Indiana University-Purdue University Indianapolis (IUPUI) / The cell membrane serves as a barrier between the interior and exterior of a living cell. Its main structural component is the lipid bilayer, which is composed of various kinds of lipids that segregate into domains. These lipid domains, distinguished in composition and physical properties from the bulk lipids that surround them, are believed to modulate the function of resident proteins by providing an appropriate lipid environment. Polyunsaturated fatty acids (PUFA) are a type of fatty acid that contain multiple C=C double bonds. They have a lot of health benefits, which may originate in part due to their incorporation into lipids in the plasma membrane. Hypotheses that PUFA-containing lipids laterally separate into domains and/or modulate the structure of existing domains have been raised to explain the fundamental role played by PUFA. In our research, we use molecular dynamics (MD) simulations to simulate model membranes composed of PUFA-containing phospholipids and to investigate their interaction with cholesterol and vitamin E that are influential membrane constituents. The presumptive function for vitamin E in membranes is to protect PUFA against oxidation. Although the chemistry of the process is well established, the role played by the molecular structure that we address with atomistic molecular dynamics (MD) simulations remains controversial. We compared the behavior of vitamin E in lipid bilayers composed of 1-stearoyl-2-docosahexaenoylphosphatidylcholine (SDPC, 18:0-22-6PC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) via all-atom MD simulations at 37° C. SDPC represents a PUFA-containing lipid, and SOPC serves as monounsaturated control. From the calculation of van der Waals energy of interaction between vitamin E and fatty acid (FA) chains, we found higher probability that the PUFA chains surround the chromanol head group on vitamin E. This is further demonstrated by probability density maps of acyl chains around vitamin E molecules. Also, an ability to more easily penetrate deep into the PUFA containing bilayer of vitamin E is detected by faster flip-flop rate of vitamin E observed in the SDPC bilayers. These results showed that the high disorder of polyunsaturated docosahexaenoic acid (DHA) chains allows vitamin E to easily tunnel down into the bilayer and often brings the PUFA chains up to the surface of the bilayer, improving the likelihood that the reactive (hydroxyl) group on vitamin E would encounter a lipid peroxyl radical and terminate the oxidation process. Thus, the simulations indicate that the molecular structure of vitamin E supports its role as an antioxidant in a PUFA-containing membrane. A subsequent study on the partitioning of vitamin E into PUFA-containing lipids was done by analyzing the binding energy of vitamin E in the corresponding lipid bilayer. The binding energy is obtained from the potential of mean force (PMF) profile of vitamin E alone the membrane normal direction (z), which is calculated from umbrella sampling MD simulations. We found the binding in SDPC is smaller in SOPC, indicating that vitamin E does not prefer PUFA-containing phospholipids. The flip-flop rate was also estimated from the PMF profile, confirming that vitamin E flip-flops across the SDPC bilayer more easily than the SOPC bilayer. From the simulations it was noted that the membrane deforms as vitamin E is pulled out, which suggests interactions between the phospholipids contribute to the binding energy of the vitamin E. In a final study, a comparison was made between the effect on membrane organization of the three types of long chain omega-3 (n-3) PUFA found in fish oils: eicosapentaenoic acid (EPA, 20:5), DHA (22:6) and docosapentaenoic acid (DPA, 22:5). MD simulations were run on lipid bilayers composed of 1-stearoyl-2-eicosapentaenoylphosphatidylcholine (EPA-PC, 18:0-20:5PC), 1-stearoyl-2-docosapentaenoylphosphatidylcholine (DPA-PC, 18:0-22:5PC), SDPC (DHA-PC, 18:0-22:6PC) and, as a monounsaturated control, SOPC (OA-PC, 18:0-18:1PC) in the absence and presence of cholesterol. By analyzing the physical properties such as membrane order and thickness, we found all three n-3 PUFAs disorder the membrane. The disordering is greatest with EPA and least with DPA. Unique among the n-3 PUFA-containing membranes, there is region of high order in the upper portion of the DPA chain. The PUFA-containing lipids were found to less favorably interact with cholesterol compared to the OA-containing lipid, which is caused by their disorder. We speculate that differences between DPA, DHA and EPA might potentially modulate their effect on lipid domain formation.

Bilayer lipid membranes (BLMs) are formed from phospholipid molecules which self-assemble into a lipid bilayer with 4 to 9 nm thickness when submerged in an aqueous solution. This is due to their amphiphilic nature, meaning that one part of the molecule is hydrophilic, or attracted to water, and one part is hydrophobic, or repelled by water. They are the primary structural component of cell membranes in living organisms and therefore are useful for modeling the properties of cell membranes since they share many of the same chemical and physical properties. The objective of this dissertation is to investigate the maximum pressure that can be withstood by a BLM formed over a porous substrate, which will be referred to as the failure pressure. This work represents the first time that this quantity has been measured and reported. The failure pressure is investigated in several complementary ways, including mechanical, electrical, and optical measurements and modeling. The phospholipids that are tested include 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphatidylcholine (SOPC) and mixtures of SOPC and cholesterol (CHOL), which was chosen because cholesterol is known to increase the strength of SOPC BLMs. A new test methodology was developed to measure the failure pressure of BLMs. A custom test fixture was used to pressurize BLMs while measuring the applied pressure with a high degree of precision and repeatability. The BLMs were tested in an electrolyte solution over substrates that contained a single pore and also substrates that contained an array of many pores. SOPC BLMs were tested over single pore substrates with pore sizes ranging from 5 to 20 microns, and this resulted in failure pressures from 67 to 19 kPa, respectively. For single pore tests, the addition of 50 mol% cholesterol to SOPC resulted in a 56% higher failure pressure on average than SOPC alone. For multi pore substrates, SOPC BLMs were tested using pore sizes between 0.05 and 10 microns, which yielded bulk failure pressures of 380 to 1.5 kPa, respectively. For multi pore tests, SOPC/CHOL-50 mol% resulted in a 47% higher bulk failure pressure on average. A model was developed to predict the pressurization curve of BLMs and was applied to both the single and multi pore tests. It was found that the failure pressure of BLMs follows a distribution which was well modeled by a Weibull distribution with a positive skew. Parameters such as the Weibull shape parameter were determined by fitting the model to the experimental pressurization curves and it was found that the shape of the Weibull distribution was nearly the same for every pore size. Using the pressurization model it was estimated that the percentage of failed BLMs that were pressurized over a multi pore substrate ranged from 4% to 33%. The model also coupled the bulk failure pressure of BLMs formed over multiple pores to the failure pressure of a single BLM, showing that the bulk failure pressure of multiple BLMs is smaller than the failure pressure of a single BLM because it represents the failure of only the weakest BLMs in a group. Electrical impedance was measured before and after pressurization of the BLMs, and these measurements were modeled by assuming that the BLMs act as a resistor and a capacitor configured in parallel. In general, the impedance magnitude dropped by two to three orders of magnitude after BLM pressurization, which was a result BLMs failing and opening conductive pathways through the subsequently empty pores. It was found that normalized conductance values for SOPC BLMs were between G / A = 4 x 10^-12 and 2 x 10^-8 S/cm^2, and normalized capacitance values varied between C / A = 3 x 10^-14 and 1 x 10^-10 F/cm^2. In the literature these values ranged from G / A = 10^-1 to 10^-9 S/cm^2 and C / A = 10^-6 to 10^-8 F/cm^2, having a wide range of values due to the many variations of experimental test procedures. Visual images of BLMs were produced using fluorescence microscopy. Images were recorded before and after pressurization of SOPC BLMs formed over a multi pore substrate. As predicted by the pressurization model, it was found that some but not all BLMs fail after pressurization. It was also found that BLMs fail over the center of a pore, and leave remnants around the perimeter of the pore. / Ph. D.

This dissertation covers a broad range of research topics all towards the ultimate goal establishing of a novel type of biosensor in which the biocompatible membrane structure reconstituted with functional transmembrane proteins is utilized as the sensing element. It focuses on 1) examining the activity of a model transmembrane protein, bovine rhodopsin (Rho) when reconstituted into stabilized lipid membranes and 2) the instrumentation of a novel type of optical spectroscopy, plasmon waveguide resonance (PWR), which is a surface sensitive technique and its application in sensing biological events.Lipid membrane play crucial roles in cell function. Their biophysical properties affect the activity of a large amount of transmembrane receptors. They are great candidates for biosensing/ biomedical coating. However, the intrinsic instability of natural or fluid membranes prevents them to be used in a device. Studies have been done to show indirect evidence that the activity of Rho maybe maintained in polymerized membrane composed of bis-SorbPC lipids. The activity of Rho reconstituted into vesicular membranes comprised of various lipids was studied by a more direct technique, UV-Vis. It was found Rho activity was maintained to 66% of that in natural Egg PC lipid in the mixture of Egg PC:(poly)bis-SorbPC (1:1 mol:mol) as opposed to minimal values in 100 % (poly)lipids.A new type of spectral PWR was developed. The working concept, technical characterization and comparisons with similar techniques were discussed and compared in this work. A modified version of angular PWR in which lipid bilayers were formed by vesicle fusion was also developed. This method excludes possible effects from a high boiling point organic solvent on either the lipid bilayer itself or the membrane proteins associated with it. A calculating program NphaseAll for PWR was developed to do predictions of waveguide properties can be made to provide guidance for waveguide design. Theoretical calculations were done for PWR and experimental results were compared with the theoretical predictions.PWR was used to detect the formation of a biological lipid membrane, the association of alpha synuclein with membranes and the binding activity of human melanarcortin to its ligands in fluid and polymerized/dried membranes.

Indiana University-Purdue University Indianapolis (IUPUI) / 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. 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. 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.

We develop a coarse-grained model of lipids and proteins in which the lipids are modelled as diblock copolymers and the proteins as rigid cylinders. The generic protein model allows the possibility of amphipathic proteins with intrinsic curvature. Self-consistent field theory (SCFT) is used to determine the morphology of the lipid bilayer in the vicinity of the proteins. In particular, we focus on the case of a long transmembrane protein inserted perpendicular to the bilayer. For this system we use SCFT to determine the mechanical properties of the membrane and the thickness profile as a function of distance from the protein inclusion. The mechanical constants are also used in an elastic theory to predict the thickness profile. Good agreement between the full SCFT and elastic theory is obtained. We also use SCFT to determine systematic trends of the boundary conditions for the thickness profile at the protein interface. Such results could be used as boundary conditions for the description of bilayers using elastic theory. We show that this system undergoes a second order wetting transition as the interaction strength between the protein and membrane is varied. / Thesis / Master of Science (MSc)

Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes compared to young thrombocytes suggesting the lipid composition may be important for differential labeling. Therefore, we tested the liposomes prepared with different ratios of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and observed that the lower amounts of PE favor the DiI-C18 labeling whereas higher concentrations of PC are less efficient. Also, in both PE and PC, increased concentrations of both resulted in decreased binding. These results are consistent with our observation that mature thrombocytes have higher concentrations GP and thus DiI-C18 may not bind to them efficiently compared to young thrombocytes.

No description available.

Within the vestibular system are otolith organs, both the utricle and saccule. The primary function of these organs is to transduce linear head accelerations and static head tilts into afferent signals that are sent to the central nervous system for the utilization of image fixation, muscle posture control, and the coordination of musculoskeletal movement in dynamic body motion. The utricle of the red ear slider turtle was studied in this dissertation. The turtle's utricle is composed of several layers. The base layer contains a set of neural receptor cells, called hair cells, and supporting cells. The three layers above the base layer compose the utricle's otoconial membrane (OM) and are: 1.) a saccharide gelatinous layer, 2.) a column filament layer, and 3.) a calcite and aragonite otoconial crystal layer. The primary goal of this research was to study the dynamic response of the turtle's OM to a variety of natural inertial stimuli in order to characterize its inherent mechanical properties of natural frequency ("n), damping ("), and shear modulus (G). The medial-lateral (ML) and anterior-posterior (AP) anatomical axes parameters were measured for the utricle. The ML axis median with 95% confidence intervals was found to be "n = 374 (353, 396) Hz, " = 0.50 (0.47, 0.53), and G = 9.42 (8.36, 10.49) Pa. The AP axis median with 95% confidence intervals was found to be "n = 409 (390, 430) Hz, " = 0.53 (0.48, 0.57), and G = 11.31 (10.21, 12.41). Nonlinearites were not found to occur in the OM for the tested inertial stimuli and no significant difference was found between the mechanical properties for the ML and AP axes. Additionally, this research presents the initial steps to form a novel bio-inspired accelerometer based on the morphology of the utricle. The primary transducer element for this possible otolith organ inspired accelerometer design is a droplet interface bilayer (DIB). A DIB is a lipid bilayer that is formed when the interface of two aqueous droplets, that contain free-floating lipids, are joined. The aqueous droplets are suspended in a nonpolar environment (oil) and the oil/water interface forms a lipid monolayer. This research developed and used an experimental test setup to characterize the mechanoelectrical characteristics of a micron-sized DIB. This information, along with examples in the text, could be used to further design the aforementioned accelerometer. / Ph. D.

Among the great breakthroughs in nanoscience and nanotechnology is the emergence of synthetic polymers that demonstrate biological activity and thus can be exploited for biomedical applications, extending from agents in therapeutics to drug delivery and tissue engineering. A key factor in the fabrication of such polymeric materials is the ability to tune and control their properties. To this end, an insight into the mode of interactions with biological systems is imperative. Computer simulations have proved to be a valuable tool that can compliment experiments and provide -otherwise inaccessible- information. In the context of this thesis, different aspects of the polymeric biological activity were investigated by studying two polymeric materials suitable for different types of applications, aiming to clarify yet undisclosed mechanisms that govern the polymers' behaviour either in solution or in conjunction with model lipid membranes. The first part of the thesis is dedicated to a nonionic amphiphilic copolymer known as Pluronic L64 that is considered as a candidate for the design of novel hybrid polymer-lipid vesicles that will act as carriers for drugs or genes. The hybrid bilayers are subjected to mechanical stress and their properties are compared to those of pure lipid bilayers. The simulations showed that the hybrid membranes can sustain increased surface tension prior to rupture, are stiffer, thicker and the polymers can induce higher lipid tail packing and also reduce the lipid mobility, rendering the membranes more ordered and less fluid. At high values of lateral pressure, which leads to pore formation, the copolymer chains decelerate the pore growth. The examination of the defect formation mechanism reveals that the hydrophilic PEO segment plays the most vital role. The same systems were also observed in varying temperatures and the impact of the inserted polymers on the phase behaviour was investigated. The data suggested that the polymers change the nature of the phase transition from a discontinuous to a continuous one. The hybrid membranes transform between the ordered and the disordered phase in a continuous manner and not at a critical melting temperature. Interestingly, the effect of polymers is different at the low and high temperature regions, as proved by the analysis of the mechanical, structural and dynamic membrane properties. The second part is focused on the study of polyhexamethylene biguanide (PHMB), a biguanide-based polyelectrolyte, that possesses remarkable biocidal properties. Even though PHMB's activity is known, the specific mode of action against bacterial membranes is still puzzling. Our work revealed that the polyelectrolyte assumes a counterintuitive behaviour in aqueous solution tending to self-organise into ordered compact structures, despite the repulsive electrostatic interactions of its positively charged segments. The formed nano-objects are thermodynamically stable, as was confirmed by free energy calculations and could be linked to PHMB's antibacterial mechanism. These findings pave the way for further computational and experimental exploration of these fascinating and promising materials that could lead to the design of novel smart biologically active nanoparticles.

Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (µL to pL), fast analysis times (s), high selectivity and efficiency, and excellent compatibility with biological samples. However, the inability of conventional CE detectors to sense biologically active compounds that are optically and electrochemically inactive limits their use for biosensing and drug screening. We have developed a highly stable electrophysiological detection platform consisting of ion channel (IC) reconstituted in synthetic bilayer membrane also known as black lipid membranes (BLM) suspended across a functionalized microaperture to be coupled to a high resolution capillary separation channel. Low energy surface modifiers were used to drastically improve the electrical, mechanical, and temporal stability of BLMs. Glass microapertures modified using tridecafluoro 1, 1, 2, 2-tetrahydrodimethylchlorosilane facilitated the rapid formation of highly stable BLMs due to the amphiphobic property (H₂O/oil repellency). Furthermore, a combination of chemically modified aperture surfaces and chemical cross-linking within the lipid membrane were used to dramatically improve BLM stability. Partial cross-linking within the bilayer maintained fluidity which allowed reconstitution of ion channel proteins while maintaining the stability of BLM-IC platform. The stable BLM-IC across glass pipette aperture was coupled to microchip electrophoresis and was shown to withstand field strength (>250 V/cm) from separation channel. Additionally, planar microapertures fabricated in SU8 were used for the formation of stable BLM-IC platform towards the construction of an integrated device. The chemical properties of the SU8 supported the formation and cross-linking within polymerizable lipid or lipid bilayer doped with polymerizable methacrylate monomers. Additionally, we expressed ion channel coupled receptor fusion protein in HEK 293 cells towards the development of ion channel sensors for wide range of ligand detection in BLM sensor platforms. The pharmacology of IC functionalized with muscarinic acetyl choline (M2-K) receptor using cell based assay by patch clamp electrophysiology showed activation by acetylcholine and inhibition by atropine. Thus this platform holds a great promise as the next-generation integrated analysis system for rapid screening of biologically active compounds (eg. glucagon) in complex matrix such as whole blood and urine for the diagnosis and management of chronic disease such as diabetes.