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Zhu, Jing. "The role of nonmuscle myosin IIA in endothelial cell". Morgantown, W. Va. : [West Virginia University Libraries], 2010. http://hdl.handle.net/10450/11006.
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Thesis (M.S.)--West Virginia University, 2010.Title from document title page. Document formatted into pages; contains viii, 37 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 33-37).
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Stevens, Richard. "Two light chains of the unconventional myosin Myo2p /". Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/9226.
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Guimard, Laurent. "Modélisation et synthèse de peptides interagissant avec une protéine cible : application au complexe calmoduline-RS20". Montpellier 1, 1995. http://www.theses.fr/1995MON1T037.
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Petzoldt, Astrid G. "DE-cadherin regulates unconventional myosin ID through myosin IC in Drosophila melanogaster". Nice, 2009. http://www.theses.fr/2009NICE4048.
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L’établissement précis de l’asymétrie G/D stéréotypée, qui est contrôlé par un programme génétique, est crucial pour le fonctionnement d’un organisme. Ce n’est que depuis récemment que le mécanisme de l’établissement de l’asymétrie G/D est étudié chez l’invertébré Drosophila Melanogaster (Hozumi et al. , 2006 ; Speder et al. , 2006). La Myosine non conventionnelle de type ID (MyoID) a été caractérisée comme un déterminant de la rotation dextrale de la plaque génitale mâme pendant le stade pupal. Afin d’identifier de nouveaux effecteurs de MyoID, nous nous sommes concentrés sur son plus proche homologue, MyoIC, car sa surexpression mime le phénotype perte de fonction de MyoID, ce qui entraîne une inversion de l’axe G/D et une rotation sinistrale des organes génitaux. Nous présentons des évidences que ce situs inversu phénotype es entraîné par l’inhibition de la fonction de MyoID par MyoIC, conséquemment nous définirons MyoIC comme un facteur anti-dextral. Il a été montré que la queue de MyoID pourrait interagir physiquement avec la beta-Catenine, (Speder et al. , 2006). Nos expériences de perte et gain de fonction de la DE-Cadhérine, un composant majeur des jonctions adhérentes, avons révélées une interaction linéaire entre DE-Cadherine et les deux Myosines. DE-Cadhérine est capable de contrôler l’expression de MyoIC en agissant comme inhibiteur de MyoIC. Car la fonctionnalité de MyoID est de réguler par l’expression de MyoIC. MyoIC fonctionne comme médiateur entre DE-Cadhérine et MyoID. Nous proposons un nouveau réseau de régulation pour l’établissement de l’asymétrie G/D dans lequel la DE-Cadhérine affecte l’activité de MyoID à travers la régulation de l’expression de LyoICThe accurate establishment of stereotyped L/R asymetry is subject to a strict genetic program and crucial for the functionality of the organism. It is only recently that the mechanism of L/R asymmetry establishment is exploited in the invertebrate species Drosphophila melanogaster (Hozumi et al. , 2006 ; Speder et al. , 2006). The unconventional type ID myosin (MyoID) has been characterised as a dextral determinant accountable for the clockwise (dextral) rotation of the male genital plate during pupae stage. In our attempt to isolate new components of the L/R mechanism, we first focussed on MyoIC, the closest homologue of genitalia, thus L/R axis inversion. We provide evidence that this situs inversus phenotype is du to an inhibition of MyoID function through MyoIC and consequently define MyoIC as an anti-dextral effector of MyoID. An interaction between MyoID and adherents junctions had been suggested by Speder et al. (2006) as the authors could show by two-hybrid screen and GST pull down that MyoID tail and beta-catenin cal physically interact. Our DE-cadherin loss and gain of function studies revealed a linear interaction between DE-cadherin zand the unconventional myosins MyoID and MyoIC. DE-cadherin controls MyoIC expression, acting as inhibitor of MyoIC. As MyoID functionality is regulated by MyoIC expression, myoIC functions as a mediator between DE-cadherin and myoID. In summary, we present in this study a new regulatory network of L/R asymmetry establishment, where DE-cadherin affects MyoID activity through regulation of MyoIC protein expression
5
Ripoll, Léa. "Role of myosin VI and actin dynamics in membrane remodeling during pigmentation". Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB102.
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Le trafic intracellulaire consiste en la formation et le transport de vésicules ou tubules qui acheminent des composants protéiques et lipidiques entre les différents organites ou avec la membrane plasmique. L’élaboration de ces tubulo-vésicules est initiée par le remodelage local d’une membrane, tout d’abord en générant une courbure puis un bourgeon qui, s’allongeant, forme la tubulo-vésicule. Enfin, la rupture de la membrane, ou scission, libère le transporteur nouvellement formé. Ces étapes repose sur un sculptage profond de la membrane. Ceci requière des forces générées par des moteurs moléculaires, lesquels s’associent aux cytosquelettes comme les microtubules ou les filaments d’actine. Afin de mieux comprendre comment le cytosquelette et leurs moteurs façonnent ces transporteurs, nous avons examiné le rôle de l’actine et de la myosine VI dans la formation de tubules membranaires aux mélanosomes. Les mélanosomes sont des organites apparentés aux lysosomes, générés dans les mélanocytes de la peau et de la choroïde de l’œil, et qui sont le lieu de synthèse et de stockage d’un pigment, la mélanine. Dans l’épiderme, ces compartiments spécialisés évoluent par différentes étapes de maturation qui aboutissent à leurs transferts aux cellules voisines, les kératinocytes. Les mélanosomes sont des organites dynamiques qui reçoivent et recyclent constamment des composants membranaires, comme la SNARE VAMP7. Nous résultats montrent que la myosine VI et son adapteur optineurine se localisent à un sous-domaine spécifique de la membrane des mélanosomes, ou elles contrôlent la scission de tubules. En effet, l’activité motrice de la myosine VI et le réseau d’actine branchée, dépendant des complexes Arp2/3 et WASH, permettent la constriction des membranes du tubule et son détachement du mélanosome. Un défaut de scission de ces tubes engendre des mélanosomes plus pigmentés, enrichis en cargos et au pH plus acide. L’altération de l’homéostasie du mélanosome affecte sa fonction, comme sa capacité à être sécrété et transféré aux kératinocytes. Nos résultats démontrent que la myosine VI en coopération avec le cytosquelette d’actine permet la constriction et fission de membranes aux mélanosomes. Les intermédiaires de transport ainsi formés recyclent des protéines cargos pour leur possible réutilisation, et participent ainsi au maintien de l’homéostasie et de la fonction de ces organitesIntracellular transport among organelles and the plasma membrane occurs through the formation and transport of vesicular and tubular membrane carriers. The formation of these carriers requires first the bending of membrane and the generation of a bud, followed by its elongation to form the tubule-vesicle. Lastly, the carrier is released from the membrane source by the scission of the membrane. Importantly, all these different steps need an accurate orchestration to properly deform the membrane. The actions exerted by molecular motors onto microtubule and actin cytoskeletons provide forces onto membrane that contribute to its remodeling during the biogenesis of carrier. Actin filaments (F-actin) and myosins are thought to participate in the initiation and the fission of carriers. However, the role of actin machinery during carrier biogenesis remains elusive. We thus decided to address the role of F-actin and the actin-based motor myosin VI in the formation of tubular intermediates at melanosome. Melanosomes are lysosome-related organelles of skin melanocytes and eye pigment cells that function in the synthesis and storage of the melanin pigment. Melanosomes originate from endosomes and progressively mature into fully pigmented compartments, which fate is to be secreted and transferred to neighboring keratinocytes. Melanosomes are dynamic organelles that constantly receive, but also recycle proteins such as the SNARE VAMP7 through the formation and release of tubular intermediates. Our work reveals that myosin VI, together with Arp2/3- and WASH-mediated branched actin localize at specific melanosomal subdomains where they promote the constriction and scission of tubular intermediates. This fission event allows the export of components such as VAMP7 from melanosomes and promotes their maturation and subsequent transfer to keratinocytes. Altogether, our results uncover a new role for myosin VI and F-actin in the constriction and scission of membrane tubules at melanosome that is required for organelle homeostasis and function
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Saeki, Nobutaka. "The Function of Myosin IX: the Ninth Class of Myosin Superfamily: a Dissertation". eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/294.
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Among 18 family members in the myosin superfamily, myosin IX is unique by possessing a GTPase activating protein (GAP) for Rho. It is also attention-grabbing since it is a single-headed processive motor, as well as a minus-end directed motor. Although many biochemical properties have been revealed, its physiological function is largely unknown. As an initial step to address this question, I attempted to find the binding partner of myosin IXb using the yeast two-hybrid screen. Through the screen using the tail domain of myosin IXb as bait I found BIG1, a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor (Arfl), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney (NRK) cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by addition of BIG1 with IC50 of 0.06 μM. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with a concentration similar to that which inhibit the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the ArfGEF, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb.
Together, the results imply that the RhoGAP activity of myosin IXb is down-regulated by BIG1 at the Golgi, where myosin IXb could be involved in the regulation of actin cytoskeleton through the Rho-signaling pathway.
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Cartón, García Fernando. "Myosin VB in intestinal pathogenesis". Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/458251.
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Miosina VB es una proteína que actúa como un motor molecular usando la energía del ATP para moverse a lo largo de filamentos de actina. Participa en el trafico intracelular de endosomas de reciclaje en la parte subapical de células polarizadas y no polarizadas. Su expresión es muy abundante en el intestino donde participa en el establecimiento y mantenimiento de la polaridad de los enterocitos. Mutaciones en MYO5B causan la enfermedad de inclusión de microvellosidades, in raro trastorno congénito que afecta a las células epiteliales del intestino cursando con diarrea acuosa persistente que suele ser fatal. Esta enfermedad se caracteriza por la presencia de alteraciones morfológicas en los enterocitos, atrofia de las vellosidades y deslocalización de proteínas del polo apical y basolateral del enterocito. Su patología molecular no se conoce, principalmente por la falta de modelos animales. En el presente estudio, describimos un versátil modelo murino con inactivación constitutiva de Myo5b e inactivación condicional en las células epiteliales intestinales inducida por tamoxifeno. En ambos casos, los animales muestras un cuadro clínico muy semejantes al de los pacientes con enfermedad de inclusión de microvellosidades, presentado diarrea y deshidratación que causan la muerte del animal. A nivel histológico, el intestino muestra las mismas alteraciones en los enterocitos que las presentes en pacientes humanos, incluyendo atrofia de vellosidades y deslocalización de marcadores proteicos. Además, la inactivación de Myo5b también provocó hiperproliferación de las criptas intestinales. Por lo tanto, el modelo animal presentado constituye una herramienta muy útil para investigar las causas moleculares de la enfermedad y ensayar de manera preclínica fármacos u otras opciones terapéuticas. Por otro lado, la pérdida de polaridad y diferenciación es también una de las señas de identidad de los carcinomas metastásicos avanzados y correlaciona con un mal pronóstico de los pacientes. En concreto, para el cáncer colorrectal, investigaciones previas llevadas a cabo en nuestro laboratorio ya han demostrado que la pérdida de miosina IA promueve la progresión la enfermedad y tiene actividad supresora de tumores. Dicha proteína es abundante en el borde en cepillo de los enterocitos, y participa en el mantenimiento de la estructura polarizada. Otros estudios han señalado la relación entre la inactivación de MYO5B con un incremento en la motilidad e invasión de células de cáncer gástrico, aunque todavía no se conoce nada de su relación con en el cáncer colorrectal. Para resolver esta cuestión, hemos diseñado modelos in vitro inducibles por doxiciclina para sobre expresar y reducir la expresión de dicha proteína en líneas celulares de cáncer de colon. Además, se ha empleado la tecnología CRISPR/Cas9 para inactivar la expresión de MYO5B en la línea de cáncer de colon Caco2-BBE. Los resultados muestran cambios en la polarización y diferenciación de dichas líneas celulares, de acuerdo con observaciones previas. También se ha observado una posible relación entre MYO5B y la capacidad de movilidad e invasión de las líneas de cáncer de colon. Sin embargo, la hiperproliferación observada en el intestino de los ratones no se reproduce en las líneas de cáncer de colon empleadas tras reducir o sobre expresar MYO5B, o en modelos xenograft subcutáneos in vivo de dichas líneas. Por otro lado, usando un microarray de tejidos con 155 muestras de tumores primarios de pacientes con cáncer colorrectal en estadio Dukes C se ha comprobado que una reducción en la expresión de MYO5B se asocia con una disminución en el tiempo de recaída y en la supervivencia total de los pacientes de cáncer de colon. Además, tumores con un grado de diferenciación bajo también expresan niveles de MYO5B significativamente reducidos. Finalmente, todos estos resultados indican que MYO5B juega un papel importante en la diferenciación del intestino normal y de las líneas de cáncer de colon. De la misma manera, MYO5B también podría desempeñar un papel en la progresión del cáncer colorrectal promoviendo movilidad e invasión de las células tumorales.Myosin VB is a molecular motor protein that uses the energy of ATP to move along actin filaments. It participates in the recycling endosomes trafficking in the subapical cytoplasmic region of non-polarized and polarized cells. It is highly expressed in the small and large intestine, where its role in the establishment of polarized function in enterocytes is also well known. Inactivating mutations of MYO5B have been associated with microvillus inclusion disease (MVID), a rare congenital disorder of the intestinal epithelial cells that presents with persistent life-threatening watery diarrhea. It is characterized by morphological enterocyte abnormalities such as microvillus atrophy and mislocalization of apical and basolateral protein transporters. The molecular pathology of the disease is not well known mainly due to the lack of animal models. In the present study, we report a versatile murine model with targeted inactivation of Myo5b. This model allowed us to generate and characterized a constitutive Myo5b knockout mice and a tamoxifen-inducible intestinal-epithelium-specific Myo5b knockout. In both cases, the mice closely resemble the phenotype of MVID patients, developing watery diarrhea and dehydration causing the death of the animal. Histological study of the intestine showed all the characteristic enterocyte defects observed in MVID patients, including microvillus atrophy and mislocalization of protein markers. Moreover, the inactivation of MYO5B also originated hyperproliferation of the intestinal crypts. Therefore, our mice constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches. In addition, hyperproliferation as well as loss of cell polarity, differentiation, and tissue architecture are hallmarks of advanced metastatic carcinomas and strongly correlate with poor patient prognosis. Specifically, for colorectal cancer, the third most common type of cancer worldwide, we have previously demonstrated that the loss of brush border MYO1A, also involved in cell polarity, promotes cancer progression and has tumor suppressor activity. Other studies have indicated a relationship between MYO5B inactivation and gastric cancer, promoting invasion and motility, but little is known regarding its role in colorectal cancer. To address this question, we have developed novel doxycycline-inducible in vitro models of MYO5B overexpression and downregulation. Moreover, we have generated MYO5B knockout Caco2-BBE cells using CRISPR/Cas9 technology. Our results showed changes in the polarization and differentiation of colon cancer cells, in agreement with previous observations in the normal intestine. Moreover, we have observed a relationship between MYO5B and the motility and invasion capacity of colon cancer cells, indicating a possible role of MYO5B in colon cancer progression. However, the effect of MYO5B loss in cell proliferation observed in our Myo5b knockout mice could not be confirmed in our models in vitro and in vivo, employing cell line-derived xenografts. In addition, using a tissue microarray containing triplicate samples from 155 primary Dukes C colorectal tumors, reduced MYO5B expression was found to be associated with shorter disease-free and overall survival of the patients. Moreover, poorly differentiated tumors showed significantly reduced expression of MYO5B. Collectively, our results indicate that MYO5B plays an important role in the differentiation of the normal intestinal epithelium and colon cancer cells, as well as a possible role in cancer progression promoting cell motility and invasion.
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Tyrrell, Graham Philip. "Modelling the myosin molecular motor". Thesis, University of York, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247144.
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Thomas, Daniel G. "The self-interaction of myosin". Thesis, University of Leicester, 1992. http://hdl.handle.net/2381/35170.
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The first event in thick filament formation must be the interaction of one myosin monomer with another to give a dimer. The energetics of the parallel apposition of the rod portion of myosin were first considered by McLachlan and Karn (Nature 299: 226-231, 1982; J. Mol. Biol. 164: 605-626, 1983). They applied a simple 'point scoring' algorithm to the periodic charge distribution of the myosin rod and suggested that there are peaks in interaction energy when the stagger between parallel rods is close to 14.3 and 43nm. We have modelled the assembly process on a more detailed basis in an attempt to understand the factors governing the structure of the thick filament. We have taken into account both the hydrodynamic and electrostatic properties of myosin. It is likely that the final state of parallel apposition of the monomers is reached via intermediate states. We identify as a likely intermediate a structure in which two monomers are bound at a single point contact to form an extended dimer, and have computed the likelihoods of formation and the stabilities of different forms of this structure. We have also computed the energetics of the pathways leading to the more stable parallel dimer. Our results suggest that selectivity for a 14.3 nm axial stagger is inherent in the pathway for dimerisation but that it is a consequence of the kinetics rather than the energetics of the assembly process. We have identified rat cardiac myosin at minimal ionic strength as a system in which later steps in the assembly process are blocked; small myosin oligomers become stable structures rather than transient intermediates and so can be trapped and characterised. The structures found are in both qualitative and quantitative agreement with the computational predictions.
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Carrington, Glenn Stuart Peter. "The flexibility of myosin 7a". Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/22504/.
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Myosin 7a is a molecular motor found in hair cells of the ear and the photoreceptor cells of the eye. Myosin 7a is comprised of an actin-binding motor domain, a lever; which is composed of 5 IQ motifs that can potentially bind 5 light chains followed by a single alpha helical (SAH) domain, and a tail composed of 2 MyTH4-FERM domains. The lever is an essential mechanical element in myosin 7a function, but an understanding of its mechanical properties and how these derive from its substructure is lacking. It has been observed in vitro that myosin 7a is able to regulate its activity through a head-tail interaction. How the flexibility of the sub-domains of the lever allows the molecule to fold up is not completely understood. To address this, the first aim of this study was to look for evidence of novel light chain binding partners in myosin 7a, which revealed calmodulin to be the preferred light chain. My second aim was to study the structure and flexibility of the lever of full-length myosin 7a using single-particle image processing of images from negative stain electron microscopy (EM). Image averaging revealed the lever to be much shorter than expected. Additionally, there was evidence of thermally-driven flexing at the motor-lever junction. A stiffness of 78 pN.nm.rad-2 for the flexing was inferred, which represents a significant compliance in the head. An investigation into lever bending analysis, by monitoring the decay of tangent-tangent correlations of the lever shapes, yielded a persistence length of 38 ± 3 nm. Finally, long time molecular dynamics (MD) simulations were compared with a novel coarse-grained (CG) simulation technique called Fluctuating Finite Element Analysis (FFEA), which treats proteins as visco-elastic continua subject to thermal noise to probe the flexibility of myosin 7a. FFEA allows sufficiently long time simulations that are computationally less expensive than corresponding all-atom MD simulations to allow myosin 7a to explore its full range of configurations. Extraction of flexibility data from all-atom MD simulations calculated the bending stiffness of the SAH domain to be 60.5 pN.nm2, with reasonable overlap of the major modes of motion between the all-atom and CG simulation types.
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Bennett, Andrew John. "Regulatory light chains of myosin". Thesis, University of Leicester, 1985. http://hdl.handle.net/2381/35131.
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Scallop myosin is unusual in that the regulatory light chains (RLC) can be reversibly dissociated on removal of divalent metal ions. In this thesis the mechanism of the RLC dissociation was studied using several approaches. The dissociation of divalent metal ions from the non-specific divalent metal ion binding site of the scallop RLC was followed, either by a pH indicator method, or by a Mn2+ displacement method. The binding and release of the RLC itself, from the heavy chain/essential light chain complex was followed using the fluorophare 8-anilino-1-naphthalene sulphonate, which was found to be specific for the RLC binding site. The evaluation of the mechanism was greatly aided by the serendipitous discovery, that Mercenaria RLC bound to the scallop heavy chain/essential light chain complex in the absence of divalent metal ions. This allowed RLC exchange experiments to be performed which suggested the nature of the proposed mechanism. The dissociation of the RLC from the scallop heavy chain/ essential light chain complex, is largely explained by a refractory state mechanism. In this mechanism, the heavy chain/essential light chain complex is envisaged to exist in two forms, a nascent state which occurs immediately after RLC dissociation and a refractory state which is favoured on a long term basis. The formation of this refractory state, is the driving force for the net dissociation of the RLC from scallop myosin and subfragments. Differences in the degree of RLC dissociation (HMM cf S1 +rd) on addition of EDTA are explained by a change in the equilibrium constant for the refractory state transition. The mechanism is discussed with reference to existing structural information on RLC denuded myosin.
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Phillips, Kelli R. "Characterization of myosin I in the inner ear". Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5140.
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Thesis (Ph. D.)--West Virginia University, 2007.Title from document title page. Document formatted into pages; contains vii, 114 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
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Schlott, Sebastian. "Proteinbiochemischer Nachweis der Exprimierung von Myosin und Myosin-Light-Chain-Kinase in Trabekelwerkszellen des Auges". [S.l.] : [s.n.], 2004. http://www.diss.fu-berlin.de/2004/207/index.html.
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Harris, Juliette May. "Gene targeting of a fast myosin promoter in muscle cells to alter myosin expression patterns". Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286625.
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Cunningham, Cynthia A. "Induction of myosin cross-reactive antibody and cytolytic T cell responses in mice with Streptococcus pyogenes". Morgantown, W. Va. : [West Virginia University Libraries], 2000. http://etd.wvu.edu/templates/showETD.cfm?recnum=1530.
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Singh, Rohit Rajendraprasad. "Stability of Myosin Subfragment-2 Modulates the Force Produced by Acto-Myosin Interaction of Striated Muscle". Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1062860/.
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Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted in a flexible myosin S2 coiled coil. The binding of MyBPC and stabilizer peptide respectively, resulted in 3.35 and 1.5 times increase in force required to uncoil the myosin S2, while the binding of destabilizer peptide resulted in 1.6 times decrease in force required to uncoil the myosin S2. The myofibrillar contractility assay was performed to test the effect of synthetic myosin S2 binding proteins on the sarcomere shortening in myofibrils. The stabilizer peptide resulted in decreased sarcomere shortening of myofibrils as a result of decreased acto-myosin interaction, on the other hand, the binding of destabilizer peptide caused an increase in sarcomere shortening. The in vitro motility assay was performed to test the effect of altered stability of myosin S2 by binding of these myosin S2 binding proteins on the motility of actin filaments sliding over myosin. The motility of actin filaments was hindered by treating myosin thick filaments with whole length skeletal MyBPC or by treating heavy meromyosin with stabilizer peptide, while the motility of actin filaments was enhanced when heavy meromyosin was treated with destabilizer peptide. This study demonstrates that the myosin S2 coiled coil stability influences the force produced by acto-myosin interaction in striated skeletal muscle. The myosin S2 coiled coil when stabilized by MyBPC and stabilizer peptide resulted in decreased force production by reduced acto-myosin interaction. While the binding of destabilizer resulted in a flexible myosin S2 coiled coil and increased force production by enhanced acto-myosin interaction. The potentially cooperative response of contractility to the instability of the S2 coiled coil promises that this biological mechanism may be the target of drugs to modulate muscle performance.
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McMichael, Brooke Kristin Trinrud. "Tropomyosin 4, myosin IIA, and myosin X enhance osteoclast function through regulation of cellular attachment structures". Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view.cgi?acc%5Fnum=osu1206052974.
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Davies, J. R. "Fish myosin stability and habitat temperature". Thesis, University of Nottingham, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234661.
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Oke, Olusola Adetayo. "Electron Microscopy of myosin V molecules". Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405799.
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Stevenson, Olivia. "Investigating myosin kinetics using optical tweezers". Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416433.
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Messer, Neil Gavin. "Protein engineering of myosin light chains". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316757.
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Knight, Alexander Edward. "The diversity of myosin-like proteins". Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337071.
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Warner, Claire Louise. "Myosin VI at the Golgi complex". Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619755.
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Lister, Ida Margaret Bonnevie. "Myosin VI : relating motility to function". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620603.
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Tesic, Ivan. "Myosin light chain kinases in Drosophila /". The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486462702466832.
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Lawson, Christopher Peter Abiodun Tevi. "The development of novel myosin inhibitors". Thesis, University of St Andrews, 2011. http://hdl.handle.net/10023/2123.
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This thesis describes a structure activity relationship (SAR) study on the recently discovered small molecule tool blebbistatin (S)-21 with particular emphasis on the development of novel synthetic protocols suitable for the rapid synthesis of libraries of blebbistatin analogues. These analogues are potentially of use as novel myosin inhibitors Chapter 1 introduces the concept of chemical biology with particular emphasis on chemical genetics. This approach has rekindled the search for new chemical tools for the investigation of biological systems. The success of blebbistatin (S)-21, which was identified in a chemical genetic study, as a research tool was also discussed. The link between several myosin classes and genetic diseases such as coeliac disease, Crohn’s disease, deafness, dermatitis, familial hypertrophic cardiomyopathy, Griscelli disease and ulcerative colitis indicate that potent inhibitors which show selectivity towards specific myosin isoforms may be of great value as tools for the study of these conditions. The plan for the SAR study around (S)-21 was outlined. Chapter 2 describes the studies undertaken to develop an efficient synthetic route to N1-alkyl analogues of (S)-21 suitable for the parallel synthesis of chemical collections. These studies culminated in the synthesis of an intermediate (S)-66 from which novel N1-alkyl analogues were synthesised. The biological evaluation of these N1-alkyl analogues was discussed. Chapter 3 describes the development of a protocol suitable for the parallel synthesis of collections of N1-aryl analogues of (S)-21 via the intermediate 66. The application of this protocol to the synthesis of a collection of racemic N1-aryl and heteroaryl analogues of (S)-21 and their biological evaluation was presented. Chapter 4 describes the successful rational design and synthesis of a novel fused thiophene ring containing inhibitor of myosin II. The structure of this compound was proposed by modelling of the existing co-crystal structure of (S)-21 bound to the metastable state of Dictyostelium discoideum myosin II (S1dC) and sought to optimise the π-π stacking interaction displayed by (S)-21 with the tyrosine 261 residue within its binding site. The biological evaluation of this novel analogue was discussed. In Chapter 5 the studies conducted to investigate the contribution of ring-C to the binding affinity of (S)-21 were described. The development of alternate routes to (S)-21, in an attempt to avoid difficulties experienced during the synthesis of some analogues of (S)-21, are described. The synthesis and biological investigation of the fluorescent dye PPBA whose binding site has been suggested to overlap with that of (S)-21 was also reported.
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Khan, Protiti. "The Role of Myosin Light Chain Kinase and Non Muscle Myosin II In Ras Signaling to ERK". Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_theses/177.
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We have previously reported that non-muscle myosin II (NMMII) and myosin light chain kinase (MLCK) are required for oncogenic Ras signaling to ERK in Ki-Ras transformed rat fibroblsasts (Helfman and Pawlak, J. Cell Biochem. 95(5), 1069-80, 2005). Here I examine if MLCK plays a role in ERK signaling in various tumor derived human epithelial cell lines. I also determined whether genetic inhibition of NMMII isoforms IIA and IIB, or MLCK also inhibits ERK activation in the MCF 10A human breast epithelial cell line expressing oncogenic H-Ras. Inhibition of MLCK by pharmacological inhibitors such as ML-7 and ML-9 was used to determine the role of MLCK in ERK signaling in an array of H/K-Ras transformed and tumor derived cell lines: T-24 bladder carcinoma, HCT 116 colon carcinoma, and MCF 10A Ras breast cancer cells. Genetic inhibition was carried out using specific siRNA targeted towards MLCK and NMMIIA or IIB. The knock down of NMM IIA and IIB did not inhibit active ERK, which suggested either a redundant function of NMM IIC or an alternate substrate for MLCK. Inhibition of MLCK by ML-7/ML-9 reduced activated ERK in all H/K-Ras transformed, or human tumor derived cell lines we tested. The possible mechanism of how MLCK could play a role in ERK signaling was tested by co-immunoprecipitation (co-IP) of MAPK scaffolding proteins with MLCK. That the ERK scaffold KSR1 regulates ERK signaling in MCF 10A Ras, was demonstrated through inhibition of KSR1 with siRNA. Moreover, KSR was shown to interact with MLCK because it was found to co-precipitate with MLCK.
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McNamara, James W., Amy Li, Nicola J. Smith, Sean Lal, Robert M. Graham, Kristina Bezold Kooiker, Dijk Sabine J. van, Cristobal G. dos Remedios, Samantha P. Harris i Roger Cooke. "Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes". ELSEVIER SCI LTD, 2016. http://hdl.handle.net/10150/621325.
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Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.
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Ruff, Christine. "Funktionsanalyse einzelner Motormoleküle mittels der kombinierten Mikronadel-Laserfallen-Technik". [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=962781282.
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Zhang, Junshan. "New model systems for the study of myosin-V mediated transport on biofunctionalized surfaces". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973923458.
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Yu, Cong. "Structural and functional characterization of the myosin VI tail /". View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BICH%202009%20YU.
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Bhat, Alka. "Les dynamiques d'agrégats de myosine et leurs rôles dans les fermetures d'epithelia". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ070.
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Les agrégats de myosine ont été observés dans plusieurs systèmes : chez la Drosophile, C. elegans, ou encore dans des expériences avec des cytosquelettes reconstitués in vitro. Cependant, leur intégration dans un cadre général manque. En théorie, les filaments d’actine et les moteurs de myosine suivent des lois génériques d’auto-organisation. Des découvertes récentes du laboratoire montrent que la dynamique de ces agrégats à l’intérieur d’un anneau de cytokinèse sont en effet associés à des fonctions biologiques : la génération de stress lorsque leurs mouvements sont radiaux, et au transport lorsque leurs déplacements sont tangentiels. Dans ce travail de thèse, nous montrons que ces règles simples sont conservées lors du processus de cicatrisation par l’anneau d’acto-myosine dans les monocouches de cellules épithéliales. En utilisant la microfabrication, la biologie cellulaire, l’imagerie quantitative et la physique théorique, nous établissons que les agrégats radiaux et tangentiels sont respectivement associés à une fermeture locale ou à un arrêt de l’anneau. La conservation de ce mécanisme entre les systèmes uni- et multi-cellulaires suggère que la dynamique de ces agrégats de myosine pourrait être utilisée comme la lecture générique pour cartographier et prédire les changements de formes des embryons en développementMyosin clusters have been reported in a variety of systems, such as Drosophila, C. elegans, and acto-myosin in vitro assays. However, their integration in a general framework is still lacking. In theory, actin filaments and myosin motors are predicted to follow generic rules of self-organisation. Recent findings from the laboratory reported that cluster dynamics within cytokinetic rings are associated with biological functions, i.e. stress generation when radial, and transport when tangential. In this study, we show that these simple rules hold as well for acto-myosin ring wound closure in epithelial monolayers. By using microfabrication, cell biology, quantitative imaging and theoretical physics, we report that radial and tangential clusters are related to local closures and stalled portions of rings, respectively. This conserved mechanism between single and multi-cellular system suggests that these myosin clusters dynamics could be used as generic read-out for mapping and predicting changes in shapes in developing embryos
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Hall, Nakiuda M. "The Relationship of Force on Myosin Subfragment 2 Region to the Coiled-Coiled Region of the Myosin Dimer". Thesis, University of North Texas, 2011. https://digital.library.unt.edu/ark:/67531/metadc103322/.
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The stability of myosin subfragment 2 was analyzed using gravitational force spectroscopy. The region was found to destabilize under physiological force loads, indicating the possibility that subfragment 2 may uncoil to facilitate actin binding during muscle contraction. As a control, synthetic cofilaments were produced to discover if the observations in the single molecule assay were due to the lack of the stability provided by the thick filament. Statistically, there was no difference between the single molecule assay data and the synthetic cofilament assay data. Thus, the instability of the region is due to intrinsic properties within subfragment 2.
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Soudan, Rahaf. "Bestämning av myosin ATPas med NADH-kopplade mätsystem jämfört med in vitro motilitet med isolerat myosin och aktin". Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-104931.
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SammanfattningSyftet med denna studie var att jämföra NADH-kopplade mätsystem och in vitro motilitets-analys (IVMA) för att bestämma aktiviteten hos isolerat myosin. Från NADH-kopplade analysmätningar bestämdes tre parameter: den maximala hastigheten med vilken myosin hydrolyserar ATP i frånvaro av F-aktin (V0), den maximala ATPas-hastigheten för myosin i närvaro av mättande aktin (kcat) och den koncentration av aktin som behövs för att nå halv maximal aktivering av myosin ATPas-aktivitet (KATPas). Från in vitro-motilitets-analys (IVMA) bestämdes två parametrar: fraktion av rörliga filament (FMF) och totala antalet rörliga filament (TMF). Från detta kunde vi uppskatta den fraktion av aktiva huvuden i myosinpreparationer som behövs för en lyckad IVMA.Myosin är ett protein som tillsammans med aktin är ansvarigt för muskelkontraktionen. I denna studie används två myosin preparationer (HMM-fragment) som vi betecknade ”bra HMM” och ”dåligt HMM” på grund av deras kvalitet för aktin motilitet. Först mättes ATPas-aktiviteten hos myosinmotorer med hjälp av ett NADH-kopplat mätsystem som bygger på övervakning av förändringen i absorbans av NADH. Därefter bestämdes V0, kcat, och KATPas för aktin-beroende av myosin-ATPast genom att mäta myosinaktivitet vid olika aktinkoncentrationer, följt av anpassning av data till Michaelis-Menten ekvationen.Parallellt utfördes IVMA-studier genom att HMM immobiliserades på ett objektglas som derivatiserats med trimetylklorsilan. Sedan observerades när HMM flyttar fram fluorescensmärkta aktinfilament i närvaro av ATP. Under samma förhållanden gav resultaten för basalt myosin ATPas aktivitet V0 värden som var ~0,03 ATP s-1 myosinhuvud -1for både dåligt och bra HMM. I en jämförelse mellan de två HMM vid olika F-aktin-koncentrationer var hastighet i ATP-förbrukningen högre för bra än för dåligt HMM. Anpassning av data till Michaelis-Menten-ekvationen gav kcat på 7,18 ATP s-1myosinhuvud-1för dåligt HMM jämfört med 11,21 ATP s-1 myosinhuvud-1för bra HMM (35 % högre). KATPas (Km) för dåligt HMM var lite högre jämfört med den för bra HMM. Vid IVMA-studierna var FMF och TMF 80 % respektive 98 % lägre för dåligt än bra HMM. Slutsatsen var att de två metoderna karakteriserar HMM-funktionen på olika sätt och med olika känslighet. Om man antar att bra HMM har nästan 100 % aktiva huvuden och eftersom man vet att uppmätt kcat är direkt proportionellt mot antalet aktiva myosinhuvuden ser man från dessa mätningar att mycket mer än 65 % av totalt myosin måste vara aktivt för att ge god aktinmotilitet i en IVMA.
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Biswas, Anindita. "Analysis of motor activity of recombinant myosin-1c". Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5522.
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Thesis (Ph. D.)--West Virginia University, 2007.Title from document title page. Document formatted into pages; contains xi, 82 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Paduano, Vanessa. "Regulation of Myosin-II activation and planar polarity during epithelial morphogenesis in Drosophila embryo". Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4102.
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Les épithéliums jouent le rôle de barrière physique et chimique chez les Métazoires. Les épithéliums subissent des remodelages pendant l’embryogénèse. La morphogénèse des tissus est dirigée par des déformations cellulaires coordonnées fonctionnant grâce à des réseaux contractiles intracellulaires constitués d’actine et de myosine. Ce réseau d’actomyosine peut être soit pulsatile, soit stable. Un exemple est l’élongation de l’ectoderme ventro-latéral par intercalation cellulaire, le long de l’axe antéro-postérieur (AP) de l’embryon de la Drosophile. Les jonctions parallèles à l’axe dorso-ventral (DV) rétrécissent et forment de manière irréversible de nouvelles jonctions parallèles à l’axe AP. Des pulsations de myosine-II (Myo-II) médio-apicale se déplacent de manière anisotrope vers les jonctions parallèles à l’axe DV. Ceci provoque le rétrécissement graduel des jonctions, rétrécissement stabilisé par une population de Myo-II polarisée dans le plan du tissu et enrichie au niveau de ces jonctions. Les mécanismes cellulaires qui régulent la pulsatilité, la stabilité et la polarité de la Myo-II restent à élucider. Lors de ma thèse, j’ai identifié de nouveaux effecteurs régulant l’activation et la polarité planaire de la voie Rho1-Rok-Myo-II aux niveaux des jonctions. J'ai d'abord caractérisé le rôle de la kinase Misshapen dans l’activation polarisée de la voie Rho1 au niveau des jonctions. Misshapen agit en aval de la signalisation GPCR afin de favoriser l’activation de Rho1 et contrôle la polarisation de cette activation en transmettant l’information des récepteurs Toll. Puis j'ai identifié Pebble comme la RhoGEF régulant Rho1 et l'accumulation de Myo-II aux jonctionsEpithelial build up strong mechanical and chemical barriers in Metazoans. Epithelia can be dramatically remodeled during embryogenesis. Tissue morphogenesis is driven by coordinated cellular deformations which are powered by intracellular contractile networks constituting actin and Myosin. Actomyosin networks can either be pulsatile or stable. One example is the elongation of the ventral-lateral ectoderm by cell intercalation, along antero-posterior (AP) axis of Drosophila embryo. Junctions parallel to the dorso-ventral (DV) axis shrink and form new junctions along AP axis. Medial apical Myosin-II (Myo-II) pulses flow anisotropically towards junctions aligned in DV axis, resulting in steps of junction shrinkage which are stabilized by a planar-polarized pool of Myo-II enriched at these junctions. Sequential deformation and stabilization drive irreversible tissue deformations akin to a ratchet. The cellular mechanisms that regulate Myo-II pulsatility, stability and polarity remained to be unfurled. During my PhD, I identified new regulators for Rho1-Rok-Myo-II pathway at junctions, and Myo-II planar polarity. On the one hand, I characterized the function of Misshapen kinase in polarized activation of Rho1 pathway at junctions. Misshapen acts downstream GPCR signaling to enhance Rho1 activation, and controls the polarization of this activation by transducing information from Toll receptors. Also, I identified Pebble as RhoGEF regulating Rho1 at junctions and Myo-II accumulation
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Choi, Myoung Soo. "Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly". University of Toledo / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532947508319487.
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Macaya, Erro Irati. "Role of myosin VI in colorectal cancer". Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/664267.
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La miosina VI (MYO6) es un motor molecular que puede anclar moléculas o proporcionar tráfico de corto alcance a lo largo del citoesqueleto de actina valiéndose de la energía obtenida de la hidrólisis de ATP. La miosina VI se mueve hacia el extremo negativo de los filamentos de actina, en dirección opuesta al resto de miosinas. Se ha demostrado que la función de transporte de la miosina VI está implicada en varios procesos celulares, como la endocitosis, el tráfico endocítico y de reciclaje de vesículas, la autofagia, la exocitosis y la transcripción nuclear; y su función de anclaje se ha visto involucrada en el mantenimiento de la estructura del complejo de Golgi, de las estereocilias de las células ciliadas cocleares, de la membrana de las microvellosidades de los enterocitos, y en el mantenimiento de las uniones adherentes. La miosina VI se expresa en la mayoría de los tejidos, pero sus niveles de expresión varían considerablemente entre ellos. Además, la miosina VI puede existir en varias isoformas que se expresan diferentemente dependiendo del tipo de tejido. Estas isoformas interactúan con diferentes moléculas y, por lo tanto, llevan a cabo diferentes funciones. La miosina VI está altamente expresada en las células epiteliales del intestino delgado y grueso, estando principalmente localizada en la membrana apical y en la basolateral, aunque también está presente de forma difusa en el citoplasma. La miosina VI ha sido relacionada con determinados tipos de cáncer. En el cáncer ovárico, el de próstata y en la leucemia linfoide, se ha visto que la miosina VI está sobre-expresada, donde se le atribuye un papel en la migración y la invasión. Otros estudios también han asociado la sobre-expresión de la miosina VI con una mayor proliferación celular en cánceres como el hepático, el de mama y el de pulmón. Sin embargo, se sabe poco sobre el papel de la miosina VI en el cáncer colorrectal, el tercer tipo más común de cáncer en el mundo. En este estudio, mediante el uso de un ‘tissue microarray’ (TMA) que contenía muestras de tejido normal de colon, tumores colorrectales primarios de estadio Dukes' C y muestras de metástasis de ganglios linfáticos, se observó que la expresión de MYO6 se reduce o se pierde con frecuencia en tumores primarios en comparación con la expresión de células epiteliales normales de colon; y que incluso aún se reduce más en las metástasis de los ganglios linfáticos, en comparación con los tumores primarios. Además, en pacientes con cáncer colorrectal localmente avanzado, la baja expresión de MYO6 en el tumor primario se asoció con una supervivencia general y una supervivencia libre de enfermedad más cortas. Para poder estudiar el posible papel de la miosina VI en el cáncer colorrectal, se diseñaron modelos celulares isogénicos inducibles por doxiciclina para reducir la expresión de MYO6. Los resultados mostraron que la inactivación de MYO6 no altera la capacidad de diferenciación/polarización de las células de cáncer de colon. Tampoco la capacidad de migración y de invasión in vitro, ni la habilidad de metastatizar en modelos de ratón, se vieron afectadas. Sin embargo, se observó que la reducción de la expresión de MYO6 incrementaba el crecimiento de las células de cáncer de colon en un modelo de xenoinjertos subcutáneos en ratones inmunodeficientes NOD/SCID, a pesar de no provocar ningún cambio en el crecimiento in vitro. Por otro lado, la ausencia de MYO6 en ratones ApcMin/+ o en los tratados con AOM, no aceleró la tumorigénesis intestinal. Colectivamente, estos resultados indican que la pérdida de MYO6 es importante para promover el crecimiento de las células de cáncer colon, lo que indica un posible papel supresor tumoral de MYO6 en el cáncer colorrectal.Myosin VI (MYO6) is a molecular motor that can provide short range trafficking along the actin cytoskeleton by using the energy from ATP hydrolysis or anchor cargoes to actin filaments. Myosin VI moves towards the minus-end of actin filaments, in the opposite direction to the rest of myosins. The trafficking function of myosin VI has been shown to be involved in several cellular processes, such as, endocytosis, endocytic trafficking and recycling of vesicles, autophagy, exocytosis and nuclear transcription; and its anchoring function has been shown to be involved in the maintenance of Golgi complex, cochlear hair-cell stereocilia, enterocytic brush border membrane and adherens-junctions. Myosin VI is widely expressed in most tissues, but its expression levels vary considerably among them. Moreover, myosin VI can exist in several isoforms that are expressed in a tissue-specific manner. The isoforms interact with different binding partners and thus, carry out different functions. Myosin VI is highly expressed in the epithelial cells of small and large intestine, being mainly localized to the apical and basolateral membranes, although it is also present diffusely in the cytoplasm. Myosin VI has been involved in certain types of cancer. In ovarian carcinoma, prostate cancer and lymphoid leukemia myosin VI is found overexpressed with a role in migration and invasion. Other studies have also associated MYO6 overexpression with an increased cell proliferation in cancers such as, hepatocellular carcinoma, breast cancer and lung cancer. However, little is known about the role of myosin VI in colorectal cancer, the third most common type of cancer worldwide. In this study, using a tissue microarray containing samples from normal colon tissues, primary Dukes’C colorectal tumors and lymph node metastases, MYO6 expression was observed to be frequently reduced or lost in primary tumors compared to normal colonic epithelial cells. Moreover, MYO6 levels were further reduced in regional lymph node metastases compared to primary tumors. Importantly, low tumor MYO6 expression was associated with shorter overall and disease-free survival in patients with locally advanced colorectal cancer. Therefore, to study the possible role of MYO6 in colorectal cancer, isogenic cell line systems with doxycycline-inducible MYO6 downregulation were engineered. The results showed that MYO6 inactivation does not alter the differentiation/polarization capability of colon cancer cells. The migration and invasion ability in vitro and the potential to metastasize in an in vivo mouse model were neither altered by MYO6 knockdown. Nevertheless, although no changes were observed in the growth in vitro, MYO6 inactivation increased the growth of colon cancer cells in a subcutaneous xenograft model in immunodeficient NOD/SCID mice. On the other hand, absence of MYO6 in ApcMin/+ or AOM-treated mice did not accelerate intestinal tumorigenesis. Collectively, our results indicate that loss of MYO6 provides a significant growth advantage to colon cancer cells, indicating a possible tumor suppressor role of MYO6 in colorectal cancer.
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Heißler, Sarah Maria [Verfasser]. "Funktionsanalyse menschlicher Myosin-Motordomänen / Sarah Maria Heißler". Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2013. http://d-nb.info/1032719583/34.
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Smith, R. C. "The regulation of thymus myosin filament assembly". Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372901.
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Batters, Christopher. "Single molecule mechanical studies of acto-myosin". Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414015.
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Smith-Palmer, Jayne. "Studies on minus end directed myosin motors". Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442658.
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Ezeigwe, Ifeoma Daramfon. "Drosophila myosin VI function in dorsal closure". Thesis, University of Kent, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589941.
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Myosin VI is a minus-end directed actin-based motor protein. In vertebrate cells, myosin VI is of importance in endocytic and exocytic-membrane trafficking pathways, and in regulating Golgi morphology. Myosin VI is also implicated in genetic diseases and is transcriptionally regulated upon DNA damage in a p53-dependent manner and shown to be over-expressed in cancer types. In Drosophila, myosin VI encoded by the jaguar (jar) gene is implicated in a range of cellular processes including dorsal closure. Although understanding of its cellular function is developing, little is known about the mechanisms regulating myosin VI. In vitro studies on vertebrate myosin VI have demonstrated that it is phosphorylated within its motor domain by the serine/threonine kinase P AK and that alters its function in a similar manner to that which occurs in myosin I. More importantly,jar has a Pak phosphorylation site in the same location as the vertebrate myosin VI. Expression of either a dominant negative Jar or a dominant negative Pak causes if not exact, overlapping dorsal phenotypes in Drosophila. The aim of this study was to investigate a working model that Jar functions during dorsal closure are dependent on Pak-mediated phosphorylation. During my thesis research a published report concluded that jar is required but not essential for Drosophila development as 40% null jar mutants survived. On the contrary, I report here that jar is of importance for development as RNA i-mediated knockdown of Jar protein level is lethal and is found to cause abnormal dorsal closure. Further delineation of jar322 mutant allele showed varied phenotypes including wing defects that implicates jar in Notch signalling and in integrin function and signalling. Importantly, I found that Jar is directly regulated by JNK transcriptional activation pathway and down-regulated by the RhoGTPase pathway. Together this work emphasises the multi functional nature of myosin VI. Further the work suggests that the pro-survival function of vertebrate myosin VI is conserved in Drosophila.
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Tacon, Daryl. "Expression, Purification & Characterization of Myosin 9b". Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503348.
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May, Karen Marie. "Molecular characterisation of fission yeast myosin II". Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263251.
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Rowe, Tony. "Molecular dissection of myosin light chain function". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282103.
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Kambara, Taketoshi. "Myosin IX: A Single-Headed Processive Motor". Digital WPI, 2005. https://digitalcommons.wpi.edu/etd-dissertations/310.
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"The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed†myosin unlike other processive myosins which have “double-headed†structure. It has been thought that “processive†motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX."
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Roberts, Rhys Clwyd. "Myosin VI and its intracellular binding partners". Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620626.
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Jackson, Andrew Paul. "The mechanism of the scallop myosin ATPase". Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35258.
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Molluscan adductor muscles display thick filament regulation. A calcium binding site (regulatory domain) near the neck of the myosin molecule is responsible for controlling the ATPase activity, hence the rate of contraction. In the absence of calcium, the ATPase activity is highly suppressed. When calcium binds to the regulatory domain the inhibition is relieved allowing contraction to occur. Steady-state measurements are insufficient to characterise the ATPase activity in detail because of the dominant contribution from unregulated myosin molecules. Therefore spectoscopic techniques, allied to transient kinetic analysis were used to determine the effect of calcium on the various steps of the HMM ATPase mechanism. ATP, ADP and calcium binding to HMM caused small, but measurable, enhancements (upto 8%) in the proteins tryptophan fluorescence. Stopped-flow fluorescence spectroscopy allowed the kinetics of binding and dissociation to be elucidated. Calcium bound to, and dissociated from, HMM rapidly (108 M-1 S-1 and 25 S-1 respectively). When calcium was bound to the regulatory domain the affinity of the active site for nucleotide was reduced, an effect seen as an increase in the rate constant for nucleotide dissociation. Fluorescent ATP analogues, based on formycin were synthesised. These nucleotides displayed large fluorescence enhancements on binding to HMM (upto 500%). Turnover of FTP by HMM was suppressed 100-fold by the removal of calcium, as determined by transient kinetic measurements. The large fluorescence enhancements seen on binding of various formycin nucleotides allowd the effect of calcium on the association and dissociation processes to be examined in great detail. Binding was found to be a complex, multistep process in which the presence of calcium increased the rate of interconversion of the various HMM/nucleotide complexes by several orders of magnitude.
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O'Neill, Stephen Charles. "Myosin and electrophysiological heterogeneity in cardiac muscle". Thesis, Connect to e-thesis, 1987. http://theses.gla.ac.uk/1012/.
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