<p>The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight.</p> <p>Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction.</p> <p>To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.</p>

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第12911号 / 論理博第1547号 / 新制||理||1590(附属図書館) / 32121 / 京都大学大学院理学研究科・物理学・宇宙物理学専攻 / (主査)講師 市川 正敏, 教授 山本 潤, 教授 田中 耕一郎 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM

The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

Lipid droplets (LDs) are organelles with many functions in cells and numerous protein interactors facilitate their biogenesis, maintenance, and turnover. The mammalian lipase responsible for LD turnover during lipophagy, LipA, has two candidate homologs in Arabidopsis: MPL1 and LIP1. One or both of these plant homologs may function in a similar manner to mammalian LipA, providing an LD breakdown pathway. To test this hypothesis, wild type (WT) Arabidopsis plants, MPL1 over-expressing (OE) mutants, and T-DNA insertion mutants of MPL1 (mpl1) and LIP1 (lip1) were examined for LD phenotypes in normal conditions and in environments where LD numbers are known to fluctuate. Plants to be imaged by confocal microscopy were exposed to heat stress and wounding to increase LD accumulation, senescence was induced in leaves to deplete lipids, and LDs were imaged throughout the day/night period to observe their diurnal regulation. The mutation of both MPL1 and LIP1 lead to an increase in LDs within the leaf mesophyll cells, although the spatial distribution of the LDs differed between the two mutants. mpl1 mutants had disrupted diurnal regulation of their LDs, but lip1 mutants did not. Alternately, lip1 mutants retained LDs during dark-induced senescence, and mpl1 mutants did not. Together these results suggest that MPL1 and LIP1 are likely both important for LD dynamics; however they appear have roles in different aspects of LD accumulation and turnover.

No description available.

Canada is a major global producer of pulse products including pulse starch, which notably contributes to a healthy diet. Strategically, Canada is taking steps to research methods of adding greater value to these crop products, and functional foods like resistant starch are particularly interesting. The primary objectives of this study were to develop an effective reactive extrusion process to produce gelatinized lipid complexed pea starches with enhanced enzyme resistance and examine the effects of bulk lipid complexing conditions on physicochemical and functional properties of extruded pea starches. One type of commercially available pea starch, Nutri-Pea, was chosen as the research subject in this study. A number of methods including; Englyst digestion method, differential scanning calorimetry (DSC), infrared spectroscopy (FTIR), contact angle, titrations, residence time distribution (RTD) and rapid visco analysis (RVA) were used to characterize the properties of extruded pea starches. The effects of feed formulation and extrusion conditions on lipid complexing and Englyst digestion profiles were systematically examined on two mixing devices. An extensive kinetics study was conducted on a lab scale twin-screw compounder, DSM-Xplore. The process was then scaled up to produce bulk lipid complexed pea starch on a Leistritz twin-screw extruder. The results showed that lipid complexing and digestion profiles were highly dependent on feed moisture and induced screw shear. Reactive extrusion of pea starches under optimized conditions achieved a significant but moderate increase in either resistant starch (RS) content (from 13.3% to 20.2%) or slowly digestible starch (SDS) content (from 7.85% to 23.3%) compared to their native counterparts. However, RS and SDS content could not be improved simultaneously based on the pea starch and extrusion process in this study. Increased degree of substitution (DS) was found for myristic acid complexed pea starches (nominal DS= ~0.8) when compared to palmitic acid complexed pea starch (nominal DS= ~0.5). Contact angle measurements, FTIR and DSC thermograms confirmed the presence of lipids. Lipid complexed starch films showed increasing hydrophobicity with increasing lipid content. As an alternative product compared to functional foods, the modified starch was considered as a biodegradable film for industrial applications. The material was produced at the highest moisture content for extruded native starch and two concentrations of lipid complexed starch using an intensive screw design. Preliminary results show that increasing lipid content and adding 1% glycerol to samples decreases the force per film thickness required to puncture films. However further investigation is required to determine effect of heat and moisture deformation. / Thesis / Master of Applied Science (MASc) / Incorporation of pulses into food products has been a major area of Canadian research for its potential to create new avenues of enzyme resistant food starches. Extrusion cooking is commonly used in industry for producing various food products such as snacks and cereals but little research has been reported on using an extruder to rapidly produce resistant pulse starches as a functional ingredient on a large scale; resistant starch is a functional food beneficial to humans in the same manner as insoluble fiber but exhibits improved textural properties. This study aimed to develop an effective reactive extrusion process to produce lipid complexed pea starches with enhanced enzyme resistance (i.e. increased slowly digestible starch (SDS) and resistant starch (RS) content) by an examination of the effects of reaction conditions on the properties of extrusion products. The lipid complexed pea starches under optimized conditions achieved a significant but moderate increase in either RS content or SDS content depending on the sample formulation compared to their native counterparts. However, RS and SDS content could not simultaneously be improved in this study.

Weltweit versuchen Wissenschaftler, künstliche Viren für den Gentransfer zu konstruieren, die nicht reproduktionsfähig sind. Diese sollen die Vorteile der natürlichen Viren besitzen (effizienter Transport von genetischem Material), jedoch keine Antigene auf ihrer Oberfläche tragen, die Immunreaktionen auslösen. <br><br> Ziel dieses Projektes ist es, einen künstlichen Viruspartikel herzustellen, dessen Basis eine Polyelektrolytenhohlkugel bildet, die mit einer Lipiddoppelschicht bedeckt ist. Um intakte Doppelschichten zu erzeugen, muss die Wechselwirkung zwischen Lipid und Polyelektrolyt (z.B. DNA) verstanden und optimiert werden. Dazu ist es notwendig, die strukturelle Grundlage der Interaktion aufzuklären. Positiv geladene Lipide gehen zwar starke Wechselwirkungen mit der negativ geladenen DNA ein, sie wirken jedoch toxisch auf biologische Zellen. In der vorliegenden Arbeit wurde daher die durch zweiwertige Kationen vermittelte Kopplung von genomischer oder Plasmid-DNA an zwitterionische oder negativ geladene Phospholipide an zwei Modellsystemen untersucht. <br><br> 1. Modellsystem: Lipidmonoschicht an der Wasser/Luft-Grenzfläche <br> Methoden:<br> Filmwaagentechnik in Kombination mit IR-Spektroskopie (IRRAS), Röntgenreflexion (XR), Röntgendiffraktion (GIXD), Brewsterwinkel-Mikroskopie (BAM), Röntgenfluoreszenz (XRF) und Oberflächenpotentialmessungen <br> Resultate:<br> A) Die Anwesenheit der zweiwertigen Kationen Ba2+, Mg2+, Ca2+ oder Mn2+ in der Subphase hat keinen nachweisbaren Einfluss auf die Struktur der zwitterionischen DMPE- (1,2-Dimyristoyl-phosphatidyl-ethanolamin) Monoschicht. <br> B) In der Subphase gelöste DNA adsorbiert nur in Gegenwart dieser Kationen an der DMPE-Monoschicht. <br> C) Sowohl die Adsorption genomischer Kalbsthymus-DNA als auch der Plasmid-DNA pGL3 bewirkt eine Reduktion des Neigungswinkels der Alkylketten, die auf einen veränderten Platzbedarf der Kopfgruppe zurückzuführen ist. Durch die Umorientierung der Kopfgruppe wird die elektrostatische Wechselwirkung zwischen den positiv geladenen Stickstoffatomen der Lipidkopfgruppen und den negativ geladenen DNA-Phosphaten erhöht.<br> D) Die adsorbierte DNA weist eine geordnete Struktur auf, wenn sie durch Barium-, Magnesium-, Calcium- oder Manganionen komplexiert ist. Der Abstand zwischen parallelen DNA-Strängen hängt dabei von der Größe der DNA-Fragmente sowie von der Art des Kations ab. Die größten Abstände ergeben sich mit Bariumionen, gefolgt von Magnesium- und Calciumionen. Die kleinsten DNA-Abstände werden durch Komplexierung mit Manganionen erhalten. Diese Ionenreihenfolge stellt sich sowohl für genomische DNA als auch für Plasmid-DNA ein. <br> E) Die DNA-Abstände werden durch die Kompression des Lipidfilms nicht beeinflusst. Zwischen der Lipidmonoschicht und der adsorbierten DNA besteht demnach nur eine schwache Wechselwirkung. Offensichtlich befindet sich die durch zweiwertige Kationen komplexierte DNA als weitgehend eigenständige Schicht unter dem Lipidfilm. <br> <br><br> 2. Modellsystem: Lipiddoppelschicht an der fest/flüssig-Grenzfläche<br> Methoden:<br> Neutronenreflexion (NR) und Quarzmikrowaage (QCM-D)<br> Resultate:<br> A) Das zwitterionische Phospholipid DMPC (1,2-Dimyristoyl-phosphatidylcholin) bildet keine Lipiddoppelschicht auf planaren Polyelektrolytmultischichten aus, deren letzte Lage das positiv geladene PAH (Polyallylamin) ist. <br> B) Hingegen bildet DMPC auf dem negativ geladenen PSS (Polystyrolsulfonat) eine Doppelschicht aus, die jedoch Defekte aufweist. <br> C) Eine Adsorption von genomischer Kalbsthymus-DNA auf dieser Lipidschicht findet nur in Gegenwart von Calciumionen statt. Andere zweiwertige Kationen wurden nicht untersucht.<br> D) Das negativ geladene Phospholipid DLPA (1,2-Dilauryl-phosphatidsäure) bildet auf dem positiv geladenen PAH eine Lipiddoppelschicht aus, die Defekte aufweist. <br> E) DNA adsorbiert ebenfalls erst in Anwesenheit von Calciumionen in der Lösung an die DLPA-Schicht.<br> F) Durch die Zugabe von EDTA (Ethylendiamintetraessigsäure) werden die Calciumionen dem DLPA/DNA-Komplex entzogen, wodurch dieser dissoziiert. Demnach ist die calciuminduzierte Bildung dieser Komplexe reversibel. / All over the world scientists are trying to engineer artificial viruses, which do not replicate, for gene delivery. These artificial viruses should have the advantages of natural viruses such as efficient transport of genetic material, but they should not carry antigens, which cause immune reactions, on their top portion.<br><br> The aim of this project is to develop an artificial virus particle that is based on a polyelectrolyte hollow capsule which is covered by a lipid bilayer. To create intact bilayers, it is crucial to understand and optimize the interaction between lipids and polyelectrolytes (e. g. DNA). Therefore the structural basis of that interaction must be elucidated. Positively charged lipids interact strongly with the negatively charged DNA but they cause toxic reactions in biological cells. Hence the present work used two model systems to study the coupling of genomic or plasmid DNA to zwitterionic or negatively charged phospholipids induced by divalent cations. <br><br> 1. Model system: Lipid monolayer at the air/water-interface <br> Methods: <br> Langmuir filmbalance in combination with IR-spectroscopy (IRRAS), X-ray reflectometry (XR), X-ray diffraction (GIXD), Brewster angle microscopy (BAM), X-ray fluorescence (XRF), and surface potential measurements<br> Results:<br> A) The presence of the divalent cations Ba2+, Mg2+, Ca2+ or Mn2+ in the subphase has no traceable influence on the structure of a zwitterionic DMPE (1,2-dimyristoyl-phosphatidyl-ethanolamine) monolayer.<br> B) DNA which is dissolved in the subphase adsorbs to the DMPE-monolayer only if divalent cations are present.<br> C) The adsorption of genomic calf thymus DNA as well as of the plasmid DNA pGL3 causes a reduction of the tilt angle of the lipid alkyl chains. The tilt reduction can be ascribed to a change in the space required by the lipid head group. This change in head group orientation increases the electrostatic interaction between the positively charged nitrogen atoms in the lipid head and the negatively charged DNA phosphates.<br> D) The adsorbed DNA exhibits an ordered structure if it is complexed by barium, magnesium, calcium or manganese ions. The spacing between parallel DNA strands depends on the size of the DNA fragments as well as on the kind of cation. The largest DNA-spacings are observed with barium ions, followed by magnesium and calcium ions. DNA-complexation with manganese ions causes the smallest spacings. This order of ions is observed for both genomic and plasmid DNA.<br> E) Compression of the monolayer does not influence the DNA spacings. Thus the interaction between the lipid monolayer and adsorbed DNA is only weak. The DNA must exist as a more or less separate layer under the lipid film.<br> <br><br> 2. Model system: Lipid bilayer at the solid/fluid-interface<br> Methods: <br> Neutron reflectometry (NR), and Quartz crystal microbalance (QCM-D)<br> Results:<br> A) The zwitterionic phospholipid DMPC (1,2-dimyristoyl phosphatidylcholine) does not form lipid bilayers on top of planar polyelectrolyte multilayers covered with the positively charged PAH (polyallylamine).<br> B) In contrast, DMPC forms a lipid bilayer with defects on top of the negatively charged PSS (polystyrolsulfonate) terminated polyelectrolyte cushion.<br> C) Genomic calf thymus DNA adsorbs only to the DMPC layer in presence of calcium ions. Different ions were not examined.<br> D) The negatively charged phospholipid DLPA (1,2-dilauryl-phosphatidic acid) also forms a lipid bilayer with defects on top of the PAH-terminated cushion.<br> E) The DNA adsorbs also to the DLPA layer only in the presence of calcium ions in the solution.<br> F) By addition of EDTA (ethylenediaminetretraacetic acid) the calcium cations are removed from the DLPA/DNA-complex and the complex dissociates. Thus the calcium induced formation of that complex is reversible.<br><br>

Call number: LD2668 .T4 1984 G664 / Master of Science

Résumé: La subérine, un polymère lipidique et complexe des plantes est retrouvé dans divers tissus dont le périderme de la pomme de terre. Le processus biologique de sa dégradation reste encore peu connu et est attribué aux champignons. Des échantillons de sol provenant d'un champ de pommes de terre ont été inoculés dans un milieu de culture contenant de la subérine comme source de carbone. Une approche métaprotéomique a été utilisée pour identifier les populations bactériennes qui se développent en présence de la subérine sur une période d'incubation de 60 jours. Le nombre de spectres normalisé (NSpC) des protéines extracellulaires produites par la communauté bactérienne du sol ont considérablement diminué du jour 5 au jour 20, puis ont augmenté lentement, révélant une succession de bactéries, où la population des bactéries du genre Pseudomonas à croissance rapide a diminué et a été remplacée par d’autres espèces bactériennes qui pouvaient se développer en présence de la subérine. La récalcitrance de la subérine a été démontrée par l'émergence de bactéries auxotrophes telles qu’Oscillatoria dans les derniers jours de la culture bactérienne. Néanmoins, l'identification de deux lipases dans le surnageant de la culture suggère qu'au moins certaines espèces bactériennes peuvent dégrader la subérine. Une des lipases (I4WGM2) a été associée à Rhodanobacter thiooxydans. Lorsque cultivée dans un milieu contenant de la subérine, la souche de R. thiooxidans LCS2 a produit trois lipases, dont I4WGM2. R. thiooxidans LCS2 a également produit d'autres protéines liées au métabolisme des lipides, des transporteurs de chaines d’acide gras et les enzymes de la [béta]-oxydation. Ceci suggère que R. thiooxydans pourrait participer à la dégradation de la subérine. / Abstract: Suberin is a complex lipidic plant polymer found in various tissues including potato periderm. The biological degradation process of suberin is poorly characterized and is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as carbon source and a metaproteomics approach was used to identify bacterial populations that develop in the presence of suberin, over a 60-day incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community drastically decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species that could develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria in the last days of the assay. Nevertheless, the identification of two putative lipases in the culture supernatants suggests that at least some bacterial species could degrade suberin. One of the lipases (I4WGM2) was associated with Rhodanobacter thiooxydans. When grown in a suberin-containing medium, R. thiooxydans strain LCS2 produced three lipases, including I4WGM2. This strain also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and [beta]-oxidation enzymes, suggesting that R. thiooxydans could participate in suberin degradation.