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1
Terry, Matthew. "Purification of wheat leaf plasma membranes and characterization of plasma membrane ATPase activity and phytochrome binding." Thesis, University of Southampton, 1990. https://eprints.soton.ac.uk/411150/.
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Großmann, Guido. "Plasma membrane compartmentation in Saccharomyces cerevisiae." kostenfrei, 2008. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1152/.
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Crooks, Kim Chantelle. "Turnover of plant plasma membrane proteins." Thesis, Oxford Brookes University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363720.
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Stanworth, Marie Helen. "Plasma membrane ATPase of Phytophthora cactorum." Thesis, University of the West of England, Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284886.
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Osman, Sangar Mahmoud. "Control of plasma membrane invagination systems." Thesis, University of Leicester, 2015. http://hdl.handle.net/2381/40507.
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The demarcation membrane system (DMS) is an extensive plasma membrane invagination system of the megakaryocyte (MK) that provides a membrane reserve for platelet generation. The properties of the DMS are poorly understood, particularly in living MKs. In this study, advanced electron microscopy, live cell confocal imaging and pharmacological tools were used to study the DMS and its connections with the extracellular environment. Confocal imaging of membrane-impermeant extracellular fluorescent indicators (HPTS, FITC-dextrans between 4 and 2000 kDa, and quantum dots) provided evidence for a discrete molecular cut-off (≈110kDa, estimated to be ≈11.0 nm by dynamic light scatter (DLS) measurements) for access to the DMS, which will prevent entry of large adhesion molecules (e.g. fibrinogen). Using extra-high resolution scanning electron microscopy (SEM) of fixed MKs, membrane invagination pores (MIPs) were observed on the MK surface that were variable in size but generally larger than predicted from live cell imaging experiments. A neck constriction was observed beneath the surface opening of the DMS using Gatan 3-view serial block face EM and may explain the rejection of molecules smaller than the surface pore. After inhibition of Cdc42, the DMS allowed entry of all indicators tested (up to 2000 kDa = reported to be ≈ 54.0 nm diameter). Using membrane-impermeant fluorescent indicators, electrophysiological capacitance measurements and transmission EM, multiple cationic amphiphilic drugs (CADs) were shown to cause complete surface detachment of the DMS. These compounds included the calmodulin inhibitor W-7, the phospholipase-C inhibitor U-73122 and anti-psychotic phenothiazines (trifluoperazine and chlorpromazine). CADs also caused loss of T tubules in cardiac ventricular myocytes and the open canalicular system of platelets. A possible explanation for this action is the ability of CADs to interfere with the interaction between PIP2 and binding of BAR domain containing proteins or the cytoskeleton. This work provides new insights into plasma membrane invagination systems.
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Kessler, Felix Ernst. "Isoforms of the plasma membrane Ca²⁺-ATPase /." Zürich, 1991. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=9630.
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Berchtold, Doris. "TOR complex 2 regulates plasma membrane homeostasis." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-146485.
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Ansa-Addo, Ephraim Abrokwa. "Plasma Membrane-derived Vesicles : Composition and Function." Thesis, London Metropolitan University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.536717.
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Quiroga, Álvarez Xarxa. "Plasma membrane mechanosensing upon stretch-induced topography remodelling." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/672367.
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Five years ago, I started walking this path that now seems like an entire life. Although everyone around tried to explain how this would feel, none of their explanations could have match what it has been in the end. And this is exactly how living systems are, at all levels. The harder the scientists try to feed our curiosity taking closer looks to them, inspecting the question from a different angle, and despite all the previous knowledge that we could gather; the more they surprise us and reveal new ways of sensing, reacting and adapting to the environment. And I think this is exactly what drove me here. I wanted to understand how this is done. I wanted to “see” it. How is it possible that a cell “understands” what is going on around? Which are the parameters that they sense? Biochemistry alone does not answer the question.
In a crowded environment, such as it is our body, cells are exposed to thousands of mechanical stimuli too. And those can be also harnessed and transformed into chemical responses as a way of signalling. While the classical biochemical inputs have long been studied, loads of questions remain open about how cells interpret those physical stimuli around them, and microscopy comes as a powerful technique to try to answer these queries.
In that sense, this thesis represents a small approach in trying to unravel how the plasma membrane, the first boundary between the cell and the extracellular media, can receive mechanical stimuli and convert them into biochemical signals amenable for the cell. To try to answer this question, this work starts with an introduction to the structure and physicochemical characteristics of the plasma membrane. An overview of the cortical component of the cytoskeleton, intrinsically interconnected to this structure, is also provided. Next, a summary of the literature available on how the plasma membrane can perceive mechanical
stimuli and which are the associated biochemical responses triggered by them is included as well. This part is based on a review article published by my colleague and co-supervisor Dr. Le Roux and myself at Philosophical Transactions B as part of the 2019 issue “Forces in cancer” [1].
After the introduction, chapter 2 describes the objectives of this study, which can be summarised as trying to unravel the way in which cells couple mechanical signals at their plasma membrane to biochemical cascades that mediate a response to those. Following, chapters 3 and 4 compose the main body of this thesis, including the methods and the experimental results coming from this research work. Both sections constitute a scientific article that has been recently submitted for publication. In chapter 3 the simplified model chosen to study the question of how cells sense and integrate mechanical stimuli at their plasma membrane is described. This consisted in submitting fibroblast to a controlled stretch-release cycle, forcing them to quickly adapt their shape, mimmicking a highly-relevant scenario in physiology. Chapter 4 recapitulates the way in which plasma membrane reacted to this mechanical perturbation. In the first place, the structure reacted by passively forming evaginations on the nanometric scale of homogeneous size and shape. These evaginations are next recognised by the I- BAR protein IRSp53, which subsequently organizes a node of actin polymerisation dependent on Rac1 and Arp2/3 that mediates the flattening of the structures. Absence of IRSp53 results, thus, in an impaired recovery of homeostasis after stretch. To reinforce the obtained experimental results, theoretical framework to model the mechanics of the system was generated in collaboration with the group of Dr. Arroyo at the Centre Internacional de Mètodes Numèrics en Enginyeria (CIMNE). The aim of this model was to describe how a network generated by the Arp2/3 complex, until now described to push, is able to generate in-plane forces that mediate the ironing of the evagination. Chapter 5 includes a discussion about the limitations of the technique, the novelty of the presented findings, the possible physiological scenarios where the described mechanochemical pathway can be of relevance and, finally, some exciting and unexplored questions that remained open after this work.
Additionally, other scientific production obtained during this thesis consisting in unexplored results or work belonging to other publications, has been added at the end of this manuscript in four appendixes. On the first one, I describe all the efforts made during the first 1 year and a half of the PhD to improve immunostaining technique for plasma membrane bound proteins in order to try
to stain endogenous BAR proteins. The second appendix gathers the findings obtained from the silencing assay of BAR candidates likely to recognise the curved shape of the stretch-release generated evaginations. The third appendix contains experimental results part of a different publication from Le Roux et al. now under review in Nature Communications [2]. Here, I studied the response of the N-BAR protein Amphiphysin after mechanical stimulation in cells. A fourth appendix including scanning electron microscopy representative images of stretch-release generated evaginations in other cell lines is also added. Finally, I included two more appendix containing the sequencing of all the IRSp53 plasmids used for the body of the work of this thesis and the MATLAB code used for analysis of evaginations flattening dynamics after stretch.
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Blixt, Ylva. "Early interaction between adenovirus type 2 and HeLa cells significance of the plasma membrane constitution /." Lund : Dept. of Microbiology, University of Lund, 1992. http://books.google.com/books?id=DzhrAAAAMAAJ.
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Ray, Koela. "Chacterization of Paramecium Tetraurelia Ciliary Membrane Plasma Membrane Calcium Pumps and Lipid Rafts." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/190.
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Paramecium, a ciliate, is an important model for studying Ca2+ signaling and understanding chemoreception and signal transduction. There are several proteins, such as plasma membrane calcium ATPases (PMCAs)/ calcium pumps, SERCA pumps, calmodulin and Ca2+ channels that play an important role in maintaining intracellular Ca2+ level and signaling in Paramecium. Isoform 2 of PMCA has been identified in both the cilia and pellicle membranes of Paramecium, the activity of which leads to hyperpolarization. Plasma and ciliary membrane of Paramecium is made up of a variety of sterols and sphingolipids which constitute lipid rafts, demonstrated by the presence of detergent resistant membranes and their distribution in sucrose and Optiprep density gradients. PMCAs are important markers of lipid rafts and PMCA 2 is found to be localized in lipid rafts of both the cilia and somatic membrane of Paramecium. Methyl-β-cyclodextrin treatment can remove up to 42% of sterols from pellicle membranes but only about 12% from cilia. Sterol depletion of pellicle perturbs the distribution of PMCA 2 and other raft proteins in pellicle which is not observed in cilia as evident from western blot analysis and immunomicroscopic studies. There is evidence that selection of gradient medium for study of lipid rafts and its associated proteins is very important in Paramecium. Glutamate receptors and adenylyl cyclase, the upstream molecules of the signal transduction pathway through PMCA have also been identified in cellular cilia, indicating that these raft molecules forms a platform for signaling in Paramecium cilia.
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Prado, Gustavo R. "Neuronal Plasma Membrane Disruption in Traumatic Brain Injury." Diss., Georgia Institute of Technology, 2004. http://hdl.handle.net/1853/7260.
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During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. Cellular events that occur at the time of and immediately after an insult are poorly understood. Immediately following traumatic brain injury (TBI), the neuronal plasma membrane may become disrupted and potentiate detrimental pathways by allowing extracellular contents to gain access to the cytosol. In the current study, neuronal plasma membrane disruption was assessed in vivo following moderate unilateral controlled cortical impact in rats using a normally cell-impermeant fluorescent compound as a plasma membrane permeability marker. This fluorescent dye was injected into the cerebrospinal fluid and was allowed to diffuse into the brain. TBI caused a widespread acute disruption of neuronal membranes which was significantly different compared to uninjured brains. Affected cells were present in cortex and hippocampal regions. These findings were complemented by an in vitro model of TBI where membrane disruption was quantified and its mechanisms elucidated. Permeability marker(s) were added to neuronal cultures before the insult as indicators for increases in plasma membrane permeability. The percentage of cells containing the permeability marker was dependent on the molecular mass, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were also positively correlated with the rate of insult. Membrane disruption was transient, evidenced by a robust resealing within the first minute after the insult. In addition, membrane resealing was found to be dependent on extracellular Ca2+, as chelation of the ion abolished a significant amount of resealing. We have also investigated the effects of mechanically-induced plasma membrane disruptions on neuronal network electrical activity. We have developed a multielectrode array system that allows the study of electrical activity before, during, and after a traumatic insult to neurons. Endogenous electrical activity of neuronal cultures presented a heterogeneous response following mechanical insult. Moreover, spontaneous firing dysfunction induced by injury outlasted the presence of membrane disruptions. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.
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Riozzi, A. "Studies of plasma membrane function in human hypertension." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/34211.
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Considerable evidence has emerged in recent years to suggest that the cell plasma membrane handles univalent and divalent cations abnormally in patients with untreated essential hypertension. Many of the phenomena originally discovered in patients with the established disease have now been found to occur in the genetically hypertension-prone offspring of hypertensive patients when their blood pressure is normal. The studies described in this thesis were designed to investigate the mechanisms which might explain these disturbances of membrane function. The first experiments were performed to investigate whether a circulating blood-borne factor might be present in excess in hypertensive patients and their relatives and by exposing cells from subjects with normal blood pressure and no family history to serum from patients and their offspring, the object was to try and reproduce the findings in hypertension. These studies were negative. Because of many reports of an overactive sympathetic nervous system in hypertension leucocytes were exposed to noradrenaline and this was found to influence sodium transport in cells from control subjects suggesting that at least some of the phenomena described in hypertension might be related to autonomic dysfunction. An alternative hypothesis to explain these abnormalities is that there is a genetically predetermined disturbance of the physicochemical structure of the plasma membrane which alters its function. The abnormality might well lie within the lipid fraction of the cell membrane. Attempts to alter this were undertaken using changes in dietary fat intake. These lowered blood pressure slightly and indeed altered sodium influx. The final series of experiments involved detailed examination of one fraction of plasma membrane phospholipids which is highly metabolically active, namely the phosphoinositides, and indeed using red cells it was possible to demonstrate that these lipids are overactive in the early stages of hypertension. These findings suggest that the plasma membrane is structurally abnormal in hypertension, the abnormality may reside in the phospho- inositide lipids and may possibly be susceptible to dietary manipulation.
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Baker, Mark Andrew 1974. "Purification and characterisation of the plasma membrane NADH:oxidoreductase." Monash University, Dept. of Biochemistry and Molecular Biology, 2002. http://arrow.monash.edu.au/hdl/1959.1/8440.
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Öyen, Mattias. "Functional studies of plasma membrane syntaxins in yeast /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish Univ. of Agricultural Sciences, 2003. http://epsilon.slu.se/a435.pdf.
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Allen, Gethyn John. "Sodium transport in wheat root plasma membrane vesicles." Thesis, Bangor University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357596.
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Drechsler, Carina [Verfasser], Heiko [Akademischer Betreuer] Heerklotz, and Rolf [Akademischer Betreuer] Schubert. "Phosphatidylserine asymmetric vesicles as eukaryotic plasma membrane model." Freiburg : Universität, 2018. http://d-nb.info/1175378763/34.
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Li, Yu. "Quality Control of Plasma Membrane Proteins: A Dissertation." eScholarship@UMMS, 1999. http://escholarship.umassmed.edu/gsbs_diss/240.
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The temperature-sensitive α-factor receptor (Ste2-3p) and arginine permease (Can1tsp) were found to provide the model substrates for quality control of plasma membrane proteins in Saccharomyces cerevisiae. When the ste2-3 mutant cells were grown at 34°C, Ste2-3p failed to accumulate at the plasma membrane and was delivered to the vacuole for degradation without traversing the plasma membrane. Upon reaching the vacuole, cytoplasmic domains of both Ste2p and Ste2-3p appeared within the vacuolar lumen. Four stp mutants were identified to suppress temperature-sensitive defects in both Ste2-3p and Can1tsp. The stp22 and STP26 mutations also caused missorting of vacuolar protein carboxypeptidase Y, and a subset of vacuolar protein sorting mutants (vps) suppressed ste2-3 mutation. In the stp22 mutant, both Ste2p and Ste2-3p accumulated in the prevacuolar compartment (PVC) and on the plasma membrane. Three independent mutations that bypassed the phenotype of stp22Δ mutant were identified and mapped to the SNF8 locus, and they were found to affect a single amino acid residue (G209D). The mutant protein, Snf8bpp, but not Snf8p, was able to compensate for the lack of functional Stp22p and to restore PVC-to-vacuole trafficking. The order of function for some VPS genes involved in PVC-to-vacuole traffic (class E) was determined by using this special snf8bp allele. In addition, a PtdIns 4-kinase encoded by the PIK1 gene was found to be involved in Ste2-3p trafficking, possibly affecting the PVC function.
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Talreja, Kavita. "Stress induced changes at the yeast plasma membrane." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321558.
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Ermolayeva, Elena. "Plasma membrane ion transport in phytochrome signal transduction." Thesis, University of York, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319767.
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Alenkvist, Ida. "Epac2 signaling at the β-cell plasma membrane." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-284638.
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Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.
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Nimeri, Ghada. "Neutrophil biology on artificial surfaces the role of adsorbed blood plasma proteins and platelets /." Göteborg : Göteborg University, 1998. http://catalog.hathitrust.org/api/volumes/oclc/68945095.html.
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Fischer, Roland. "Molecular cloning of a human plasma membrane Ca²⁺-ATPase /." Zürich, 1989. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=8811.
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Kojima, Kiyohide. "Molecular Aspects of the Plasma Membrane in Tumor Cells." 名古屋大学医学部, 1993. http://hdl.handle.net/2237/6164.
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MacDonald, James Innes Scott. "Atpases in plasma membrane enriched fractions from Dictyostelium discoideum." Thesis, University of British Columbia, 1986. http://hdl.handle.net/2429/27141.
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Evidence is presented for the existence of an ATPase activity in D. discoideum plasma membranes. This activity was distinct from the mitochondrial ATPase in that it was insensitive to azide and oligomycin. The ATPase was stimulated by Mg⁺² and to a lesser extent by Ca⁺² and was not affected by equimolar Na⁺/K⁺ or ouabain. Vanadate, DES, thimerosal and DCCD all proved to be partially inhibitory.
Enzyme activity was solubilized with a wide variety of detergents, with lysolecithin giving the best results. Concomitant with solubilization was a partial loss of DES sensitivity which was shown to be due to the presence of a labile DES sensitive ATPase in addition to a stable DES insensitive activity. The DES sensitive ATPase was stimulated by Mg⁺² but only somewhat by Ca⁺² whereas the DES insensitive enzyme was stimulated equally by either. The DES sensitive enzyme also displayed Michaelis-Menten kinetics when enzyme activity was measured as a function of the ATP concentration while the DES insensitive ATPase displayed kinetics which were indicative of a sigmoidal relationship between substrate concentration and enzyme activity.
Fractionation of solubilized plasma membranes by ion exchange chromatography resolved two DES insensitive ATPase activities, designated peaks I and II. Peak I was insensitive to vanadate and expressed optimum activity with pyrophosphate. Optimal activity was at alkaline pH values. Peak II was purified by two different procedures. The first involved an initial separation on DEAE-Sephace1 followed by centrifugation through a linear glycerol gradient. The second involved an initial chromatographic fractionation by Sephacryl S-300 gel filtration followed by DEAE-Sephacel ion exchange chromatography of the ATPase containing fractions. Both procedures resulted in preparations that contained a single major component of apparent molecular weight 6 4 kDa, as assessed by SDS-polyacrylamide gel electrophoresis.
The purified ATPase was sensitive to vanadate and fluoride but insensitive to DCCD , thimerosal and N-ethylmaleimide. The enzyme was activated equally by either Mg⁺² or Ca⁺² in millimolar concentrations. ATP hydrolysis was also stimulated by millimolar concentrations of Mn⁺², Zn⁺² or Cu⁺². The ATPase displayed sigmoidal kinetics when assayed as a function of ATP concentration in the absence of any divalent cation. Addition of 1 or 10 mM Mg⁺² or Ca⁺² increased the substrate affinity of the enzyme, while 100 mM divalent cation proved inhibitory. The enzyme was not stimulated by low concentrations of Ca⁺² or by Ca⁺²-calmodulin, suggesting that it was probably not a Ca⁺² pump.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Brown, Claire M. "Distribution and co-localization of plasma membrane associated proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ31134.pdf.
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Shadan, Sadaf. "Cholesterol dynamics in the plasma membrane of mammalian spermatozoa." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615713.
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Ibrahim, Z. A. J. "Studies of the plasma membrane of malignant melanoma cells." Thesis, University of Strathclyde, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382392.
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Crofts, Alan. "Anion efflux across the plasma membrane of Chara corallina." Thesis, University of York, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358101.
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Robinson, Lucy Elizabeth. "Regulation of the P2X7 receptor by plasma membrane cholesterol." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709439.
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Piguet, Joachim. "Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics." Doctoral thesis, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-178147.
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Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.QC 20151217
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Nazemidashtarjandi, Saeed. "Interactions of Engineered Nanomaterials with the Cell Plasma Membrane." Ohio University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1617363923755762.
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Wilcock, Carol. "The effects of nitrogen mustard on plasma membrane function." Thesis, Aston University, 1987. http://publications.aston.ac.uk/12553/.
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The antitumour bifunctional alkylating agent nitrogen mustard (HN2) inhibited the unidirectional influx of the potassium congener, 86 rubidium, into murine PC6A plasmacytoma cells and L1210 leukaemia cells. The proliferation of L1210 cells in vitro was characterised and shown to be sentitive to HN2. 86Rubidium influx into cells from rapidly-dividing cultures was more sensitive to inhibition by HN2 than that of cells from stationary cultures. Three components of unidirectional 86Rb+ & K+ influx into proliferating L1210 cells were identified pharmacologically: approximately 40% was active to the Na+ K+ ATPase inhibitor ouabain (10-3M), 40% was sensitive to the `loop' diuretics bumetanide (10-4M) and furosemide (10-3M) and the remainder was insensitive to both ouabain and furosemide. HN2 (10-5M) selectively inhibited the diuretic-sensitive component, which was entirely dependent upon extracellular Na+ and Cl- ions, and was presumed to represent Na+ K+ Cl- cotransport activity. The system did not mediate K+ /K+ exchange or unidirectional 86Rb+ efflux; accordingly, 86Rb+ efflux was insensitive to HN2. Inhibition of 86Rb & K+ influx by 10-5M HN2 was accompanied by approximately 35% of cell volume under isosmotic conditions; thus intracellular Na+ and K+ concentrations remained unchanged. These effects followed lethal damage to the cells but preceded actual cell death; other cellular functions were maintained including accumulation of cycloleucine, transmembrane potential, permeability to trypan blue, intracellular pH, total intracellular glutathione and calcium concentrations. No evidence was found that elevated cAMP levels or reduced ATP levels were involved in modulation of 86Rb+ & K+ influx. However, the Na+ - depedent transport of an amino acid was inhibited in a manner which appeared to be independent of 86Rb & K+ influx. An HN2-resistant L1210R cell line was also resistant to furosemide, and lacked a component of 86Rb+ & K+ influx which was sensitive to furosemide (10-3M). The results strongly suggest that the Na+ K+ Cl- costransporter of L1210 cells is a cellular target for HN2. This lesion is discussed with reference to the cytotoxic effects of the agent.
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Walsh, Ciara. "The regulation of STIM1 translocation to the plasma membrane." Thesis, University of Liverpool, 2010. http://livrepository.liverpool.ac.uk/1482/.
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A rise in intracellular Ca2+ concentration is key to controlling both short term and long term Ca2+ dependent processes which include secretion, metabolism and gene expression, cell growth and proliferation. Store operated Ca2+ channels (SOCs), which are activated by the depletion of Ca2+ from internal Ca2+ stores, the main store being the endoplasmic reticulum (ER), are the major route for Ca2+ influx in non-excitable cell types. Stromal interacting molecule 1 (STIM1) is a Ca2+ sensing protein located in the endoplasmic reticulum (ER). Depletion of ER calcium stores triggers oligomerisation and subsequent translocation of STIM1 from its reticular location to specialized endoplasmic reticulum-plasma membrane (ER-PM) junctions where it forms STIM1 puncta and interacts with the SOC channel, Orai1. This induces the clustering of Orai1 into a functional tetrameric pore which is permeable to Ca2+ ions, enabling Ca2+ entry into the cell. The precise mechanism by which STIM1 is recruited to the plasma membrane to activate SOCs and the plasma membrane components involved in targeting STIM1 to the plasma membrane are largely unknown. In this study the mechanisms underlying movement of STIM1 to the plasma membrane and its accumulation at ER-plasma membrane junctions was explored in HeLa cells. In the initial part of this study I investigated whether the movement of STIM1 to the plasma membrane is an ATP-dependent process. I found that depletion of cytosolic ATP can stimulate STIM1 puncta formation in HeLa cells and that the formation of STIM1-Orai1 complexes at the plasma membrane is unaffected in these conditions. Inhibition of ATP synthesis also initiated the loss of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) from the plasma membrane. ATP depletion did not affect the structure of the microtubule cytoskeleton. These results suggest that the translocation of STIM1 and the formation of STIM1-Orai1 complexes is an ATP independent process which is not due to the disruption of microtubules and support a diffusional model for STIM1 puncta formation. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with plasma membrane phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai1. I investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE in response to store depletion. Treatment of HeLa cells with inhibitors of the phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1-EYFP into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also partially inhibited SOCE. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and under these conditions SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER-PM junctions with a second translocation mechanism involving direct STIM1/Orai1 interactions. It was recently reported that STIM1 and Orai1 may function within a macromolecular complex involving other unidentified proteins. In this study I have identified that Golli-BG21, a member of the myelin basic protein (MBP) family, can directly interact with STIM1. Golli interacts with the C-terminal domain of STIM1 in both in vitro and in vivo binding assays and this interaction may be modulated by intracellular Ca2+ concentration. Golli also colocalises with full length STIM1 and Orai1 complexes in HeLa cells following store depletion. Overexpression of Golli reduces SOCE in HeLa cells but this inhibition is overcome by overexpressing STIM1. We therefore suggest that Golli binds to STIM1-Orai1 complexes to negatively regulate the activity of SOCs.
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Bhuyan, Nihar Ranjan. "Characterization of transplasma membrane electron transport system and plasma membrane bound p-type ATPase in leishmania donovani promastigote." Thesis, University of North Bengal, 2009. http://hdl.handle.net/123456789/1409.
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Dhaliwal, Kanwaljit. "A study of membrane choline transport and renal failure." Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300380.
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Masilamani, A. P. "Membrane glycohydrolases are involved in the production of ceramide in the plasma membrane of fibroblasts." Doctoral thesis, Università degli Studi di Milano, 2008. http://hdl.handle.net/2434/60936.
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Siddiqua, Ashia. "Studies of the Ca²⺠ATPases in human platelets and DAMI cells." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251772.
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Karley, Alison Jane. "Differential ion accumulation and ion fluxes in the mesophyll and epidermis of barley." Thesis, University of York, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298388.
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Hawkins, Kirstie. "Salt tolerance and chloride transport in three beet subspecies." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270062.
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Ladha, Shabirali. "The effect of changes in plasma membrane lipid composition on the heat sensitivity of hepatoma tissue culture cells and selected plasma membrane enzymes." Thesis, Durham University, 1990. http://etheses.dur.ac.uk/6197/.
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Hepatoma Tissue Culture (HTC) cells grown in the presence of 60µM arachidonic acid for 24, 36 and 48 hours became progressively more thermosensitive than control cells. However, this difference in thermal sensitivity was only detectable with the clonogenic assay and not with the colorimetric assay. Attempts were also made to manipulate cellular cholesterol levels. Firstly, some cells were incubated with phosphatidylcholine liposomes to deplete the plasma membrane of cholesterol: Secondly, another group of cells were treated with 25 hydroxycholesterol, an inhibitor of cholesterol synthesis, to lower cholesterol levels: Finally, a third group of cells were supplemented with cholesterol hemisuccinate, a hydrophilic ester of cholesterol. The first two approaches did not enhance the thermal sensitivity of HTC cells. Supplementation with cholesterol hemisuccinate, which was predicted to partition in to the plasma membrane and reduce membrane fluidity, resulted in increased thermal sensitivity of the cells. Thus, the thermal sensitivity of HTC cells could be enhanced by supplementation with either arachidonate or cholesterol hemisuccinate. A rapid plasma membrane isolation procedure was developed which generated plasma membranes in relatively high yield and purity. The plasma membrane- enriched fraction was also assayed for contaminating intracellular membranes by determining marker enzyme activities associated with these membranes. Using this method, plasma membranes were prepared from HTC cells grown m 60µM arachidonic acid for 36 hours and from cells grown in normal medium. Analysis of the plasma membrane showed that the arachidonic acid content of the phospholipid fatty acyl groups had been significantly increased in cells grown in the presence of this fatty acid. There was no change in the cholesterol/phospholipid molar ratio or cholesterol concentration relative to amount of protein in the plasma membranes from the two cell populations. The measurement of fluidity using DPH fluorescence polarisation revealed that the increase in the arachidomc acid content of the plasma membrane phospholipid acyl groups was associated with enhanced plasma membrane fluidity when compared to control plasma membranes. This increase in plasma membrane fluidity correlated with the enhanced thermal sensitivity of the cells grown in arachidonic acid-containing medium when compared to cells grown in normal medium. Furthermore, the thermal sensitivity of Na(^+), K(^+) –ATPase and alkaline phosphodiesterase I were assessed in plasma membranes derived from arachidonic acid-supplemented and control cells. The enhanced fluidity of plasma membranes derived from arachidonate-supplemented cells also correlated with increased thermosensitivity of alkaline phosphodiesterase I.
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Torres, Mariana Andrade. "Efeito da adição de plasma seminal nas características da motilidade e na fertilidade de espermatozoides criopreservados de suínos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-16092016-142035/.
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A criopreservação do sêmen suíno ainda é um desafio devido a extensão dos danos causados pelo choque-frio. Os espermatozoides oriundos da fração rica do ejaculado suíno possuem a membrana plasmática mais estável, são mais resistentes ao choque-frio e a reação acrossomal precoce do que aqueles oriundo da fração total do ejaculado. Com isso, o uso do plasma seminal oriundo dessa fração do ejaculado suíno também poderia aumentar a criotolerância dos espermatozoides suínos e/ou reverter os danos oriundo da criopreservação. Entretanto, a grande maioria das técnicas de avaliação espermática inclui apenas um parâmetro de funcionalidade espermática. Por outro lado, quanto mais características espermáticas analisadas, mais fiel se torna a avaliação quanto ao potencial fertilizante da amostra. Nesse contexto, esse trabalho objetivou a validação de uma técnica de análise espermática por citometria de fluxo para a avaliação simultânea da integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial. Além de avaliar os efeitos da adição/manutenção do plasma seminal oriundo da fração rica do ejaculado suíno sobre a cinética espermática, integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial, fluidez e peroxidação das membranas espermáticas, fosforilação do aminoácido tirosina, taxa de fertilidade e de prenhez precoce. A técnica sugerida em nosso trabalho é capaz de detectar (R2 = 0,9356; p < 0,01) simultaneamente os espermatozoides com a membrana plasmática e acrossomal integras e alto potencial de membrana mitocondrial (PIAIA). O plasma seminal da fração rica do ejaculado suíno gera benefícios para a cinética espermática melhorando a motilidade total e progressiva dos espermatozoides descongelados (p < 0,05). Por outro lado, a manutenção/adição de plasma seminal oriundo da fração rica do ejaculado suíno, não é capaz de alterar (p > 0,05) os espermatozoides PIAIA, a fosforilação do aminoácido tirosina, a fluidez e a peroxidação das membranas espermáticas. Entretanto, essas características devem ser cuidadosamente interpretadas, uma vez que, a fluidez e a peroxidação das membranas espermáticas são alteradas (p < 0,05) pelo processo de centrifugação. As taxas de fertilidade e de prenhez precoce também não foram influenciadas (p > 0,05) pela adição de plasma seminal oriundo da fração rica do ejaculado suíno. Portanto, podemos concluir que o plasma seminal da fração rica possui efeito benéfico sobre a fisiologia dos espermatozoides pós-descongelação, melhorando a criopreservação do sêmen suíno.Boar semen cryopreservation is still a challenge due to the extension of cold shock damage. Boar spermatozoa arising from sperm-rich ejaculate fraction are reported to have a more stable plasma membrane, more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Thus, seminal plasma arising from whole sperm-rich fraction can increase cryotolerance of boar spermatozoa, and in other domestic species it has the ability to reverse cryopreservation damage. However, the majority of actual sperm evaluation techniques include a single sperm parameter, which makes the sperm characterization of a single population difficult. In this context, this work was performed to validate a sperm flow cytometry fourfold coloration technique for the simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. Furthermore, we evaluate the seminal plasma arising from whole sperm-rich fraction effects on sperm kinetics, plasma and acrosomal membrane integrity and mitochondrial membrane potential, tyrosine phosphorylation, membrane fluidity and peroxidation, fertility and early pregnancy rate. The proposed technique is able (R2 = 0.9356; p < 0.01) to simultaneous detection of plasma and acrosomal membrane integrity and high mitochondrial membrane potential (IPIAH). Seminal plasma arising from whole sperm-rich fraction is able to improve (p < 0.05) total and progressive motility. Furthermore, seminal plasma arising from whole sperm-rich fraction maintenance/addition was not able to improve (p > 0.05) the IPIAH sperm population, tyrosine phosphorylation, membrane fluidity and peroxidation. However, these sperm characteristics need to be carefully interpreted, once, membrane fluidity and peroxidation could be influenced (p < 0.05) by centrifugation. Fertility and early pregnancy rate we not improved (p > 0.05) by seminal plasma addition. Therefore, we concluded that sperm fourfold coloration can be used to evaluate simultaneously plasma and acrosomal membranes integrity and mitochondrial membrane potential and that whole sperm-rich fraction seminal plasma can be beneficial to post-thawed sperm physiology, thereby improving boar semen cryopreservation.
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Campbell, Lauryl Elizabeth. "Membrane Properties Involved in Calcium-Stimulated Microparticle Release from the Plasma Membranes of S49 Lymphoma Cells." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3301.
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The mechanism of microparticle shedding from the plasma membrane of calcium-loaded cells has been investigated in erythrocytes and platelets. Recent studies have revealed the physiological and clinical importance of microparticle release from nucleated cells such as lymphocytes and endothelium. The experiments of this study were designed to address whether simple mechanisms discovered in platelets and erythrocytes also apply to the more complex nucleated cells. Four such mechanisms were addressed: potassium efflux, transbilayer phosphatidylserine migration, cytoskeleton degradation, and membrane lipid order. The rate and amount of microparticle release in the presence of a calcium ionophore, ionomycin, was assayed by light scatter at 500 nm. To inhibit the calcium-activated potassium current, cells were exposed to 1 mM quinine or a high-potassium buffer. Both interventions substantially attenuated microparticle shedding induced by ionomycin. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated phosphatidylserine migration to the cell surface. This result indicated that such phosphatidylserine exposure is also required for microparticle shedding. The importance of cytoskeletal rearrangement was evaluated through the use of E64-d, a calpain inhibitor, which appeared to have no affect on release. Thus, if cytoskeleton degradation is important for microparticle release, a different enzyme or protein must be involved. Finally, the effect of membrane physical properties was addressed by varying the experimental temperature (32–42 °C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammoniumdiphenylhexatriene and patman revealed significant differences in the level of apparent membrane order along that temperature range. Ionomycin treatment appeared to cause further disordering of the membrane, although the magnitude of this change was minimally temperature-sensitive. Thus, it was concluded that microparticle release depends more on the initial level of membrane order than on the change imposed by calcium uptake. In general, mechanisms involved in particle release from platelets and erythrocytes appeared relevant tolymphocytes with the exception of the hydrolytic enzyme involved in cytoskeletal degradation.
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Köster, Darius Vasco. "Role of Caveolae in Membrane Tension." Doctoral thesis, Universitätsbibliothek Leipzig, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-63103.
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Caveolae sind charakteristische Plasmamembraneinstülpungen, die in vielen Zelltypen vorkommen und deren biologische Funktion umstritten ist. Ihre besondere Form und ihre Häu gkeit in Zellen, die stets mechanischen Belastungen ausgesetzt sind, führten zu der Annahme, dass Caveolae die Plasmamembran vor mechanischen Belastungen schützen und als Membranreservoir dienen. Dies sollte mit dieser Dissertation experimentell geprüft werden. Zunächst wurde der Ein uss der Caveolae auf die Membranspannung von Zellen im Normalzustand untersucht. Dann wurden die Zellen mechanisch belastet. Mit Fluoreszensmikroskopie wurde das Verschwinden von Caveolae nach Strecken der Zellen oder nach einem hypo-osmotischen Schock beobachtet. Messungen der Membranspannung vor und unmittelbar nach dem hypo-osmotischem Schock zeigten, dass Caveolae einen Anstieg der Membranspannung verhindern, unabhängig von ATP und dem Cytoskelett. Die Erzeugung von Membranvesikel mit Caveolae erlaubte es, diesen Effekt der Caveolae in einem vereinfachten Membransystem zu beobachten. Schliesslich wurden Muskelzellen untersucht. Zellen, die genetisch bedingt weniger Caveolae haben und mit Muskelschwundkrankheiten in Verbingung stehen, waren mechanisch weniger belastbar als gesunde Zellen. Zusammenfassend wird mit dieser Dissertation die These bestärkt, dass Caveolae einem Anstieg der Membranspannungen entgegenwirken. Dass dies in Zellen und in Vesikeln unabhängig von Energie und Cytoskelett geschieht, lässt auf einen passiven, mechanisch getriebenen Prozess schliessen. Diese Erkenntnis trägt zum Verständnis der Rolle von Caveolae in Zellen bei und kann dem besseren Verständnis von Krankheiten bedingt durch Caveolin-Mutationen, wie z.B. Muskelschwundkrankheiten, dienenCaveolae, the characteristic plasma membrane invaginations present in many cells, have been associated with numerous functions that still remain debated. Taking into account the particular abundance of caveolae in cells experiencing mechanical stress, it was proposed that caveolae constitute a membrane reservoir and bu er the membrane tension upon mechanical stress. The present work aimed to check this proposition experimentally. First, the in uence of caveolae on the membrane tension was studied on mouse lung endothelial cells in resting conditions using tether extraction with optically trapped beads. Second, experiments on cells upon acute mechanical stress showed that caveolae serve as a membrane reservoir bu ering surges in membrane tension in their immediate, ATP- and cytoskeleton-independent attening and disassembly. Third, caveolae incorporated in membrane vesicles also showed the tension bu ering. Finally, in a physiologically more relevant case, human muscle cells were studied, and it was shown that mutations with impaired caveolae which are described in muscular dystrophies render muscle cells less resistant to mechanical stress. In Summary the present work provides experimental evidence for the hypothesis that caveolae bu er the membrane tension upon mechanical stress. The fact that this was observed in cells and membrane vesicles in an ATP and cytoskeleton independent manner reveals a passive, mechanically driven process. This could be a leap forward in the comprehension of the role of caveolae in the cell, and in the understanding of genetic diseases like muscular dystrophies
Cavéoles sont des invaginations caractéristiques de la membrane plas- mique présents dans beaucoup de types cellulaires. Ils sont liées à plusieurs fonctions cellulaires, ce qui sont encore débattues. Prenant compte de l importance des cavéoles dans les cellules soumises au stress mécanique, les cavéoles sont proposées de constituer un réservoir membranaire et de tamponner la tension membranaire pendant des stresses mécaniques. Cette étude a eu le but de tester cette hypothèse expérimentalement. En premier, l in uence des cavéoles sur la tension membranaire au repos a été étudiée sur des cellules endothéliales du poumon de la souris. Puis, on a montré que les cavéoles tamponnent l augmentation de la tension membranaire après l application d un stress mécanique. En suite, la réalisation des vésicules membranaires contenant des cavéoles a permit de montrer leur rôle comme réservoir membranaire dans un système simpli é. Finalement, dans un contexte physiologiquement plus relevant, l étude des cellules musculaires a montrée que les mutations du cavéolin associées aux dystrophies musculaires rendent les cellules moins résistante aux stresses mécaniques. En conclusion, cette étude supporte l\'hypothèse que les cavéoles tamponnent la tension membranaire pendant des stresses mécaniques. Le fait que cela se passe dans les cellules et les vésicules indépendamment d ATP et du cytosquelette révèlent un processus passif et mécanique. Cela pourrait servir à une meilleure compréhension du rôle des cavéoles dans la cellule et les maladies génétiques comme les dystrophies musculaires
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Hernandez, Lopez Agustin. "Plasma membrane sterols and fatty acids : effects on membrane properties and H'+-ATPase of Ustilago maydis." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336825.
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de, la Haba Fonteboa Carlos. "Effects of oxidative stress on plasma membrane fluidity: biological consequences." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/311421.
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El estrés oxidativo (OS) es característico de muchas enfermedades y se produce cuando hay un desequilibrio entre oxidantes y antioxidantes, lo cual favorece un estado oxidativo que genera especies reactivas de oxígeno y de nitrógeno. Los lípidos de la membrana plasmática son dianas preferentes del OS y ello tiene como consecuencia la peroxidación lipídica. Este proceso modifica propiedades de la membrana tales como su fluidez, característica física muy importante conocida por modular la localización de las proteínas de membrana y las uniones receptor-ligando.
Objetivos: 1) Evaluar el efecto del OS en la regionalización de la fluidez en la membrana plasmática de células vivas tales como macrófagos THP-1 y linfocitos MEC-1, de manera individualizada. 2) Analizar, en estas células, la relación entre la peroxidación lipídica y la fluidez de membrana. 3) Estudiar el efecto del OS sobre la unión receptor-ligando y sobre la fluidez de membrana: lipopolisacárido/receptores de tipo Toll (TLR2/4) en macrófagos y progesterone-induced blocking factor (PIBF)/PIBF-receptor en linfocitos.
Material y Métodos: Se estandarizó la metodología two-photon microscopy por primera vez en la Universidad Autónoma de Barcelona, para analizar la fluidez de membrana en células vivas individuales. Conjuntamente se ha desarrollado un nuevo software capaz de medir el tamaño y el número de los dominios lipídicos de membrana. El OS se indujo mediante H2O2 y se empleó la sonda fluorescente Laurdan para detectar las diferencias de fluidez en la membrana plasmática. Se utilizaron LPS y PIBF soluble en macrófagos y linfocitos respectivamente, para analizar interacciones receptor-ligando en condiciones OS.
Resultados:
En los macrófagos se observó un aumento significativo, dependiente de la concentración de H2O2, en la frecuencia de regiones lipídicas rígidas principalmente compuestas por dominios lipid raft, a expensas de las regiones de fluidez intermedia. Asimismo, se detectó en condiciones de OS, un mayor número, aunque no un mayor tamaño, de dominios lipid raft. La activación de macrófagos con LPS incrementó la frecuencia de regiones fluidas en las membranas, efecto que fue inhibido en condiciones de OS. En cuanto a la función de los macrófagos, se detectó una disminución en la secreción de TNFα en condiciones oxidantes.
En los linfocitos se observó un aumentó significativo en la frecuencia de regiones lipídicas rígidas, a expensas de las regiones fluidas, en condiciones de OS. Por otro lado, la unión del PIBF a su receptor, provocó un aumentó en la rigidez de la membrana plasmática debido al clustering de dominios lipid raft. Por el contrario, cuando se indujo OS en linfocitos en presencia de PIBF, se inhibió el clustering de dominios lipid raft y también disminuyó el reconocimiento del receptor de PIBF a su ligando.
Conclusiones: 1) Se ha evaluado en células vivas, de forma individual, la dinámica lipídica de la membrana plasmática. 2) Una consecuencia general importante es que, durante el OS, tanto en macrófagos como en linfocitos la membrana plasmática se vuelve más rígida. 3) La fluidez de membrana cambia de forma distinta en los dos tipos celulares estudiados, como consecuencia de las interacciones receptor-ligando: durante la unión LPS-TLR2/4 se observó un aumento en la fluidez de la membrana plasmática de los macrófagos y, por el contrario, durante la unión PIBF/PIBF-R la membrana de los linfocitos se rigidificó, aumentando el clustering de los dominios lipid raft. 4) No obstante, en ambos casos el OS inhibió los cambios en la fluidez de membrana inducidos por la unión receptor-ligando.Oxidative stress is present in many diseases and it is produced in cells when an imbalance between oxidants and antioxidants occurs, favoring an oxidant status which produce reactive oxygen and nitrogen species. Lipids in plasma membrane are one of the preferential targets giving rise to lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and receptor-ligand binding. Aims: 1) To evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages and MEC-1 lymphocytes. 2) To analyze, in these cells, the relationship between lipid peroxidation and membrane fluidity. 3) To study the effect of oxidative stress on receptor-ligand binding and membrane fluidity: lipopolysaccharide/toll-like receptors (TLR2/4) in macrophages and progesterone-induced blocking factor (PIBF)/PIBF-receptor in lymphocytes. Material and Methods: Two-photon microscopy was standardized for the first time in Universidad Autónoma de Barcelona by our laboratory, to analyze membrane fluidity in single living cells. It was also developed a new software application to analyze membrane lipid domain size and number. Cellular oxidative stress was induced by H2O2; the fluorescent probe Laurdan was applied to evaluate plasma membrane fluidity changes. LPS in macrophages or soluble PIBF in lymphocytes were used to analyze receptor-ligand interactions under oxidative stress. Results: Macrophages showed a significant H2O2 concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. Under oxidative stress conditions, an increase in number, but not in size, of lipid raft domains was detected. Macrophage activation by LPS increase the frequency of fluid regions, which was inhibited by oxidative stress. Concerning macrophage function, secretion of TNFα under oxidative conditions was decreased. Lymphocytes showed a significant increase in the frequency of rigid lipid regions, at the expense of fluid regions, under oxidative stress conditions. Upon PIBF binding to its receptor, lymphocyte plasma membrane became more rigid due to clustering of lipid rafts. However, when PIBF bound lymphocytes were placed in oxidizing conditions, lipid raft clustering was inhibited and PIBF binding to its receptor was also decreased. Conclusions: 1) In single living cells plasma membrane lipid dynamics was evaluated. 2) An important general consequence of oxidative stress is that both in macrophages and lymphocytes plasma membrane becomes more rigid. 3) Receptor-ligand interactions have an effect on membrane fluidity, which vary greatly between the two cell types studied: macrophages and lymphocytes. Upon receptor-ligand binding, macrophage plasma membrane became more fluid while lymphocytes plasma membrane became more rigid. Our results suggest that lipid raft clustering is linked to cell function: upon PIBF binding to its receptor lipid raft clustering occurs in lymphocytes; however, upon LPS/TLR2/4 lipid raft clustering does not occur in macrophages. 4) Nevertheless, the effect induced by receptor-ligand binding on membrane fluidity was inhibited during oxidative stress in both cases.
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Stauffer, Thomas P. "Isoforms and alternative splicing of the plasma membrane calcium ATPase /." [S.l.] : [s.n.], 1995. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=11053.
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Mahammad, Saleemulla. "Cholesterol in T cells homeostasis, plasma membrane organization and signaling /." Doctoral thesis, Stockholm : The Wenner-Gren Institute, Stockholm University, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-38357.
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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.
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Hofmann, Francesco. "Structure-function studies on the human plasma membrane Ca²⁺ATPase /." Zürich, 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10272.
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Degani, Nikhat. "Altered plasma membrane cholesterol in Niemann-Pick type II disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq24831.pdf.
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