Lipid molecules are amphiphilic in nature consisting of a hydrophilic head group and hydrophobic hydrocarbon tails. The lipid bilayer consists of two layers of lipid molecules arranged with their hydrophobic tails facing each other and their hydrophilic head groups solvated by water. Lipid bilayers with hydrophilic polymer chains grafted onto the head groups have applications in various fields, such as stabilization of liposomes designed for targeted drug delivery, synthesis of supported bilayers for biomaterial applications, surface modification of implanted medical devices to prevent biological fouling and design of in vitro biosensors. The focus of this thesis lies in understanding the effects of polymer grafting on the thermodynamics and mechanical properties of lipid bilayers. Dissipative particle dynamics (DPD) has evolved as a promising method to study complex soft matter systems. The basic DPD algorithm, and its implementation are discussed in Chapter 2 of this thesis. It is important to achieve a tensionless state while studying phase transitions and deducing the mechanical properties of the bilayer. We proposed a modification of the Andersen barostat which can be incorporated in a DPD simulation to achieve the tensionless state as well as carry out simulations at a prescribed tension. In Chapter 3 of this thesis the effect of polymer grafting on single tailed lipid bilayers is studied. Simulations are carried out by varying the grafting fraction, Gf, defined as the ratio of the number of polymer molecules to the number of lipid molecules. At lowGf, the bilayer shows a sharp transition from the gel (Lβ) to the liquid crystalline (Lα) phase. This main melting transition temperature is lowered as Gf is increased. Corresponding to this, an increase in the area per head group is also observed. Above a critical value of Gf the interdigitated, LβI phase is observed prior to the main transition for the longer lipid tails. The analysis for two tailed lipids as a function of polymer chain length is extensively studied in Chapter 5. For the case of two tailed lipids, an intermediate interdigitated phase was not observed and the decrease in the melting temperature is more pronounced as the length of the polymer chain is increased. The scaling for fractional change in the area per head group, as well as the decrease in transition temperature as a function of polymer grafting are in good agreement with mean field theory predictions. The bending modulus (k) and area stretch modulus (kA) are essential for determining the shape and the mechanical stability of biological cells or lipid based vesicles. In simulations, the bending modulus k is evaluated from the Fourier transform of the out-of-plane fluctuations of the bilayer mid-plane. In Chapter 4 of this thesis, we illustrate that a surface representation based on Delanuay triangulation provides a robust parameter free representation of the bilayer surface. By evaluating the bending modulus for single tail lipids of different tail lengths, the continuum scaling relation d2 is verified. To our knowledge this is the first systematic investigation and verification of this scaling relationship using computer simulations. Using the continuum relation, =kAd2/ we find that α depends weakly on the tail lengths of the bilayer. Nevertheless we illustrate that a value of α=130 can be used to reliably estimate the bending modulus from the area stretch modulus for polymer free bilayers. Using our method, we are also able to capture the low q scalings and obtain the bending modulus of the gel (Lβ) phase. Grafted polymer was found to increase the value of the bending modulus for single tail lipids. Although the presence of polymer directly increases the area per head group, the suppressed height fluctuations dominate and the bending modulus increases for the single tail lipids. For two tail lipids a small decrease in the bending modulus was observed at low grafting fractions and short polymer chains. For large polymer lengths the bending modulus was found to increase monotonically.

In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD accumulation observed in the mutants, that this phenotype occurs in above ground tissues and is present throughout the early growth stages of the plant. Further examination of lrt1 mutant plants has allowed a preliminary understanding of the role LRT1 may play in LD regulation. Taken together, the results of this study point towards some promising strategies to increase LD content in plant tissues.

This work is devoted to understanding the formation of supported lipid bilayers (SLBs) on curved surfaces as a function of lipid properties such as headgroup charge/charge density and alkyl chain length, and nanoparticle properties such as size and surface characteristics. In particular, the formation of SLBs on curved surfaces was studied by varying the size of the underlying substrate SiO2 nanoparticles with size range from 5-100 nm. Curvature-dependent shift in the phase transition behavior of these supported lipid bilayers was observed for the first time. We found that the phase transition temperature, Tm of the SLBs first decreased with decreasing the size of the underlying support, reached a minimum, and then increased when the size of the particles became comparable with the dimensions of the lipid bilayer thickness; the Tm was above that of the multilamellar vesicles (MLVs) of the same lipids. The increase in Tm indicated a stiffening of the supported bilayer, which was confirmed by Raman spectroscopic data. Moreover, Raman data showed better lipid packing and increased lateral order and trans conformation for the SLBs with increasing the curvature of the underlying support and decrease of the gauche kinks for the terminal methyl groups at the center of the bilayer. These results were consistent with a model in which the high free volume and increased outer headgroup spacing of lipids on highly curved surfaces induced interdigitation in the supported lipids. These results also support the symmetric lipid exchange studies of the SLBs as a function of the curvature, which was found to be slower on surfaces with higher curvature. Further, the effect of surface properties on the formation of SLBs was studied by changing the silanol density on the surface of SiO2 via thermal/chemical treatment and monitoring fusion of zwitterionic lipids onto silica (SiO2) nanoparticles. Our findings showed that the formation of SLBs was faster on the surfaces with lower silanol density and concomitantly less bound water compared to surfaces with higher silanol density and more bound water. Since the two SiO2 nanoparticles were similar in other respects, in particular their size and charge (ionization), as determined by zeta potential measurements, differences in electrostatic interactions between the neutral DMPC and SiO2 could not account for the difference. Therefore the slower rate of SLB formation of DMPC onto SiO2 nanoparticles with higher silanol densities and more bound water was attributed to greater hydration repulsion of the more hydrated nanoparticles. Lastly, we have investigated the effect and modulation of the surface charge of vesicles on the formation of SLBs by using different ratios of zwitterionic and cationic DMPC/DMTAP lipids. Through these studies we discovered a procedure by which assemblies of supported lipid bilayer nanoparticles, composed of DMPC/DMTAP (50/50) lipids on SiO2, can be collected and released from bilayer sacks as a function of the phase transition of these lipids. The lipids in these sacks and SLBs could be exchanged by lipids with lower Tm via lipid transfer. / Chemistry

Thesis (M.S.)--University of North Carolina Wilmington, 2008. / Title from PDF title page (viewed September 24, 2008) Includes bibliographical references (p. 58-59)

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The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ₄₂) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understanding is the major challenge to our efforts to elucidate not only the interactions of the Aβ42 with the lipid membranes, but also the behaviour of these systems towards the changes of the environmental conditions (pH, concentration, temperature). Our results suggest that there are various different methods, such as AFM, CARS microscopy and Raman spectroscopy as well as neutron scattering that are capable of fast imaging. Overall, all these techniques contributed in a complementary study of Aβ₄₂ aggregation states under extreme and physiological conditions as well as to image Aβ₄₂ interactions with lipid bilayers consisted of specific lipids.

G-protein coupled receptor 50 (GPR50) is a genetic risk factor for psychiatric illness. It is a member of the melatonin receptor family, which includes the well characterised melatonin receptors 1 and 2 (MT1 and MT2). However, the ligand for GPR50 remains elusive and little is known about GPR50 signalling pathways. Despite this, GPR50 is known to enhance neurite outgrowth and inhibit the actions of the neurite outgrowth inhibitor NOGO-A. Existing evidence also indicates a role in lipid metabolism; GPR50 knockout mice displayed abnormalities in energy homeostasis and weight control, whilst sequence variants are associated with altered lipid levels in humans. Further, a yeast-2-hybrid screen identified SREBF2 and ABCA2, regulators of lipid homeostasis, as GPR50 interactors. This thesis explores the role of GPR50 in neuronal development and lipid metabolism. The work presented in this thesis shows that GPR50 promotes neuronal differentiation. Overexpression significantly increased the number of neurites per cell in SH-SY5Y cells. Further, dendritic branching was enhanced by GPR50 transfection in hippocampal and cortical neurons (DIV 14). In hippocampal neurons, GPR50 transfection also lead to a shift towards spine maturity although it had no effect on spine morphology, suggesting GPR50 enhances spine development but may not alter synaptic strength. The effect of GPR50 on neuronal morphology may be driven by actin remodelling. Immunocytochemistry showed an enrichment of GPR50 in highly dynamic regions of the membrane, i.e. the lamellipodia and dendritic spines. Overexpression in SH-SY5Y cells also resulted in an increase in WAVE-2 and phosphorylated RAC1/CDC42, key modulators of actin dynamics. Additionally, GPR50 transfection altered the protein level and localisation of α- catenin, another regulator of actin organisation, in HEK293 and SH-SY5Y cells respectively. An involvement of GPR50 in lipid metabolism has also been demonstrated in this thesis. Verification of the Y2H study suggested GPR50 does not physically interact with SREBF2 or ABCA2. However, ABCA2 appears to induce the intracellular localisation of GPR50 in several cell lines. In SH-SY5Y cells, this was mimicked by the inhibition of cholesterol trafficking, suggesting the translocation of GPR50 to the plasma membrane is dependent on cholesterol transport. Further, the depletion of lipoproteins resulted in the downregulation of GPR50, indicating a responsiveness to lipid levels. Finally, GPR50 increased lipid metabolism, as seen by a decrease in intracellular lipid droplets upon GPR50 overexpression. The data presented here extends previous work indicating a role of GPR50 in neurodevelopment. It also highlights a potential mechanism by which GPR50 regulates neuronal morphology, i.e. via actin remodelling. Reports that GPR50 is involved in energy homeostasis is also supported in this thesis, further, results presented here suggest GPR50 is specifically involved in lipid metabolism. These processes are often disrupted in mental illness, thus this work may provide a functional link between GPR50 and psychiatric disorders.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Previous studies in yeast in this laboratory have found carnitine to be both protective against oxidative stress induced by hydrogen peroxide and to increase the detrimental effect of dithiothreitol. These phenotypes were found to be independent of the role of carnitine within the carnitine shuttle. A screen for suppressor mutations for these carnitine-dependent phenotypes identified, among others, Δcho2 and Δopi3. Cho2p and Opi3p catalyse the sequential methylation reactions in the formation of phosphatidylcholine from phosphatidylethanolamine. Therefore, this study aimed to investigate the relationship between choline, phosphatidylcholine and the carnitine phenotypes. Liquid growth assays of Δcho2 and Δopi3 cultures revealed that addition of choline can restore the protective effects of carnitine against hydrogen peroxide. The connection between the cellular phospholipid composition and the carnitine-dependent shuttleindependent phenotypes was also investigated. Analysis of the lipid composition of cells by LCMS showed that Δcho2 and Δopi3 had a largely different lipid composition compared with the wild type, most notably, a reduction in phosphatidylcholine and an increase in triacylglycerol content were observed for both mutants. These changes were reversed by supplementation with choline. However, no effects on the lipid composition of cells in response to carnitine treatment were observed, either when supplemented alone or in combination with DTT and hydrogen peroxide. Carnitine has also been investigated in mammalian systems for its potential to protect cells from oxidative stress, an effect which would be of benefit in various neurodegenerative disorders. Several studies have documented the positive effects of carnitine against oxidative stress in mammalian cells however the mechanism behind this action remains unknown. It is therefore thought that, provided similar effects for carnitine can be shown in mammalian cells as was observed in yeast, it would be beneficial to use yeast as a model system for the study of the molecular changes induced by carnitine. In view of this, the effects of carnitine on toxicity induced by oxidative stress in mammalian neural cells were compared to that which has been observed in yeast. For this purpose the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, a measure of reductive capacity of cells, was used. However, no effects for carnitine were observed in the MTT assay in combination with either dithiothreitol or paraquat. / AFRIKAANSE OPSOMMING: Vorige studies op gis in hierdie laboratorium het bevind dat karnitien beskermend is teenoor oksidatiewe stres wat deur waterstofperoksied geïnduseer word en ook die nadelige effek van ditiotreitol verhoog. Hierdie fenotipes is gevind om onafhanklik te wees van die rol van karnitien binne die karnitien-pendel. Die sifting vir onderdrukker-mutasies van hierdie karnitienafhanklike fenotipes het onder andere Δcho2 en Δopi3 geïdentifiseer. Cho2p en Opi3p kataliseer die opvolgende metileringsreaksies tydens die vorming van fosfatidielcholien vanaf fosfatidieletanolamien. Hierdie studie het dus gepoog om die verhouding tussen cholien, fosfatidielcholien en die karnitienfenotipes te ondersoek. Vloeistofanalises van Δcho2- en Δopi3-kulture het aangedui dat die byvoeging van cholien die beskermende effekte van karnitien teenoor waterstofperoksied kan herstel. Die verband tussen die sellulêre fosfolipiedsamestelling en die karnitienafhanklike pendel-onafhanklike fenotipes is ook ondersoek. Die analise van die lipiedsamestelling van selle deur middel van LCMS het getoon dat Δcho2 en Δopi3 ‘n grootliks verskillende samestelling het in vergelyking met die wilde tipe, en daar is veral ‘n afname in fosfatidielcholien en ‘n verhoging in triasielgliserol-inhoud vir beide mutante waargeneem. Hierdie veranderinge is omgekeer deur aanvulling met cholien. Geen effekte op die lipiedsamestelling van die selle is egter in reaksie op die karnitienbehandelings waargeneem nie, hetsy toe dit alleen aangevul is of in kombinasie met ditiotreitol en waterstofperoksied. Karnitien is ook in soogdierstelsels ondersoek vir sy potensiaal om selle teen oksidatiewe stres te beskerm, ‘n effek wat groot voordeel sal inhou vir verskeie neurodegeneratiewe steurings. Verskeie studies het reeds die positiewe effekte van karnitien teen oksidatiewe stres in soogdierselle opgeteken, hoewel die meganisme agter hierdie werking nog onbekend is. Daar word dus vermoed dat, gegewe dat soortgelyke effekte vir karnitien in soogdierselle getoon kan word as wat in gis waargeneem is, dit voordelig sou wees om gis as ‘n modelsisteem vir die studie van die molekulêre veranderinge wat deur karnitien geïnduseer word, te gebruik. In die lig hiervan is die effekte van karnitien op giftigheid wat deur oksidatiewe stres in soogdiersenuselle geïnduseer is, vergelyk met dít wat in gis waargeneem is. Om hierdie rede is die 3-[4,5-dimetieltiasool-2-iel]-2,5-difeniel tetrasoliumbromied (MTT) essaiëring, ‘n meting van die verminderende kapasiteit van selle, gebruik. Geen effekte vir karnitien is egter met die MTT essaiëring in kombinasie met óf ditiotreitol óf parakwat waargeneem nie.

Lipid rafts are operationally defined as cholesterol-rich membrane microdomains resistant to solubilization in nonionic detergents at low temperatures. Lipid rafts, which are quite different in lipid composition from the surrounding membranes, are of great importance to signal transduction, protein sorting and membrane transport. They have been implicated in a range of biosynthetic and endocytic processes and systems-signaling, molecular trafficking, diseases as well as being involved in the immune, vascular, digestive and reproductive systems. Dietary nutrients like fatty acids and vitamins of different types also play a critical and decisive role in the regulation of lipid rafts. / text