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Dinamica molecular de articaina em membranas POPC / Molecular dynamics of articaine in POPC membranesPrates, Erica Teixeira, 1985- 08 March 2009 (has links)
Orientadores: Munir Salomão Skaf, Monica Andrea Pickholz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T17:06:24Z (GMT). No. of bitstreams: 1
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Previous issue date: 2009 / Resumo: Neste trabalho foi feito o estudo das interações da articaína, um anestésico local de ampla aplicação médico-odontológica, com membranas modelo de POPC (palmitoil-oleil-fosfatidilcolina) em condições próximas às biologicamente relevan- tes empregando-se simulações computacionais de dinâmica molecular. Em uma primeira etapa, empregamos métodos quânticos para modelar a articaína com base no campo de força CHARMM. Das simulações de equilíbrio da articaína em POPC, foi possível obter informações como o seu comportamento conformacional e sua posi- ção transversal na bicamada, assim como suas interações especícas com os lipídios. Os estudos foram realizados para os estados neutro e protonado da articaína, consi- derando também seus isômeros ópticos. Estas análises, em conjunto com resultados experimentais de H-RMN realizados pela Prof. Eneida de Paula (IB-UNICAMP) e pelo Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP), demonstram que a articaína, em sua forma neutra, posiciona-se preferencial- mente na interface membrana/água, onde interage frequentemente com os lipídios através de ligações de hidrogênio. Através de ferramentas como perfil de densidade eletrônica do sistema, da parte teórica, e perfil do tempo de relaxação longitudinal para diferentes regiões dos lipídios, da parte experimental, foi discutida a lipossolubilidade da articaína em relação a outros anestésicos. Também foram realizadas simulações de não equilíbrio, utilizando a técnica de Dinâmica Molecular de Caminho Induzido, em que uma molécula de articaína foi removida do interior da membrana para o meio aquoso, através de uma força aplicada em seu centro de massa. Com a aplicação da igualdade de Jarzynski a estas simulações, foi possível estimar a energia livre de partição da ATC neutra (forma mais potente) entre os estados em que encontra-se no seio aquoso e no interior da membrana POPC. / Abstract: We studied the interactions of articaine - a local anesthetic widely used for me- dical and odontological applications - with model membranes of POPC (palmitoyl-oleyl-phosphatidylcholine) at biological relevant conditions. We have employed molecular dynamics technique, which allowed us to investigate the system at molecular level. Firstly, we applied quantum mechanical methods to parametrize articaine molecule based on CHARMM force field. We have done extensive molceular dynamics simulations, taking into account the different ionization states of the drug (neutral and protonated) as well as its optical isomers. From the equilibirum simulations of articaine in POPC membranes, we investigated the conformational behaviour of the drug, its tranversal position and its specific interactions with the lipids and water molecules. Our results show a preferential orientation of the articaine molecule within the membrane. Neutral articaine was mainly found at the lipid head/water interface, in very good agreement with H-RMN experimental results from Prof. Eneida de Paula (IB-UNICAMP) and Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP) and from literature (C. Song et al, 2008). By studying properties like electronic density prole and longitudi- nal time relaxation for different regions of the lipid molecules, we discussed the lipossolubility of articaine in comparison to other local anesthetics. We have also performed non-equilibrium simulations, using steered molecular dynamics (SMD) technique. A single articaine molecule was extracted from the membrane to the wa- ter phase, by applying an external force in its mass centre. Coupling the Jarzynski identity to the SMD simulations, we estimated the partition free energy of the neutral drug (the most potent specie) in POPC membranes. / Mestrado / Físico-Química / Mestre em Química
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Cell membrane softening in human breast and cervical cancer cellsHändel, Chris, Schmidt, B.U. Sebastian, Schiller, Jürgen, Dietrich, Undine, Möhn, Till, Kießling, Tobias R., Pawlizak, Steve, Fritsch, Anatol W., Horn, Lars-Christian, Briest, Susanne, Höckel, Michael, Zink, Mareike, Käs, Josef A. 12 August 2022 (has links)
Biomechanical properties are key to many cellular functions such as cell division and cell motility and
thus are crucial in the development and understanding of several diseases, for instance cancer. The
mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial
systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and
membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this
study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the
plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell
blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be
shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This
can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the
reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.
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Electrostatics and binding properties of Phosphatidylinositol-4,5-bisphosphate in model membranesGraber, Zachary T. 24 November 2014 (has links)
No description available.
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Structure of charged two-component lipid membranes and their interaction with colloids studied by different X-ray and microscopy techniques / Struktur der geladenen Zwei-Komponenten-Lipidmembranen und ihre Interaktion mit Kolloiden studierte durch verschiedene Röntgenstrahl- und MikroskopietechnikenNovakova, Eva 09 July 2008 (has links)
No description available.
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Microscopia de varredura por sonda aplicada a materiais biológicosLorite, Gabriela Simone, 1983- 23 March 2007 (has links)
Orientador: Monica Alonso Cotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-09T02:40:20Z (GMT). No. of bitstreams: 1
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Previous issue date: 2007 / Resumo: Apresentamos nesta dissertação o estudo realizado em cristais de proteínas e membranas modelo (bicamadas lipídicas) utilizando microscopia de força atômica (AFM) como principal técnica de análise. Para os cristais de proteína foram adquiridas imagens topográficas e óticas com a finalidade de obtermos maiores informações sobre o processo de cristalização dos mesmos. No caso das membranas modelo investigamos sua interação com o anestésico local dibucaína (DBC) através de imagens de topografia e fase durante a adição gradual da DBC à solução em contato com a bicamada, além de medidas adicionais de cinética de adsorção e elasticidade.
Os cristais de proteína forma preparados pelo método de difusão de vapor em gota posicionada. Para a realização das imagens dos cristais em sua solução de crescimento (altamente viscosa) foi desenvolvida uma metodologia com a finalidade de evitar o amortecimento da vibração da alavanca. Imagens topográficas do cristal de proteína da bactéria patogênica Mycobacterium Tuberculosis) mostraram que a técnica de AFM permite avaliar o grau de ordem cristalina, bem como a existência de defeitos. No cristal da proteína da bactéria Xylella Fastidiosa, foi observada a presença de uma superfície suave com alguns terraços e degraus com altura de aproximadamente 3nm. Também foram observadas formações anisotrópicas na superfície do cristal, em forma de nanofios, sugerindo uma rota diferente de cristalização em condições de baixa concentração de proteína. A partir dessa observação, variamos as condições de crescimento, revelando mudanças significativas nas taxas de crescimento ao longo das diferentes direções do cristal que favoreciam o aumento da anisotropia de forma.
As bicamadas lipídicas foram preparadas a partir dos fosfolipídios: fosfatidilcolina de ovo (EPC) e dimiristoilfosfatidilcolina (DMPC) pelo método de fusão de vesículas. Imagens de topografia e fase foram adquiridas por AFM, tanto para caracterização topográfica, bem como para observação da ação da DBC (adicionada gradualmente durante a aquisição) sobre a bicamada. As imagens AFM mostram que as bicamadas EPC se formam em domínios sobre a mica e não apresentam um formato típico nem distribuição uniforme. Por outro lado, bicamadas de DMPC se formam em domínios extensos com multi camadas e de maneira mais homogênea quando comparadas com as bicamadas de EPC.
No caso da EPC, observamos a ação da DBC em função do aumento de sua concentração e ao longo do tempo. A seqüência de imagens topográficas mostra o desaparecimento de arranjos lipídicos com o aumento da concentração de DBC. Também observamos que para concentrações elevadas (5mM) o efeito da DBC na membrana é muito drástico. Já para bicamadas de DMPC, observamos as alterações morfológicas para única concentração de DBC (5mM) ao longo do tempo. Neste caso, apesar da concentração elevada, o desaparecimento das bicamadas ocorreu lentamente em comparação com as bicamadas de EPC. Em ambos os casos, as imagens de fase mostram alterações na superfície da bicamada, indicando a alterações nas propriedades elásticas da bicamada na presença de DBC. Medidas de cinética de adsorção e elasticidade superficiais em monocamadas destes lipídios na presença de DBC pelo método de gota pendente corroboram esta hipótese / Abstract: In this work we report the study on protein crystallization mechanisms and the interaction of local anesthetic dibucaine (DBC) with lipids domains in model membranes by Atomic Force Microscopy (AFM). We have also used optical microscopy for crystals analysis as well as adsorption kinetics and elasticity measurements for the dibucaine-membrane interaction.
Crystallized proteins were prepared by the sitting drop vapor diffusion method. Experimental procedures were developed for imaging the crystal in the very viscous solution where they grow, in order to prevent strong dampening of the cantilever vibration. Topographic images of the protein crystal of the pathogenic bacterium Mycobacterium Tuberculosis show that AFM can provide an evaluation of the crystalline quality and information on existing defects. Moreover, protein crystals of the phytopathogenic bacterium Xylella Fastidiosa were more thoroughly analyzed. The AFM topography of this protein crystal shows smooth surfaces with terraces and step edges about ~3nm high. We also observed anisotropic structures on the surface (nanowires), indicating a possible different route for crystallization at lower protein supersaturations. Based on this interpretation, we have changed growth conditions, thus altering growth rates along the different directions and obtaining a more anisotropic crystal shape.
Supported egg phosphatidylcholine (EPC) and dimyristoylfosfatidylcholine (DMPC) were formed on mica using the vesicle fusion method. Topography and phase images were acquired on the lipid membrane in the absence or presence of DBC. The AFM images show irregularly distributed and sized EPC domains on mica. On the other hand, DMPC formation presents extensive bilayer (on mica) with multi-bilayer domains.
For EPC bilayers, we have observed a progressive decrease in size of the original EPC domains with increasing DBC concentration. At higher concentrations (5mM), the DBC effect was more drastic, causing disruption of the EPC bilayer. In the DMPC bilayer case, at 5mM DBC concentration, we observed a progressive disruption of the domains with time, but more slowly than in the EPC case. In both cases, phase images show the formation of small structures on the bilayer surface, indicating changes in the elastic properties of the bilayers when DBC is present. Adsorption kinetics and elasticity measurements of EPC and DMPC monolayers in the presence of DBC by pendant drop method confirm this hypothesis. A discussion of possible mechanisms for these effects is presented. / Mestrado / Física / Mestre em Física
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Interactions of Quercetin-Uranium Complexes with Biomembranes and DNAAttia, Enas 21 July 2014 (has links)
Uranium decontamination gains a great importance with the spread of nuclear waste in both soil and water systems across the planet. All known remediation methods of uranium can be exclusively based either on synthetic materials with high adsorbent power and known physical chemistry or life organisms by which the uranium eventually accumulated inside their tissues. In the present thesis, it was attempted to design a rational approach for uranyl removal primarily from waters using the reducing potential of quercetin, which is a plant-derived small organic molecules, along with its photochemical activities. Such approach, which is neither a fully synthetic nor an organism-based approach, was chosen here to avoid disadvantages with both traditional strategies. Here, complexation experiments were designed to assess the use of uranyl-quercetin complexes for the photoreduction of water-soluble U(VI) to insoluble U(IV) by comparing absorption properties of uranyl-quercetin complexes in acetone, water, and hydrophobic bilayer lipid vesicles.
The UV-vis data show that uranyl quercetin complex can form in both hydrophobic and hydrophilic environments. In both cases the B-ring band in quercetin structure becomes reduced, red shifted and a pronounced absorption arises in the 400-500 nm range. Such data suggests that U(VI) binds at the 3-OH and 4-carbonyl of ring C of quercetin.
Interestingly, the results of UV-Vis spectroscopy part hint at a crucial role of a stable or transiently ionized hydroxyl for the efficient uranyl-dependent photodegradation of quercetin. FTIR spectroscopy absorption changes further demonstrates that the UV-vis-spectroscopic changes are indeed accompanied by changes in the chemical structure of the complex as expected for a uranyl-dependent photodegradation. IR data thus suggest that U(VI) becomes reduced by the photoreaction, rather than merely changing its coordination shell. The frequency shifts in the C=C and C=O absorption range on the other hand are consistent with changes in force constants rather than bond breakage. Upon illumination condition, uranyl quercetin complex in water forms a dark precipitate. Uranyl precipitation and the disappearance of U(VI) IR absorption bands upon illumination further demonstrate that uranyl acts as a redox partner rather than a catalyst in the photoreaction of quercetin.
The formation of uranyl-quercetin complexes in the presence of lipidic phases has been addressed experimentally. The complex is partitioned into the hydrophilic/hydrophobic interface of liposomes. Its electronic absorption properties are influenced by the degree of hydrophobicity provided by the adjacent lipid headgroups. The preference of quercetin to associate with hydrophobic microenvironments can thus be exploited to transfer uranyl to the lipid water biomolecular interface. Illumination of the uranyl-quercetin complex in the presence of different liposomes has been performed in this study for the first time, to the best of my knowledge. The data provide evidence that again uranyl is a redox partner for the photodegradation of quercetin also in this microenvironment. Uranyl in an oxidation state smaller than VI is unsoluble in water.
Therefore, its quercetin-mediated photoreduaction of uranium provides a method to transfer soluble uranium to the liposome and stabilize the reduced photoproduct. Thereby, uranyl could be removed from solution in an insoluble form using cheap natural compounds.
The binding site assignment of uranyl-quercetin complex in acetone have been verified here using NMR spectra and DFT theory. NMR Spectra showed that the observations of broadened and narrow bands in the NMR spectra of quercetin, upon complexation with uranyl, support an intramolecular exchange or site exchange within the quercetin molecule. Moreover, the complexation takes place around the carbonyl group with U(VI) exhibiting two possibly coordination modes, involving the carbonyl and the adjacent O(H) groups. This has been also confirmed from the DFT calculations.
Finally, interaction experiments of uranyl-quercetin complex with DNA have been performed to assess an alternative uranyl-trapping and photoreduction system. The data show that consecutive addition of quercetin and uranyl destabilizes DNA. However, a preformed uranyl quercetin complex has very little effect on DNA structure. On the other hand, quercetin and uranyl appear to bind to DNA as a preformed complex in the loop portion of hairpin DNA. Therefore, also HP DNA is expected to be a suitable but less effective trapping system for the uranyl quercetin complex and its potential photoproducts.
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Fluctuations and Oscillations in Cell Membranes / Fluktuationen und Oszillationen in ZellmembranenHändel, Chris 29 March 2016 (has links) (PDF)
Zellmembranen sind hochspezialisierte Mehrkomponentenlegierungen, welche sowohl die
Zelle selbst als auch ihre Organellen umgeben. Sie spielen eine entscheidende Rolle bei
vielen biologisch relevanten Prozessen wie die Signaltransduktion und die Zellbewegung.
Aus diesem Grund ist eine genaue Charakterisierung ihrer Eigenschaften der Schlüssel
zum Verständnis der Bausteine des Lebens sowie ihrer Erkrankungen. Besonders Krebs
steht im engen Zusammenhang mit Veränderungen der biomechanischen Eigenschaften
vom Gewebe, Zellen und ihren Organellen. Während Veränderungen des Zytoskeletts
von Krebszellen im Fokus vieler Biophysiker stehen, ist die Bedeutung der Biomechanik
von Zellmembran weitgehend unklar. Zellmembranen faszinieren Wissenschaftler jedoch
nicht nur wegen ihrer biomechanischen Eigenschaften. Sie sind auch Beispiele für eine
selbstorganisierte und heterogene Landschaft, in der Prozesse fernab des Gleichgewichtes,
wie z.B. räumliche und zeitliche Musterbildungen, auftreten. Die vorgelegte Dissertation
untersucht erstmals umfassend die zentrale Rolle der Zellmembran und ihrer molekularen
Architektur für die Signalübertragung, die Biomechanik und die Zellmigration. Hierfür
werden einfache Modellmembranen aber auch komplexere Vesikel und ganze Zellen
mittels etablierter physikalischer Methoden analysiert. Diese reichen von Fourier-
Analysen zur Charakterisierung von thermisch angeregten Membranundulationen über
Massenspektrometrie und ‘Optical Stretcher’ Messungen von ganzen Zellen bis hin
zur Filmwaagentechnik. Des Weiteren wird ein Modellsystem vorgestellt, welches
sowohl einen experimentellen als auch einen mathematischen Zugang zum ‘ME-switch’
ermöglicht. Die vorgelegte Dissertation bietet neue Einblicke in wichtige Funktionen
von Zellmembranen und zeigt neue therapeutische Perspektiven in der Membran- und
Krebsforschung auf.
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Applications of droplet interface bilayers : specific capacitance measurements and membrane protein corrallingGross, Linda C. M. January 2011 (has links)
Droplet Interface Bilayers (DIBs) have a number of attributes that distinguish them from conventional artificial lipid bilayers. In particular, the ability to manipulate bilayers mechanically is explored in this thesis. Directed bilayer area changes are used to make precise measurements of the specific capacitance of DIBs and to control the two dimensional concentration of a membrane protein reconstituted in the bilayer. Chapter 1 provides a general introduction to the role of the lipid membrane en- vironment in the function of biological membranes and their integral proteins. An overview of model lipid bilayer systems is given. Chapter 2 introduces work carried out in this laboratory previously and illustrates the experimental setup of DIBs. Some important bilayer biophysical concepts are covered to provide the theoretical background to experiments in this and in later chapters. Results from the characterisation of DIBs are reported, and an account of the development of methods to manipulate the bilayer by mechanical means is given. Chapter 3 describes experiments that apply bilayer area manipulation in DIBs to achieve precise measurement of specific capacitance in a range of lipid systems. Chapter 4 reports results from experiments investigating the response of bilayer specific capacitance to an applied potential. Chapter 5 covers the background and experimental setup for total internal fluo- rescence microscopy experiments in DIBs and describes the expression, purification and characterisation of the bacterial β-barrel membrane protein pore α-Hemolysin. Chapter 6 describes experiments that apply the mechanical manipulation of bilayer area in DIBs to the corralling and control of the surface density of α-Hemolysin.
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Fluctuations and Oscillations in Cell MembranesHändel, Chris 22 February 2016 (has links)
Zellmembranen sind hochspezialisierte Mehrkomponentenlegierungen, welche sowohl die
Zelle selbst als auch ihre Organellen umgeben. Sie spielen eine entscheidende Rolle bei
vielen biologisch relevanten Prozessen wie die Signaltransduktion und die Zellbewegung.
Aus diesem Grund ist eine genaue Charakterisierung ihrer Eigenschaften der Schlüssel
zum Verständnis der Bausteine des Lebens sowie ihrer Erkrankungen. Besonders Krebs
steht im engen Zusammenhang mit Veränderungen der biomechanischen Eigenschaften
vom Gewebe, Zellen und ihren Organellen. Während Veränderungen des Zytoskeletts
von Krebszellen im Fokus vieler Biophysiker stehen, ist die Bedeutung der Biomechanik
von Zellmembran weitgehend unklar. Zellmembranen faszinieren Wissenschaftler jedoch
nicht nur wegen ihrer biomechanischen Eigenschaften. Sie sind auch Beispiele für eine
selbstorganisierte und heterogene Landschaft, in der Prozesse fernab des Gleichgewichtes,
wie z.B. räumliche und zeitliche Musterbildungen, auftreten. Die vorgelegte Dissertation
untersucht erstmals umfassend die zentrale Rolle der Zellmembran und ihrer molekularen
Architektur für die Signalübertragung, die Biomechanik und die Zellmigration. Hierfür
werden einfache Modellmembranen aber auch komplexere Vesikel und ganze Zellen
mittels etablierter physikalischer Methoden analysiert. Diese reichen von Fourier-
Analysen zur Charakterisierung von thermisch angeregten Membranundulationen über
Massenspektrometrie und ‘Optical Stretcher’ Messungen von ganzen Zellen bis hin
zur Filmwaagentechnik. Des Weiteren wird ein Modellsystem vorgestellt, welches
sowohl einen experimentellen als auch einen mathematischen Zugang zum ‘ME-switch’
ermöglicht. Die vorgelegte Dissertation bietet neue Einblicke in wichtige Funktionen
von Zellmembranen und zeigt neue therapeutische Perspektiven in der Membran- und
Krebsforschung auf.:1 Introduction
2 Background
2.1 The Cell Membrane
2.1.1 Lipids in Cell Membranes
2.1.2 Membrane Proteins
2.1.3 An Overview on Membrane Models
2.1.4 Lipid Rafts
2.2 Model Membranes – An Experimental Access to Cell Membranes
2.2.1 Surface Tension and Thermodynamic Equilibrium
2.2.2 Langmuir Monolayer
2.2.3 The Polymorphism of Langmuir Monolayers
2.2.4 Membrane Vesicles
2.3 Biological Membranes as Semiflexible Shells
2.3.1 Elasticity of Soft Shells
2.3.2 Helfrichs Theory About Bending Deformations
2.3.3 Membrane Undulation
2.4 Membranes in Cell Signaling
2.4.1 Signal Transduction Fundamentals
2.4.2 Phosphoinositides
2.4.3 Phosphatidylinositol Signaling Pathway
2.4.4 The Myristoyl-Electrostatic Switch
2.5 Reaction-Diffusion Systems
2.5.1 Diffusion
2.5.2 Michaelis-Menten Kinetics
2.5.3 Reaction-Diffusion Systems
3 Methods, Materials and Theory
3.1 Optical Microscopy
3.1.1 Fluorescence Microscopy
3.1.2 Phase Contrast Microscopy
3.2 Cell Culture and GPMV Formation
3.2.1 Tumor Dissociation and Cell Culturing of Primary Cells
3.2.2 Cell Lines and Cell Culturing
3.2.3 Preparation of Giant Plasma Membrane Vesicles
3.3 Optical Stretcher
3.4 Fourier Analysis of Thermally Excited Membrane Fluctuations
3.4.1 The Quasi-Spherical Model – Membrane Fluctuations
3.4.2 Determination of the Bending Rigidity
3.5 Mass Spectrometry
3.5.1 MALDI-TOF Mass Spectrometry
3.5.2 ESI Mass Spectrometry
3.6 Migration, Invasion and Cell Death Assays
3.7 Langmuir-Blodgett Technique
3.7.1 Langmuir Troughs and Film Balances
3.7.2 Experimental Setup and Monolayer Preperation
3.7.3 Phospholipids, Dyes and Buffer Solutions
4 Fluctuations in Cell Membranes
4.1 Cell Membrane Softening in Human Breast and Cervical Cancer Cells
4.1.1 Bending Rigidity of Human Beast and Cervical Cell Membranes
4.1.2 MALDI-TOF Analysis of Lipid Composition
4.1.3 Summary and Discussion
4.2 Targeting of Membrane Rigidity – Implications on Migration
4.2.1 ESI Tandem Analysis of Lipid Composition
4.2.2 Biomechanical Behavior of Whole Cells and Membranes
4.2.3 Migration and Invasion Behavior
4.2.4 Summary and Discussion
5 Oscillations in Cell Membranes
5.1 Mimicking the ME-switch
5.1.1 DPPC/PIP2 monolayers at the presence of MARCKS
5.1.2 Lateral organization of PIP2 in DPPC/PIP2 monolayers
5.1.3 Translocation of MARCKS
5.1.4 Phosphorylation of MARCKS by PKC
5.1.5 Summary and Discussion
5.2 Dynamic Membrane Structure Induces Temporal Pattern Formation
5.2.1 Mechanism of the Oscillation
5.2.2 Modeling the ME-switch
5.2.3 Time Evolution
5.2.4 Phase Diagrams and Open Systems
5.2.5 Summary and Discussion
6 Conclusion and Outlook
Appendix
Bibliography
List of Figures
List of Abbreviations
Acknowledgement
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