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No description available.

No description available.

Every day, general anaesthetics are given to a large number of patients around the world but the cellular mechanisms of how anaesthetics act are still not clear. General anaesthetics cause the intended unconsciousness, amnesia and immobility in patients, but also side effects such as a decrease in mean arterial pressure and arrhythmia, both of which contribute to complications such as heart damage and stroke. With more knowledge of the mechanism of anaesthetic drugs, these complications could be reduced. It has been shown that anaesthetics cause a disruption of the thalamocortical connectivity and brain network connectivity. How the network communication is disrupted however is not known. Propofol and thiopental are both intravenous anaesthetic drugs used widely in clinical anaesthesia. They bind to the GABAA receptor and enhance its function. The cytoskeleton helps the cell to maintain its shape and participate in cellular movement and transport. Cellular transport to and from a neuron’s cell body and periphery is performed by motor proteins that move vesicles, organelles and proteins along cytoskeletal tracks. We have previously shown that propofol causes a reorganisation of the cytoskeleton protein actin in neurons, but we were further interested to study the effects of propofol and thiopental on the cytoskeletal function of cultured cortical rat neurons. Our results show that propofol and thiopental cause neurite (axon and dendrite) retraction. Propofol’s effects were time- and dose-dependent, and can be reversed when propofol is removed. We were able to inhibit propofolinduced neurite retraction if we stabilised actin by blocking either the motor protein myosin II or the GABAA receptor. We have previously shown that a small GTP-binding protein, RhoA, inhibits propofol-caused actin reorganisation. Propofol-induced neurite retraction was mediated via a downstream effector of RhoA, ROK, which induces phosphorylation of the myosin light chain and increases contractility. Furthermore, we have shown that propofol causes a switch from anterograde to retrograde transport and increases the average velocity of the moving vesicles in neurites. The propofol induced retrograde vesicle transport was GABAA receptor-mediated. Orexin A is a neuropeptide which regulates the sleep/awake cycle and has also been shown to reduce anaesthesia in animals when given intracerebroventricularly. We found that orexin A reverses propofol and thiopental-induced neurite retraction and actin reorganisation. Moreover, we have shown that the orexin A inhibition of propofol-induced neurite retraction is mediated via the PLD/PKC intracellular signalling pathway. Propofol and thiopental decreased the tyrosine phosphorilation of the intermediate cytoskeletal protein vimentin which is reversed by orexin A. Taken together, these results suggest that propofol causes a time- and dose-dependent, reversible and GABAAreceptor-mediated neurite retraction in cultured cortical rat neurons. Propofol also causes a switch from anterograde to retrograde vesicle transport in neurites. Orexin A reverses propofol and thiopental-induced neurite retraction and cytoskeletal reorganisation. Orexin A inhibits propofol-induced neurite retraction via the PLD/PKC intracellular signalling pathway.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 71-88).

Embryogenesis was observed through light and transmission electron microscopy to elucidate the role of the cytoskeleton in the cellularization of the coenocytic endosperm in Helianthus annuus. Microtubules were not seen throughout the development of the endosperm despite the fact that nuclear division occurred frequently. The positioning of organelles in the cytoplasm in close proximity to the developing cell plate as well as the crooked appearance of the growing endosperm cell wall suggests that a traditional microtubular system, in which a phragmoplast controls wall formation, is not present in this species. The growth patterns exhibited by endosperm walls, similar to that of tip growing cells, may indicate that actin filaments are controlling the cellularization of the endosperm. To further investigate the role of microtubules in the development of the endosperm of H. annuus, inhibitory studies using 100 µM, 300 µM and 1 mM colchicine were conducted on ovules both in-vivo and in-vitro. Enlarged nuclei, partial collapse of the integumentary tapetum, and loss of organelles were observed in colchicine treated cells which indicates that colchicine affected the development of the endosperm. However, since little difference was seen between the various concentrations of colchicine and the cellularization of the endosperm was still observed in treated cell, this may indicate that either colchicine did not affect the cytoskeleton controlling endosperm development or that it did not successfully penetrate the embryo sacs. The minor effect seen by the application of colchicine may support the idea that actin plays a role in the cellularization of endosperm in H. annuus. / Thesis (Master, Biology) -- Queen's University, 2008-11-03 10:18:50.557

Interactions Between the Actin Cytoskeleton and Anthrax Toxin Receptor 1 (ANTXR1) Kristopher M. Garlick, Doctor of Philosophy, 2012, Department of Laboratory Medicine and Pathobiology, University of Toronto ABSTRACT Anthrax is caused by the bacterium Bacillus anthracis, which secretes three proteins that assemble to form a toxic complex at the host cell surface. These proteins are known as protective antigen (PA), lethal factor (LF), and edema factor (EF), and together constitute the major virulence factor of the bacterium. PA binds to one of two cell surface receptors, ANTXR1 or ANTXR2, and facilitates the entry of LF and EF into the cell where they exert various toxic activities. Studying anthrax toxin receptor interactions with PA is thus important for understanding anthrax pathogenesis as well as discerning their natural functions in cells. In this thesis I sought to investigate how interactions with intracellular proteins regulate ligand binding by ANTXR1. Previously it was demonstrated that ANTXR1 associates with the actin cytoskeleton, and this association correlated with low levels of PA binding. It was suggested that this interaction is most likely mediated by adaptor proteins. However, in this thesis I demonstrate that the cytoplasmic domain of ANTXR1 binds directly to actin. Further, I provide evidence that this direct link is sufficient to regulate interactions of the receptor with PA ligands. Finally, I demonstrate that it is the interaction with actin filaments, rather than monomers that accounts for the lower PA binding properties of ANTXR1 compared with receptors that do not associate with actin.

Interactions Between the Actin Cytoskeleton and Anthrax Toxin Receptor 1 (ANTXR1) Kristopher M. Garlick, Doctor of Philosophy, 2012, Department of Laboratory Medicine and Pathobiology, University of Toronto ABSTRACT Anthrax is caused by the bacterium Bacillus anthracis, which secretes three proteins that assemble to form a toxic complex at the host cell surface. These proteins are known as protective antigen (PA), lethal factor (LF), and edema factor (EF), and together constitute the major virulence factor of the bacterium. PA binds to one of two cell surface receptors, ANTXR1 or ANTXR2, and facilitates the entry of LF and EF into the cell where they exert various toxic activities. Studying anthrax toxin receptor interactions with PA is thus important for understanding anthrax pathogenesis as well as discerning their natural functions in cells. In this thesis I sought to investigate how interactions with intracellular proteins regulate ligand binding by ANTXR1. Previously it was demonstrated that ANTXR1 associates with the actin cytoskeleton, and this association correlated with low levels of PA binding. It was suggested that this interaction is most likely mediated by adaptor proteins. However, in this thesis I demonstrate that the cytoplasmic domain of ANTXR1 binds directly to actin. Further, I provide evidence that this direct link is sufficient to regulate interactions of the receptor with PA ligands. Finally, I demonstrate that it is the interaction with actin filaments, rather than monomers that accounts for the lower PA binding properties of ANTXR1 compared with receptors that do not associate with actin.

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 71-88). Also available in print.

La migration cellulaire est une étape clé intervenant à un stade précoce de la dissémination des cellules cancéreuses dans l’organisme, et est donc responsable de la formation des métastases qui tuent environ nonante pourcent des patients atteints de cancer. De plus, ces cellules migrantes résistent à l’apoptose grâce à l’activation constitutive de voies de signalisation anti-apoptotiques, et développent donc une résistance vis-à-vis des traitements anti-cancéreux actuels qui sont généralement pro-apoptotiques. Nous avons pris pour cible ce processus de migration cellulaire dans l’espoir d’identifier des agents anti-migratoires qui permettraient de lutter contre la formation des métastases et de restaurer chez les cellules migrantes une certaine sensibilité aux traitements pro-apoptotiques. Dans la première partie de notre travail, nous avons analysé les effets anti-angiogéniques et anti-migratoires des agents anti-tubuline. Nous avons confirmé que le Taxol® présentait une action anti-angiogénique à des concentrations non-cytotoxiques. Nous avons ensuite démontré que d’autres agents anti-tubuline exerçaient la même action que le Taxol®, et que cette action leur était spécifique. Nous avons montré que certains de ces agents étaient également capables de réduire la migration de lignées cellulaires tumorales, toujours à des concentrations non-cytotoxiques, et que cette action pouvait s’exercer via une affectation du cytosquelette d’actine. Dans la deuxième partie du présent travail, nous avons démontré l’importance de la mise au point d’une approche pharmacologique originale permettant l’identification de composés à action anti-migratoire puisque l’outil utilisé par le U.S. National Cancer Institute pour le criblage de nouvelles molécules anti-cancéreuses ne permet pas de discerner l’activité anti-migratoire des molécules testées. Enfin dans la troisième partie de ce travail, après avoir souligné la raison du choix de l’actine comme cible pour inhiber la migration cellulaire, nous avons développé une stratégie pharmacologique in vitro originale de découverte de composés anti-actine à activité anti-migratoire. Grâce à une approche divisée en plusieurs étapes, à savoir un essai de cytotoxicité, une étude de la dynamique de la polymérisation d’actine en tubes ou sur cellules entières, et des essais de migration bidimensionnelle sur cellules individuelles ou sur population cellulaire, nous avons montré d’une part que des molécules connues pour affecter le cytosquelette actinique étaient capables d’affecter la migration cellulaire, et d’autre part que la méthodologie que nous avons développée permettait bien l’identification de composés affectant l’actine et capables de réduire la migration de cellules tumorales. En conclusion, cette stratégie in vitro pourrait être utilisée dans l’identification de nouvelles molécules à activité anti-migratoire pour lutter contre le cancer.