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Huang, Wei. "Polycystin-1 and Bone Mechanotransduction." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10279.
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Bone mechanotransduction is a fundamental process underlying the remarkable ability of bones to perceive surrounding physical cues and adapt their mass, structure and overall strength to their mechanical environment. Therefore, it is central to many aspects of bone biology and disease. The key to a mechanistic understanding of this process lies in better knowledge of critical signaling molecules that relay the mechanical information inside bone cells. In this thesis, we investigate the role of polycystin-1 (PC1), a proposed fluid flow sensor in kidney epithelial cells, in transducing mechanical signals in bone cells. Loss of PC1 in osteoblast lineage cells using osterix-Cre (Osx-Cre) causes mild osteopenia in mice with reduced calvarial and trabecular bone formation, and markedly attenuated anabolic bone formation responses to in vivo mechanical loading of long bones. Loss of PC1 in limb bud mesenchymal cells at an early stage causes mildly increased bone formation and a tendency to exhibit enhanced anabolic responses to in vivo mechanical loading of long bones. These findings suggest that PC1 has a complex role in different bone cell populations both during development and in bone mechanotransduction. PC1 has been shown to mediate tensile force-induced proliferation in osteoprogenitor cells (OPCs) in craniofacial sutures. To investigate the role of PC1 in periosteal osteoprogenitor mechanotransduction, we establish a shockwave-induced periosteum mechanical stress model. Shockwave treatment triggers dramatically increased cell proliferation, potent osteogenic activity, and intramembranous new bone formation in the periosteum. We show that loss of PC1 in periosteal cells (Prx1-Cre) does not affect periosteal mechanoresponsiveness to shockwave mechanical stress. These findings suggest that the role of PC1 in OPCs is likely tissue or force dependent. Fluid shear stress (FSS) in the lacunar-canalicular network is a major force element that osteocytes experience and respond to in vivo. To study the role of PC1 in FSS-mediated osteocyte/osteoblast mechanotransduction, we establish a laminar FSS system with custom-made flow chambers and a PC1-deficient osteoblast cell line. Our data show that PC1 is essential for regulation of FSS-induced initial \(Ca^{2+}\) influx in osteoblasts and mediates osteoblast FSS responses in a COX-2 and AP-1 independent manner.
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Chronopoulos, Antonios. "Mechanotransduction in health and disease." Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/56622.
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Physical forces regulate cellular behaviour and function during all stages of life. Mechanotransduction, the process by which cells convert mechanical stimuli into biochemical signalling events is central to a number of physiological and pathological processes. The first part of this work focuses on the effect of retinoid therapy on the mechanobiology of pancreatic cancer. Pancreatic cancer is characterised by a persistent activation of stromal fibroblasts, known as pancreatic stellate cells (PSCs), which can perturb the biomechanical homeostasis of the tumour microenvironment to favour tumour invasion. Using biophysical and biological techniques, we report that all-trans retinoic acid (ATRA), an active vitamin A metabolite restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-mediated downregulation of actomyosin (MLC-2) contracility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. We thus suggest that ATRA may serve as a stroma reprogramming agent for the treatment of pancreatic cancer. In the second part of this work, we focus on syndecan-4 (Syn-4) - a ubiquitous transmembrane proteoglycan receptor. We identify Syn-4 as a cellular mechanotransducer that tunes cell mechanics by eliciting a global mechanosignalling response. We outline a mechanotransduction model whereby localised tension on Syn-4 triggers a synergistic cell-wide activation of β1 integrins, in a PI3K-dependent manner, to subsequently activate the RhoA pathway and induce adaptive cell stiffening. Furthermore, syndecan-4 mediated mechanosensing is required for YAP activation and downstream changes in gene expression. We propose that this newly identified mechanotransductive ability of Syn-4 should have direct implications for the field of mechanobiology.
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Pucker, Andrew David. "Mechanotransduction in the Ciliary Muscle." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1460647729.
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Malone, Amanda Michelle Dolphin. "Mechanotransduction mechanisms in bone cells /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Kuck, Jan L. "Mechanotransduction in red blood cells." Thesis, Griffith University, 2023. http://hdl.handle.net/10072/421118.
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Red blood cells (RBC), the oxygen-carriers within blood, eject their nuclei and other organelles to optimise cellular mechanics for gas exchange in capillary networks. Lack of organelles, however, strictly limits circulatory longevity of these cells, due to the inability to repair damaged cellular components. Given the turnover of RBC, the cell population within blood is inherently heterogenous, comprising RBC across the whole spectrum of in vivo age. Moreover, surrender of translational capacity restricts cellular signalling within RBC to modifications of existing proteins and/or flux of ions through membrane-embedded channels,
rather than alterations in protein expression.
The traversal of the cardiovascular system for the purpose of gas exchange exposes RBC to varying mechanical forces. Exposure to mechanical force physically deforms the RBC membrane, which, upon cessation of force exposure, readopts its native bi-concave disc chape. Novel observations support that these mechanical forces also activate biochemical pathways that may acutely and transiently alter RBC mechanics. The molecular machinery facilitating these mechanotransduction processes in RBC, however, is largely undescribed.
The aim of the present body of work was thus to elucidate i. mechanotransductive pathways in mature, enucleated RBC; ii. the contribution of mechanically-activated signalling to the regulation of RBC mechanics; and iii. the impact of sub-populations of RBC with abnormal mechanical properties on blood fluid behaviour.
The salient findings of the present dissertation support the presence of a relevant post-translational signalling network in circulating, enucleated RBC, some of which is sensitive to activation by mechanical forces. The cation channel Piezo1 appears to be a central mechanism of ‘force sensing’ in these cells. That is, opening of Piezo1 in response to mechanical force facilitates influx of calciumions, which regulate RBC mechanics via diverse mechanisms, including acute shifts in cell volume, selective removal of susceptible cells within a given RBC population, and initiation of nitric oxide production.
Collectively, the herein presented results enhance the current understanding of fundamental RBC physiology by elucidating hitherto unrecognised signalling pathways. Given the demonstrated relevance of these processes to the regulation of RBC mechanical properties, which determine blood fluid properties and effective gas exchange, components of mechanically-activated signalling in these cells may provide novel therapeutic targets. Moreover, adverse complications arising in scenarios where blood is exposed to mechanical forces far exceeding those investigated here, for example during transit of mechanical circulatory support devices or dialysis machines, may be linked to overactivation of mechanically-sensitive signalling.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sci & Soc Wrk
Griffith Health
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Bays, Jennifer McQuown. "Mechanisms of E-cadherin mechanotransduction." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5711.
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Cells experience force throughout their lifetimes. Cells sense force via adhesion receptors, such as the cadherins, which anchor cells to neighboring cells, and integrins, which tether cells to the underlying matrix. Both adhesion receptors respond to force by activating signaling pathways inside the cell. These pathways trigger growth of adhesion complexes and reinforcement of the cytoskeleton in order to resist the force. These activities are energetically costly. Thus, mechanisms are needed to couple force transmission and energy production.
In this thesis, I demonstrated force on cadherins activates a master regulator of energy homeostasis known as AMP-activated kinase (AMPK). In response to force, AMPK was recruited to the cadherins. AMPK promoted growth of the adhesion complex and cytoskeletal reinforcement by stimulating energy production in the cell. Additionally, AMPK formed a complex with vinculin—a protein that is recruited to both cadherins and integrins. I observed AMPK activation of vinculin dictates whether vinculin joins the cadherin complex. Conversely, AMPK activation has no bearing on vinculin recruitment to integrins.
This work provides three novel contributions: (1) the first link between energy production and force transmission, (2) a molecular mechanism for how AMPK increases adhesion complex growth, and (3) an explanation for how vinculin discriminates between cadherins and integrins.
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Bouclet, Adrien. "Evolutionary implication of mechanotransduction in development." Phd thesis, Université René Descartes - Paris V, 2014. http://tel.archives-ouvertes.fr/tel-01071238.
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In this thesis, I first focused on the testing of the hypothesis of the mechanotransductive activation of the apical accumulation of Myosin-II (Myo-II) that leads to Drosophila embryos mesoderm invagination, in response to the active cell apex pulsations preceding gastrulation in the mesoderm. This hypothesis was proposed on the basis of previous experiments realized in my host lab, having consisted in the rescue of mesoderm invagination in pulsation and invagination defective mutants, in response to a simple mechanical indent of the mesoderm. Here I demonstrated quantitatively the plausibility of such mechanical trigger of the active apical accumulation of Myo-II leading to subsequent mesoderm invagination, in response to the mechanical strains developed by the endogenous pulsative movements of mesoderm cell apexes, in silico. In a second part, I tested experimentally the role of the mechanical strains developed by the very first morphogenetic movements of zebrafish (Danio rerio) and Drosophila embryos, in the early specification of mesoderm cells identity. Specifically, to test this hypothesis, I developed magnetic biophysical tools to mimic the epiboly morphogenetic movements in epiboly defective zebrafish embryos. We found the beta-catenin (B-cat) Y667 phosphorylation as the common mechano-transductive pathway involved in earliest mesoderm genes expression notail and twist respectively, in response to the very first morphogenetic movements of embryogenesis in both species, epiboly and mesoderm invagination, respectively. This allowed to suggest such mechanotransduction pathway as conserved from the last common ancestor of both species, namely the last common ancestor of bilaterians, therefore possibly involved in the origins of mesoderm emergence in the ancestor, which represents a currently important opened question of evo-devo. In a third part, I developed experiments of mechanical indent of Drosophila embryos germ cells, and demonstrated the production of generational heritable developmental defects induced on at least 3 generations. These experiments suggest accidental mechanical perturbation of germ cells as a putative new motor mode of heritable modulations in the genetic developmental program of embryogenesis, with the molecular mechanism underlying such transmission being currently in progress.
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Aragona, Mariaceleste. "Role of YAP/TAZ in Mechanotransduction." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422159.
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Cells perceive their microenvironment not only through soluble signals but also in term of physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, including growth, differentiation and cancer malignant progression; but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP and TAZ as nuclear relays of mechanical signals exerted by ECM rigidity and cell-shape. This regulation requires Rho activity and tension of the acto-myosin cytoskeleton but is independent from the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behavior. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.Le cellule percepiscono il loro microambiente non solo attraverso molecole segnale e fattori solubili ma anche attraverso stimoli fisici e meccanici. Le cellule traducono questi stimoli in segnali biochimici attraverso un processo definito meccanotrasduzione, in grado di regolare numerosi aspetti del comportamento cellulare, tra cui crescita, differenziamento e progressione tumorale. Tuttavia, non è ancora noto come la percezione dei segnali meccanici si traduca nell’attivazione di specifici fattori di trascrizione a livello nucleare. Questo lavoro individua YAP (Yes-associated protein), e TAZ (transcriptional coactivator with PDZ-binding motif, anche noto come WWTR1), omologhi di Yorkie in Drosophila, quali fattori di trascrizione in grado di rispondere ai segnali meccanici generati dalla rigidità della matrice extracellulare e dalla forma propria di ogni singola cellula. Questa regolazione richiede l’attivazione della GTPase Rho e la presenza di un citoscheletro di actina contrattile, ma è indipendente dall’attività della via di segnale delle chinasi Hippo e LATS. Non solo YAP/TAZ vengono regolati da segnali meccanici, ma sono anche funzionalmente richiesti per il differenziamento delle cellule staminali mesenchimali indotto dalla stiffness (elasticità o rigidità) della matrice e per la sopravvivenza delle cellule endoteliali regolata dalla geometria cellulare. In maniera complementare, l’espressione di una forma attivata di YAP domina sull’azione degli stimoli fisici nel determinare il destino cellulare. Queste scoperte identificano YAP/TAZ come sensori e mediatori degli stimoli meccanici indotti dal microambinete cellulare.
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Dutour, Provenzano Gaëlle. "Role of intermediate filaments in mechanotransduction." Electronic Thesis or Diss., Sorbonne université, 2021. http://www.theses.fr/2021SORUS364.
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Les cellules s'adaptent en permanence à leur microenvironnement. En particulier, elles modifient leur morphologie, leur croissance, leur division et leur motilité en fonction des propriétés biochimiques et physiques de la matrice extracellulaire (MEC). Elles sont équipées de structures adhésives appelées plaques d’adhérences, permettant aux cellules d'interagir avec les protéines de la MEC via les protéines transmembranaires appelées intégrines et de détecter la nature et la rigidité de la MEC. Le signal est transduit par les protéines des plaques d’adhérences et résulte par exemple en une modification de la tension mécanique induite par l'acto-myosine. Les voies de signalisation en aval peuvent également atteindre le noyau. L'expression des gènes peut alors être modifiée, ce qui peut en retour affecter la composition des plaques d’adhérences et de la MEC pour une réponse cellulaire adaptative. Nous avons émis l'hypothèse qu'en plus des voies de signalisation, un couplage mécanique direct entre les événements se produisant à la périphérie de la cellule et le noyau pourrait participer à la transmission de signaux mécaniques. Bien que les filaments intermédiaires (FIs) aient des propriétés mécaniques extrêmement intéressantes et résistent à des charges de tension élevées, leur implication dans les voies de mécanotransduction est encore mal connue. En utilisant l'astrocyte comme modèle, en raison de sa combinaison spécifique de FIs : vimentine, GFAP, nestine et synémine, nous avons d'abord étudié l'effet de la rigidité du substrat sur la morphologie, la structure et les fonctions du noyau, ainsi que sur l'organisation des FIs autour du noyau. Nous avons ensuite étudié l’impact de l’absence de FI les changements nucléaires observés en réponse à la rigidité du substrat. En utilisant une combinaison de techniques de microfabrication, de méthodes biochimiques et de microscopie, nous avons montré que la rigidité du substrat affecte la forme, le volume du noyau, la structure de la chromatine et le recrutement des facteurs de transcriptions (YAP). Nos résultats suggèrent que les FI forment une structure en forme de cage autour du noyau d'une manière dépendante de la rigidité : un substrat plus rigide induit la formation d’une cage de vimentine et de nestine. Cette interaction avec le noyau pourrait expliquer les modifications nucléaires observées en réponse à la rigidité du substrat. Au total, les résultats obtenus au cours de notre étude permettent de mieux comprendre le rôle des filaments intermédiaires dans les réponses nucléaires aux propriétés mécaniques du substratCells continuously adapt to their microenvironment. In particular, they modulate their morphology, growth, division, and motility according to the biochemical and physical properties of the extracellular matrix (ECM). Cells are equipped with adhesive structures called FAs, allowing them to interact with ECM proteins through the core transmembrane proteins called integrins and to sense the nature and the rigidity of the ECM. This information are transduced by FA proteins and lead, for instance, to changes in acto-myosin-mediated mechanical tension. Downstream signalling pathways also reach the nucleus; gene expression is then modified and may, in return, affect the composition of FAs or of the ECM proteins for adaptative cell response. Here, we hypothesized that, in addition to signalling pathways, a direct mechanical coupling between the events occurring at the cell periphery and the nucleus may participate in the transmission of mechanical cues and the regulation of nuclear functions. Although intermediate filaments (IFs) have extremely interesting mechanical properties and resist high tension load, their involvement in mechanotransduction pathways remains elusive. Using astrocyte as a model, due to its specific combination of IFs: vimentin, GFAP, nestin, and synemin, we studied first the effect of substrate rigidity on the nucleus morphology and function, and on the organisation of IFs around the nucleus. Then, we investigated the role of IFs in rigidity-induced nuclear changes. Using a combination of microfabrication techniques, biochemical and microscopy methods, we showed that substrate rigidity affects the nucleus shape, volume, and structure of the chromatin and the recruitment of transcription factor (YAP) and IFs are mediating these changes. Our results suggest that IFs form a cage-like structure around the nucleus in a rigidity-dependent manner: stiffer substrates promote the formation of a cage of vimentin and nestin. In the absence of IFs, the nuclear changes induced by rigidity are different than with IF. The nucleus increases its size in soft substrate, together with an increase in tension measured by YAP localising in the nucleus. The structure of the chromatin is changed. Altogether, the results obtained during our investigation give a better understanding of the role of intermediate filaments in the mechanosensitive nuclear responses
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Huesa, Carmen. "Mechanotransduction in cells of the osteoblast lineage." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25468.
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Pardo, Pastor Carlos 1989. "Piezo ion channels in cancer cell mechanotransduction." Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/664209.
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The mechanical dependence of transformation and metastasis is an emerging field, but the role of mechanosensitive channels has been largely omitted. This thesis focuses on the roles played by the mechanosensitive ion channels Piezo1 and Piezo2 in the transduction of mechanical stimuli (confinement, adhesion, substrate rigidity, adhesive ligand concentration) by cancer cells.
In a first chapter, we show that confinement triggers Piezo1-mediated calcium entry. This activates phosphodiesterase 1, reducing cAMP levels and, consequently, PKARac1 activity, relieving Myosin II from its inhibition. We also find a parallel, direct activation of Myosin II by confinement. As a combined result, cells stiffen and optimize their adhesion-free migration mode, usually responsible for in vivo migration during metastatic invasion. Piezo1 knockdown supresses confinement-induced calcium entry and impairs the underlying circuitry in ovarian epithelial (CHO) or melanoma (A375) cells. As a result, siPiezo1 cells show reduced migratory capacity under confinement.
In the second chapter, we discover an essential role for Piezo2 as a transducer of environmental mechanical cues into RhoA activation to modulate the mechanobiological responses of MDA-MB-231-BrM2 brain metastatic breast cancer cells. Piezo2 knockdown disturbs stress fibre formation, adhesion orientation, force transmission and nuclear accumulation of the malignant co-transcriptional activator YAP, and this is phenocopied by extracellular calcium suppression. Promoting Actin polymerization with jasplakinolide or by over-expressing constitutively active forms of Rho or mDia1 restores stress fibres and nuclear YAP accumulation. In addition, Piezo2 knockdown disrupts several pro-metastatic functions: cell proliferation, migration, invadopodia formation, extracellular matrix degradation, and secretion of SERPINB2, a protein needed for protecting invasive cells from brain parenchymal defence mechanisms.
The works presented in this thesis unveil important roles for Piezo channels as a first line of mechanical input detectors in distinct cells. These discoveries are relevant for several fields, e.g. cancer research, and highlight the importance of ion channels as transducers of environmental stimuli.La dependència mecànica de la transformació i la metàstasi és un camp d’estudi / de recerca emergent, però el paper que hi juguen els canals iònics mecanosensibles s’ha omès fins ara. Aquesta tesi se centra en els rols dels canals Piezo1 i Piezo2 en la transducció d’estímuls mecànics per cèl·lules canceroses, com ara confinament, adhesió, rigidesa del substrat, concentració de lligands adhesius. En un primer capítol, mostrem que el confinament dispara l’entrada de calci per mitjà de Piezo1. Això activa la fosfodiesterasa 1, que redueix els nivells d’AMPc i, en conseqüència, l’activitat PKARac1, que deixen d’inhibir Miosina II. També trobem una activació paral·lela de Miosina II directament per confinament. Com a resultat final, les cèl·lules guanyen rigidesa i optimitzen el seu mode migratori independent d’adhesions, que és el preponderant in vivo durant la invasió metastàtica. Reduir els nivells de Piezo1 suprimeix l’entrada de calci induïda per confinament i desactiva el circuit subjacent en cèl·lules ovàriques epitelials (CHO) i de melanoma (A375). Això minva la capacitat migratòria de les cèl·lules siPiezo1. En un segon capítol, descobrim un rol essencial per a Piezo2 com a activador de RhoA en resposta a estímuls mecànics. Això modula les respostes mecanobiològiques de les cèl·lules MDA-MB-231-BrM2, de càncer de mama metastàtic a cervell. La reducció dels nivells de Piezo2 destorba la formació de fibres d’estrès, l’orientació de les adhesions, la transmissió de forces i l’acumulació nuclear del regulador transcripcional prometastàtic YAP. Suprimir el calci extracel·lular fenocòpia aquests resultats. Promoure la polimerització d’Actina amb jasplaquinolida o mer mitjà de la sobreexpressió de formes constitutivament actives de RhoA o mDia1 restableix les fibres d’estrès i l’acumulació nuclear de YAP. A més, la reducció de Piezo2 suspèn diverses funcions prometastàtiques: proliferació cel·lular, migració, formació d’invadopodis, degradació de la matriu extracel·lular i secreció de SERPINB2, una proteïna necessària per protegir les cèl·lules invasores dels mecanismes de defensa del parènquima cerebral. Els treballs presentats en aquesta tesi desvelen rols importants pels canals Piezo com a una primera línia de detectors d’estímuls mecànics en diferents tipus cel·lulars. Aquests descobriments són rellevants per a diversos àmbits, com ara la recerca en càncer, i remarquen la importància dels canals iònics com a transductors d’estímuls ambientals.
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Al-Rekabi, Zeinab. "Investigating Mechanotransduction and Mechanosensitivity in Mammalian Cells." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30256.
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Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.
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Lammerding, Jan 1974. "Quantitative analysis of subcellular biomechanics and mechanotransduction." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/18039.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references.
Biological cells such as endothelial or muscle cells respond to mechanical stimulation with activation of specific intracellular and extracellular signaling pathways and cytoskeletal remodeling, a process termed mechanotransduction. Intracellular mechanosensors are thought to be activated by conformational changes induced by local cellular deformations. Since these mechanosensors have been speculated to be located in several cellular domains including the cell membrane, the cytoskeleton, and the nucleus, it is necessary to achieve a detailed understanding of subcellular mechanics. In this work, we present novel methods to independently quantify cytoskeletal displacements, mechanical coupling between the cytoskeleton and the extracellular matrix, and nuclear mechanics based on high resolution tracking of cellular structures and receptor bound magnetic beads in response to applied strain or microscopic forces. These methods were applied to study the effects of several human disease associated mutations on subcellular mechanics and to examine the interaction between known protein function and specific changes in cellular mechanical properties and mechanotransduction pathways. Initial experiments were targeted to the role of membrane adhesion receptors. Experiments with cells expressing a mutant form of the integrin-associated molecule tetraspanin CD151 revealed that CD151 plays a key role in selectively strengthening α6βl integrin-mediated adhesion to laminin-1. We then studied cytoplasmic behavior using cells from mice with an αB-Crystallin mutation (R120G) that causes desmin-related myopathy. These studies showed impaired passive cytoskeletal mechanics in adult mouse cardiac myocytes. Finally, we studied cells deficient in the nuclear envelope
(cont.) protein lamin A/C and showed that lamin A/C deficient cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain, suggesting that the tissue specific effects observed in laminopathies such as Emery-Dreifuss muscular dystrophy or Hutchinson-Gilford progeria may arise from varying degrees of impaired nuclear mechanics and transcriptional regulation. In conclusion, our methods provide new and valuable tools to examine the role of subcellular biomechanics on mechanotransduction in normal and mutant cells, leading to improved understanding of disease mechanisms associated with altered cell mechanics.
by Jan Lammerding.
Ph.D.
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Pitts-Yushchenko, Svetlana. "Mechanism of mechanotransduction in the Pacinian corpuscle." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/15429.
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Touch perception is important in most living organisms and extremely sensitive detection systems have evolved to meet this need. Pacinian corpuscles (PCs) are primary mechanoreceptors. In the human, they are found in the skin (where they act as touch receptors), in the joints, in muscles and in many organs (where they act as motion sensors). The purpose of the work described in this thesis is to investigate how the performance of the PC is achieved, with reference to structure, mechanical properties and possible transduction mechanisms. PCs were obtained from the equine hoof and their distribution and clustering were investigated. Corpuscles were located in the frog area of the hoof (the digital cushion); they were found to be surrounded by adipose tissue and often closely associated with blood vessels. The physiological implications of these observations are discussed. The structure and composition of corpuscles was investigated using confocal microscopy with histological stains for collagen, proteoglycans and lipids. Nonlinear microscopy was also used to investigate the distribution of collagen (by second-harmonic generation), elastin (by intrinsic two-photon fluorescence) and membrane 4 lipids (by coherent Raman imaging). These techniques provided novel insights into the three-dimensional structure of the intact corpuscle, demonstrating: (i) three clearly distinguishable zones – the outer zone, the inner zone, and the core; (ii) blood vessels running through the outer lamellae and the core; (iii) the presence of proteoglycans – less in the outer zone than in the inner zone; (iv) two types of collagen fibres (one type associated with the lamellae and the other forming a complex fibre network through the inter-lamellar spaces); (v) occasional elastic fibres; (vi) a sheath of adipose tissue closely associated with the corpuscle’s outer surface. Mechanical testing by micro indentation, micropipette aspiration and osmotic challenge showed that the outer zone was stiff and able to quickly restore its original shape after distortion. Dynamic mechanical properties were investigated over a range 50 to 400 Hz. Observations of lamellar displacement (amplitude and phase) were consistent with the predictions of the Loewenstein-Skalak model. This model includes 30 lamellae; however, the same overall frequency response could be replicated in a single-lamella model with suitably chosen parameters. The benefits of a lamellar structure for transduction of mechanical signals therefore remain unresolved. 5 The permeability of the corpuscle to water and solutes was investigated using osmotic swelling and fluorescence tracer techniques. Both revealed unexpected complexity in the pathways of uptake to the inner core and demonstrated the presence of an impermeable boundary between the inner and outer zones, whose implications for mechanotransduction and nutrition in the corpuscle remain to be determined.
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Cappelli, Holly. "TRPV4 Mechanotransduction in Vascular Growth and Integrity." Kent State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=kent1487764322127302.
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Lee, Herng-Sheng. "Integrin-mediated mechanotransduction in human articular chondrocytes." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23083.
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Mechanical forces influence chondrocyte metabolism and function. It has been shown that 0.33 Hz cyclical mechanical stimulation results in membrane hyperpolarization of normal human articular chondrocytes by activation of Ca2+ -dependent K+ (SK) channels. Using pharmacological inhibitors of intracellular signalling molecules, stretch-activated ion channels (SAC) and specific antibodies against integrins, integrin-associated protein, cytokines and cytokine receptors the data show that membrane hyperpolarization is the result of mechanically-induced secretion of IL-4. Secreted IL-4 binds to type II IL-4 receptors and stimulates a signal cascade involving PLC and PKC leading to activation of SK channels. Specific blockade of a5b1 integrins, SAC and tyrosine kinases block mechanically-induced membrane hyperpolarization but have no effect on the hyperpolarization of exogenously added IL-4 consistent with a role for these molecules upstream of IL-4 release in the mechanotransduction pathway. Studies of OA chondrocytes demonstrate that unlike normal chondrocytes, these cells show a membrane depolarization response to 0.33 Hz mechanical stimulation as a result of activation of tetrodotoxin-sensitive Na+ channels. The mechanotransduction pathway involves a5b1 integrins, SAC, tyrosine kinases, PLC, PI 3-kinase and cytokines secretion but the actin cytoskeleton and PKC, which are important in the membrane hyperpolarization response in normal chondrocytes, are not necessary for membrane depolarization in OA chondrocytes following mechanical stimulation. The tyrosine phosphorylation events in the mechanotransduction pathway have been investigated in detail. The results show tyrosine phosphorylation of three major proteins, p125, p90, and p70 within 1 min of onset of mechanical stimulation. Immunoblotting and immunoprecipitation show these to be FAK, b-catenin, and paxillin, respectively. Tyrosine phosphorylation of all three proteins is inhibited by RGD containing oligopeptides and gadolinium, which is known to block SAC. b-catenin coimmunoprecipitates with FAK and is colocalized with a5 integrin and FAK. These results indicate a previously unrecognized role for an integrin-b-catenin signalling pathway in human articular chondrocyte responses to mechanical stimulation.
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Miller, Gregory J. "Mechanoregulation and mechanotransduction in skeletal tissue differentiation." Thesis, Boston University, 2013. https://hdl.handle.net/2144/11147.
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Thesis (Ph.D.)--Boston UniversityMechanical factors play a critical role in the development, maintenance and repair of skeletal tissues. Mechanical stimulation can alter the course of healing by directing the differentiation of mesenchymal progenitor cells into the cells that form the various skeletal tissues, and can enhance or impair the repair of orthopaedic injuries. Several mechanoregulatory hypotheses describing the relationships between mechanical stimuli and skeletal tissue differentiation have been proposed; however, these hypotheses have not been fully tested, nor have the underlying mechanisms been established. Identification of the specific mechanical stimuli and molecular mechanisms that direct the differentiation of mesenchymal progenitor cells would provide insight for treating injuries. The focus of this dissertation was to further our understanding of the mechanobiology of skeletal tissue differentiation by identifying the mechanisms that regulate the differentiation of mesenchymal progenitor cells. The first part of this dissertation identified consistent associations between the patterns of the formation of skeletal tissues (bone, cartilage, fibrocartilage and fibrous tissues) and the magnitudes of strains (shear and principal strains) in a mechanically-stimulated bone defect in vivo. The second part of this dissertation found evidence that the Rho-family GTPases, as well as adhesion receptors and their associated focal adhesion proteins, may be possible mediators of the mechanotransduction mechanisms involved in the decisions of cell fate of the mesenchymal progenitor cells within the stimulated tissues. Finally, in an anticipation of the next steps in research on mechano-regulation of tissue differentiation, a microindentation technique was developed to determine the poroelastic material properties of the soft tissues forming in the callus. The values of Young's modulus, Poisson's ratio and permeability of articular cartilage were measured at the microscale and compared to those determined using standard macroscale techniques. Together, the findings of this dissertation further our understanding of the mechanoregulation of skeletal tissue differentiation, and can be used to inform and improve the various hypotheses regarding the mechanoregulation of tissue differentiation. Clinically, these results could potentially direct the development of therapies to improve treatment outcomes and reduce recovery time.
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ULLOA, SEVERINO LUISA. "Mechanobiology and mechanotransduction in propathological cellular system." Doctoral thesis, Università degli Studi di Trieste, 2018. http://hdl.handle.net/11368/2919813.
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Cellule e tessuti rispondono alle force ambientali tramutandole in segnali biochimici. Questa proprietà cellulare ha indotto un largo interesse nell'area biochimica a studiare la possibilità di usare le proprietà meccaniche come un marker per la diagnosi precoce di malattie e per controllare il destino cellulare attraverso l'utilizzo di nuovi materiali prostetici. In questa tesi io ha mostrato che l'AFM può essere un eccellente strumento per valutare le proprietà meccaniche di differenti sistemi cellulari. In particolare io ho studiato la biomeccanica nella calcificazione della valvola aortica (CAVD), nel tumore associato alle mutazioni missenso di p53 a nella miogenesi.
CAVD è la più comune cardiomiopatia ed è la causa della stenosi aortica. La principale causa per la calcificazione aortica è la differenziazione delle cellule valvolari interstiziali nel fenotipo patologico. L'altra caratteristica è il cambiamento dell'organizzazione, composizione eproprietà meccaniche della matrice che porta alla disfunzione valvolare. In questo contesto io ho studiato le proprietà biomeccaniche e molecolari delle cellula valvolari interstiziali umane e porcine cresciute su substrati lisci, ma a diversa stiffness e su nanotubi di carbonio che simulano la morfologia della matrice, rispettivamente.
Successivamente, ho focalizzato i miei studi sulla meccanobiologia dei mutanti p53 associati ai tumori. In questa parte del mio lavoro ho studiato i processi cellulari e i percorsi biochimici che stabilizzano le cellule mutanti di p53. Ho eseguito una caratterizzazione della spettroscopia di forza AFM delle cellule dopo il trattamento con diversi farmaci e ho confermato una riduzione della rigidità nelle cellule trattate determinata da un processo di de-polimerizzazione dell'actina e una riduzione dei livelli di p53. In seguito, ho studiato lo stesso processo in un modello ex vivo sfruttando la procedura di nano-indentazione AFM. Alla fine della mia tesi, ho caratterizzato un nuovo polipeptide della famiglia HELP, HELPc, sintetizzato all'Università di Trieste tramite AFM imaging.In seguito, HELPc è stato utilizzato come substrato per la crescita di mioblasti C2C12. I risultati hanno mostrato che l'aggiunta di una sequenza all'isoforma di α2 di collagene di tipo IV, contenente motivi RGD, determina un miglioramento dell'attività cellulare combinando diversi metodi, come l'immunofluorescenza, la microscopia a forza atomica e l'imaging Ca2 +.Cells and tissues respond to environmental forces transducing them into biochemical signals. This cellular property has induced a large interest in biomedical area to study the possibility of using cell (or tissue) mechanical properties (e.g., their stiffness) as a marker for early diagnosis of pathologies or as trigger point to modulate cell/tissue behavior via new, ad hoc designed, prosthetic materials. In this thesis work I have shown that AFM could represent an excellent tool to evaluate the mechanical properties of different cellular systems. In particular, I have studied the biomechanics in the onset of calcific aortic valve disease, in the tumor associated p53 missense mutants and in myogenesis. Calcific aortic valve disease is the most common cardiomyopathy and is the main cause of aortic stenosis. The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a disease-associated phenotype. Another characteristic of this disease is the significant change in the organization, composition and mechanical properties of the ECM that beside being the result of the dysfunction of the valve cells. In this context, I have initially investigated on the variation of mechanical and morphological properties in hVICs when grown on flat polyacrylamide gels at different stiffness. I performed this task using both AFM force spectroscopy and molecular biology essays. My results show that there is a strong direct dependence of cellular rigidity by that of the underlying substrate, resembling a sort of flat matrix. In addition, I observed that inhibition of Rho kinase will result in a preclusion of actin polymerization followed by a reduced cellular stiffness and YAP activation. I have performed the same AFM characterisation on a more complex experimental model that involves thin sections of explanted stenotic calcific human valves. After this, I moved my studies on the effect of a nanostructured extra-cellular matrix like substrate could have on VICs. I did that taking advantage of multi-walled CNTs carpets grown on glass slides above which I subsequently seeded porcine Valvular Interstitial cells. Initially I performed a morphological characterization pointing out that CNTs have a positive effect on pVICs, in particular the myofibroblat percentage in VICs developed above CNTs is similar to that of a healthy valve. I performed cell stiffness measurements via AFM and I discovered that myofibroblast stiffness is not significantly altered by CNTs. I associated this result to the low density of focal adhesions expressed on these cells by the nanostructures substrate. At this point I could hypothesize that CNTs, piercing and pinching the plasma membrane, are able to facilitate the creation of clusters of FAs that, at the very end, will increase the cellular rigidity. Subsequently, I focused my studies to the mechanobiology of tumor associated p53 mutants. In this part of my work I studied the cellular processes and biochemical pathways stabilizing mutant p53 cells. I performed an AFM force spectroscopy characterization of cells after treatment with different drugs and I confirmed a stiffness reduction in treated cells determined by an actin de-polymerization process and a reduction of p53 levels. Following, I studied the same process in a ex vivo model taking advantage of AFM nano-indentation procedure. At the end of my thesis, I have characterized a new HELP family polypeptide, HELPc, synthetized at the University of Trieste. By characterizing HELPc coating and hydrogel via AFM imaging. Following, HELPc was used as a substrate for C2C12 myoblasts growth. The results showed that the addition of a sequence to from the α2 isoform of collagen type IV, containing to RGD motives, determines an improve of cells activity by combining several methods, as immunofluorescence, Atomic Force Microscopy and Ca2+ imaging.
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Shin, Jung-Bum. "Identification of genes involved in sensory neuron mechanotransduction." [S.l. : s.n.], 2002. http://www.diss.fu-berlin.de/2003/68/index.html.
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Jokl, Elliot. "Mechanotransduction at the Z-disc of skeletal muscle." Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/19324/.
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The importance of the transglutaminase-like protein kyphoscoliosis peptidase (KY) in skeletal muscle was discovered in a KY-deficient mouse model of hereditary kyphoscoliosis (ky/ky). The ky/ky pathology primarily affects muscles experiencing high tension, e.g., the soleus, which undergoes regeneration and shows persistent hallmarks of structural damage, including the aberrant localisation of the z-disc crosslinker Filamin C (FLNC) – a known KY interaction partner. However, muscles are globally smaller in size, and fast-twitch muscles (e.g., the extensor digitorum longus or EDL) show an inability to undergo hypertrophy in response to elevated tension. A robust function for KY has not been identified. Recently reported human cases of KY-deficient myopathy share hallmarks with the mouse pathology and underscore the value of increasing our understanding of KY and its function. Chaperone Assisted Selective Autophagy (CASA) is a tension-induced mechanism for FLNC turnover understood to be critical for muscle maintenance. This thesis hypothesises that the absence of KY disrupts CASA, thereby indicating that KY might have a role in CASA. This thesis examines CASA in the ky/ky mouse model and describes the generation and analysis of novel cellular (C2C12) and zebrafish models of KY-deficiency. These indicate that constitutive upregulation of CASA markers may be a primary hallmark of KY-deficiency. Additionally, data may indicate inefficient turnover of the CASA complex in tissues experiencing high tension. It is also shown that in vivo overexpression of recombinant KY is not sufficient to drive hypertrophy in normal mice, but may partially rescue fibre size in ky/ky mice.
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Karcher, Hélène. "The mechanics of mechanotransduction : analyses of cell perturbation." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38239.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references.
Cells sense mechanical stimuli and respond by changing their phenotype, e.g. shape, gene expression, motility. This process, termed mechanotransduction, was investigated using computational and theoretical approaches, as well as comparisons with experiments. As a first step, a three-dimensional viscoelastic finite element model was developed to simulate cell micromanipulation by magnetocytometry. The model provided a robust tool for analysis of detailed strain/stress fields induced within a single cell or cell monolayer produced by forcing one tethered microbead. On the assumption of structural homogeneity, stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending less than 10tm from the bead. Modification of the model to represent experimental focal adhesion attachments supported a non-uniform force transmission to basal surface focal adhesion sites. Proteins in identified zones of high stresses in the cell are candidate mechanosensors and their molecular response to force was hence investigated, A generic model of protein extension under external forcing was created inspired by Kramers theory for reaction rate kinetics in liquids.
(cont.) The protein was hypothesized to have two distinct conformational states: a relaxed state, Ci, preferred in the absence of external force, and an extended state, C2, favored under force application. Appearance and persistence of C2 was assumed to lead to transduction of the mechanical signal into a chemical one. While the level of applied force and the energy difference between states largely determined equilibrium, the dominant influence on the extension time was the height of the transition state. Force-induced distortions in the energy landscape were also shown to have a significant influence on extension time, however, exhibiting a weaker force dependence than exponential. Finally, the link between membrane receptors and the extracellular matrix -- or the bead in magnetocytometry experiments -- was investigated as a primary path for force transduction to the cell interior. To shed light on the role of bonds formed by membrane receptors on measurements of cellular rheology, we modeled the process by which a forced, cell-tethered microbead translates and rotates as influenced by the stochastic formation and. rupture of adhesion bonds. We show that this process is crucial in the inference of cell mechanical properties from microbead experiments.
by Hélène Karcher.
Ph.D.
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McVittie, Anna K. (Anna Kathleen) 1977. "Pathways for mechanotransduction in pressurized airway epithelial cells." Thesis, Massachusetts Institute of Technology, 2001. http://hdl.handle.net/1721.1/89315.
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Drew, Liam John. "Mechanisms of mechanotransduction by dorsal root ganglia neurons." Thesis, University College London (University of London), 2004. http://discovery.ucl.ac.uk/1446740/.
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The molecular mechanisms that mediate mammalian sensory mechanotransduction are poorly understood. Detection of mechanical events by sensory neurons of the dorsal root ganglia (DRG) is the primary event in the senses of touch, pressure-induced pain and proprioception. Recent work has demonstrated that the somatic membrane of cultured DRG neurons is a suitable system for studying physical transduction. In this thesis the responses of cultured DRG neurons to focal mechanical stimulation were investigated. It was shown that mechanical stimulation activated non-selective cation channels in these cells. The response properties of different subclasses of sensory neurons were characterised and were consistent with the presumed in vivo phenotypes of these cells. A number of antagonists of mechanically activated currents, with affnity in the low micromolar range, were identified; these included the pore blocking compounds gadolinium and ruthenium red and FMl-43 acted as a permeant blocker of mechano- sensitive channels. Modulation of mechanically activated currents by extracellular calcium was observed and it was shown that currents were regulated by the actin cytoskeleton and the extracellular matrix protein laminin. Investigation of null mutant mice revealed that the acid sensing ion channels 2 and 3, which are widely hypothesised to function in mammalian mechanosensation, did not contribute to mechanically activated currents. Venom of the marine snail Conus ventricosus was found to block mechanically activated currents although the active component of this venom is yet to be identified. Overall, this work has shown that cultured DRG neurons are a useful system for studying mechanotransduction and has revealed a number of functional and pharmacological properties of the ion channels that underlie this process.
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McNamara, Laura Elizabeth. "Molecular and cellular analysis of topography-induced mechanotransduction." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/2207/.
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Edited Abstract: Mechanotransduction is the process by which cells convert mechanical stimuli into an adaptive gene- and protein-level response, via signalling cascades or direct physical effects of the cytoskeleton on the nucleus, and appropriate mechanosignalling is crucial for tissue development and function. Most techniques currently used to study cellular mechanoresponses are relatively damaging to the cells. In contrast, topographically structured substrates, such as microgrooves, have great potential for use as non-invasive mechanostimuli. In this study, quartz microgrooved substrata (2 μm depth x 25 μm pitch) were used as platforms for the confinement and alignment of cells. A multi-layered approach was adopted to begin to integrate the changes induced by the topographical mechanostimulus at the chromosome, small RNA, transcript, protein and structural levels. Together, the results provide insight into multiple facets of topography-induced mechanotransduction, which should contribute to understanding of mechanotransduction and cell-material interactions.
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Dahl, Anna Caroline E. "Membrane protein mechanotransduction : computational studies and analytics development." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:67798647-8ed5-46e0-bde9-c71235fe70ba.
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Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.
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Heayn, Michelle Diane. "The Role of Caveolae in PECAM-1 Mechanotransduction." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/263778.
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PhysiologyPh.D.
Altered fluid flow, which is found in branches and curvatures of arteries, results in abnormal forces on the endothelial cells (EC). These forces have been shown to alter EC gene expression and phenotype and to activate several cellular structures including G-proteins, ion channels, adhesion molecules, and caveolae. Recently, PECAM-1 has been implicated as the primary sensor of hemodynamic forces in EC. Shear stress rapidly induces tyrosine phosphorylation of PECAM-1 and the recruitment of SHP-2. These events appear to contribute to shear-activation of ERK1/2. Additionally, PECAM-1 has been shown to form a mechanosensory signaling complex with VE-cadherin, VEGFR2, and βcatenin which plays a role in adhesion molecule expression and regulation of NF-κB. Past work has shown that caveolae membrane domains also serve as mechanotransduction sites that regulate many of these same second messengers. Based on these novel observations, we hypothesize that the PECAM-1 mediated mechanotransduction requires caveolar membrane domains to effectively propagate mechano-signals. In this study, we intended to specifically test this hypothesis by 1) evaluating the role of caveolae in shear stress-induced PECAM-1 tyrosine phosphorylation, recruitment of SHP-2, and formation of a signaling complex with VE-cadherin, VEGFR2, and βcatenin and 2) determining the functional significance of PECAM-1 compartmentalization within caveolae with regard to changes in endothelial cell phenotype induced by atherogenic patterns of flow. Here, we have identified a pool of PECAM-1 which localizes within lipid rafts and caveolar membranes. This pool of PECAM-1 was shown to be activated by tyrosine phosphorylation and recruitment of mechanosignaling complex members in response to shear stress. We were also able to demonstrate complex formation in an in vivo model of disturbed blood flow. The significance of PECAM-1 compartmentalization to these membrane microdomains was demonstrated in endothelial cells treated with raft/caveolae disrupting compounds where shear stress-induced PECAM-1 tyrosine phosphorylation was markedly attenuated. Finally, we attempted to generate an adenovirus expressing a mutant form of PECAM-1 which was unable to target to lipid rafts in order to determine the importance of PECAM-1 localization in lipid rafts and caveolae on its downstream signaling in response to shear stress. Results from these studies provide new knowledge as to how endothelial cells respond to changing hemodynamic parameters, which could provide greater insight into how flow influences vascular homeostasis.
Temple University--Theses
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FARRIS, FRANCESCO. "AMYLOID FIBRILS INDUCE GLYCOCALYX MEDIATED MECHANOTRANSDUCTION IN MELANOMA." Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/946381.
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PMEL is an amyloidogenic protein found overexpressed in melanoma compared to healthy skin. PMEL expression correlates with unfavorable prognosis, but the mechanism beyond the adverse disease outcome is still unknown. Recently, our lab established a link between the presence of PMEL amyloid fibrils in the metastatic melanoma secretome and YAP activation, driving cancer proliferation and drug resistance.
In this study, we show that PMEL amyloid fibrils, secreted by cancer cells, are component of the extracellular matrix and modify its stiffness thus activating mechanosignalling.
We highlight the signaling pathway that connects PMEL fibrils to YAP activation. In particular, we show that Agrin is part of the extracellular amyloid plaques, interacts with PMEL amyloid fibrils and it is necessary to drive PMEL dependent YAP activation. To further dissect the signaling cascade triggered by amyloids we also overexpressed LATS1 or/and LATS2, key members of Hippo pathway downstream of Agrin. The overexpression of LATS2 does not affect YAP activation, while LATS1 overexpression abrogates PMEL dependent YAP activation indicating that this kinase is a downstream inhibitor of the amyloid mediated mechano-response. We demonstrate that PMEL amyloid fibrils activate YAP in a ROCK independent fashion. Finally, we demonstrated that the presence of amyloid fibrils in the extracellular space enhances the migration and invasion capacity of melanoma cells.
Our study uncovers the mechanism by which amyloid fibrils drive YAP activation impacting on migration and invasion of melanoma cells. These data suggest that inhibitors capable to reduce the formation of extracellular amyloid fibrils could interfere with melanoma progression.
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Schwartz, Christine. "Muscle LIM protein and Nesprin-1 in Mechanotransduction." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066374/document.
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J’ai étudié trois protéines qui participent à deux vois différentes de méchano-transduction qui est la conversion des stimuli physiques en un signal biochimique.Dans une culture cellulaire en 2D, lorsque les cardiomyocytes sont étirés, MLP est transloqué vers le noyau. Sans translocation, les cellules ne parviennent pas à répondre à la stimulation. Les patients porteurs de mutations dans MLP développent une cardiomyopathie comme les souris MLP knock-out (MLP-/-). Mon objectif a été d’élucider le rôle de MLP dans ces cardiomyopathies en surexprimant des mutations de MLP dans les cardiomyocytes isolés des souris MLP-/- néonataux. Dans les cultures 2D mais pas 3D, MLP n’était pas transloqué vers le noyau après l’étirement des cellules. Bien que je n’aie pas pu résoudre ce problème, j’ai mis au point les expériences nécessaires à la poursuite de ce projet.Nesprins s’intègrent dans un complexe transmembranaire de l’enveloppe nucléaire (EN), le LINC complexe, qui connecte le cytosquelette à l’intérieur du noyau. Les myoblastes isolés des patients porteurs des mutations de Nesprin ou de Lamin, qui est associé au LINC complexe, ont présenté des noyaux déformés ainsi que des anomalies de réponses méchanosensibles : Si cultivées sur supports mous, les cellules affichaient un niveau élevé de fibres musculaires stressées et d’adhésions focales. Le knock-down de FHOD, une cible en aval de ROCK et SRC, qui également étaient actives dans ces myoblastes, a réduit ce phénotype. Bien que l’on ait émis l’hypothèse que les mutations dans Nesprins et Lamins conduisent à une instabilité mécanique de l’EN, ces résultats indiquent que les voies de signalisation par l’EN sont perturbées aussiI studied three striated muscle proteins that are participating in two different pathways of mechanotransduction, which is the translation of a physical stimulus into a biochemical signal.When isolated cardiomyocytes are stretched in 2D, MLP shuttles to the nucleus. Without shuttling MLP, these cells fail to respond to the stretch stimulus. Human patients with MLP-mutations develop cardiomyopathies, as well as mice with a knock-out of MLP (MLP-/-). By expressing mutated MLP in neonatal cardiomyocytes of MLP-/- mice, I wanted to elucidate the role of mutant MLP. Surprisingly, MLP did shuttle after stretching of 2D but not 3D cell cultures. Although I could not solve this issue, I prepared the setup for subsequent experiments.Nesprins are part of the nuclear envelope (NE) spanning LINC complex, which connects the cytoskeleton with the nucleus. Myoblasts from patients with mutations in Nesprins or LINC-associated Lamins displayed deformed nuclei and had defects in mechanosensitive responses with an elevated level of stress fibers and focal adhesions on soft surfaces. This phenotype could be rescued by knock-down of formin FHOD1, a downstream target of ROCK and SRC, which also were highly active in the mutant cells. While mutations in Nesprins and Lamins are thought to lead to mechanical instability of the NE, these results indicate that signaling pathways through the NE are disturbed as well
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Schwartz, Christine. "Muscle LIM protein and Nesprin-1 in Mechanotransduction." Electronic Thesis or Diss., Paris 6, 2016. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2016PA066374.pdf.
Full textAbstract:
J’ai étudié trois protéines qui participent à deux vois différentes de méchano-transduction qui est la conversion des stimuli physiques en un signal biochimique.Dans une culture cellulaire en 2D, lorsque les cardiomyocytes sont étirés, MLP est transloqué vers le noyau. Sans translocation, les cellules ne parviennent pas à répondre à la stimulation. Les patients porteurs de mutations dans MLP développent une cardiomyopathie comme les souris MLP knock-out (MLP-/-). Mon objectif a été d’élucider le rôle de MLP dans ces cardiomyopathies en surexprimant des mutations de MLP dans les cardiomyocytes isolés des souris MLP-/- néonataux. Dans les cultures 2D mais pas 3D, MLP n’était pas transloqué vers le noyau après l’étirement des cellules. Bien que je n’aie pas pu résoudre ce problème, j’ai mis au point les expériences nécessaires à la poursuite de ce projet.Nesprins s’intègrent dans un complexe transmembranaire de l’enveloppe nucléaire (EN), le LINC complexe, qui connecte le cytosquelette à l’intérieur du noyau. Les myoblastes isolés des patients porteurs des mutations de Nesprin ou de Lamin, qui est associé au LINC complexe, ont présenté des noyaux déformés ainsi que des anomalies de réponses méchanosensibles : Si cultivées sur supports mous, les cellules affichaient un niveau élevé de fibres musculaires stressées et d’adhésions focales. Le knock-down de FHOD, une cible en aval de ROCK et SRC, qui également étaient actives dans ces myoblastes, a réduit ce phénotype. Bien que l’on ait émis l’hypothèse que les mutations dans Nesprins et Lamins conduisent à une instabilité mécanique de l’EN, ces résultats indiquent que les voies de signalisation par l’EN sont perturbées aussiI studied three striated muscle proteins that are participating in two different pathways of mechanotransduction, which is the translation of a physical stimulus into a biochemical signal.When isolated cardiomyocytes are stretched in 2D, MLP shuttles to the nucleus. Without shuttling MLP, these cells fail to respond to the stretch stimulus. Human patients with MLP-mutations develop cardiomyopathies, as well as mice with a knock-out of MLP (MLP-/-). By expressing mutated MLP in neonatal cardiomyocytes of MLP-/- mice, I wanted to elucidate the role of mutant MLP. Surprisingly, MLP did shuttle after stretching of 2D but not 3D cell cultures. Although I could not solve this issue, I prepared the setup for subsequent experiments.Nesprins are part of the nuclear envelope (NE) spanning LINC complex, which connects the cytoskeleton with the nucleus. Myoblasts from patients with mutations in Nesprins or LINC-associated Lamins displayed deformed nuclei and had defects in mechanosensitive responses with an elevated level of stress fibers and focal adhesions on soft surfaces. This phenotype could be rescued by knock-down of formin FHOD1, a downstream target of ROCK and SRC, which also were highly active in the mutant cells. While mutations in Nesprins and Lamins are thought to lead to mechanical instability of the NE, these results indicate that signaling pathways through the NE are disturbed as well
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Chowdhury, Tina Taneer. "Role of nitric oxide and PGEâ‚‚ in chondrocyte mechanotransduction." Thesis, Queen Mary, University of London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246308.
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Liao, Pinhu. "Mechanotransduction in alveolar epithelial cells subjected to mechanical strain." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479153.
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Liu, Jie. "Mechanotransduction in Endothelial Cells:Cell Growth, Angiogenesis and Wound Healing." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1274392778.
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Balasubramanian, Lavanya. "Integrin Mediated Mechanotransduction in Renal Vascular Smooth Muscle Cells." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002227.
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Summerour, Sonya R. "Static equibiaxial stretch mediated mechanotransduction in adult cardiac fibroblasts /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1999. http://wwwlib.umi.com/cr/ucsd/fullcit?p9952652.
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Vanderploeg, Eric James. "Mechanotransduction in Engineered Cartilaginous Tissues: In Vitro Oscillatory Tensile Loading." Diss., Available online, Georgia Institute of Technology, 2006, 2006. http://etd.gatech.edu/theses/available/etd-05192006-110158/.
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Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2007.Radhakrishna, Harish, Committee Member ; LaPlaca, Michelle, Committee Member ; Nerem, Robert, Committee Member ; Garcia, Andres, Committee Member ; Levenston, Marc, Committee Chair.
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Lian, Ian Yu-Zen. "The roles of E-Tmod in mechanotransduction and cardiac myofibrillogenesis." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3249659.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed March 23, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 123-134).
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Schwartz, Christine [Verfasser]. "Muscle LIM Protein and Nesprin-1 in Mechanotransduction / Christine Schwartz." Berlin : Freie Universität Berlin, 2017. http://d-nb.info/112815062X/34.
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Fitzgerald, Jonathan Basil. "Chondrocyte gene expression and intracellular signaling pathways in cartilage mechanotransduction." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33869.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.Includes bibliographical references (p. 152-167).
Chondrocytes respond to in vivo mechanical loads by regulating the composition of the cartilage extracellular matrix. This study utilized three loading protocols that span the range of forces and flows induced by in vivo loading. Constant (static) compression of cartilage explants induces a transient hydrostatic pressure buildup and fluid exudation from the compacted matrix until relaxation leads to a new equilibrium compressed state. Dynamic compression induces cyclic matrix deformation, hydrostatic pressures, fluid flows, and streaming currents. Dynamic tissue shear causes cyclic matrix deformation only. After applying these loading protocols to intact cartilage explants for 1 to 24 hours, we used real-time PCR to measure the temporal expression profiles of selected genes associated with cartilage homeostasis. In concurrent experiments, we assessed the involvement of intracellular signaling pathways using molecular inhibitors. In order to interpret the results we developed two techniques that reliably clustered intermediate-sized datasets using principal component analysis and k-means clustering. Mechanical loading regulated a variety of genes including matrix proteins, proteases, protease inhibitors, transcription factors, cytokines, and growth factors. Static compression transiently upregulated matrix proteins, however, mRNA levels were suppressed by 24 hours.
(cont.) Dynamic compression and dynamic shear increased matrix protein transcription particularly after 24 hours. In contrast, matrix proteases were upregulated by all 24 hour loading regimes, particularly static compression. Taken together these results demonstrate the functionally-coordinated regulation of chondrocyte gene transcription in response to mechanical forces, and support the hypothesis that dynamic loading is anabolic for cartilage and static loading is anti-anabolic. Intracellular calcium release, cAMP activation of protein-kinase-A, and the phosphorylation of MAP kinases (ERK1/2, p38), were all identified as signaling events necessary for mechanically-induced transcription. In addition, we measured the immediate, transient increase in mRNA levels of transcription factors downstream of the MAP kinase pathway (c-Fos and c-Jun), in response to all three loading types. The prevention of protein synthesis during static compression suppressed mechanically-induced transcription suggesting that signaling molecules are synthesized in response to mechanical forces. Comparison of this well characterized model of normal cartilage mechanotransduction to what occurs within diseased cartilage will hopefully provide insight into the mechanisms driving the progression of osteoarthritis.
by Jonathan Basil Fitzgerald.
Ph.D.
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Rabodzey, Aleksandr. "Flow-induced mechanotransduction in cell-cell junctions of endothelial cells." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/41586.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (leaves 86-92).
Endothelial cells show an unexpected behavior shortly after the onset of laminar flow: their crawling speed decreases ~40% within the first 30 min, but only in a confluent monolayer of endothelial cells, not in subconfluent cultures, where cell-cell interactions are limited. This led us to study early shear effects on cell-cell adherens junctions. We found a 30±6% increase in the number of VE-cadherin molecules in the junctions. The strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin protein also increased after laminar flow. These observations suggest that endothelial cell junction proteins respond to flow onset. The process of clustering may induce diffusion of monomers to the junction area, resulting in an overall increase in VE-cadherins in the junctions. To directly confirm the role of adherens junctions in the decrease in cell crawling speed, we used siRNA-knockdown technique to produce cells lacking VE-cadherin. These cells showed no decline in crawling speed under flow. Our interpretation is consistent with previous data on junction disassembly 8 hr after flow onset. The speed of endothelial cell crawling returns to the original level by that time, and junctional disassembly may explain that phenomenon. In order to understand better the change in VE-cadherin distribution under flow and during junction formation and remodelling, we developed a mathematical model of VE-cadherin redistribution in endothelial cells. This model allowed us to develop a quantitative framework for analysis of VE-cadherin redistribution and estimate the amount of protein in the junctions and on the apical surface. In addition to that, the model explains rapid junction disassembly in the leukocyte transmigration and junction formation in subconfluent cells.
(cont.) These studies show that intercellular adhesion molecules are important in the force transmission and shear stress response. Their role, however, is not limited to flow mechanotransduction. Intercellular force transmission has an important application - organ development and, specifically, angiogenesis. We studied the role of VE-cadherin in vessel development in HUVECs and showed that VE-cadherin-null cells do not form vessels in the in vitro assay. This observation confirms the important role of intercellular force transmission in response to external force caused by flow or exerted by other cells.
by Aleksandr Rabodzey.
Ph.D.
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Kojić, Nikola 1978. "Mechanotransduction via airway epithelial cells : the effect of compressive stress." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40870.
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Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007."June 2007."
Includes bibliographical references (leaves 153-157).
A classic finding in asthma is a change in the structural organization of the airway epithelium. This complex process known as airway remodeling is not fully understood, and we believe that the forces accompanying airway constriction activate the epithelium and contribute to airway remodeling. To better understand this mechanotransduction mechanism we used an in vitro system of cultured normal human bronchial epithelial cells that could simulate compressive stresses experienced by the epithelium during bronchoconstriction. The application of a transcellular pressure gradient (10-50cmH20) for 10 minutes resulted in transient activation of the epidermal growth factor receptor (EGFR) - MAP kinase (ERK) signaling pathway. Furthermore, specialized real-time high-speed imaging revealed an exponential decrease in the volume of the compliant lateral intercellular space (LIS) separating neighboring cells. The measured LIS volume collapse curves were directly inputted into 2-D and 3-D numerical finite element models, whose output was EGFR-ligand concentration dynamics in the LIS.
(cont.) During the first three minutes under pressure, the calculated increase in ligand concentration (specifically HB-EGF, which is made by the cells and shed into the LIS, thereby constituting an autocrine loop with the EGFR) matched the measured phosphorylated EGFR (pEGFR) dynamics. The model thus provided crucial insight into how an observed change in LIS geometry esulted in activation of the EGFR signaling pathway. This insight, coupled to EGFR signaling models, could one day be applied to the design of novel pharmacogenetic therapeutics aimed at preventing airway over-activation and potentially hindering airway remodeling progression in asthmatic patients.
by Nikola Kojić.
Ph.D.
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Jonas, Maxine. "Fluorescence laser tracking microrheology for quantitative studies of cytoskeletal mechanotransduction." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39911.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references (p. 111-127).
To shed light on the cell's response to its mechanical environment, we examined cell rheology at the single cell level and quantified it with nanometer spatial and microsecond temporal resolutions over a five-decade frequency range (- 0.5 Hz to 50 kHz). To this end, we developed and optimized an instrument for fast fluorescence laser tracking microrheology (FLTM). This novel method aims at experimentally deriving cellular viscoelastic properties from the passive monitoring of fluorescent microspheres undergoing Brownian motion inside the tested sample. Further instrument enhancement even broadens the FLTM frequency span up to seven decades by modulating data acquisition speed or complementing FLTM with a two-particle microrheology modality. In living cells, FLTM accurately characterizes the solid-like vs. liquid-like cytoskeletal behavior from measurements based on endocytosed micron-sized beads, independently of probe size or surface chemistry. FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint.
(cont.) FLTM also demonstrates the existence of two distinct rheological regimes on the cell surface and in the cell interior: While the former surface investigations show power-law frequency variations of the complex shear modulus G*(co), the latter intracellular experiments identify multiple time and length scales affecting cell rheological features. Finally, FLTM evaluates frequency-specific stretch-induced cell mechanics and thus promises to broaden and diversify the scientific knowledge on mechanotransduction, from a molecular and cellular standpoint.
by Maxine Jonas.
Ph.D.
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Mahajan, Kalpesh D. "Development of Nanodevices for Bio-detection, Separation, Therapy, and Mechanotransduction." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376446892.
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Servin, Vences Martha Rocio [Verfasser]. "Ion-channel mediated mechanotransduction in chondrocytes / Martha Rocio Servin Vences." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1154766608/34.
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Childs, Peter Geoffrey. "Cellular mechanotransduction : development of a nanovibrational bioreactor for cellular stimulation." Thesis, University of the West of Scotland, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739388.
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Saini, Navpreet S. "Cell Type and Substrate Dependence of Fibronectin Properties and Mechanotransduction." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5806.
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Fibronectin is an important protein that is able to bind to other fibronectin molecules and to cell surface receptors. In doing so, the interactions fibronectin can perform is important for the processes of cell migration and tissue formation. Understanding the properties of fibronectin and fibril assembly is useful for areas such as wound healing, where fibronectin molecules are assembled to protect the tissue and to perform other tasks. Because of these reasons, it is important to understand how fibronectin is assembled and how its properties affect the fibril assembly, which in return affects the way the cell matrix operates. Previously published papers illustrate that the properties of fibronectin affect the mechanotransduction process, the cell conversion of mechanical stimulus to chemical, and this leads to various changes of the fibril assembly. However, the question that now comes to focus is what variables affect the fibril assembly? The two main variables that come into question is the substrate stiffness (ksub) (pN/nm) and the actin velocity (Vu) (nm/s). In order to test this hypothesis, several fibril assembly simulations were performed via MATLAB based upon the Weinberg-Mair-Lemmon Fibronectin Model. These simulations were performed by varying the parameters of substrate stiffness and actin velocity as well as fibril size, which affect the various measurements of the fibronectin, such as stretched length, relaxed length, etc. Through these various experiments, it was determined that the actin velocity and fibril size had the greatest impacts in affecting the fibronectin’s properties and its assembly.
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Fulzele, Keertik S. "ROLE OF ACTIN CYTOSKELETON FILAMENTS IN MECHANOTRANSDUCTION OF CYCLIC HYDROSTATIC PRESSURE." MSSTATE, 2004. http://sun.library.msstate.edu/ETD-db/theses/available/etd-07122004-171347/.
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This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.
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Roberts, Susan Read. "Mechanotransduction pathways associated with intracellular calcium in chondrocytes within 3D constructs." Thesis, Queen Mary, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270626.
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Polacheck, William J. (William Joseph). "Mechanotransduction of interstitial fluid stresses and effects on tumor cell migration." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/85531.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013."September 2013." Cataloged from PDF version of thesis.
Includes bibliographical references (pages 93-106).
Breast cancer incidence in the United States is I in 8, and over 90% of breast cancer related deaths are due to metastases, secondary tumors at a site distant from the primary tumor. Metastasis formation requires carcinoma cells to navigate through the tumor microenvironment and invade the surrounding stroma. Migration is a highly orchestrated process in which cells are guided by both internal signals and signals from the microenvironment. Hence, understanding the mechanisms that guide cell migration in response to various stimuli in the tumor and stromal microenvironments is key to developing therapies that prevent tumor cell migration and render cancer more treatable. Osmotic and hydrostatic pressure gradients within the extracellular matrix (ECM) drive flow of interstitial fluid through the ECM. Elevated osmotic pressure, lymphatic collapse, solid stress, and increased microvascular permeability contribute to elevated interstitial fluid pressure (IFP) during carcinoma progression, and high intratumoral IFP leads to pressure gradients at the tumor margin, which drive fluid flow that emanates from the tumor core to drain in the surrounding stroma. In this thesis, we explore the effect of interstitial flow (IF) on tumor cell migration. We developed a microfluidic platform to apply repeatable, robust IF through tissue constructs consisting of human metastatic breast cancer cells embedded within a 3D collagen type I matrix. We implemented the microfluidic device to validate CCR7-mediated autologous chemotaxis as a mechanism that guides downstream migration in response to IF. However, we identified a separate competing pathway that drives cell migration upstream (rheotaxis). Rheotaxis results from asymmetry in matrix adhesion stress that is required to balance fluid drag imparted by IF on tumor cells. Thus, autologous chemotaxis, mediated by chemical transport, and rheotaxis, mediated by fluid stresses, compete to direct cell migration downstream or upstream in response to IF. Our results provide insight into mechanotransduction in 3D porous media and into the mechanisms by which asymmetries in matrix adhesion tension guide cell migration. Furthermore, our results demonstrate that the consideration of IF is crucial for understanding and treating metastatic disease. Key words: Interstitial flow, mechanotransduction, tumor cell migration, microfluidics.
by William J. Polacheck.
Ph. D.
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Patel, Sagar. "THE MECHANOTRANSDUCTION OF PRIMARY CILIA IN TUMOR PROGRESSION OF LUNG ADENOCARCINOMA." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3071.
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The objective of this study was to investigate primary cilia and their mechanotransduction role in lung adenocarcinoma tumor progression. The main focus investigated the effect of primary cilia on cell cycle progression, survival, adhesion and migration analysis of these cells and the role of sonic hedgehog signaling pathway in mechanotransduction. Human Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma biopsies contain more primary cilia than non-tumor lung sections. To observe the effects of primary cilia presence in lung cancer cells in-vitro, formation of primary cilia is inhibited using small interfering RNA. A549 cells with intact primary cilia observe less cell cycle progression than cells deficient in primary cilia under static and cyclic stretch conditions. Primary cilia cause higher cell survival and adhesion. Increase in cell adhesion also increases the migration and wound closure rates in control samples compared to samples treated with inhibition of IFT88, thereby increasing the metastasis of these cells. Several downstream regulatory genes in sonic hedgehog signaling pathway observe significantly decreased gene expressions in primary cilia deficient cells, thus indicating inefficient mechanotransduction. Therefore, cancer cells need primary cilia to survive, adhere and migrate and continue tumor progression.
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Stephenson, Natalie. "Mechanotransduction of the Notch signalling pathway via the negative regulatory region." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/mechanotransduction-of-the-notch-signalling-pathway-via-the-negative-regulatory-region(c13c0f01-3095-4895-a536-1dfc324d9899).html.
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The Notch receptor is part of a highly conserved omnipresent developmental pathway that has crucial roles in developing and self-renewing tissues. During activation of the signalling pathway Notch binds to its ligand, presented on a neighbouring cell. It is thought that this results in a conformational change within the Negative Regulatory Region (NRR) unmasking a key proteolytic site (S2) and allows for metalloprotease cleavage. This facilitates further cleavage by gamma-secretase, initiating downstream events. Thus far, the molecular mechanism by which the S2 site is revealed has not been defined, though indirect evidence favours a model whereby transendocytosis of the Notch extracellular domain into the ligand bearing cell results in mechanical unfolding of the NRR. Research presented here suggests the NRR of human Notch2 (hN2) unfolds within a mechanosensing force range. Furthermore, through the application of a force (200 pN) the hN2-NRR was shown to unfold sufficiently to expose the S2 site allowing cleavage by metalloproteases. Molecular dynamics (MD) simulations offer insight into the unfolding process of the hN2-NRR, revealing near-sequential unfolding of its constituent LNR and HD domains. Removing the linker region between LNR’s A and B appears to be the first force ‘barrier’ in the unfolding pathway, producing the largest increase in solvent accessibility at the S2 cleavage site. Through docking simulations, this unfolding event was shown to expose the S2 cleavage site sufficiently to allow access to the metalloprotease TACE. Removing coordinated metal ions from the hN2-NRR structure resulted in a dramatic decrease in the forces required for unfolding during AFM experiments, highlighting their role in increasing the resistance of the hN2-NRR to forced unfolding. Removal of disulphide bonds within the structure resulted in a loss of detectable LNR unfolding, highlighting their role in LNR stabilisation.Six HD destabilising mutants, characterised through their role in the hN1 disease, T-cell Acute Lymphoblastic Leukemia, showed three key changes to the unfolding pathway of the hN2-NRR. Firstly, mutants A1647P, L1573P and V1623D showed a dramatic decrease in force required for unfolding in AFM experiments. MD simulations highlighted a lack of force required for the unfolding the LNRA:B linker previously characterised as the key event in removing NRR autoinhibition. Secondly, all the mutants studied here showed changes to the stability of the alpha3-helix (within the HD domain) resulting in transient shifts or bending during unfolding of the LNRA:B linker and the LNRB. Finally, changes were observed within the LNRC of A1647P and L1566P. Within these mutants the LNRC was observed to be unfolding, an event not present during wild-type unfolding. Within mutant L1566P this is thought to be due to the disruption of the conserved salt-bridge occurring between Arg1567 (HD domain) and Asp1506 (LNRC). Within mutant A1647P this is likely due to widespread domain destabilisation. Overall, research presented here has provided the first direct evidence that the NRR is mechanosensing and that mechanical force can allow for cleavage at the S2 site. Further characterisation has been performed to analyse the unfolding pathway through ion chelation, disulphide oxidation and mutagenesis studies.