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1
Connell, E. J. "Protein-lipid interactions in synaptic vesicle exocytosis". Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597894.
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The fusion of neurotransmitter-filled synaptic vesicles with the pre-synaptic membrane in response to calcium influx is exquisitely regulated. Synaptic vesicle exocytosis is energetically demanding and the neuronal SNARE proteins syntaxin, SNAP25 and synaptobrevin have come to prominence as the driving engines behind this process. Resident on both vesicular and pre-synaptic membranes they form a stable four-helical bundle, the assembly of which contributes to membrane fusion. However, SNAREs do not act in isolation during synaptic vesicle exocytosis but are instead regulated by a complex web of interactions with other proteins including synaptotagmin, a calcium-sensing component of the vesicle itself, and Munc18, a highly-conserved cytosolic protein. In addition, changes in the lipid environment surrounding the SNAREs play a critical role. In this thesis I report the results of two lines of investigation, into both synaptotagmin’s and Munc18’s action. Firstly, I consider the significance of the cytoplasmic double C2 domain structure of synaptotagmin. Using several strategies including a novel real-time absorbance assay, I show that these tandem C2 domains, but neither domain alone, rapidly cross-link lipid membranes in the presence of calcium. This property is conserved. Cross-linking ability can be masked in full-length synaptotagmin, via an electrostatic interaction with the membrane in which it is embedded. Finally, I address the mechanism of arachidonic acid action on syntaxin/Munc18, showing that this lipid activates Munc18-bound syntaxin and that a Munc18/syntaxin/SNAP25 assembly exists in brain. Arachidonic acid also activates free syntaxin, defining a molecular target for the reported role of this lipid in the promotion of vesicle fusion. My data are incorporated into a revised model of the protein-lipid interactions underlying synaptic vesicle exocytosis.
2
Hamada, Tsutomu. "Morphological dynamics and biological functions in a cell-sized lipid vesicle". 京都大学 (Kyoto University), 2006. http://hdl.handle.net/2433/144128.
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Kyoto University (京都大学)0048
新制・課程博士
博士(理学)
甲第12078号
理博第2972号
新制||理||1444(附属図書館)
23914
UT51-2006-J73
京都大学大学院理学研究科物理学・宇宙物理学専攻
(主査)教授 吉川 研一, 助教授 瀬戸 秀紀, 教授 小貫 明
学位規則第4条第1項該当
3
Kausik, Ravinath, Brandon D. Armstrong i Songi Han. "Study of local diffusion coefficients of the hydration layer of lipid vesicle bilayers". Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-192351.
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Water molecules near the surface of vesicle bilayers exhibit slow dynamics with respect to that of pure bulk water as they belong to the hydration layer. We present a unique analysis tool for the selective detection of local water of the hydration layer on the surface
of unilamellar vesicles and the determination of its diffusion coefficients. We utilized stable nitroxide radicals covalently attached to the hydrophilic head groups of DOPC lipid chains that incorporate along with other lipids into vesicles. Through the use of dynamic
nuclear polarization (DNP) the 1H NMR signal of local water interacting with the radical is amplified, and we present here an analysis of the local diffusion coefficients of this hydration layer.
4
Kausik, Ravinath, Brandon D. Armstrong i Songi Han. "Study of local diffusion coefficients of the hydration layer of lipid vesicle bilayers". Diffusion fundamentals 10 (2009) 27, S. 1-4, 2009. https://ul.qucosa.de/id/qucosa%3A14118.
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Water molecules near the surface of vesicle bilayers exhibit slow dynamics with respect to that of pure bulk water as they belong to the hydration layer. We present a unique analysis tool for the selective detection of local water of the hydration layer on the surface
of unilamellar vesicles and the determination of its diffusion coefficients. We utilized stable nitroxide radicals covalently attached to the hydrophilic head groups of DOPC lipid chains that incorporate along with other lipids into vesicles. Through the use of dynamic
nuclear polarization (DNP) the 1H NMR signal of local water interacting with the radical is amplified, and we present here an analysis of the local diffusion coefficients of this hydration layer.
5
Rouhvand, Bahar. "Vesicle-Protein Diffusion and Interaction Study Using Time Resolved Fluorescence Correlation Spectroscopy". University of Akron / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=akron1503261462042903.
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Ashrafzadeh, Parham. "Exploring Cellular Dynamics : From Vesicle Tethering to Cell Migration". Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-306174.
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Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.
7
Schwamborn, Miriam. "Establishment of a fluorescence assay for characterization of protein-mediated vesicle fusion and acidification". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3E83-7.
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Dun, Alison. "Spatial and temporal control of regulated exocytosis by protein and lipid interactions". Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8087.
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Cellular communication requires the transport of chemical messengers between intracellular compartments and from cell to cell. The regulated exocytosis of a secretory vesicle at the plasma membrane involves the merger of two bilayers, with markedly different lipid composition, within a millisecond time scale. The spatial and temporal control of the protein and lipid complement at these fusion sites is essential. A highly conserved family of proteins are known to drive this fusion event; SNAP-25 and syntaxin-1 (t-SNAREs) associate at the plasma membrane in a 1:1 stoichiometry to provide a binding site for the vesicle-membrane protein synaptobrevin (v-SNARE). The formation of this complex and subsequent fusion requires accessory proteins for efficient calcium-triggered exocytosis; which of these proteins facilitate the initial attachment of vesicle to the plasma membrane prior to fusion is still under debate. Specific sites for vesicle fusion have been proposed and the organisation of lipids and proteins at these fusion sites has been extensively investigated with limited spatial and temporal resolution; however the presence of raft-forming lipids at these sites as well as the arrangement of SNARE proteins at the molecular level is still under contention. The data presented within this thesis aims to elucidate the protein and lipid environment at the fusion site using super-resolution microscopy and advanced vesicle tracking. Under diffraction-limited microscopy the t-SNAREs are visualised as 200 nm homogenous clusters; however I have used single molecule localisation microscopy to reveal a more complex heterogeneous molecular arrangement. Quantification of lipid order exclusively at the plasma membrane provided insight into the influence of cholesterol-induced lipid arrangement on SNAP-25 localisation. In addition the t-SNARE interaction was investigated using TCSPC-FLIM identifying two lipid-order-dependent conformations in distinct clusters at the plasma membrane. Extensive vesicle tracking at optimum sampling rates demonstrated the ‘sampling’ behaviour of LDCVs and allowed characterisation of vesicle fusion sites. In summary I find that vesicles exhibit preference for residence and probably fusion at regions of plasma membrane with a low t-SNARE density; these proteins appear to exert control over exocytosis by adopting alternative conformations that are under cholesterol-induced regulation.
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Harman, Alison. "A Molecular Dynamics Simulation of Vesicle Deformation and Rupture in Confined Poiseuille Flow". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26127.
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Vesicles are simple structures, but display complex, non-linear dynamics in fluid flow. I investigate the deformation of nanometer-sized vesicles, both fully-inflated and those with excess area, as they travel in tightly confined capillaries. By varying both channel size and flow strength, I simulate vesicles as they transition from steady-state to unstable shapes, and then rupture in strong flow fields. By employing a molecular dynamics model of the vesicle, fluid, and capillary system one is able to rupture the lipid bilayer of these vesicles. This is unique in that most other numerical methods for modelling vesicles are unable to show rupture. The rupture of fully-inflated vesicles is applicable to drug delivery in which the release of the encapsulated medicine needs to be controlled. The deformation and rupture of vesicles with excess area could be applicable to red blood cells which have similar rheological properties.
10
Kuhlmann, Jan Wilhelm. "Modulation of lateral membrane tension and SNARE-mediated single vesicle fusion on pore spanning membranes". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3F13-E.
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Ferreira, Vasconcelos Luis Daniel. "Oligonucleotide Complexes with Cell-Penetrating Peptides : Structure, Binding, Translocation and Flux in Lipid Membranes". Licentiate thesis, Stockholms universitet, Institutionen för neurokemi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-109299.
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The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.
12
Nikolaus, Jörg. "Hemifusion and lateral lipid domain partition in lipid membranes of different complexity". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16437.
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Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden.Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.
13
Asghari, Adib Ali. "Interactions of Engineered Silica Nanoparticles with Cell Membrane Models". Ohio University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1501764587639053.
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Ho, Chian Sing. "Inquiry of Lipid Membranes Interacting with Functional Peptides and Polyphenol Drug Molecules". Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6255.
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Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives modulate the phase behavior of free-standing GUVs, as well as the miscibility transition temperature (Tm). We find that M2TM increases Tm and causes vesicle budding; polyglutamine aggregates disrupt lipid membranes; and the three polyphenol compounds exert disparate effects on GUV Tm.
15
Jackson, James R. "On the influence of PI(4,5)P2 and PI(3,4,5)P3-enriched lipid microdomains on exocytosis". University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1367937501.
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Henneberry, Annette Lisa. "Molecular analysis of lipid synthesis and its role in vesicle trafficking, de novo cloning, expression, and characterization of two human cholinephosphotransferases". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ66672.pdf.
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Groth, Mike Christopher. "Investigation of the Linker Region of Coiled Coil SNARE-Analoga and Membrane Composition on Vesicle Fusion". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://hdl.handle.net/21.11130/00-1735-0000-0005-159B-5.
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Sinn, Cornelia G. "Ion binding to polymers and lipid membranes in aqueous solutions : Ionenbindung an Polymeren und Lipidmembranen in wässrigen Lösungen". Phd thesis, Universität Potsdam, 2004. http://opus.kobv.de/ubp/volltexte/2005/186/.
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Ziel dieser Arbeit ist die Untersuchung der Ionenbindung an Polymeren und Lipidmembranen in wässrigen Lösungen. Im ersten Teil dieser Arbeit wurde der Einfluss verschiedener anorganischer Salze und Polyelektrolyte auf die Struktur des Wassers mit Hilfe Isothermer Mikrotitrationskalorimetrie (ITC) erforscht. Die Verdünnungswärme der Salze wurde als Maß für die Fähigkeit der Ionen, die geordnete Struktur des Wassers zu stabilisieren oder zu zerstören, verwendet. Die Verdünnungswärmen konnten auf Hofmeister Effekte zurückgeführt werden. Im Anschluss daran wurde die Bindung von Ca2+ an Natrium- Poly(acrylsäure) (NaPAA) untersucht. Mit Hilfe von ITC und einer Ca2+- selektiven Elektrode wurde die Reaktionsenthalpie und Bindungsisotherme gemessen. Es wurde gezeigt, dass die Binding von Ca2+ - Ionen an NaPAA stark endotherm und daher entropiegetrieben ist. Anschließend wurde die Bindung von Ca2+ an die eindimensionale Polymerkette mit der an ein Lipidvesikel mit denselben funktioniellen Gruppen verglichen. Es wurde beobachtet, dass die Ionenbindung –wie auch im Fall des Polymers- endotherm ist. Ein Vergleich der Ca2+- Bindung an die Lipidmembran mit der an das Polymer konnte zeigen, dass das Ion schwächer an die Membran bindet. Im Zusammenhang mit diesen Experimenten wurde auch beobachtet, dass Ca2+ nicht nur an geladene, sondern auch an zwitterionische Lipidvesikel bindet. Schließlich wurde die Wechselwirkung zweier Salze, KCl and NaCl, mit einem neutralen Polymergel, PNIPAAM, und dem geladenen Polymer PAA untersucht. Mit Hilfe von Kalorimetrie und einer kaliumselektiven Elektrode wurde beobachtet, dass die Ionen mit beiden Polymeren wechselwirken, unabhängig davon, ob diese Ladungen tragen, oder nicht.The goal of this work was to study the binding of ions to polymers and lipid bilayer membranes in aqueous solutions. In the first part of this work, the influence of various inorganic salts and polyelectrolytes on the structure of water was studied using Isothermal Titration Calorimetry (ITC). The heat of dilution of the salts was used as a scale of water structure making and breaking of the ions. The heats of dilution could be attributed to the Hofmeister Series. Following this, the binding of Ca2+ to poly(sodium acrylate) (NaPAA) was studied. ITC and a Ca2+ Ion Selective Electrode were used to measure the reaction enthalpy and binding isotherm. Binding of Ca2+ ions to PAA, was found to be highly endothermic and therefore solely driven by entropy. We then compared the binding of ions to the one-dimensional PAA polymer chain to the binding to lipid vesicles with the same functional groups. As for the polymer, Ca2+ binding was found to be endothermic. Binding of calcium to the lipid bilayer was found to be weaker than to the polymer. In the context of these experiments, it was shown that Ca2+ not only binds to charged but also to zwitterionic lipid vesicles. Finally, we studied the interaction of two salts, KCl and NaCl, to a neutral polymer gel, PNIPAAM, and to the ionic polymer PAA. Combining calorimetry and a potassium selective electrode we observed that the ions interact with both polymers, whether containing charges or not.
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ACCATTATIS, FELICE MARIA. "MAY A PHARMACOLOGICAL MODULATION OF LIPIDS ALTER PROTUMORIGENIC SIGNALING OF EXTRACELLULAR VESICLES? A LONG JOURNEY IN EVS DIMENSIONAL, LIPIDIC AND PROTEIC CHARACTERIZATION FROM A METASTATIC MELANOMA CELL LINE'". Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/831719.
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New insights into size, protein and lipid composition may help in characterizing functionality of Extracellular Vesicles (EVs) and infer with their fast development as delivery tools. Unfortunately, despite latest size-based classifications which divide EVs in small (50-80nm) or large (80-120nm) exosomes, microvesicles (<1000nm) and the new smallest (<50nm) population (exomeres), overlapping of different EV populations and unproper separation methods impair the comprehension of their biological role. In addition (EVs) are a very attractive pharmacological target, due to their involvement in cell-cell communication in physiological and pathological conditions. Among lipids, cholesterol and BisMonoacylGlyceroPhosphate (BMP) play a paramount role in EV biogenesis since their interplay drive endosomes towards the secretory or recycling pathway. We used as model a lymph node metastatic cell line (LM-16).
1)We set up a reproducible ultracentrifugation (UC) method for a size-based separation of different EV populations by 5 UC steps, in which physical and dynamic parameters are determined by an algorithm developed by Livshits et al. 2) In vitro treatment LM-16 cells with simvastatin and/or KT182, respectively inhibitors of cholesterol biosynthesis and of BMP degradation, was carried for 3 days with 10% FCS and for 3 days in serum free conditions. In this case we only isolated two fractions according to canonical EVs isolation method, namely 10K (microvesicles) and 100K (exosomes). EVs were further characterized by Nanosight, Zetasizer, Confocal and Electron microscopy. Quantitative proteomic analysis was performed by mass spectrometry and results analyzed by Ingenuity Pathway Analysis (IPA) while lipid analysis was carried out by gas-liquid chromatography.
1) Zetasizer and TEM analysis documented the existence of 5 different EV populations, whose relative % in saturated fatty acids gradually and continuously increased from larger to smaller EV (from 37.21±0.21 to 64.79±9.47). Proteomics identified a total of 2003 proteins differentially distributed (or even unique) among the 5 EV populations (n=697, 819, 1079, 1621 and 1654, respectively). IPA analysis of these distributions revealed different characteristics signaling pathways. 2) Treatment with simvastatin (0.1µM) and KT182 (50nM) alone or in combination did not affect cell proliferation, and EVs size and counts. Proteomics identified 1294 and 1192 proteins respectively for 100K and 10K, showing different significant expression patterns, compared to control, with several proteins up- and down regulated. IPA showed that treatments specifically decreased the expression of proteins involved in cellular movement, migration, proliferation and cytoskeleton arrangement, both in 100K and 10K fractions, with typical and striking differences and patterns.
Melanoma-released EVs include vesicles of different size, fatty acid and protein composition. These differences may translate into distinct behaviors and functions in biological fluids and help to define the role of specific EV populations in physiological and pathological processes. Cholesterol and BMP modulation of melanoma cells dramatically alters the protein content of released EVs, possibly leading to altered EVs functionality, suggesting the potential of lipid modulation in reshaping tumor cell-released EVs to develop new therapeutic approaches. Finally, these results pave the road to new pharmacological treatments to modulate EVs functions or to use EVs as pharmaceutical tools or melanoma biomarkers.
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Subramaniam, Varuni. "Preparation and Characterization of Novel Lipid and Proteolipid Membranes from Polymerizable Lipids". Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/194889.
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The work described here has focused on two types of supramolecular assemblies, supported lipid bilayers (SLBs) and giant vesicles (GVs) from polymerizable lipids. SLBs are explored extensively as structural models in biophysical studies of cell membranes and biosensor coatings. With regard to implementation as biocompatible scaffoldings for receptor-based molecular devices, fluid SLBs lack chemical, thermal and mechanical stability as lipids are self-organized by weak, noncovalent forces. One possible solution is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how polymerization affects transmembrane protein structure and activity. Specifically it is unclear if lipid cross-linking can be achieved without adversely affecting the activity of incorporated proteins. In this work the effect of lipid polymerization on transmembrane protein activity was studied with rhodopsin. The protein was reconstituted into SLBs composed of polymerizable lipids, bis-SorbPC, bis-SorbPC:mono-SorbPC, bis-DenPC and bis-SorbPC:mono-SorbPE. Rhodopsin photoactivity was monitored using plasmon waveguide spectroscopy. The results show that reconstitution of rhodopsin into SLBs composed of phosphatidylcholine with the polymerizable moiety in the acyl chain terminus, followed by photoinduced cross-linking of the lipids, does not significantly perturb protein function. A possible explanation is that a bilayer with relatively low Xn retains sufficient elasticity to accommodate the membrane deformation that accompanies the conformational change associated with rhodopsin photoactivation when polymerized in the acyl chain terminus. GVs have diameters ranging from several to few hundred micrometers and thus can be observed by optical microscopic methods. This allows manipulation of individual vesicles and observation of their transformations in real time. GVs have attracted attention as microcontainers for enzymes and drugs, and as biosensors. With the aim of increasing stability for these types of applications, GVs were prepared from synthetic dienoyl lipids that can be polymerized to form robust vesicles. The stability of these vesicles after polymerization was investigated by surfactant treatment, drying and rehydration, and temperature variations. The structure of poly(GVs) was largely retained under these conditions which destroy unpolymerized vesicles. Permeability studies on poly(GVs) suggests that they could be potentially used in a variety of technological applications, including sensors, macromolecular carriers, and microreactors.
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Mann, Jamie Ferguson Sajjan. "Oral vaccine delivery using lipid vesicles". Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431816.
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Liu, Shuk Yi. "Encapsulation of magnetosomes in lipid vesicles". HKBU Institutional Repository, 2004. http://repository.hkbu.edu.hk/etd_ra/615.
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Lau, Kent G. "Formulation of novel double-chain lipid vesicles". Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272376.
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Bandegi, Sanaz. "INTERACTION OF FLUORESCENT LIPID DYES WITH LIPID VESICLES AND SUPPORTED LIPID BILAYERS AND THEIR APPLICATIONS". Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/584744.
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ChemistryPh.D.
Lipophilic dye probes are widely used for labelling of cells, organelles, liposomes, viruses and lipoproteins. The lipophilic dye diffuses in the membrane and stains the cell and cells even tolerate the lipophilic dye in high concentration. The fluorescence of styryl dyes increases after insertion into the hydrophobic environment of the lipid membrane compared their fluorescence in the aqueous phase solution. The alkyl chains of the fluorescent styryl dye probe insert into membranes and are used to understand their biophysical properties and their behavior in lipid bilayers. The mechanism of incorporation of the dyes into cell membranes, or vesicle model systems, is not resolved. In this study we used a modified dialkylaminostyryl fluorescent lipid, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA), replacing the I- counterion with the Cl- anion to make DiA-Cl increase hydration of the polar head and to enable self-assembling in water and formation of vesicles. Vesicles composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine)/DiA, DPPC (1,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine) /DiA, DSPC (1,2-distearoyl-sn-glycero-3- phosphatidylcholine) /DiA, DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine)/DiA, DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine)/DiA and DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)/DiA have been prepared in mole ratios between 100/0 to 0/100, in order to investigate the effects of chain length and headgroup type on chain packing and phase separation in these mixed amphiphilic systems, using nanocalorimetry, dynamic light scattering and fluorescence data, as well as confocal laser scanning microscopy (CLSM) and cryo-transmission electron microscopy (Cryo-TEM). In addition, we report the self-assembly of DiA-Cl, to form H-aggregates of lipid bilayers in aqueous solution, beyond a critical vesicle concentration. Lipid bilayers can be fused onto silica nanoparticles (NPs) to form supported lipid bilayer (SLB)-NPs. (SLB)-NPs have a varous interdisciplinary applications from medicine to environmental fields and agriculture sciences. Here, the lipids on the nanoparticles were used for two applications. One was to adsorb polycyclic aromatic hydrocarbons (PAHs) from the environment and the other was as vehicles for foliar delivery of nutrients to plants. Silica SLB nanoparticles can increase the solubility of Benzo[a]Pyrene (BaP) in order to extract the BaP from soil for in situ biodegradation. Initial studies were begun on the effect of foliar application of silica SLBs nanoparticles on plants. The SLBs to be used were prepared using both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and DiA, in order to determine whether the lipid increased the entry of the silica into the plant leaves and whether the lipids also entered.
Temple University--Theses
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Hargrave, Peter Charles. "Correlated photon studies of radio-labelled lipid vesicles". Thesis, Queen Mary, University of London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286457.
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Peters, Jeffrey. "Formation of Vesicles in Lipid-Liquid Crystal Colloidal Mixtures". Digital WPI, 2014. https://digitalcommons.wpi.edu/etd-theses/622.
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The formation, phase ordering, and evolution has been studied in lipid and liquid crystal (LC) colloidal aqueous mixtures as a function of LC concentration and thermal history. The lipid used was 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) while the liquid crystal was pentylcyanobiphenyl (5CB). POPC is a naturally occurring lipid in eukaryotic cell membranes and mimics many of the properties of human cell walls. 5CB is a polar liquid crystal that exhibits a thermodynamically stable orientationally ordered (nematic) state at room temperature. Colloidal dispersions were made at various 5CB and POPC concentrations in water and studied via optical microscopy (phase contrast, confocal, florescence, and cross-polarizing) to probe phase order and evolution as well as by calorimetry to study phase transformations. Very large vesicles (larger than 100 micrometers) were observed to form that appear to use the phase separated 5CB droplets as scaffolds. Also, there appears a unique promotion of dye (used to image the lipid bilayers) crystallization within liquid crystal domains well above room temperature.
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Klemm, Robin. "Lipid rafts in protein sorting and yeast cell polarity". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1184754346185-43377.
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The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.
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Ravoo, Bart Jan. "Membrane fusion of vesicles of oligomerisable lipids". [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 1998. http://irs.ub.rug.nl/ppn/291236774.
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Eleiwa, M. M. "Magnetic field effects in phospholipid vesicles measured by light scattering". Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235243.
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Monem, A. S. M. A. "Angular light scattering from phospholipid vesicles and the effect of magnetic fields". Thesis, University of Southampton, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373565.
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Patty, Philipus Josepus. "Studies of the mechanical properties of lipid vesicles using extrusion methods". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ61479.pdf.
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Stöckl, Martin Thomas. "Giant vesicles". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15869.
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In der vorliegenden Arbeit wird ein neuer Ansatz vorgestellt, um Lipiddomänen, die Bindungsorte peripherer und integraler Membranproteine darstellen können, zu charakterisieren. Insbesondere wurde die Analyse der Fluoreszenzlebenszeiten von NBD-markierten Lipidanaloga benutzt, um Lipiddomänen in Giant unilamellar vesicles (GUV) und darauf aufbauend, in der Plasmamembran von Säugerzellen zu untersuchen. Das typische Zeitfenster von Fluoreszenzlebenszeiten im Bereich von Nanosekunden ermöglicht es, auch sehr kurzlebige Lipiddomänen nachzuweisen. Mit Hilfe des Fluorescence lifetime imaging (FLIM) wurden für die liquid disordered (ld) und liquid ordered (lo) Domänen in GUV jeweils spezifische Werte für das Abklingen der Fluoreszenz gemessen. Sogar die Existenz von submikroskopischen Domänen in GUV konnte nachgewiesen werden. Die Fluoreszenzlebenszeit des Lipidanalogs C6-NBD-PC zeigte in der Plasmamembran von Säugerzellen eine breite Verteilung. Dies legt in Übereinstimmung mit FLIM-Experimenten an aus der Plasmamembran von HeLa-Zellen gewonnenen Giant vesicles nahe, dass in der Plasmamembran von Zellen eine Vielzahl verschiedener submikroskopischer Lipiddomänen existiert. Darauf aufbauend wurde die Fluoreszenzmikroskopie an GUV angewendet, um die Bindung von fluoreszenzmarkiertem alpha-Synuclein an mittels FLIM charakterisierte Lipiddomänen zu untersuchen. Die Experimente zeigten, dass das Protein mit hoher Affinität an negativ geladene Phospholipide unter der Vorraussetzung bindet, dass diese sich in ld Domänen befinden. Im Gegensatz dazu erfolgt keine Bindung wenn diese Lipide in lo Domänen lokalisiert sind. Im Vergleich zum wildtypischen alpha-Synuclein zeigte die Variante A30P eine geringere Affinität zur Membran, während die E46K-Variante eine stärkere Bindung zeigte. Dies deutet darauf hin, dass bei den erblichen Formen des Morbus Parkinson eine veränderte Assoziation des alpha-Synucleins mit der Membran eine Rolle spielen kann.In the present study a novel approach to characterize lipid domains, which may provide binding sites for peripheral or integral membrane proteins, is demonstrated. In particular, analysis of fluorescence lifetimes of NBD-labeled lipid analogues was used to study lipid domains in Giant unilamellar vesicles (GUV) and – based on the GUV results – in the plasma membrane of mammalian cells. As fluorescence decays in a few nanoseconds it is possible to to detect also very short-lived lipid domains. Fluorescence Lifetime Imaging (FLIM) revealed that the fluorescence decay of NBD-lipid analogues showed domain dependent decay times in the liquid disordered (ld) and the liquid ordered (lo) phase of GUV. Even the existence of submicroscopic domains in lipid membranes could be detected by FLIM. A broad distribution of the fluorescence lifetime was found for C6-NBD-PC inserted in the plasma membrane of mammalian cells. In agreement with FLIM studies on lipid domain forming Giant vesicles derived from the plasma membrane of HeLa-cells this may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact mammalian cells. Based on that, fluorescence microscopy was used on GUV to study the binding of fluorescently labeled alpha-synuclein at lipid domains previously characterized by FLIM. The experiments suggested that alpha-synuclein binds with high affinity to negatively charged phospholipids, when they are embedded in a ld as opposed to a lo environment. When compared with wildtype alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that an altered association of alpha-synuclein with membranes may play a role in the inherited forms of Parkinson’s disease.
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Webb, Murray S. "Interactions and permeability properties of vesicles of thylakoid lipids". Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/29759.
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The large-scale purification of the major spinach thylakoid lipids by a combination of silica and carboxymethyl-cellulose chromatography is described. Yields of hundreds of milligrams of the lipids, representing 25-40% of the original lipid, have been obtained. In addition, routine purities in excess of 99.7% of the isolated lipids has been demonstrated. The structures of the purified lipids have been confirmed by fatty acid analysis, thin layer chromatography, and ¹³C-NMR. Some minor reassignments to previously published ¹³C-NMR for these compounds are described. In addition, the ¹H-NMR spectra for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), are shown. The resonance assignments for MGDG and SQDG have been obtained by a combination of off-resonance decoupling experiments and by two-dimensional COSY ¹H-NMR experiments. Similar experiments with DGDG have failed to resolve the proton assignments due to extensive overlapping of the proton resonances.
Interbilayer interactions between large unilamellar vesicles of DGDG in aqueous salt solutions have been examined by light scattering, freeze-fracture electron microscopy, and X-ray diffraction. When suspended in aqueous salt solutions, vesicles of 100 nm diameter were found to aggregate in a rapid and reversible manner to yield aggregates greater than 1000 nm in diameter. Freeze-fracture electron microscopy showed these aggregates to consist of appressed, but not fused, vesicles. Quasi-elastic light scattering and turbidity experiments showed that aggregation was not due to charged impurities of the lipid behaving in accordance with electrostatic double layer theory. Experiments testing the efficacies of various chloride salts indicated a strong correlation existed between ionic radius and ability of the salt to promote aggregation. Similar experiments examining the effect of sodium salts, glycerol, and pH on vesicle aggregation implicate an interaction between the DGDG head group and structured water as underlying the aggregation process.
The effect of additions of other lipids on the extent of DGDG aggregation has been examined. Addition of 0.5 to 5.0% of either anionic lipid phosphatidylglycerol (PG) or SQDG inhibited the aggregation of DGDG vesicles, probably by the development of an electrostatic potential. Different effects of PG and SQDG on the concentration of Mg²⁺ required for aggregation indicated that PG may form a bidentate ligand with Mg²⁺ at ≥ 5 mol% PG. SQDG did not show this behavior, indicating that its negatively charged sulfonate group is unavailable for cation complex formation. Addition of MGDG to DGDG up to 50 mol% had no effect on the Mg²⁺ requirement for aggregation, but at ≥ 25 mol% triggered irreversible vesicle aggregation. This suggests that the MGDG head group is as effective at causing aggregation as the DGDG head group. Further, MGDG probably triggers vesicle fusion at ≥ 25 mol%. The results suggest that the galactolipids may contribute to the close approach of thylakoids in higher plant chloroplasts.
The permeability properties of large unilamellar vesicles of DGDG to ⁸⁶Rb⁺, ³⁶Cl⁻, and ³H-glucose have been determined. In addition, the permeabilities of binary, ternary, and quaternary mixtures of thylakoid lipids to ⁸⁶Rb⁺ have also been measured. Vesicles of DGDG were found to be 60-130 fold more permeable to Rb⁺ and 46-76 fold less permeable to CI⁻ than phosphatidylcholine vesicles. Vesicles of DGDG and PC were similar in glucose permeability. Electron spin resonance measurement of DGDG bilayer fluidity indicated that fluidity differences could not account for the observed differences in ion permeability. The addition of 50 mol% of MGDG to DGDG vesicles had no effect on Rb⁺ permeability, suggesting that the HII phase preference of MGDG does not increase bilayer permeability. The addition of SQDG led to a large increase of Rb⁺ permeability. The calculated permeability coefficient to Rb⁺ for a DGDG/MDGD/SQDG/PG (1/2/0.5/0.5) mixture similar to that of thylakoid membranes was 2.0-10⁻⁹ cm-s⁻¹. This value is three orders of magnitude higher than that for phospholipid systems, and ten-fold higher than that for vesicles of pure DGDG. It is concluded that the permeability properties of thylakoid lipids are dominated by oriented surface dipoles and not by bilayer fluidity or acyl chain packing considerations.
It is also proposed that the high permeability of thylakoid lipids to cations is the main cause of low observed thylakoid membrane electrical potentials, and large proton gradients across thylakoid membranes.
It has been proposed previously that the high proportions of saturated phosphatidylglycerols (ie. DPPG) found in chilling-sensitive plants may promote the formation of gel phase lipid, and cause increased metabolite leakage, in the thylakoids of these species at chilling temperatures. The leakage of ⁸⁶Rb⁺ from large unilamellar vesicles of thylakoid lipids containing proportions of disaturated PG (as DPPG) mimicking those of chilling-sensitive and chilling-resistant plants has been measured. This data indicated that no increase in Rb⁺ permeation occurred between any of the tested vesicles systems between 7° and 30° C. Differential scanning calorimetry showed no heat flow indicative of gel to liquid- crystalline phase separation in any of the lipid mixtures, even with DPPG levels as high as 12 mol%. It is concluded that a direct effect of disaturated PG on chilling injury in sensitive plants by an increase of low-temperature thylakoid permeability is unlikely.Science, Faculty of
Botany, Department of
Graduate
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Merrill, Allan Rodney. "Lipid-protein interactions and calcium uptake in intestinal brush border membrane vesicles". Thesis, University of Ottawa (Canada), 1988. http://hdl.handle.net/10393/5127.
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Lamont, Michael Robert Elsdon. "Detection of shape fluctuations in large lipid vesicles using diffusing-wave spectroscopy". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0027/MQ51385.pdf.
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Bartelt, Solveig Mareike [Verfasser]. "Light-controlled adhesion and dynamic processes in lipid vesicles / Solveig Mareike Bartelt". Mainz : Universitätsbibliothek Mainz, 2019. http://d-nb.info/1177739577/34.
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Yeh, Martín Noël. "Cellular mimics within lipid vesicles and in thermal out-of-equilibrium chambers". Doctoral thesis, University of Trento, 2018. http://eprints-phd.biblio.unitn.it/3468/1/No%C3%ABl_Yeh_Martin_PhD_Thesis_final_for_upload.pdf.
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The absence of clear criteria to recognize life and evaluate attempts at building a cell from component parts has slowed progress towards the construction of cellular mimics that fully display the properties of natural living cells. In the first part of this PhD thesis, a method to objectively quantify progress is proposed. In the second part of the thesis, preliminary results are shown and discussed for the construction of out-of-equilibrium cellular mimics generated by thermal gradients that do not rely on compartments made from lipid membranes.
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Cama, Jehangir. "Quantifying passive drug transport across lipid membranes". Thesis, University of Cambridge, 2016. https://www.repository.cam.ac.uk/handle/1810/254296.
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Antibiotic resistance has emerged as one of the World's leading public health challenges. The inexorable emergence of drug resistant pathogens, combined with a steep decline in antibacterial drug discovery, has led to a major crisis. One of the most common drug resistance mechanisms involves bacteria adapting to reduce intracellular drug accumulation. To understand these resistance mechanisms, one needs quantitative information about the membrane permeability of drugs. In this Thesis, we develop a novel optofluidic permeability assay that allows us to quantify the permeability coefficient of drugs crossing lipid membranes. Lipid vesicles are used as model systems and drug molecules are tracked directly using their autofluorescence in the ultraviolet. The permeability coefficient of the drug is inferred by studying the increase in drug autofluorescence intensity within vesicles as they traverse a microfluidic network while exposed to the drug for well defined times. This provides a novel platform from which we can develop membrane models for understanding drug permeability. We incorporate the Escherichia coli outer membrane protein OmpF in vesicles and quantify its role in the transport of fluoroquinolone antibiotics. We provide direct visualisation of OmpF mediated fluoroquinolone transport. We study the pH dependence of antibiotic transport both through pure phospholipid membranes and through OmpF, and present a physical mechanism to explain the pH dependence of E. coli fluoroquinolone susceptibility. We also show the importance of lipid composition on drug permeability - changing the lipid composition of the membrane is shown to change antibiotic permeability by over an order of magnitude. Finally, we report on the discovery of a novel signalling mechanism in E. coli that relies on the transport of small drug-like molecules, and discuss the role it plays in stress response in the microbial community.
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Maleki, Karyak Mohsen. "Modeling and analysis of lipid bilayers with applications to vesicles and lipoprotein particles". Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121455.
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Continuum approaches for modeling lipid bilayers are developed and applied to two-phase lipid vesicles and discoidal high-density lipoprotein (HDL) particles. First, relying on a three-dimensional model, the mechanics of a lipid bilayer with spontaneous curvature is considered. Kinematics, material symmetry, stress relations, and coherency of lipid bilayer leaflets are discussed. Treating a lipid bilayer as a thin structure, the areal energy density of a lipid bilayer with spontaneous curvature is obtained using a dimension-reduction procedure. Attention is paid on the source of spontaneous curvature in the well known Canham–Helfrich energy density. Also, the effect of constitutive asymmetry of the leaflets on the areal energy density of a lipid bilayer is highlighted.Considering a two-phase vesicle as system of coexisting spherical domains, its equilibrium is studied using a simple continuum model. Multidomain and ground-state configurations are considered. Whereas in the former case multiple budded lipid domains coexist on a vesicle, in the latter case the vesicle is composed of two large lipid domains. Variations of the net potential-energy of a multidomain vesicle with the number of lipid domains and osmotic pressure are studied. Based on an energy comparison argument, two ground-state configurations corresponding to minimum energy levels are identified: pinched-off and complete sphere configurations. The results indicate that osmotic pressure and initial excess radius play key roles in the final shape of attaining ground-state configurations. The critical values of these parameters are identified. Lastly, the equilibrium and stability of a discoidal HDL particle are studied. A model in which the lipid bilayer and double-belt apoA-I components of discoidal HDL particle are represented by a material surface and a material curve perfectly bonded to the edge of the surface is proposed. The curvature energy and surface tension of lipid bilayer and the bending energy of apoA-I chain are included. Adopting a variational scheme, nonlinear equilibrium equations of a discoidal HDL particle in a general configuration are derived using both direct, geometrically-based and parametrized formulations. The linearized equilibrium equations of a flat circular HDL particle are obtained and its linear stability is investigated using the second variation method. An energy comparison method is applied and is found to offer a handy approach for ascertaining linear stability. Numerical results are provided for the equilibrium and stability of flat circular HDL particle. A stability plane indicating different stable and unstable regions of underlying dimensionless input parameters is provided. Possible pathways of stability change and instability mode shapes are identified. It is shown that the first transverse and planar instability modes resemble nonplanar saddle-like and planar elliptic shapes, respectively.Des méthodes de milieux continus pour la modélisation de bicouches lipidiques sont développées et appliquées à des vésicules lipidiques à deux phases et à des particules discoïdes de lipoprotéines de haute densité (HDL). Tout d'abord, en s'appuyant sur un modéle tridimensionnel, la mécanique d'une bicouche lipidique possédant une courbure spontanée est considérée. La Cinématique, la symétrie matérielle, les relations de stress, et la cohérence de bicouches lipidiques sont discutées. En traitant une bicouche lipidique comme une structure mince, la densité d'énergie surfacique d'une bicouche lipidique ayant une courbure spontanée est obtenue à l'aide d'une procédure de réduction de dimension. L'attention est portée sur la source de courbure spontanée de la densité d'énergie bien connue de Canham–Helfrich. En outre, l'effet de l'asymétrie constitutive des sur la densité d'énergie surfacique d'une bicouche lipidique est mis en évidence. Considérant une vésicule à deux phases comme système de domaines sphériques coexistants, son équilibre est étudié à l'aide d'un modèle simple de milieu continu. Des configurations multi-domaines et de l'état fondamental sont considérées. Alors que, dans le premier cas, plusieurs domaines lipidiques bourgeonnés coexistent sur une vésicule, dans le dernier cas, la vésicule est composée de deux grands domaines lipidiques. La variation de l'énergie potentielle nette d'une vésicule multi-domaine en fonction du nombre de domaines lipidiques et de la pression osmotique est étudiée. En se basant sur la comparaison de l'énergie, deux configurations de l'état fondamental correspondant à des niveaux d'énergie minimaux sont identifiés: la configuration étranglée et la sphère complète. Les résultats indiquent que la pression osmotique et le rayon excédentaire initial jouent un rôle clé dans la forme finale des configurations à l'état fondamental. Les valeurs critiques de ces paramètres sont identifiées. Enfin, l'équilibre et la stabilité d'une particule HDL discoïde sont étudiés. Un modèle dans lequel la bicouche lipidique et les composants d'ApoA-I à double bande de la particule de HDL discoïde sont représentées par une surface de matériau et une courbe de matériau parfaitement collée sur le bord de la surface est proposé. L'énergie de courbure et la tension de surface de la bicouche lipidique ainsi que l'énergie de flexion de la chaîne apoA-I sont incluses. En adoptant un schéma variationnel, les équations d'équilibre non-linéaire d'une particule de HDL discoïdale dans une configuration générale sont calculées d'après des formulations directes, basées sur la géométrie, ou paramétrées. Les équations d'équilibre linéarisées d'une particule de HDL circulaire plane sont obtenues et sa stabilité linéaire est étudiée en utilisant la seconde méthode de variation. Une méthode de comparaison de l'énergie est appliquée et se trouve à offrir une approche pratique pour déterminer la stabilité linéaire. Des résultats numériques sont présentés pour l'équilibre et la stabilité des particules de HDL circulaires planes. Un plan de stabilité indiquant différentes régions stables et instables des paramètres d'entrée adimensionnels sous-jacents est fourni. Certaines possibilités de changement de stabilité et les formes modales d'instabilité sont identifiées. Il est démontré que les premiers modes d'instabilité transversale et plane ressemblent aux formes de selle non planes et d'ellipse plane, respectivement.
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Blochel, Andreas. "Adsorption of halogenated phenolate ions to egg-phosphatidylcholine vesicles". PDXScholar, 1992. https://pdxscholar.library.pdx.edu/open_access_etds/4271.
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Takashima, Kouhei. "Regulation of Lipid Biosynthesis by Golgi-to-ER Retrograde Traffic via COPI-coated Vesicles". 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/175226.
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Nilsson, Martin. "GIANT UNILAMELLAR VESICLES FOR PEPTIDE-MEMBRANE INTERACTION STUDIES USING FLUORESCENCE MICROSCOPY". Thesis, Linköpings universitet, Biofysik och bioteknik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167467.
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Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.
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Ngo, Thien-An. "Tuning the electronic properties of conjugated polyelectrolytes via complexation with lipid vesicles or silica nanobeads". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97001.
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This thesis describes luminescent nanostructures involving the anionic poly(phenylene vinylene) MPS-PPV in complex with lipid vesicles or silica beads. The vesicular encapsulation of light-emitting polymer produced a "liposome beacon," whose fluorescence response to the electron-transfer quencher methyl viologen or an energy-accepting carbocyanine dye (DiD) reported on the extent of encapsulation via ensemble spectroscopy and single-particle imaging of immobilized vesicles. Divalent cation was furthermore used to modulate interactions between MPS-PPV and zwitterionic lipid vesicles. Binding to the lipid vesicles disrupted polymer aggregates, enhancing and shifting the polymer's fluorescence. Fluorescence resonance energy transfer (FRET) elucidated the effect of divalent cation in promoting lipid-polymer association. Finally, MPS-PPV was adsorbed onto silica nanoparticles via electrostatic interactions. The effects of silica bead adsorption on MPS-PPV photophysical properties were investigated via ensemble spectroscopy and single-particle total-internal-reflection fluorescence microscopy. The MPS-PPV-coated silica bead exhibited similar sensitivity to methyl viologen as the free polyelectrolyte, and was resistant to the fluorescence effects of nonspecific interactions. Our results provide insight towards tuning the sensitivity of fluorescent water-soluble conjugated polyelectrolytes and contributing to the development of new applications for conjugated poly(phenylene vinylene)s in fluorescent assays.Les polymères conjugués (PC) ont une fluorescence amplifiée mais facilement supprimée, ce qui les rend utile pour la détection d'une vaste gamme de molécules. Ici on a crée des nouvelles structures luminescents basées sur les interactions entre la PC (poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) et des liposomes ou des particules de silice de diamètre environ 100 nm. L'interaction entre la PC et les lipides est vérifié avec la spectroscopie. Ajoutant une autre fluorophore aux lipides, on voit fluorescence resonance energy transfer (FRET) de la PC au fluorophore. L'encapsulation de la PC est vérifié par la réponse de la fluorescence des complexes PC / lipide au méthyle viologène, et la conjugaison spécifique des complexes contenant des lipides biotinylées aux surfaces couvert de streptavidin. Le cation divalent de calcium a été utilisé pour modifier les interactions lipide-PC, ce qui était indiquée par FRET. En outre, MPS-PPV a été adsorbée sur les particules de silice via des interactions électrostatiques. Les effets de l'adsorption sur les caractéristiques photophysiques des complexes MPS-PPV / silice ont été étudiés avec la spectroscopie et la microscopie de fluorescence par réflexion totale interne (TIRFM). Leur réponse au méthyle viologène est aussi amplifiée que celui du polymère libre, mais leur luminescence résiste les effets des interactions hydrophobiques. Cette recherche donne des nouveaux aperçus sur la modification de la fluorescence amplifiée des PC, vers continuer le développement des systèmes utilisant des polyvinyles de para-phénylène pour la détection luminescent biologique et chimique.
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Eriksson, Hanna M. "Intracellular vesicles induced by monotopic membrane protein in Escherichia coli". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-29070.
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The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.
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Yokoyama, Fumiaki. "Studies on Production Mechanisms of Extracellular Membrane Vesicles of Cold-Adapted Bacteria". Kyoto University, 2020. http://hdl.handle.net/2433/253343.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第22507号
農博第2411号
新制||農||1078(附属図書館)
学位論文||R2||N5287(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 植田 充美, 教授 山口 信次郎
学位規則第4条第1項該当
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CASTANGIA, INES. "New lipid nanovesicles as topical delivery sistems for ant-inflammatory drugs". Doctoral thesis, Università degli Studi di Cagliari, 2014. http://hdl.handle.net/11584/266437.
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The purpose of the present study was to formulate new vesicular carriers to optimize the topical delivery of anti-inflammatory drugs: diclofenac, quercetin and curcumin.
In the first work concentrated and interconnected penetration enhancer containing vesicles (PEVs) are proposed as carriers for dermal delivery of diclofenac. PEVs were prepared by using a commercial phosphatidylcholine mixture (180 mg/m) and transcutol in different amounts. Conventional liposomes were also prepared and tested as control. All vesicles showed a mean size ranging from 75 to 253 nm with fairly narrow size distribution, negative zeta potential value, and drug loading capacity between 48 and 70%. SWAXS studies showed that composition affected vesicle structure and morphology: 10 and 30% transcutol PEVs were unilamellar while liposomes and 20% transcutol PEVs were multilamellar. Rheological studies demonstrated that control liposomes and 10 and 30% transcutol containing PEVs behaved as Newtonian fluids while 20% transcutol containing PEVs showed a plastic behavior. Ex vivo (trans)dermal delivery experiments showed an improved skin deposition of diclofenac when PEVs were used. Vesicle toxicity and
uptake of fibroblasts, target of inflammation treatment, were evaluated by MTT test and fluorescence
microscopy. Control liposomes and PEVs were both able to interact and being internalized by the 3T3
fibroblasts at all time exposure tested. Furthermore, PEVs showed to be able to reduce the in vitro drug
toxicity.
The aim of the second study was to improve the knowledge of drug–glycol–phospholipid-interactions and their effects in lamellar vesicle suitability as drug delivery systems. Liposomes were prepared using hydrogenated soy phosphatidylcholine (P90H, 60 mg/ml) and diclofenac sodium salt at two con-centrations (5–10 mg/ml). To obtain innovative vesicles two permeation enhancers with glycol group, diethyleneglycol monoethyl ether and propylene glycol, were added to the water phase at different ratios (5%, 10%, and 20%). Vesicle organization was deeply investigated by physico-chemical characterization, including differen-tial scanning calorimetry and small-angle diffraction signal analysis while macroscopic structure behavior was evaluated by rheological studies. Results evidenced that the presence of the penetration enhancer and diclofenac sodium salt led to structural rearrangements within and among vesicles forming a tridi-mensional and complex architecture in which vesicles were closely packed and interconnected. This new design allowed a change in the physical state of dispersions that became highly viscous liquid or soft-solid-like, thus forming an ideal system for topical application able of both adhering to the skin and delivering the drug. © 2013 Elsevier B.V. All rights reserved.
In the last work biocompatible quercetin and curcumin nanovesicles were developed as a novel approach to prevent and restore skin tissue defects on chronic cutaneous pathologies. Stable and suitable quercetin- and curcumin-loaded phospholipid vesicles, namely liposomes and penetration enhancercontaining vesicles (PEVs), were prepared. Vesicles were made from a highly biocompatible mixture of phospholipids and alternatively a natural polyphenol, quercetin or curcumin. Liposomes were obtained by adding water, while PEVs by adding polyethylene glycol 400 and Oramix_CG110 to the water phase. Transmission electron microscopy, cryogenic-transmission electron microscopy and small- and wideangle X-ray scattering showed that vesicles were spherical, oligo- or multilamellar and small in size (112–220 nm). In vitro and in vivo tests underlined a good effectiveness of quercetin and curcumin nanovesicles in counteracting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced lesions and inflammation. Myeloperoxydase activity, used to gauge inflammation, was markedly inhibited by quercetin liposomes (59%) and curcumin liposomes and polyethylene glycol (PEG)-PEVs (_68%). Histology showed that PEG-PEVs provided an extensive re-epithelization of the TPA-damaged skin, with multiple layers of thick epidermis. In conclusion, nanoentrapped polyphenols prevented the formation of skin lesions abrogating the various biochemical processes that cause epithelial loss and skin damage.
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Sarri, Barbara Claire Mireille Annick. "Spectrin-lipid interactions and their effect on the membrane mechanical properties". Thesis, University of Exeter, 2014. http://hdl.handle.net/10871/15976.
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This thesis presents the experimental work performed on the spectrin protein. The aim of the work was to study the direct interactions of spectrin, the cytoskeleton of RBCs, with membrane lipid to determine its effects on the mechanical properties of the lipid bilayer. Motivation for this work came from a lack of unanimity in the field of spectrin, and the hypothesized potential of the protein to perforate giant unilamellar vesicles. The work aimed to investigate and determine how spectrin-lipid interactions influence membrane mesoscopic morphology and biophysics in ways that could ultimately be important to cellular function. For this purpose, a protocol was implemented to take into account the different aspects of the binding. Direct visualisation of the spectrin-lipid interaction and distribution was achieved using confocal fluorescence microscopy. Changes in the mechanical properties of the membrane were investigated using the micropipette aspiration technique. Finally the thermodynamics of the interaction were considered with isothermal titration calorimetry experiments. This allowed evaluation of the protein-lipid interaction in a complete and coherent manner. Experiments were also performed on another elastic protein, alpha-elastin, for comparison. In addition to its similarities with spectrin (both possess hydrophobic domains and entropy elasticity), elastin is auto-fluorescent which makes it an attractive model protein. Elastin was also used as a sample model to implement new techniques using nonlinear optics microscopy.
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Hunter, David Gregory. "The effects of lipid composition and extrusion pressure and temperature on the properties of phospholipid vesicles". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq24162.pdf.
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Al-Ahmady, Z. S. "Design, engineering and biological performance of responsive lipid vesicles for enhanced drug delivery by mild hyperthermia". Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1410930/.
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The design of a delivery system that specifically delivers anticancer drug to the tumour site avoiding normal tissues damage has always been a challenge. In this thesis we describe the engineering and biological performance of novel temperature-sensitive liposomes (TSL) that have both a substantial in vivo stability and an efficient content-release by mild hyperthermia (HT). First, we explain the development of novel lipid-peptide hybrids (Lp-Peptide) by anchoring leucine zipper temperature-sensitive peptide within the liposomal lipid bilayer. We characterized this system by studying its physicochemical properties and the interaction of the peptide with the lipid bilayer. Then we examined its potential to retain and trigger the release of the anticancer drug, doxorubicin, in vitro at physiological temperatures and after exposure to mild HT. In addition, the blood kinetics, tumour and other tissues accumulation were explored when we studied the system in vivo. Our data suggested that Lp-Peptide hybrids can increase both immediate and long-term drug accumulation in the tumour. Therefore, we studied their therapeutic activity comparing two different heating protocols to mimic intravascular and interstitial drug release. The last chapter of this thesis explored the opportunities of increasing the therapeutic specificity of TSL by designing anti-MUC-1 targeted vesicles based on the traditional TSL (TTSL) to trigger drug release after specific uptake into cancer cells. The system was evaluated by studying the in vitro cellular binding, uptake and therapeutic efficacy. Taking this system a step further, its biodistribution and therapeutic potential were also examined. Different protocols were applied to explore the effect of HT on the accumulation of targeted TTSL into the tumour and their therapeutic efficacy. In summary, our studies demonstrate the critical factors to consider in the design of clinically relevant TSL and the importance of matching the heating protocol to their physicochemical and pharmacokinetic parameters to maximise therapeutic benefits.
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Ariöz, Candan. "Exploring the Interplay of Lipids and Membrane Proteins". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-102675.
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The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.