This family includes three isoforms PKN1, PKN2 and PKN3. Especially it deals with the kinase PKN3 in more detail. These are kinases related to protein kinase C, belonging to the AGC superfamily. PKN kinases are activated via small G proteins of the Rho GTPase family or unsaturated fatty acids. PKN kinases are involved in many cellular processes, such as the regulation of cytoskeletal rearrangements, affect cell adhesion, cell movement, embryonic development and the cell cycle. Expression of PKN3 is particularly increased in cancer cells but is only present in small amounts in normal body cells. Therefore, PKN3 appears to be a very interesting therapeutic target for the treatment of cancer. Studies have shown that PKN3 has a significant effect on the motility of cancer cells, thus contributing to their migration and ability to form metastases.
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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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Regulation of microtubule dynamics revealed by single-molecule TIRF and IRM microscopy
Zhernov, Ilia ; Lánský, Zdeněk (advisor) ; Cifra, Michal (referee) ; Varga, Vladimír (referee)
The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...
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Protein composition of the cytoskeleton of protists
Švagr, Eva ; Hampl, Vladimír (advisor) ; Pánek, Tomáš (referee)
While we have a good understanding of the roles of actin and tubulin filaments in the cell cytoskeleton, intermediate filaments (IFs) are often overlooked. However, the importance of IFs becomes quite apparent, as proteins similar to IF proteins have been identified in many protist cells. This holds particularly for the cells of protists, where even the functions of some members of the of actin and tubulin superfamilies remain unclear. Intermediate filaments are still not well established as components of protist cytoskeletons, in contrast to their more thoroughly studied counterparts in Metazoa. Protist and metazoan IF proteins are dissimilar in their sequence; however, they share similarities in structure, and they assemble autonomously into analogous filaments. IF-like proteins have been localized to striated fibers or unique cytoskeletal components in several organisms, even though not much is known about the protein composition of these striated fibers to date. This suggests that IF-like proteins might be a universally present component of these striated fibers often seen in protist cells. Keywords: Protists, cytoskeleton, microtubules, microfilaments, intermediate filaments, fibrils, protein composition
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Evolutionary-developmental study of membrane proteins
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Baluška, František (referee) ; Štorchová, Helena (referee)
Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...
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Molecular mechanism of mechanoreception in plants
Jelínková, Barbora ; Martinek, Jan (advisor) ; Fendrych, Matyáš (referee)
Plant, as sedentary organism, does not have many possibilities to physically escape it's unpleasant surroundings, instead it adapts oneself. One of many plant senses that are crucial for tracking environment changes is mechanoreception. Plant senses and differentiates between many mechanical cues, some of them affecting plant immunity and morphogenesis. The whole plant cell reacts to mechanical cues and many cellular structures are involved in mechanoreception. Any change in cell wall - a borderline between the cell and it's surroundings - is transduced to plasma membrane, then to the cytoskeleton and potentially to other structures. Concept of this cell wall-plasma membrane-cytoskeleton continuum and it's use as an instrument to illuminate molecular mechanisms of mechanoreception in plants are the key topics of my thesis.
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Interakce viru klíšťové encefalitidy s cytoskeletem hostitelských buněk
PRANČLOVÁ, Veronika
This thesis is focused on the role of host cytoskeleton, primarily microtubules and microfilaments, during tick-borne encephalitis virus infection in human neuroblastoma cell line SK-N-SH and tick cell line IRE/CTVM19. The importance of cytoskeletal integrity and dynamics to the viral replication cycle were examined using specific chemical inhibitors showing the virus utilizes studied structures in both cell lines. Immunofluorescence microscopy revealed structural changes in the actin cytoskeleton during late infection in SK-N-SH cells. Moreover, differences in expression of cytoskeleton-associated genes in both cell lines were compared. Several genes with up-regulated expression in SK-N-SH cells were identified during late infection.
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Analysis of CESA complexes dynamics in plant cytoskeletal mutants
Dubenecká, Kamila ; Schwarzerová, Kateřina (advisor) ; Malínská, Kateřina (referee)
The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.
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Molecular mechanism of microtubule severing by katanin
Podhájecký, Roman ; Lánský, Zdeněk (advisor) ; Libusová, Lenka (referee)
Microtubule cytoskeleton is a dynamic filamentous network, whose reorga- nization underpins important cellular processes, such as cell motility or cell di- vision. This remodeling highly depends on microtubule-associated proteins that can remodel individual microtubules, such as katanin. Katanin is a microtubule- associated protein that employs ATP hydrolysis to sever microtubules. This function can rapidly reorganize microtubule networks by providing fast amplification of the microtubule polymer number as well as microtubule fast degradation. The exact mechanism of microtubule severing and its regulation by katanin is largely dis- cussed nowadays. In this project I would like to describe katanin's mechanism of action and its regulation on a molecular level. Key words: cytoskeleton, remodelling of microtubule networks, microtubules, microtubule se- vering enzymes, katanin 1
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The influence of aluminum toxicity on the dynamics of plant cortical microtubules
Pohl, Jana ; Schwarzerová, Kateřina (advisor) ; Pejchar, Přemysl (referee)
Aluminium toxicity is the main factor limiting plant growth on acid soils. Aluminium inhibits root growth within few minutes after aluminium treatment. The mechanism and primary target of his action is still unknown. In this diploma thesis the effect of aluminium toxicity on dynamics of cortical microtubules WT and pldα1 plants was studied using the EB1a-GFP marker. Polymerization rate in both the transition and the elongation zone increased immediately after the aplication of aluminium. Nevertheless, microtubules in the transition zone are much more sensitive to aluminium, because the aluminium-induced increase in the polymerization rate was higher than in the elongation zone. Plants lacking PLDα1 showed higher dynamics on plus ends of cortical microtubules compared to WT during aluminium stress, which enabled them to react faster to stress stimuli. Mutants showed lower sensitivity to aluminium and 100 μM concentration of aluminium ions has beneficial effect on root growth in pldα1. These results suggest that PLDα1 influences microtubule dynamics. Microtubules in pldα1 plants were more dynamic and they polymerized faster in the response to aluminium, which was accompanied by decreased sensitivity to aluminium stress compared to WT. Changes in microtubule dynamics may play a role in aluminium...
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