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Application of tensegrity structures in modelling of mechanical behaviour of smooth muscle cells
Bauer, David ; Fuis, Vladimír (referee) ; Burša, Jiří (advisor)
The master’s thesis deals with the computational modelling of the mechanical testing of isolated smooth muscle cells. The main aims are to create computational model of a cell, to simulate single-axis tensile test and to modify the model so that the model reflects real mechanical response. The model of the cell includes cytoplasm, nucleus, cell membrane and cytoskeleton which is modelled as a tensegrite structure. On this model the tensile test was simulated in case of the cell with cytoskeleton and the cell with distributed the cytoskeleton. Force-elongation curves, which were obtained from this simulation, were compared with experimental data which were taken from literature. Tensile properties were measured on freshly isolated cells from rat thoracic aorta, cultured cells, and cells treated with cytochalasin D to disrupt their actin filaments. It was found that the cytoskeleton influence on the cell load in computational model was smaller than in the real cell. Therefore the model was modified by changing material propreties and geometry so that the model of the cell corresponded with the different types of experimentally measured cells.
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Computational Simulation of Mechanical Tests of Isolated Animal Cells
Bansod, Yogesh Deepak ; Kučera,, Ondřej (referee) ; Florian, Zdeněk (referee) ; Canadas, Patrick (referee) ; Burša, Jiří (advisor)
Buňka tvoří složitý biologický systém vystavený mnoha mimobuněčným mechanickým podnětům. Hlubší pochopení jejího mechanického chování je důležité pro charakterizaci její odezvy v podmínkách zdraví i nemoci. Výpočtové modelování může rozšířit pochopení mechaniky buňky, která může přispívat k vytvoření vztahů mezi strukturou a funkcí různých typů buněk v různých stavech. Za tímto účelem byly pomocí metody konečných prvků (MKP) vytvořeny dva bendotensegritní modely buňky v různých stavech: model vznášející se buňky pro analýzu její globální mechanické odezvy, jako je protažení nebo stlačení, a model buňky přilnuté k podložce, který vysvětluje odezvu buňky na lokální mechanické zatížení, jako třeba vtlačování hrotu při mikroskopii atomárních sil (AFM). Oba zachovávají základní principy tensegritních struktur jako je jejich předpětí a vzájemné ovlivnění mezi komponentami, ale prvky se mohou nezávisle pohybovat. Zahrnutí nedávno navržené bendotensegritní koncepce umožňuje těmto modelům brát v úvahu jak tahové, tak i ohybové namáhání mikrotubulů (MTs) a také zahrnout vlnitost intermediálních filament (IFs). Modely předpokládají, že jednotlivé složky cytoskeletu mohou měnit svůj tvar a uspořádání, aniž by při jejich odstranění došlo ke kolapsu celé buněčné struktury, a tak umožňují hodnotit mechanický příspěvek jednotlivých složek cytoskeletu k mechanice buňky. Model vznášející se buňky napodobuje realisticky odezvu síla-deformace během protahování a stlačování buňky a obě odezvy ilustrují nelineární nárůst tuhosti s růstem mechanického zatížení. Výsledky simulací ukazují, že aktinová filamenta i mikrotubuly hrají klíčovou úlohu při určování tahové odezvy buňky, zatímco k její tlakové odezvě přispívají podstatně jen aktinová filamenta. Model buňky přilnuté k podložce dává odezvu síla-hloubka vtlačení ve dvou různých místech odpovídající nelineární odezvě zjištěné experimentálně při AFM. Výsledky simulací ukazují, že pro chování buňky je rozhodující místo vtlačení a její tuhost určují aktinová povrchová vrstva, mikrotubuly a cytoplazma. Navržené modely umožňují cenný vhled do vzájemných souvislostí mechanických vlastností buněk, do mechanické úlohy komponent cytoskeletu jak individuálně, tak i ve vzájemné synergii a do deformace jádra buňky za různých podmínek mechanického zatížení. Tudíž tato práce přispívá k lepšímu pochopení mechaniky cytoskeletu zodpovědné za chování buňky, což naopak může napomáhat ve zkoumání různých patologických podmínek jako je rakovina a cévní choroby.
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The role of cytoskeleton in cell chirality
Jandjuková, Anna ; Libusová, Lenka (advisor) ; Soukup, Vladimír (referee)
The cytoskeleton is one of the key structures inside the cell. The cytoskeleton includes microfilaments, microtubules, intermediate filaments, and more recently, septins. Cytoskeletal proteins play a role in intracellular transport, cell movement, cell division, and other structural functions. A chiral object is one that is not identical to its mirror image. The concept of chirality is most commonly used to describe chemical molecules. Cells of living organisms are composed of molecules, most of which are chiral. In recent years, cellular chirality has been observed, likely involving the cytoskeleton. Cellular chirality refers to the cell shape, organelle positioning, stable cell movement and directionally biased growth of cell culture. The latest findings suggest a probable influence of cell chirality on the development of right-left asymmetry during the embryonic development of animals. This work summarizes current knowledge regarding the role of the cytoskeleton in establishing cellular chirality and its likely connection to the development of right-left asymmetry during embryonic development. Key words Cell chirality, cytoskeleton, embryonal development
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Mammalian septins in cellular processes
Hrbáč, Patrik ; Libusová, Lenka (advisor) ; Zelená, Marie (referee)
Septins are cytoskeletal GTP-binding proteins with unique properties that can be found in species ranging from single-cell eukaryotes to mammals with the exclsuion of vascular plants. Septins are well conserved across species in both their structure and function. They are located in the cytoplasm, where they can assemble into filaments and higher order structures. Moreover, septins can interact with membranes, actin filaments, microtubules, and various proteins. Changes in septin expression often result in numerous defects in cellular processes and are, therefore, linked to a multitude of diseases. For that reason, mammalian septins appear to be of clinical importance. With the ever-increasing number of discovered cellular roles, septins are becoming a point of interest for many and the amount of information known about them grows rapidly. In this review, I attempt to summarise knowledge about the structure, assembly and function of mammalian septins, as well as their possible practical use.
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Intermediate filaments in mammalian cell motility
Čermáková, Kateřina ; Libusová, Lenka (advisor) ; Pelantová, Markéta (referee)
Cell migration is crucial for the formation and maintenance of a multicellular mammalian organism, contributing to important processes such as embryonic development, tissue renewal, and immune surveillance. It is a complex phenomenon involving a plethora of processes, including relevant signalization. An impairment of those processes could be projected into innumerable pathological states, from wound healing disruption to tumour metastasis and invasiveness, thus the interest of many researchers has turned toward migration. The migration of mammalian cells is dependent on a cytoskeleton, which is being considerably rearranged in motile cells. This thesis aims to summarise the role of intermediated filaments in cell motility - the less understood cytoskeletal network in this context. Cytoplasmic, as well as nuclear intermediate filaments, due to their unique mechanical properties, affect cell mechanics. They protect against physical stresses (as cells squeeze through confined pores in a complex intercellular microenvironment) and modulate and direct actomyosin-generated forces, which are the main driving force of migration. In addition, they contribute to important migration- involved steps, such as cell polarisation, cell adhesion to surrounding surfaces, cohesion in collective migration, and...
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Ezrin-Radixin-Moesin (ERM) proteins: The link between tetraspanin web and cytoskeleton in gametes
Picková, Jana ; Frolíková, Michaela (advisor) ; Lánská, Eva (referee)
Ezrin, which belongs to the ERM protein family along with radixin and moesin, plays an important role in linking membrane proteins to the actin cytoskeleton. In sperm, ezrin was described so far in bovine and humans, where it probably contributes in actin polymerization during capacitation. During the acrosome reaction, actin plays a significant role in relocation of protein from the inner acrosomal membrane to the equatorial segment, which is an essential process for successful fertilization. One of the proteins that relocates to the equatorial segment, the site of initiation of gamete fusion, is protein CD9. This protein probably stabilizes protein interactions between sperm and oocyte during gamete fusion. In this diploma thesis, we focused on the role of ezrin in linking the CD9 protein to the actin cytoskeleton in mouse sperm. Using indirect immunofluorescence, we detected ezrin, CD9 and actin in acrosome intact mouse sperm. The co-localization of these proteins in the apical acrosomal region suggests their interactions. However, this was not confirmed by our co-immunoprecipitation experiments. Nevertheless, in the apical acrosomal region we also detected EWI-2 protein, which could act as linker between CD9 and ezrin. In this thesis, we also investigated changes in levels of active...
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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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Axon, in development and injury
Polčanová, Zuzana ; Kárová, Kristýna (advisor) ; Novák, Ondřej (referee)
The cytoskeletal structure of growth cones plays an important role in both the development of the nervous system and during periods of axon re- generation. The growth cone is a highly dynamic structure located at the tip of growing axons, providing navigation and movement. Signalling cascades are activated that lead to the regulation of the growth cone cytoskeleton, defining its displacement, rotation, or collapse. Despite advances in under- standing guidance cues and their mechanisms of action, knowledge of what happens to the nervous system after injury is lacking. Unlike axons in the peripheral nervous system (PNS), that are able to regenerate after neuronal injury, axons in the central nervous system (CNS) loose regenerative ability as they mature. Unravelling the mechanisms of axon guidance, together with their behaviour after axotomy and regeneration, is extremely important for the understanding of CNS injuries and to provide treatment of these injuries in the future.
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Role of NAV3 in glioblastoma cells invasiveness
Legátová, Anna ; Brábek, Jan (advisor) ; Libusová, Lenka (referee)
The invasion of tumor cells from the primary lesion and the formation of metastases are the main reasons for the severe impact of cancer diseases. An option for dealing with this poor impact is the development of drugs (so-called migrastatics) that would target these processes and thus limit the spread of tumor cells from the site of the primary tumor. However, to develop such drugs, it is essential to clarify the molecular mechanisms that control or promote cell migration. One of the possible strategies for migrastatics development is the targeting of cytoskeletal structures, which play an indispensable role in cell migration. This work is focused on Neuron navigator 3 (NAV3), a protein that binds to + ends of microtubules (MTs), participates in their stabilization, and is able to mediate crosstalk between MTs and the actin network. The function of NAV3 is important for directing MTs into growing axons and proper neurite outgrowth, which is necessary for brain development. The results of this thesis suggest that NAV3 could act as a pro-tumor factor, which localizes not only to the + ends of MTs, but also to the cell protrusions, and whose presence supports the cell expansion and increases the invasive potential of glioblastoma cell lines. Key words: neuron navigator 3, microtubules, cancer,...
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Intermediate filament proteins of Preaxostyla flagellates
Švagr, Ezra ; Hampl, Vladimír (advisor) ; Varga, Vladimír (referee)
5 Abstract Monocercomonoides exilis and Paratrimastix pyriformis are protists from within the group Preaxostyla (Metamonada), they possess an excavate morphology that is presumed to be an ancestral cytoskeletal organisation of eukaryotic cells. A significant part of eukaryotic but specifically excavate morphology is fibers composed of unidentified proteins. The hypothesis on which this thesis builds upon is that these fibers are composed of Intermediate Filament proteins (IF proteins). IF proteins are a polyphyletic group of proteins involved in the assembly of mechano-elastically important fibers in eukaryotes. The most widespread group of these proteins is a family called SF-assemblins, homologues of which were identified first in Chlamydomonas reinhardii, and giardins a protein family first discovered in Giardia intestinalis, which were also found to be related to this group. Nested into the bigger hypothesis is an idea, that SF-assemblins are present in M. exilis and P. pyriformis, further strengthening their position as a universally present eukaryotic feature. The goal of this work was to find support for the morphological hypothesis that LECA possessed an excavate morphology in protein composition of the cytoskeleton. Two approaches were employed. First, identifying proteins in cytoskeletal fraction...
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