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Lane, D. C. "A mathematical investigation of a mechanochemical model for the cytoskeleton." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379843.
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Dewolf, Christine Elizabeth. "Properties of model biological membranes." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244082.
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Muddana, Hari Shankar. "Integrated biomechanical model of cells embedded in extracellular matrix." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1074.
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Monreal, Gretel. "Ventricular Remodeling in a Large Animal Model of Heart Failure." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1210007937.
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Lewis, Sara Ann. "Functions of Drosophila Pak (p21-activated kinase) in Morphogenesis: A Mechanistic Model based on Cellular, Molecular, and Genetic Studies." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/594389.
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Intellectual disability (ID) is a common phenotype of brain-development disorders and is heterogeneous in etiology with numerous genetic causes. PAK3 is one gene with multiple mutations causing ID. Affected individuals have microcephaly, and other brain-structure defects have been reported. Additionally, PAK3 is in a genetic network with eighteen other genes whose mutations cause ID, suggesting the molecular mechanisms by which PAK3 regulates of cognitive function may be shared by other genetic ID disorders. Studies in rodent models have shown that the orthologs of PAK3 are important for regulating dendrite spine morphology and postnatal brain size. In Drosophila melanogaster, the morphological processes of oogenesis, dorsal closure during embryogenesis, and salivary gland-lumen formation require Pak, the Drosophila ortholog of PAK3. Additionally, Pak is important for development of the subsynaptic reticulum of the neuromuscular junction, sensory axon pathfinding and terminal arborization in the Drosophila central nervous system (CNS). However, the role of Pak in mushroom body (MB) structure and intrinsic neurite arbor morphogenesis, as well as details of the underlying cellular and molecular mechanisms are unknown. To address this gap, I used Drosophila models of PAK3 gene mutations, Pak, and a combination of immunostaining, primary cell culture, and genetic interaction studies to elucidate these mechanisms. I performed a detailed characterization of the previously reported adult Pak phenotypes of decreased survival as well as leg and wing morphology. I found that decreased survival is a low-penetrance phenotype that is enhanced by chromosomes from the same mutagenesis. Defects of the adult wing include folding and misalignment between the layers, blisters, and missing or partial cross veins. The Pak-mutant legs are short and often misdirected in the pupal case with morphological defects in the shape of the leg segments themselves. The mushroom bodies are important insect learning and memory brain structures whose lobes are composed of axon bundles with individual axons bifurcating to form the α and β lobes. Mutations in Pak cause defects in the length, thickness, and direction of the MB α and β lobes. These defects increase in severity during metamorphosis, when neurogenesis and differentiation of these structures occur, suggesting that Pak stabilizes the branches of the α/β mushroom body neurons. Pak-mutant cultured neurons have reduced neurite arbor size with defects in neurite caliber. Initial outgrowth was normal, followed by a decrease in neurite branch number, again supporting the role of Pak in neurite-branch stability. There are defects in the cytoskeleton in growth cones at six hours post-plating as well as in neurons after three days in vitro. The Pak-mutant phenotype severity depends on the phosphorylation status of myosin regulatory light chain, supporting the mechanistic hypothesis that Pak regulates neurite-branch stability by inhibiting myosin light chain kinase. The neuronal phenotype of decreased branch stability suggests a mechanism of excessive retraction as the cellular pathogenesis underlying PAK3 mutation-associated brain disorders. I used western blotting to characterize the protein products of four nonsense mutations in Drosophila Pak to interpret genotype-phenotype relationships. Each allele has molecularly unique consequences: Pak¹¹, stop-codon read through and truncated protein; Pak¹⁶, no read through, but truncated protein; Pak⁶, read through with no truncated protein; Pak ¹⁴, neither readthrough nor truncated protein. Truncated proteins produced by Pak¹¹ and Pak¹⁶ alleles retained partial function for survival, wing blistering, leg morphology, and neurite length. Conversely, truncated protein increased the severity of the mushroom body defects. Truncated proteins have no effect on neuron branch number, wing folding, or vein defects. Together, these results demonstrate a role of Pak in regulating epithelial morphology, brain structure, and neurite arbor size and complexity. These closely resemble features of the human disorder, providing evidence that this is a good genetic model for this cause of ID.
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Bauer, David. "Využití tensegritních struktur pro modelování mechanického chování hladkých svalových buněk." Master's thesis, Vysoké učení technické v Brně. Fakulta strojního inženýrství, 2011. http://www.nusl.cz/ntk/nusl-229836.
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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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Yadav, Preeti [Verfasser], and Michael [Gutachter] Sendtner. "Studying Neuronal Cytoskeleton Defects and Synaptic Defects in Mouse Model of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy / Preeti Yadav. Gutachter: Michael Sendtner." Würzburg : Universität Würzburg, 2016. http://d-nb.info/1113535075/34.
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Kovari, Daniel T. "Investigations of the spreading and closure mechanisms of phagocytosis in J774a.1 macrophages." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54882.
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Phagocytosis is the process by which cells engulf foreign bodies. It is the hallmark behavior of white blood cells, being the process through which those cells ingest and degrade pathogens and debris. To date a large amount of research has focused on documenting the existence and role of biochemical components involved with phagocytosis. Scores of signaling molecules have been implicated in the complex signal cascade which drives the process. These molecules are small (typically no larger than 5 nanometers) and operate in a crowded, chemically “noisy,” environment, yet they coordinate the cell's activity over comparatively expansive distances (as large as 20 micrometers). How these molecular processes scale-up to coordinate the activities of the cell over such massive distances is largely unknown. Using a planar analog of phagocytosis termed “frustrated phagocytosis,” we experimentally demonstrate that phagocytosis occurs in three distinct phases: initial cell-antigen binding, symmetric spreading, and late-stage contraction. Initial binding and symmetric spreading appears to be both mechanically and chemically similar to the quasi-universal cellular behaviors of adhesion and migration. Adhesion and migration have received extensive attention from the biophysics community in recent years. Leveraging these similarities, we adapt the biomechanical frameworks used in models of migration to phagocytosis. We show that macroscopic properties such as a cell's effective viscosity and membrane cortical tension can be used to model cell behavior during phagocytosis. Our experiments reveal that late-stage contraction distinguishes frustrated phagocytosis from other spreading behaviors. This contraction is myosin dependent. Additionally we demonstrate, for the first time, that late-stage contraction corresponds with formation of a contractile F-actin belt. Based on the dynamic contraction model (DC) developed to explain actin structure during cell migration we propose a DC model of phagocytosis which posits that contractile belt formation is the result of a late-stage myosin activity coupled with F-actin.
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Ganay, Thibault. "Caractérisation du modèle murin de la Neuropathie à Axones Géants : rôle de la gigaxonine dans la survie neuronale et l'organisation du cytosquelette." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22075.
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La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophiqueGiant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases
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Henninger, Nils. "Inhibiting Axon Degeneration in a Mouse Model of Acute Brain Injury Through Deletion of Sarm1." eScholarship@UMMS, 2017. http://escholarship.umassmed.edu/gsbs_diss/900.
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Traumatic brain injury (TBI) is a leading cause of disability worldwide. Annually, 150 to 200/1,000,000 people become disabled as a result of brain trauma. Axonal degeneration is a critical, early event following TBI of all severities but whether axon degeneration is a driver of TBI remains unclear. Molecular pathways underlying the pathology of TBI have not been defined and there is no efficacious treatment for TBI.
Despite this significant societal impact, surprisingly little is known about the molecular mechanisms that actively drive axon degeneration in any context and particularly following TBI. Although severe brain injury may cause immediate disruption of axons (primary axotomy), it is now recognized that the most frequent form of traumatic axonal injury (TAI) is mediated by a cascade of events that ultimately result in secondary axonal disconnection (secondary axotomy) within hours to days.
Proposed mechanisms include immediate post-traumatic cytoskeletal destabilization as a direct result of mechanical breakage of microtubules, as well as catastrophic local calcium dysregulation resulting in microtubule depolymerization, impaired axonal transport, unmitigated accumulation of cargoes, local axonal swelling, and finally disconnection. The portion of the axon that is distal to the axotomy site remains initially morphologically intact. However, it undergoes sudden rapid fragmentation along its full distal length ~72 h after the original axotomy, a process termed Wallerian degeneration.
Remarkably, mice mutant for the Wallerian degeneration slow (Wlds) protein exhibit ~tenfold (for 2–3 weeks) suppressed Wallerian degeneration. Yet, pharmacological replication of the Wlds mechanism has proven difficult. Further, no one has studied whether Wlds protects from TAI. Lastly, owing to Wlds presumed gain-of-function and its absence in wild-type animals, direct evidence in support of a putative endogenous axon death signaling pathway is lacking, which is critical to identify original treatment targets and the development of viable therapeutic approaches.
Novel insight into the pathophysiology of Wallerian degeneration was gained by the discovery that mutant Drosophila flies lacking dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously recapitulated the Wlds phenotype. The pro-degenerative function of the dSarm gene (and its mouse homolog Sarm1) is widespread in mammals as shown by in vitro protection of superior cervical ganglion, dorsal root ganglion, and cortical neuron axons, as well as remarkable in-vivo long-term survival (>2 weeks) of transected sciatic mouse Sarm1 null axons. Although the molecular mechanism of function remains to be clarified, its discovery provides direct evidence that Sarm1 is the first endogenous gene required for Wallerian degeneration, driving a highly conserved genetic axon death program.
The central goals of this thesis were to determine (1) whether post-traumatic axonal integrity is preserved in mice lacking Sarm1, and (2) whether loss of Sarm1 is associated with improved functional outcome after TBI. I show that mice lacking the mouse Toll receptor adaptor Sarm1 gene demonstrate multiple improved TBI-associated phenotypes after injury in a closed-head mild TBI model. Sarm1-/- mice developed fewer beta amyloid precursor protein (βAPP) aggregates in axons of the corpus callosum after TBI as compared to Sarm1+/+ mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phosphorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after TBI. Strikingly, whereas wild type mice exhibited a number of behavioral deficits after TBI, I observed a strong, early preservation of neurological function in Sarm1-/- animals. Finally, using in vivo proton magnetic resonance spectroscopy, I found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1-/- mice compared to controls immediately following TBI. My results indicate that the Sarm1-mediated prodegenerative pathway promotes pathogenesis in TBI and suggest that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after TBI.
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Petrov, Eugene P., Senthil Arumugam, Jens Ehrig, and Petra Schwille. "Cytoskeletal pinning prevents large-scale phase separation in model membranes." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-182836.
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Petrov, Eugene P., Senthil Arumugam, Jens Ehrig, and Petra Schwille. "Cytoskeletal pinning prevents large-scale phase separation in model membranes." Diffusion fundamentals 20 (2013) 40, S. 1, 2013. https://ul.qucosa.de/id/qucosa%3A13607.
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Roberts-Pilgrim, Anna M. Phillips Charlotte L. "Glomerulosclerosis in the Col1a2-deficient mouse model homotrimer pathogenesis and MMP expression /." Diss., Columbia, Mo. : University of Missouri--Columbia, 2009. http://hdl.handle.net/10355/6155.
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Title from PDF of title page (University of Missouri--Columbia, viewed on Feb. 20, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Charlotte L. Phillips. Vita. Includes bibliographical references.
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Kumar, Ravinash Krishna. "Cytoskeletal-like assemblies within model protocells : en route towards synthetic cellular constructs." Thesis, University of Bristol, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619256.
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The aim of this thesis was to rationally design, construct and test novel protocells equipped with primitive cytoskeletons, which could be produced by integration of simple chemical systems. This was achieved by the non-covalent assembly of a functionalised amino acid, N-fluorenylmethyloxycarbonyl-tyrosine (FMOC-Tyr), to yield highly structured nanofilament morphologies within the aqueous interiors of micron-sized, membrane-bounded compartments. Initially. the formation of the non-covalently assembled FMOC-Tyr nanofilaments was studied in isolation. Significantly, at a critical filament concentration and appropriate pH, the formation of supramolecular hydrogels was ascertained. Moreover, by substituting FMOC-Tyr with the hydrophilic analogue N-fluorenylmethyloxycarbonyl- tyrosine-( O)-phosphate (FMOC-Tyr-POH), cleavage of the phosphate group by alkaline phosphatase or cerium oxide nanopal1icles resulted in the formation of gel networks with tuneable (PH or temperature) gel-sol and sol-gel transitions. After the experimental design parameters for hydrogel formation were determined, the FMOC-Tyr nanofilaments were grown within two contrasting model compartments: phospholipid vesicles and inorganic colloidosomes. Significantly, FMOC-Tyr supramolecular hydrogel-containing vesicles showed enhanced resistance to mechanical deformation, osmotic pressure and increased structural persistence. Moreover, primitive cytoskeletal function was further demonstrated via heat-induced structural deformations of vesicles, where morphological transformations were driven by disassembly of the internalised nanofilaments. Growth of FMOC-Tyr cytoskeletal-like filaments within inorganic colloidosomes resulted in silica membrane-directed nanofilament growth from the interior of the inorganic surface towards the centre of the microcapsule. In this instance, it was possible to demonstrate that temperature-dependent filament assembly and disassembly could be used to modulate the interior viscosities of colloidosomes, which in tum regulated intra-protocellular enzyme kinetics. The work presented in this thesis describes the first examples of protocells equipped with functional artificial cytoskeletons comprising self-assembled synthetic building blocks. The incorporation of these bio-inspired scaffolds within protocells not only provides the structural persistence that is central in all living cells, but also a mechanism to re-structure interior environments and control compartment shape.
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Genty, Christian. "Réponse in vitro des cellules ostéoformatrices aux stimuli mécaniques : étude en microgravité réelle et sur les modèles animaux après surcharge et décharge mécanique." Saint-Etienne, 1993. http://www.theses.fr/1993STET4013.
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Les variations de contraintes mécaniques appliquées sur l'os entrainent des modifications du métabolisme osseux. La diminution des contraintes mécaniques telle qu'elle existe dans le modèle du rat suspendu, induit une perte osseuse rapide dès le début de la suspension qui se stabilise par la suite. L'augmentation des contraintes par un exercice de type dynamique (la course sur tapis roulant) induit un gain osseux. Les mécanismes cellulaires responsables de ces modifications sont encore mal connus. Grâce à la mise au point d'une méthode d'isolement des cellules ostéoformatrices à partir du tibia de rat, nous avons pu isoler les cellules des facteurs environnementaux et les maintenir en culture primaire. Les études in vitro sur les différents modèles animaux précités, ont permis de montrer que la diminution de contrainte entraine une inhibition de la maturation des ostéoblastes conduisant à une perte osseuse, qui cesse dès que les contraintes sur l'os se retrouvent au niveau initial. Inversement, l'exercice stimule la différenciation des ostéoblastes en ostéoblastes matures aboutissant à un gain osseux qui diminue les contraintes mécaniques de l'os. Nous avons pu étudier l'effet direct de la microgravité sur les cellules ostéoblastiques grâce au développement d'un module de culture et de techniques d'investigations adaptées aux conditions expérimentales du vol spatial biocosmos x. Les résultats de cette expérience ont montré que la cellule ostéoblastique est capable de s'adapter à son nouvel environnement physique en modifiant son activité cellulaire et l'organisation de son cytosquelette. Nous pensons que les contraintes mécaniques pourraient avoir une influence sur le cytosquelette des ostéoblastes qui modifierait l'activité cellulaire, permettant à l'os de s'adapter à son nouvel environnement
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Koch, Matthias [Verfasser], and Alexander [Akademischer Betreuer] Rohrbach. "Biomechanics of prokaryotic & eukaryotic cytoskeletal model systems probed by time-multiplexed optical tweezers." Freiburg : Universität, 2015. http://d-nb.info/1119246539/34.
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García, Arguinzonis Maísa Inés. "Analysis of signal transduction pathways and the cytoskeleton in VASP-deficient cell lines and mouse models." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969694741.
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Na, Sungsoo. "Effects of mechanical forces on cytoskeletal remodeling and stiffness of cultured smooth muscle cells." Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1704.
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Roth, Alexander. "Nano-mechanics of biomimetic models of the actin based cytoskeleton from single molecules to complex composite structures /." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972309322.
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Belbahri, Mohamed Reda. "Rôle des protéines réticulantes de l'actine dans la mécanique de l'endocytose chez la levure." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0209.
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L'objet de cette thèse porte sur l'étude du rôle des protéines réticulantes dans la génération de force des réseaux d'actine branchés lors de l'endocytose. Une première étape consiste en une quantification in-vivo de la dynamique des patchs d'endocytose dans des levures (S. cerevisiae) sauvages et dans des levures mutantes n'exprimant pas une ou plusieurs protéines réticulantes. En mettant ces résultats en parallèle avec des données mécaniques déjà établies par une ancienne doctorante, nous avons pu démontrer une corrélation entre l'élasticité d'un réseau d'actine et l'efficacité de l'endocytose. Cette étude, publiée en 2019, nous proposons une chaîne de causalité pour expliquer les phénotypes observés.En parallèle de ces travaux, et à partir d'une technique élaborée au PMMH, j'ai travaillé sur le développement d'un outil de mesure de la mécanique et de la rhéologie de gels d'actine in-vitro : les micro-cylindres magnétiques. Avec cet outil et l'expertise de l'IBDM sur les extraits cellulaires, j'ai pu analyser la rhéologie des gels d'actine reconstitués à partir d'extraits cellulaires de levure comportant (ou pas) les agents réticulants. La rhéologie de ces gels d'actine semble être un élément de plus en plus important à considérer pour expliquer l'efficacité de l'endocytose.La comparaison des résultats in vivo et in vitro nous a permis de déterminer l’effet des protéines de réticulations sur ces gels, d’observer des corrélations entre certains paramètres mécaniques et l’efficacité de l’endocytose dans la cellule et enfin de mieux comprendre le rôle des réseaux d’actine dans la génération de force durant l’endocytoseThe subject of this thesis is to study the role of actin cross-linking proteins in force generation during endocytosis. First, we characterized in-vivo in yeast (S. cerevisiae) the dynamics of endocytic patches in wild and mutant cells which do not express one or several crosslinkers. Then, we developed an experiment to measure in-vitro the rheology of actin gels reconstituted from cellular extracts of these strains, using magnetic microcylinders. With these results, we have been able to determine the effect of cross-linking proteins on actin gels, to establish correlations between certain mechanical parameters and the efficiency of endocytosis in cells, and finally to better understand how actin networks are able to generate force during endocytosis
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Palmer, Jeffrey Shane. "Microstructurally-based constitutive models of cytoskeletal networks for simulation of the biomechanical response of biological cells." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44752.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (v. 2, p. 335-375).
The elastic and viscoelastic stress-strain behavior of cytoskeletal networks, important to many cellular functions, is modeled via a microstructurally-informed continuum mechanics approach. The force-extension behavior of the individual filaments is captured with a new analytical expression of the MacKintosh worm-like chain relationship for semiflexible filaments. The filament expression is used in the Arruda-Boyce eight-chain network model to capture the 3D stress-strain behavior, quantifying the effects of isotropic network prestress and tracking microstructural stretch and orientation states during large deformations. The network model captures the initial stiffness of the network as well as the nonlinear strain stiffening observed at large stresses in shear rheological data of bundled/unbundled in vitro F-actin networks. The cytoskeletal network model has also been extended to include the internal energy based mechanical contributions at the filament and network levels from torsional crosslink deformations as well as from direct axial stretching of filaments. This enhanced model effectively captures the stress-strain behavior of F-actin networks cross-linked with two different types of actin binding proteins (filamin and streptavidin). The enhanced model is also used to evaluate the influence of the cross-links' torsional stiffness on the entropic bending configuration space of the cytoskeletal filaments. The 3D constitutive network model provides a framework for capturing time-dependent spatial diffusion of cytosol within a porous, visco hyperelastic filament network. The poroelastic behavior is coupled with the hyperelastic network behavior through a 3D biphasic theory that includes network swelling effects for finite deformations.
(cont.) The mechanical response of the cytoskeletal network due to the localized swelling is captured by employing multiplicative decomposition of mechanical and swelling stretches. Nonlinear shear viscoelasticity is also included to create a 3D network model capable of capturing the time-dependent response of cytoskeletal networks on short and long time scales. The model captures the nonlinear time dependent behavior of in vitro actin-filamin and actin-avidin networks observed in shear rheological experiments. The constitutive models are evaluated in a finite element model with a cellular geometry (including membrane and nucleus submodels) and the ability to spatially vary network properties throughout the cell.
by Jeffrey Shane Palmer.
Ph.D.
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Wurm, Christine [Verfasser], Andreas [Akademischer Betreuer] Bausch, and Joachim O. [Akademischer Betreuer] Rädler. "Structure, Mechanics and Dynamics of Cytoskeletal Model Systems / Christine Wurm. Gutachter: Andreas Bausch ; Joachim O. Rädler. Betreuer: Andreas Bausch." München : Universitätsbibliothek der TU München, 2011. http://d-nb.info/1012186806/34.
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Lombardo, Andrew Thomas. "Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/860.
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Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need.
To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system.
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Kaushik, Azad Kumar. "Etude idiotypique, structurale et analyse du repertoire des autoanticorps dans deux modeles d'autoimmunite naturelle chez la souris : autoanticorps reagissant avec les globules rouges de souris bromelises, autoanticorps reagissant avec l'adn et les." Paris 7, 1987. http://www.theses.fr/1987PA077074.
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Haga, Raquel Brandão. "Inibição da migração mediada pelo gene RECK em modelo de glioma humano através de alterações no citoesqueleto e adesão focal." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-11092012-134839/.
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Gliomas são tumores altamente invasivos, resistentes aos tratamentos disponíveis atualmente e com alta taxa de mortalidade. A superexpressão de RECK na linhagem de glioma humano T98G comprometeu a capacidade das células de migrar e invadir in vitro, com rearranjo do citoesqueleto e alteração na distribuição espacial de FAK fosforilado. Entretanto, o possível mecanismo envolvido na inibição da migração mediada por RECK não foi desvendado. Para estudarmos os mecanismos envolvidos nesta alteração da capacidade migratória, as células T98G foram transfectadas com o vetor plasmidial pCXN2-hRECK (RECK+). A via das integrinas, a atividade de alguns membros da família das RhoGTPases e elementos do citoesqueleto foram avaliados através de imunoblotting, imunomarcação e ensaios de pull-down para as células RECK+ em comparação com células T98G não-transfectadas (WT), células T98G transfectadas com vetor pCXN2 na ausência do gene RECK (vetor) e fibroblastos primários humanos (FF287). Nossos resultados mostram um aumento na expressão de integrina β1 e uma diminuição da fosforilação de FAK no sítio de auto-fosforilação Tyr397 que, juntamente com o aumento das fibras de estresse e a diminuição dos lamelipódios, sugerem um fenótipo menos migratório da célula. Porém, quando avaliada a atividade de Rac1, esta se mostrou aumentada, embora uma das vias de ativação de Rac1 seja através da fosforilação de FAK levando à formação dos lamelipódios. A hipótese é que RECK inibe a quebra das adesões focais que participam do processo de migração, dificultando a mobilidade celular. Como as células continuam recebendo o estímulo para migrar, estas ativam Rac1 através de uma via independente de FAK. Além disso, a imunomarcação de paxilina mostrou um aumento no tamanho das adesões focais nas células RECK+, indicando que RECK pode influenciar nas estruturas responsáveis pelo contato célula-matriz.Gliomas are highly invasive, treatment-resistant and lethal tumors. Overexpression of RECK in human glioma cell line T98G decreased cell migration and invasion in vitro, lead to cytoskeleton rearrangement and caused changes in phospho-FAK distribution. However, the pathway involved in RECK-mediated inhibition of cell migration has not been elucidated yet. To study the mechanisms by which RECK affects cell motility, T98G cells were transfected with pCXN2-hRECK vector (RECK+). Some proteins involved in the integrin pathway, activity of some proteins of RhoGTPase family and cytoskeleton proteins were analyzed through immunoblotting, immunostaining and pull-down assay in RECK+ cells and compared with non-transfected T98G cells, T98G transfected with pCXN2 without RECK gene and human primary fibroblasts (FF287). Our results showed an increase in integrin β1 expression and a decrease in FAK phosphorylation in the Tyr397 site, which together with the increase of stress fibers and decrease of lamellipodia, suggest a less migratory phenotype. Despite this, Rac1 activity was increased even though one of Rac activation pathways is through phospho-FAK, leading to lamellipodium formation. Our hypotheses is that RECK affects focal adhesion turnover, diminishing cell motility. As cells are still receiving a positive signal to migrate, they activate Rac1 through a FAK-independent pathway. Besides that, paxillin immunostaining showed that focal adhesions are larger in RECK+ cells, indicating that RECK can influence structures related with cell-matrix contact.
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Maier, Matthias [Verfasser], Andreas [Akademischer Betreuer] Bausch, and Matthias [Akademischer Betreuer] Rief. "Living Cells and Cytoskeletal Model Systems: Mechanics and Responses to Dynamic Shear / Matthias Maier. Gutachter: Andreas Bausch ; Matthias Rief. Betreuer: Andreas Bausch." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1052995489/34.
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Chamcheu, Jean Christopher. "Disease-causing Keratin Mutations and Cytoskeletal Dysfunction in Human Skin : In vitro Models and new Pharmacologic Strategies for Treating Epidermolytic Genodermatoses." Doctoral thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-123071.
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Epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI) are rare skin fragility diseases characterized by intra-epidermal blistering due to autosomal dominant-negative mutations in basal (KRT5 or KRT14) and suprabasal (KRT1 or KRT10) keratin genes, respectively. Despite vast knowledge in the disease pathogenesis, the pathomechanisms are not fully understood, and no effective remedies exist. The purpose of this work was to search for keratin gene mutations in EBS patients, to develop in vitro models for studying EBS and EI, and to investigate novel pharmacological approaches for both diseases. We identified both novel and recurrent KRT5 mutations in all studied EBS patients but one which did not show any pathogenic keratin mutations. Using cultured primary keratinocytes from EBS patients, we reproduced a correlation between clinical severity and cytoskeletal instability in vitro. Immortalized keratinocyte cell lines were established from three EBS and three EI patients with different phenotypes using HPV16-E6E7. Only cell lines derived from severely affected patients exhibited spontaneous keratin aggregates under normal culture conditions. However, heat stress significantly induced keratin aggregates in all patient cell lines. This effect was more dramatic in cells from patients with a severe phenotype. In organotypic cultures, the immortalized cells were able to differentiate and form a multilayered epidermis reminiscent of those observed in vivo. Addition of two molecular chaperones, trimethylamine N-oxide dihydrate (TMAO) and sodium 4-phenylbutyrate (4-PBA), reduced the keratin aggregates in both stressed and unstressed EBS and EI keratinocytes, respectively. The mechanism of action of TMAO and 4-PBA was shown to involve the endogenous chaperone system (Heat shock proteins e.g. Hsp70). Besides, MAPK signaling pathways also seemed to be incriminated in the pathogenesis of EBS. Furthermore, depending on which type of keratin is mutated, 4-PBA up-regulated Hsp70 and KRT4 (possibly compensating for mutated KRT1/5), and down-regulated KRT1 and KRT10, which could further assist in protecting EBS and EI cells against stress. In conclusion, novel and recurrent pathogenic keratin mutations have been identified in EBS. Immortalized EBS and EI cell lines that functionally reflect the disease phenotype were established. Two pharmacologic agents, TMAO and 4-PBA, were shown to be promising candidates as novel treatment of heritable keratinopathies in this in vitro model.
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Amaral, Jonatas Bussador do. "Células MCF-7 como modelo 3D no estudo de câncer de mama humano." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-21072011-134443/.
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O diferencial da cultura de células em 3-dimensões é permitir que as células explorem as 3-dimensões do espaço, aumentando assim as interações com o ambiente e entre as células. Em estudos relacionados à biologia do câncer de mama, vem ganhando espaço a utilização de esferóides para estudos que visam à compreensão da morfogênese do espaço luminal. Neste trabalho foi mostrado que as células MCF-7 reorganizam-se em estruturas tubulares e acinares. Em ambas as situações, a formação do lúmen veio acompanhada pelo estabelecimento de uma camada de células polarizadas, arranjo este muito semelhante ao encontrado em glândulas mamárias. Os resultados apresentados apontam para a existência de uma população de células na linhagem MCF-7 que não estão totalmente comprometidas ao fenótipo tumoral. Mantidos diferenciados, os esferóides de células MCF-7 apontam como um novo modelo para estudos relacionados à formação do lúmen, permitindo assim explorar o papel de diferentes vias como as relacionadas a apoptose, autofagia, diferenciação e sobrevivência celular.As a particularity, a 3D cell culture permits cells to explore the three dimensions of the space thereby increasing cell-cell interactions, as well as interaction with the environment. In studies related to breast cancer biology, spheroids are becoming widely used in the aim to comprehend luminal space morphogenesis. We showed that MCF-7 cells reorganize themselves in tubular and acinar structures. In both situations, lumen formation was accompanied by the establishment of a layer of polarized cells, an arrangement that is very similar to that of breast glands. The presented results suggest the existence of an MCF cell line population not completely committed to the tumor phenotype. When maintained as differentiated, MCF-7 cell spheroids can be a new model for studies regarding lumen formation, thereby exploring the role of diiferent pathways, such as those related to cell apoptosis, autophagy, differentiation and survival.
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Mefti, Nacim. "Mise en oeuvre d'un modèle mécanique de l'adhésion cellulaire : approche stochastique." Thesis, Vandoeuvre-les-Nancy, INPL, 2006. http://www.theses.fr/2006INPL099N/document.
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L'adhésion cellulaire est un phénomène important en biologie. Le but de ce travail est le développement d'un modèle mécanique décrivant des phénomènes d'adhésion cellulaire à différentes échelles. La première échelle, microscopique, a pour objet la description des phénomènes cinétiques moléculaires durant le rolling. La seconde échelle, mésoscopique, est relative à la modélisation des déformations actives de la cellule durant la motilité. La troisième échelle, dite macroscopique, concerne la description de l'évolution dans le temps de l'adhésion d'une population de cellules. Les simulations réalisées mettent en évidence le rolling, et la déformation active de la celluleCell adhesion is an important phenomenon in biology, especially in the immune defence and tissue growth.We focus in this work on the development of a mechanical model for the description of the cell adhesion in a multiscal context. The first one is microscopic scale, which describes the molecular rupture and adhesion kinetics.At the mesoscopic scale, we model the active deformation of the cell during the motility phenomenon. At the macroscopic scale, we model the time evolution of the adhesion of cell population, under the action of the fluid. Numerical simulations emphasize the rolling phenomenon and the active deformation of a cell
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Braunger, Julia. "Ezrin activation in vitro: Investigation of ezrin's conformation and the interaction between ezrin and F-actin." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2013. http://hdl.handle.net/11858/00-1735-0000-0022-609D-5.
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Taniguchi, Leandro Utino. "Avaliação imunohistoquímica das alterações do citoesqueleto na parede alveolar em modelo experimental de lesão pulmonar induzida pela ventilação mecânica em ratos." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5159/tde-07122009-180201/.
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INTRODUÇÃO: A ventilação mecânica é uma terapia importante, mas com possíveis complicações. Uma das mais relevantes é a lesão pulmonar induzida pelo ventilador (VILI do inglês Ventilator-induced lung injury). Devido à hiperdistensão alveolar, o pulmão inicia um processo inflamatório, com infiltrado neutrofílico, formação de membrana hialina, fibrogênese e prejuízo de troca gasosa. Nesse processo, a mecanotransdução do estímulo da hiperdistensão celular se faz através do citoesqueleto da célula e de suas interações com a matriz extracelular e com as células vizinhas. Apesar desse papel fundamental no processo da VILI, não existem estudos in vivo sobre as alterações do citoesqueleto e de suas proteínas associadas durante esse processo patológico. O objetivo desse estudo foi descrever as alterações no citoesqueleto e em duas de suas principais proteínas associadas (FAK e paxilina) durante esse processo. MÉTODOS: Nesse estudo experimental foram feitos três grupos (n = 4 6): um controle e dois ventilados por quatro horas com PEEP de 5 cmH2O. Um grupo foi ventilado com volume corrente de 8 ml/kg (BV) e o outro com 24 ml/kg (AV). Dados de mecânica respiratória foram calculados no início e no final do período experimental. Os pulmões foram avaliados por histomorfometria quanto à área proporcional de parênquima, índice de infiltrado neutrofílico e índice de edema perivascular, quanto à quantidade de fosfo-FAK, fosfo-paxilina, paxilina total, actina músculo liso e alfa-tubulina por Western Blot, quanto à imunofluorescência para paxilina total com microscopia confocal a laser e com microscopia eletrônica de transmissão. RESULTADOS: os grupos foram semelhantes nas características basais. Houve aumento da elastância dinâmica (Edin) no grupo BV e redução no grupo AV (Edin inicial e final: 0,76 ± 0,4 vs 1,02 ± 0,47 respectivamente, em cmH2O/ml; p = 0,001). Não houve diferença na área proporcional de parênquima ou índice de edema perivascular entre os grupos estudados. A ventilação mecânica induziu infiltrado neutrofílico pulmonar nos animais, tanto no grupo BV como no AV em relação ao controle (p < 0,001). O infiltrado foi mais importante no grupo AV que no BV (p = 0,003). Houve um aumento de 40% na fosfo-FAK pelo Western Blot no grupo AV em relação ao controle (p=0,069) e aumento significativo de fosfo-paxilina no grupo AV em relação ao controle (p<0,001) e ao BV (p<0,001). Não se observaram diferenças para paxilina total, actina músculo liso e alfa-tubulina. A microscopia confocal demonstrou marcação para paxilina total nos septos alveolares. A microscopia eletrônica sugeriu reorganização do citoesqueleto nas zonula adherens do grupo AV. CONCLUSÕES: A ventilação mecânica promove lesão pulmonar com infiltrado neutrofílico numa relação dose-dependente. A ventilação com alto volume corrente promove fosforilação da FAK e de paxilina. As alterações no citoesqueleto em modelo in vivo de VILI são possíveis de serem descritas utilizando-se de métodos de microscopia confocal, Western Blot e microscopia eletrônica.INTRODUCTION: Mechanical ventilation is an important therapy, but is associated with complications. One of the most relevant is ventilator-induced lung injury (VILI). Due to alveolar hyperdistension, the lung initiates an inflammatory process, with neutrophilic infiltration, hyaline membrane formation, fibrogenesis and gas exchange impairment. In this process, cellular mechanotransduction of the overstretching stimulus is mediated through the cytoskeleton and its cell-cell and cell-matrix interactions. But, although the cytoskeleton has this important role in the pathogenesis of VILI, there are no in vivo models for the research of cytoskeletal and cytoskeleton-associated proteins modifications during this pathological process. Our objective was to describe the immunohistochemical modifications during this process on the cytoskeleton and on two of its associated proteins (FAK and paxillin). METHODS: in this experimental study, three groups (n = 4 6) were studied: a control group and two ventilated for four hours with PEEP of 5 cmH2O. One group was ventilated with tidal volume of 8 mL/kg (LV) and the other with 24 mL/kg (HV). Data of respiratory mechanics were obtained at the beginning and the end of the experimental period. The lungs were evaluated with histomorphometry for parenchymal proportional area, neutrophilic infiltrate and perivascular edema, with Western Blot for phospho-FAK, phospho-paxillin, total paxillin, alpha-smooth muscle actin and alpha-tubulin, with confocal laser scanning microscopy for total paxillin, and with transmission electron microscopy. RESULTS: the groups were similar at the baseline. Dynamic elastance (Edin) increased in LV group and decreased in HV group (Edin initial to final: 0.76 ± 0.4 vs. 1.02 ± 0.47 respectively, in cmH2O/ml; p = 0.001). There was no difference in the parenchymal proportional area or the perivascular edema in the three groups. Mechanical ventilation induced pulmonary neutrophilic infiltration, both in the LV group and the HV group in comparison with control (p < 0.001). The infiltrate was more important in the HV group than in the LV group (p = 0.003). Phospho-FAK increased 40% in the HV group in Western Blot in comparison with control (p=0.069). Phosphopaxillin increased significantly in HV group compared with control (p<0.001) and with LV (p<0.001). Total paxillin, alpha-smooth muscle actin and alpha-tubulin did not show any differences. Confocal microscopy showed total paxillin labeling at alveolar septa. Electron microscopy suggested cytoskeleton reorganization at the zonula adherens in the AV group. CONCLUSIONS: Mechanical ventilation induces pulmonary injury with neutrophilic infiltrate in a dose-dependent relationship. Ventilation with high tidal volume promotes FAK and paxillin phosphorilation. The alterations in cytoskeleton in an in vivo model of VILI are possible to be studied with confocal microscopy, Western Blot and electron microscopy.
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Campos, Luciene Cristina Gastalho. "Identificação e caracterização de proteínas modificadas em enxertos de veias safenas humanas arterializadas no modelo ex vivo." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5131/tde-16122008-174047/.
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A revascularização cardíaca utilizando a ponte de safena é um procedimento bastante utilizado para restabelecer o fluxo coronariano. Apesar do sucesso deste procedimento, a patência deste enxerto pode chegar a menos de 50% em 10 anos. Atribui-se parte deste insucesso a variações no processo adaptativo à nova condição hemodinâmica, onde o shear stress e o estiramento aumentados podem estar interferindo na função endotelial e vascular. Este processo envolve a participação de diversas proteínas e o estudo de como elas participam conjuntamente é uma importante abordagem para entender as alterações fisiológicas e patológicas que ocorrem no enxerto vascular. Neste trabalho, tecnologias proteômicas, gel 2-D e ICAT, foram utilizadas para identificar as proteínas que são modificadas nas fases precoces da arterialização do enxerto venoso. Foi utilizado um sistema ex vivo de perfusão controlada, desenvolvido em nosso laboratório, onde a veia safena humana foi cultivada tanto em regime hemodinâmico venoso (5 mL/min) e arterial (50 mL/min, 80 mmHg) por 24 horas. Dentre as proteínas identificadas, a maioria apresenta funções estruturais como, por exemplo, -actina de músculo liso, CRP1, colágeno VI, tropomiosina, miosina, desmina e vimentina. Para avaliação funcional foram selecionadas a -SMA e a CRP. A -SMA mostrou-se diminuída nas fases mais precoces da arterialização venosa, com quase desaparecimento após 3 dias da cirurgia, seguido de um aumento nos períodos subseqüentes. A CRP3 mostrou-se com expressão predominantemente arterial tanto em amostra humana como de rato. A arterialização de segmentos venosos induziu a expressão da CRP3, sendo dependente do aumento do estiramento (stretch) nas células musculares lisas e não do aumento do shear stress na superfície endotelial. Coletivamente, neste trabalho caracterizamos duas proteínas que foram modificadas durante o processo de arterialização e/ou adaptação da veia à condição hemodinâmica arterial. As proteínas identificadas contribuirão para o melhor entendimento do processo de arterialização venosa e poderão ser testadas como novos alvos terapêuticos para melhorar a patência destes enxertosCoronary artery bypass surgery by saphenous vein graft is still widely used to revascularization of ischemic heart. Despite the success of this procedure, about 50% occlude after 5-10 years. The vein graft is subjected to increased tensile stress and the adaptive vein response to the arterial hemodynamic condition may predispose to bypass occlusion. Several proteins are modulated during arterialization, the understanding of the molecular changes of this process may be useful to new therapeutics approaches development attempting to increase vein graft patency. In this work, proteomics plataform, gel 2-D and ICAT, were used to identify the proteins that are modified in the early stages of vein graft rterialization. Human saphenous vein were cultured in an ex vivo flow through system in both venous (5 ml / min) and arterial (50 ml / min, 80 mm Hg) hemodymanic conditions for 24 hours. The identified proteins were related to cell structural function, such as -SMA, CRP1, collagen VI, tropomyosin, myosin, desmin and vimentin. To functional characterization, -SMA and CRP were selected. In rat vein arterialization model, - SMA showed to be decreased during the early stages of arterialization and almost disappeared after 3 days of surgery. Later on, -SMA-positive cells increase reaching similar expression levels of normal jugular vein. The expressiom of CRP3 showed to be predominantly to arterial beds both in human and rat. When vein segment were submitted to arterial hemodynamic condition, it was observed a significant induction of CRP3 expression. Interestingly, the increase of CRP3 is dependent of stretch stimulus in smooth muscle cells while shear stress did not modify its expression in endothelial cells. Collectively, we successfully identified proteins differentially expressed during the vein arterialization by using proteomic technique. -SMA and CRP3 were modified in vein segments exposed to arterial hemodynamic condition and efficiently discriminate smooth muscle cell phenotype. The identified proteins will contribute to the better understanding of the venous arterialization process and may be tested as new therapeutic targets for improving the patency these grafts
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Lee, Hannah Yun Young. "Calcium homeostasis in lens transparency and the involmement of calpains in cataract." Lincoln University, 2006. http://hdl.handle.net/10182/1897.
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The absolute clarity of the lens of the eye is vital in the visual system. The unique structural and physiological properties of the lens are tightly integrated with highly ordered protein content to allow the lens to remain transparent. Consequently, any alteration or disturbance of these highly ordered proteins can affect the optical properties of the lens. In humans, cataracts are the major cause of blindness, yet the exact aetiology of cataract formation (cataractogenesis) is not fully understood. The purpose of the current research was to investigate whether deregulation of the Ca²⁺-dependent enzyme, calpains, following changes in lens Ca²⁺ homeostasis, is a key mechanism leading to undesired cleavage of a number of proteins that are linked with maintaining lens transparency and contributing to cataractogenesis. An ovine lens culture (in vitro) system and the heritable ovine cataract (in vivo) model were used to test the research hypothesis. The Ca²⁺ ionophore, ionomycin, was used to induce a Ca²⁺ overload and in vitro opacification during lens culture. Opacity in the lens was graded by a computer image analysis program. Protein profile (SDS-PAGE, 2-DE and Western detection), calpain activity (casein zymography), lens structure (microscopic view) and cytotoxicity level (LDH leakage assay) were analysed in Ca²⁺-induced opaque lenses. The involvement of calpain during opacification was further examined by applying synthetic exogenous calpain inhibitors to the in vitro system. Two novel exogenous calpain inhibitors were also assessed for their therapeutic potential in preventing the progression of cataracts in the in vivo cataract model by topical administration of the inhibitor direct to the sheep's eye over a 11 week period. HPLC was used to detect the penetration of these compounds into ocular tissues. Sustained Ca²⁺ influx into cultured lenses caused dense opacification. The opacity was characterised by formation of a turbid fraction and cell death in the outer cortex of the ovine lens. There was increased calpain autolysis associated with the progress of opacification, indicating increased calpain activity. Major degradation of the cytoskeletal proteins, spectrin and vimentin, was observed whilst there was limited degradation of the lens structural soluble proteins, crystallins, in response to a Ca²⁺ flux. Lens proteins were protected from degradation by adding synthetic calpain inhibitors to the culture medium. Topical administration of novel anti-calpain molecules failed to retard the progression of cataractogenesis in the ovine inherited cataract model. Further investigation of drug penetration showed that efficacy of inhibitory compounds was limited by permeability of these molecules across the cornea and the ability of the molecules to reach and penetrate into the lens. The ovine lens Ca²⁺-induced opacification (OLCO) model in this thesis has provided a model to understand the role of Ca²⁺ homeostasis in lens transparency. With sustained intracellular Ca²⁺ level, the degradation of cytoskeletal elements is highly correlated with calpain activity. Cataractogenesis is the pathological response to the loss of lens Ca²⁺ homeostasis in this model. The current results support the hypothesis that the deregulation of calpain activity is a trigger for a series of cascading events, leading to death of the cells in the lens.
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Lomasko, Tatiana. "One-hit Stochastic Decline in a Mechanochemical Model of Cytoskeleton-induced Neuron Death." Thesis, 2008. http://hdl.handle.net/1807/16801.
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Much experimental evidence shows that the cytoskeleton is a downstream target and effector during cell death in numerous neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's diseases. However, recent evidence indicates that cytoskeletal dysfunction can also trigger neuronal death, by mechanisms as yet poorly understood. We studied a mathematical model of cytoskeleton-induced neuron death in which assembly control of the neuronal cytoskeleton interacts with both cellular stress levels and cytosolic free radical concentrations to trigger neurodegeneration. This trigger mechanism is further modulated by the presence of cell interactions in the form of a diffusible toxic factor released by dying neurons. We found that, consistent with empirical observations, the model produces one-hit exponential and sigmoid patterns of cell dropout. In all cases, cell dropout is exponential-tailed and described accurately by a gamma distribution. The transition between exponential and sigmoidal is gradual, and determined by a synergetic interaction between the magnitude of fluctuations in cytoskeleton assembly control and by the degree of cell coupling. We concluded that a single mechanism involving neuron interactions and fluctuations in cytoskeleton assembly control is compatible with the experimentally observed range of neuronal attrition kinetics. We also studied the transit of neurons through states intermediate between initial viability and cell death. We found that the stochastic flow of neuron fate, from viability to cell death, self-organizes into two distinct temporal phases. There is a rapid relaxation of the initial neuron population to a more disordered phase that is long-lived, or metastable, with respect to the time scales of change in single cells. Strikingly, cellular egress from this metastable phase follows the one-hit kinetic pattern of exponential decline now established as a principal hallmark of cell death in neurodegenerative disorders. Intermediate state metastability may therefore be an important element in the systems biology of one-hit neurodegeneration. Further, we studied the full spatiotemporal dynamics of death factor pulses released from dying neurons to emphasize the effects of the cell-to-cell coupling strength on neuron death rates. The rate of neuron cell loss monotonically increased with increased diffusion-dependent intercellular communication. Death factor diffusion effects may therefore be important moderators of one-hit neurodegeneration.
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Silva, Marina Isabel Oliveira da. "Dissecting the role of cytoskeleton remodelling in a Drosophila model of TTR-induced neurodegeneration." Dissertação, 2015. https://repositorio-aberto.up.pt/handle/10216/82245.
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Silva, Marina Isabel Oliveira da. "Dissecting the role of cytoskeleton remodelling in a Drosophila model of TTR-induced neurodegeneration." Master's thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/82245.
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Yadav, Preeti. "Studying Neuronal Cytoskeleton Defects and Synaptic Defects in Mouse Model of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy." Doctoral thesis, 2016. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-138093.
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Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature. Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMADie Amyotrophe Lateralsklerose und die spinale Muskelatrophie sind die beiden häufigsten Formen der Motoneuronerkrankungen. Sie sind charakterisiert durch eine Destabilisierung der Axonendigungen, durch Axondegeneration und durch Änderungen im neuronalen Zytoskelett. Eine Anhäufung von Neurofilamenten konnte in einigen neurodegenerativen Erkrankungen beobachtet werden. Der genaue Mechanismus, welcher zu einer Destabilisierung des Axons führt, ist bis heute jedoch unklar. Hiermit zeige ich, dass eine gesteigerte Expression von Neurofilamenten in motorischen Nerven von pmn mutierten Mäusen zu einer Störung der Mikrotubuli – Dynamik führt. Ein Neurofilamentabbau durch Nefl knockout steigert die Anzahl an neu wachsenden Mikrotubuli in pmn mutierten Motoneuronen und führt zu erneutem Axonwachstum. Dieser Effekt wird durch eine Interaktion zwischen dem Neurofilament und dem Stathmin Komplex vermittelt. Ein Abbau des Neurofilaments führt zu einer Erhöhung der Stathmin-Stat3 Interaktion und zu einer Stabilisierung der Mikrotubuli. Demzufolge ist die Versorgung der Axone verbessert und die pmn mutierten Mäuse überleben länger. Wir vermuten, dass dieser Mechanismus auch für andere neurodegenerative Erkrankungen, bei denen Neurofilamentanhäufung ein dominantes Merkmal ist, relevant sein könnte. Des Weiteren studierte ich mit Hilfe des Smn-/-;SMN2 Mausmodels, den molekularen Mechanismus der sich hinter dem Synapsenverlust bei SMA verbirgt. SMA ist charakterisiert durch eine Degeneration der unteren -Motoneuronen im Rückenmark. Es ist jedoch unklar, wie ein Verlust des ubiquitär exprimierten SMN Proteins zu einer MN-spezifischen Degeneration führt. Smn ist involviert in den Prozess des pre-mRNA Splicing (Pellizzoni, Kataoka et al. 1998) und ein Verlust des Proteins führt zu einer Störung des Splicing. Eine Denervierung der motorischen Endplatte führt zu einer Neurodegeneration in SMA. Es gibt jedoch keinen Hinweis auf eine kausale Verbindung zwischen anomalem Splicen von stromabwärts gelegenen Transkripten des Smn und einer Reduktion präsynaptischer Axone, wie man es bei SMA beobachten kann. In dieser Studie konnten wir beobachten, dass Expression und Splicing von Nrxn2, welches für ein präsynaptisches Protein kodiert, in SMA Mäusen betroffen ist und dass Nrxn2 ein Kandidat sein könnte, der eine Verbindung zwischen Störungen im Splice Prozess und synaptischen Motoneuron-Defekten in der SMA herstellen könnte
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Saal, Kim Ann. "Modulation of the ROCK pathway in models of Parkinson´s disease." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0028-8665-9.
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Petr, Martin. "Jaderný myosin 1 a jeho role v regulaci tenze cytoplazmatické membrány." Master's thesis, 2014. http://www.nusl.cz/ntk/nusl-332401.
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Myosin 1c (Myo1c) is a molecular motor involved in regulation of tension-gated ion channels, exocytosis, endocytosis, motility and other membrane-related events. Moreover, it acts as a dynamic linker between the cell membrane and cortical actin network, contributing to the maintenance of plasma membrane tension. In contrast, nuclear myosin 1 (NM1), an isoform of Myo1c, has been described only in the nucleus where it participates in various nuclear processes, including transcription or chromatin remodeling. However, although traditionally regarded as exclusively cytoplasmic or nuclear, all myosin 1c isoforms participate in nuclear functions and they are present in the cytoplasm as well. The main focus of this study was to characterize the functional significance of NM1 in the cytoplasm. We have found that NM1 localizes to plasma membrane and shows a uniform punctuated distribution with a high concentration at the cell periphery. Moreover, atomic force microscopy measurements of mouse NM1 KO fibroblasts revealed a significant increase in an overall plasma membrane elasticity in comparison to WT cells, indicating a disruption in the regulation of plasma membrane tension caused by the loss of NM1. Since a higher membrane elasticity and deformability is a characteristic marker of cancer cells,...
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García, Arguinzonis Maísa Inés. "Analysis of signal transduction pathways and the cytoskeleton in VASP-deficient cell lines and mouse models." Doctoral thesis, 2003. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-6195.
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The mammalian Vasodilator Stimulated Phosphoprotein (VASP) is a founding member of the Ena/VASP family of proteins that includes Drosophila Enabled (ena), the mammalian Ena homologue (Mena) and the Ena-VASP-like protein (Evl). VASP was initially discovered and characterized as a substrate for cGMP- and cAMP-dependent protein kinases (cGKs and cAKs). Ena/VASP proteins are involved in Actin-filament formation, plasma membrane protrusion, acceleration of Actin-based motility of Listeria and the establishment of cell-cell adhesion. Moreover, Ena/VASP proteins have been implicated as inhibitory factors in repulsive axon guidance and inhibition of plasma membrane activity and random motility in fibroblast. In order to study the physiological function of VASP, VASP-deficient mice had been generated in the laboratory by homologous recombination. VASP-/- mice showed hyperplasia of megakaryocytes in the bone marrow and spleen and a two-fold increase in thrombin- and collagen-induced platelet activation. To further investigate the cellular function of VASP, I established cardiac fibroblast cell lines derived from both wild type and VASP-/- mice. Both cell lines presented similar growth rates and normal contact dependent-growth inhibition but showed differences in morphology, migration and adhesion. Adherent VASP-/- cells, despite normal Mena and Evl expression levels, were highly spread. VASP-/- cells covered about twice the substrate surface area as wild type cells, while the cell volumes were unchanged. This shape difference suggests that VASP is involved in the regulation of spreading. Since the small GTPases Rac and Cdc 42 and their effector p21-activated kinase (Pak) are key regulators of lamellipodia formation and cell spreading, I analyzed this signalling pathway in VASP-/- cells stimulated with Platelet Derived Growth Factor-BB (PDGF-BB) or fetal calf serum. In wild type cells Rac and Pak were rapidly and transiently activated by PDGF or serum; however, in the absence of VASP both Rac and Pak activation was dramatically prolonged. The Rac/Pak pathway is known to play an essential role in cell motility. VASP deficient cells showed compromised migration and reorientation in a wound healing assay, probably due to enhanced Rac activity. The spreading phenotype, compromised migration and the effect observed on the Rac and Pak activities were reverted in VASP-/- cells stably transfected with full lenght human VASP, indicating a VASP dependent modulation of the Rac/Pak pathway and Rac/Pak regulated processes. Moreover, adhesion and detachment of VASP-deficient cells were significantly slower when compared to wild type cells. Preincubation of VASP+/+ cells with a cGMP analog accelerated adhesion. This acceleration did not take place in the VASP-/- cells, suggesting a VASP dependent effect. The second part of this work focused on VASP function in platelets. On the one hand I investigated the possibility of VASP-dependent Rac regulation in mouse platelets. Murine platelets are a good model for studying Rac regulation since they express high levels of VASP but not Mena/Evl and since VASP-deficient platelets show an increased platelet activation. Rac was activated by platelet agonists which was inhibited by preincubation with cGMP and cAMP analogs. Initial results which need to be extended showed that the cGMPcaused inhibition of Rac activation was VASP-dependent. Finally, in vivo platelet adhesion (platelet-vessel wall interactions) was studied using VASP-deficient mice. These studies demonstrated in-vivo that VASP down regulates platelet adhesion to the vascular wall under both physiological and pathophysiological conditionsDas Säugerprotein Vasodilator Stimulated Phosphoprotein (VASP) ist ein Gründungsmitglied der Ena/VASP Proteinfamilie, die das Drosophila Enabled (ena), das homologe Säugerprotein ena (Mena) und das Ena-VASP-like Protein (Evl) einschließt. VASP wurde ursprünglich als ein Substrat von cGMP- und cAMP abhängigen Proteinkinasen (cGKs und cAKs) entdeckt und charakterisiert. Ena/VASP Proteine sind bei der Polymerisation von Aktinfilamenten, bei der Protrusion von Plasmamembranen, der Beschleunigung von Aktinbasierter Beweglichkeit von Listerien und bei der Ausbildung von Zell-Zell-Adhäsionen beteiligt. Außerdem wurde gezeigt, dass Ena/VASP-Proteine hemmende Faktoren bei der repulsiven Axonführung sind und sowohl die Plasmamembranaktivität als auch die ungerichtete Fibroblastenbeweglichkeit hemmen. Um die physiologische Funktion von VASP zu untersuchen, wurden VASP-defiziente Mäuse im Labor durch homologe Rekombination generiert. VASP-/- Mäuse zeigten eine Hyperplasie der Megakaryozyten im Knochenmark und in der Milz sowie eine zweifache Erhöhung der durch Thrombin und Kollagen induzierten Plättchen-Aktivierung. Um die zelluläre Funktion von VASP weiter aufzuklären, etablierte ich kardiale Fibroblasten- Zelllinien sowohl von Wildtyp als auch von VASP-/- Mäusen. Beide Zelllinien zeigten gleiche Wachstumsraten und eine normale, kontaktabhängige Wachstumshemmung, hatten aber Unterschiede in ihrer Morphologie, Wanderung und Adhäsion. Adhärente VASP-/- Zellen waren trotz normaler Mena und Evl Expression stark ausgebreitet. VASP-/- Zellen bedeckten eine ungefähr zweimal so große Substratoberfläche wie Wildtyp-Zellen, während das Zellvolumen unverändert war. Diese Formunterschiede lassen vermuten, dass VASP bei der Regulation der Ausbreitung involviert ist. Da die kleinen GTPasen Rac und Cdc 42 und ihr Effektorsystem p21-aktivierte Kinase (Pak) Schlüsselregulatoren der Lamellipodienformierung und der Zellausdehnung sind, untersuchte ich diesen Signalweg in VASP-/- Zellen, die mit Platelet Derived Growth Factor-BB (PDGF-BB) oder fetalem Kälberserum stimuliert wurden. In Wildtypzellen wurden Rac und Pak schnell und transient durch PDGF oder Serum aktiviert, in der Abwesenheit von VASP war die Aktivierung von Rac und Pak jedoch dramatisch verlängert. Der Rac/Pak Signalweg ist dafür bekannt, dass er eine essentielle Rolle bei der Zellbeweglichkeit spielt. VASP defiziente Zellen zeigten, wahrscheinlich wegen der erhöhten Rac Aktivität, eine veränderte Wanderung und Reorientierung in einem Wundheilungs-Versuch. Der ausgebreitete Phänotyp, die veränderte Wanderung und die beobachteten Effekte bei den Rac und Pak Aktivitäten wurden in VASP-/- Zellen, die stabil mit humanem VASP transfiziert wurden, normalisiert, was eine VASP abhängige Steuerung des Rac/Pak Signalwegs und der Rac/Pak regulierten Prozesse vermuten läßt. Weiterhin waren die Adhäsion und die Ablösung von VASP-defizienten Zellen signifikant langsamer als in den Wildtyp-Zellen. Die Vorinkubation von VASP+/+ Zellen mit einem cGMP-Analog beschleunigte die Adhäsion. Diese Beschleunigung fand in VASP-/- Zellen nicht statt, was einen VASP-abhängigen Effekt vermuten läßt. Der zweite Teil dieser Arbeit konzentrierte sich auf die VASP Funktion in Thrombozyten. Einerseits untersuchte ich die VASP-abhängige Regulation von Rac in murinen Thrombozyten. Diese sind dafür besonderes gut geeignet, da sie VASP aber nicht Mena/Evl exprimieren und da VASP-defiziente Thrombozyten verstärkt aktiviert werden. Rac wurde durch Thrombozyten-Agonisten aktiviert, was durch eine Präinkubation mit cGMP- und cAMP-Analoga gehemmt wurde. Erste Ergebnisse, die noch einer weiteren Bestätigung bedürfen, zeigten, daß die cGMP-vermittelte Hemmung der Rac-Aktivierung VASP-abhängig war. Abschließend wurde auch die in-vivo Plättchen-Adhäsion (Thrombozyten-Gefäßwand- Interaktion) unter Einsatz von VASP-defizienten Mäusen untersucht. Diese Ergebnisse zeigten für in-vivo-Bedingungen, daß VASP die Thrombozyten-Adhäsion an die Gefäßwand sowohl unter physiologischen als auch pathophysiologischen Bedingungen unterdrückt
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Roth, Alexander [Verfasser]. "Nano-mechanics of biomimetic models of the actin based cytoskeleton : from single molecules to complex composite structures / Alexander Roth." 2004. http://d-nb.info/972309322/34.
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García, Arguinzonis Maísa Inés [Verfasser]. "Analysis of signal transduction pathways and the cytoskeleton in VASP-deficient cell lines and mouse models / vorgelegt von Maísa Inés García Arguinzonis." 2003. http://d-nb.info/969694741/34.
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Tsitkov, Stanislav. "Emergent Properties of Biomolecular Organization." Thesis, 2021. https://doi.org/10.7916/d8-dmje-n287.
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The organization of molecules within a cell is central to cellular processes ranging from metabolism and damage repair to migration and replication. Uncovering the emergent properties of this biomolecular organization can improve our understanding of how organisms function and reveal ways to repurpose their components outside of the cell. This dissertation focuses on the role of organization in two widely studied systems: enzyme cascades and active cytoskeletal filaments.Part I of this dissertation studies the emergent properties of the spatial organization of enzyme cascades. Enzyme cascades consist of a series of enzymes that catalyze sequential reactions: the product of one enzyme is the substrate of a subsequent enzyme. Enzyme cascades are a fundamental component of cellular reaction pathways, and spatial organization of the cascading enzymes is often essential to their function. For example, cascading enzymes assembled into multi-enzyme complexes can protect unstable cascade intermediates from the environment by forming tunnels between active sites.
We use mathematical modeling to investigate the role of spatial organization in three specific systems. First, we examine enzyme cascade reactions occurring in multi-enzyme complexes where active sites are connected by tunnels. Using stochastic simulations and theoretical results from queueing theory, we demonstrate that the fluctuations arising from the small number of molecules involved can cause non-negligible disruptions to cascade throughput. Second, we develop a set of design principles for a compartmentalized cascade reaction with an unstable intermediate and show that there exists a critical kinetics-dependent threshold at which compartments become useful. Third, we investigate enzyme cascades immobilized on a synthetic DNA origami scaffold and show that the scaffold can create a favorable microenvironment for catalysis.
Part II of this dissertation focuses on the organization of active cytoskeletal filaments. Many mechanical processes of a cell, such as cell division, cell migration, and intracellular transport, are driven by the ATP-fueled motion of motor proteins (kinesin, dynein, or myosin) along cytoskeletal filaments (microtubules or actin filaments). Over the past two decades, researchers have been repurposing motor protein-driven propulsion outside of the cell to create systems where cytoskeletal filaments glide on surfaces coated with motor proteins. The study of these systems not only elucidates the mechanisms of force production within the cell, but also opens new avenues for applications ranging from molecular detection to computation.
We examine how microtubules gliding on surfaces coated with kinesin motor proteins can generate collective behavior in response to mutualistic interactions between the filaments and motors, thereby maximizing the utilization of system components and production. To this end, we used a microtubule-kinesin system where motors reversibly bind to the surface. In experiments, microtubules gliding on these reversibly bound motors were unable to cross each other and at high enough densities began to align and form long, dense bundles. The kinesin motors accumulated in trails surrounding the microtubule bundles and participated in microtubule transport.
In conclusion, our study of the emergent properties of the spatial organization of enzyme cascades and the mutualistic interactions within active systems of motor proteins and cytoskeletal filaments provides insight into both how these systems function within cells and how they can be repurposed outside of them.