The synthesis of the three 6”-deoxy-6”-thio glycolipid analogues β-D-Gal-(1→6)-β-D-Gal-(1→4)-β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane, β-D-Gal-(1→4)-β-D-Glu-(1→4)- β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane and β-D-Gal-(1→4)-β-D-Glu-(1→4)-β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-octadecane is presented here. Glycosylation at position O-4’ of a propargyl cellobioside glycosyl acceptor and position O-6’ of a propargyl lactoside glycosyl acceptor with a 6-thio-6-deoxy galactosyl donor gave rise to two unique trisaccharides that in turn underwent copper-catalyzed azide-alkyne cycloadditions with either 1-azidododecane or 1-azidooctadecane. The potential for each of these analogues to function as tethers of lipid bilayers to Au(111) was assessed primarily by differential capacitance experiments. Deposition of a bilayer of DMPC/cholesterol (70:30) by Langmuir-Blodgett (LB) transfer followed by Langmuir-Schaefer (LS) touch to a self-assembled monolayer of the O-6’ linked analogue, diluted with 1-β-D-thioglucose, failed. This led to simplifying the target architecture to diagnose the quality of the monolayers. A monolayer of the known monosaccharide 1-octadecane-4-(6-thio-β-D-galacto-pyranosyloxymethyl)-[1,2,3]-triazole1 prepared by LB transfer was found to support a lipid monolayer deposited by LS touch and this bilayer had the lowest minimum capacitance observed of 0.9 µF/cm2. An attempt to produce a bilayer by the same method using the trisaccharide bearing the C-18 alkane chain failed and this was attributed to high water solubility, which gave rise to poor organization at the air-water interface. A self-assembled monolayer of this variant went forward to produce a poor quality bilayer with a minimum capacitance of 7.1 µF/cm2, which was the lowest value obtained for the trisaccharide series of analogues. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.

The work presented on this thesis is focused on studies of the kinetics of electron transfer in bilayer lipid membranes (BLMs). Three different types of BLM were studied: i) tethered, ii) pore suspended (commonly known as ‘black’) and iii) based on the avidin – biotin interaction (these are part of the wider group of polymer cushioned BLMs). In order to produce tethered BLMs (tBLMs) of the best quality possible, self – assembled monolayers (SAMs) of a thiolipid (1,2 dipalmitoyl-sn-glycero-phosphothioethanol (DPPTE)) and of the same thiolipid mixed with L α phosphatidylcholine (EggPC) were characterised and their behaviour compared to that of SAMs of two alkanethiols (1 – heptanethiol and 1 – dodecanethiol). The SAMs that were formed by a mixture of lipids (DPPTE+EggPC) presented better kinetic parameters and were the chosen to produce tBLMs. Tethered BLMs were made by using the SAM described above as the lower leaflet; the second leaflet was deposited by vesicle fusion, the vesicles were made of EggPC. tBLMs are commonly used as model membranes, however in biophysical studies free-standing membranes or ‘black’ lipid membranes are more realistic models of cellular processes. The rates of electron transfer in both types of bilayer lipid membranes are compared. These BLMs were modified using two very important mitochondrial membrane associated molecules – ubiquinone-10 (UQ10) and α-tocopherol (VitE). The studies involved the use three redox couples, Fe(CN)_6^(3-/4-), Ru(NH_3 )_6^(3+/2+) and NAD+/NADH using cyclic voltammetry and electrochemical impedance spectroscopy. The NAD+/NADH couple is of particular interest as it is the key to several important biochemical processes. The last type of BLM that was studied was the BLMs based on the avidin – biotin interaction. Avidin was deposited on a platinum surface by electrodeposition and then vesicles composed of EggPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt) (DOPE(B)) are burst by applying +0.7V (vs. Ag/AgCl, KCl 3.5M), leading to the formation of a supported BLM. The vesicles used had methylene blue (MB) inside; its release, when the vesicles burst, was monitored by cyclic voltammetry and UV-Vis. The kinetic parameters were determined based on the EIS measurements using Fe(CN)_6^(3-/4-) and Ru(NH_3 )_6^(3+/2+) as redox couples.

No description available.

Lipid rafts are nanoscale entities in the membranes of eukaryotic cells which provide a mechanism for the functional membrane segregation vital for several cellular processes. This lateral segregation of specific lipid and protein components provides the facilitative platforms for a variety of signaling and trafficking events at the plasma membrane and in the Golgi. Rafts are distinguished from the surrounding membranes by their physical properties and composition - they are relatively tightly packed and enriched in saturated lipids, sterols, and lipid-anchored proteins. Although the existence of rafts has been conclusively confirmed by several independent techniques, questions concerning various aspects of membrane heterogeneity are still to be addressed. Typical experiments investigating raft composition have been designed to evaluate the affinity of a given component for raft domains. In such experiments, the results are usually interpreted in a Boolean fashion, i.e., the component is either a raft molecule, or not. However, this binary point of view overlooks potential complexity that may underlie the nature of membrane heterogeneity. In this work, we systematically investigated the nature of functional cellular membrane heterogeneity. We started by characterizing the model membranes and fluorescent lipid analogs widely used in research into membrane domains. After extensively evaluating the potentials/limits of these approaches and the artifacts that must be avoided or alternatively could be exploited, we applied these tools to understand whether the cell membrane has multiple kinds of raft domains with distinct compositions and physical properties, rather than only one. We found that cell membranes have the potential to form various kinds of functional domains having different physicochemical properties, compositions, and functional outputs. Therefore, we propose continuously variable lipid packing as the principle of the functional membrane lateral heterogeneity. According to this principle, the membrane is not composed of a single variety of raft domain with strictly defined properties coexisting alongside a specific and uniform non-raft environment; rather it is composed of entities having continuously variable lipid packing. Finally, we show that this spectrum of membrane packing modulates the orientation of membrane lipid receptors, which ultimately influences their specific bioactivity. Our results showing continuously variable lipid packing and its ability to fine-tune the activity of membrane molecules comprise a novel model for the structure and function of eukaryotic membranes.

<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>

No description available.

No description available.

Hair cell structures are one of the most common forms of sensing elements found in nature. In humans, approximately 16,000 auditory hair cells can be found in the cochlea of the ear. Each hair cell contains a stereocilia, which is the primary structure for sound transduction. This study looks to develop and characterize a bilayer lipid membrane (BLM) operated artificial hair cell sensor that resembles the stereocilia of the human ear. To develop this sensor, a flexible substrate with internal compartments for hosting the biomolecules and mating cap are constructed and experimentally characterized. The regulated attachment method (RAM) is used to form bilayers within the sealed device. Capacitance measurements of the encapsulated bilayer show that the sealing cap slightly compresses the bottom insert and reduces the size of the enclosed bilayer. Single channel measurements of alamethicin peptides further verify that the encapsulated device can be used to detect the gating activity of transmembrane proteins in the membrane. The flexible substrate was incorporated into a low-noise, portable test fixture. The response of the sensor and tip velocity of the hair were measured with respect to an impulse input on the test fixture and several frequency response functions (FRFs) were created. The FRF between the sensor and the tip velocity was used to show that the hair vibration was transmitted to the bilayer for certain hair lengths. The transfer function between the sensor and the input was used to show the effect of membrane potential on sensor response. / Master of Science

Applications in controlled release and delivery calls for a good understanding of molecular transport within the carrier material and the dominating release mechanisms. It is clear that a better understanding of hindered transport and diffusion of guest molecules is important when developing new porous materials, e.g., surfactant templated silica spheres, for biotechnological applications. Confocal laser scanning microscopy was used to quantify the bulk release and intraparticle transport of small charged fluorescent dyes, and fluorescently-tagged neutral dextran, from mesoporous silica spheres. The time dependent release and the concentration profiles within the spheres have been used to analyze the release mechanisms using appropriate models. While the small, non-adsorbing anionic dye is released following a simple diffusion driven process, the concentration of the cationic dye varies radially within the spheres after loading. The release of the cationic dye is controlled by diffusion after an initial period of rapid release, which could be due to a significant fraction of the cationic dye that remains permanently attached to the negatively charged walls of the mesoporous silica spheres. The diffusion of dextran and the resulting flat concentration profiles could be related to the complex structural feature of the cylindrical pores close to the surface, and a possible conformational change of the dextran with the concentration. The stability and leaching of a catalytic enzyme, lipase, immobilized in hydrophobilized mesoporous support has also been quantified. Colloidal monodisperse mesoporous silica spheres were synthesized and transmission electron microscopy showed that the inner pore structure display a radially extending pores. The mesoporous spheres were used as solid supports for a lipid membrane incorporated with a multi-subunit redox-driven proton pump, which was shown to remain functional. / Synthesis, functionalisation and controlled release of mesoporous materials