Supported lipid bilayers (SLBs) are unique model systems for biological membranes. SLBs can be formed by fusing liposomes on solid substrates, which can be characterized by a variety of surface analytical techniques, such as Atomic Force Microscopy (AFM), X-ray diffraction, Quartz Crystal Microbalance (QCM), etc. In this study we used AFM to investigate the dynamic process of the formation of SLBs from liposomes in solutions containing metal ions and phase separation between different lipids as a function of temperature. Divalent cations, Ni2+ in particular, was found to be critical to the deposition of bilayers. Lipid rafts are plasma membrane microdomains rich in sphingolipid and cholesterol forming a liquid ordered phase surrounded by a liquid disordered phase. Lipid rafts are insoluble in cold non-ionic detergents, also called Detergent Resistant Membranes (DRMs). The interaction behaviors between detergent (Triton X-100) and mixed bilayers (DOPC/DPPC and DOPC/SpM) were studied by AFM. The way lipid bilayers were solubilized by Triton X-100 was quite different below and above its critical micelle concentration (CMC), and the SpM domains were found to be resistant to detergent extraction in the cold.

The amyloid beta peptide particularly the 40 and 42 amino acid residues are the responsible for plaque formation in Alzheimer's disease (AD) patients. Extra cellular plaque formation has been recognized after incessant investigations along with the formation of intracellular tau protein tangles as the hallmarks of AD. Furthermore, the plaque formation has been linked mostly as a cause of the disease and the tangles mostly as a consequence. Our investigation is focused on studying the formation of AD plaques. The amyloid beta (A[beta]) is a physiological peptide secreted from neurons under normal conditions, along with other soluble forms cleaved from the amyloid precursor protein (APP). These soluble forms of APP have neuroprotective and neurotrophic functions, while the A[beta] is considered an unwanted by-product of the APP processing. Under normal conditions there is an anabolic/catabolic equilibrium of the A[beta] peptide; therefore, it is believed that the formation of the plaque does not take place. On the other hand, the neurons' surface may play an important role in the adhesion mechanisms of the A[beta] peptide. Our experiments show that the neuron surfaces along with the media conditions may be the most important causes for progressive formation of plaques. We have incubated rigid supports (mica) and soft biomimetic substrates (lipid bilayers on top of a PEG cushion layer drafted onto a silica surface) with the three different conformations of the A[beta] peptide (monomeric, oligomeric and fibrils structures) to determine the adhesion mechanisms associated with in situ plaque formation. The soft biomimetic substrates have been assembled first by depositing and activating a thin film of silica (i.e., to create surface silanol groups). This film is then reacted with polyethylene glycol (PEG), which is a biocompatible polymer, to create a cushion-like layer that supports and allows the lipid bilayer to have high mobility. A lipid bilayer is then deposited on this soft support to reproduce a cell membrane using the Langmuir Blodgett deposition technique. The characterization of such biomimetic membranes has been studied by using Atomic Force Microscopy (AFM) in liquid environments. Our results show that these lipid bilayers are highly mobile. Additionally the structure and topography characteristics of the A[beta] conformations have been followed with atomic force microscopy (AFM). The kinetics and rates of adhesion have been measured with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Our results show the progress of the plaques' formation with time where simple monomers deposit on the substrates and allow the development of oligomeric species.

This research focuses on the modeling of dendrimer molecules for their application as delivery vectors within drug and gene therapy systems. We examine how the architecture and composition of dendrimers affect their drug and gene binding efficacies along with their interactions with anionic bilayers. We specifically focus on how the weakly basic nature of dendrimer monomers and the addition of neutral grafts to dendrimer surface groups affect their interactions with drugs, linear polyelectrolytes, and bilayers. By using polymer self-consistent field theory (SCFT) to model such systems, we develop a computationally efficient means to provide physical insights into these systems, which are intended to guide dendrimer design for delivery applications.We study the conformational properties of weakly basic (annealed) polyelectrolyte dendrimers by developing a SCFT model that explicitly accounts for the acid-base equilibrium reaction of the weakly basic monomers. We specifically focus on the role of local counterion concentration upon the charge and conformations of the annealed polyelectrolyte dendrimers. We compare our results to existing polymer scaling theories and develop a strong stretching theory for the dendrimer molecules.We extend the previous study to model the interactions between weakly basic dendrimers and weakly acidic, hydrophobic drug molecules. We specifically examine the effects of excluded volume, electrostatic, and enthalpic interactions on the binding efficacies between dendrimers and drugs under a variety of dendrimer generations, solution pOH conditions, drug sizes, and Bjerrum length values.We study the role of neutral dendrimer grafts on the conformations and drug binding efficacies of dendrimers. We then elucidate how the observed conformational changes affect the charge of the dendrimers. Furthermore, we examine how the presence of grafts affects the steric, electrostatic, and hydrophobic interactions between the drugs and dendrimers under a variety of solution conditions. We compare our results with the binding efficacies observed for non-grafted dendrimers to delineate the conditions under which the grafted dendrimers are better suited as drug hosts.We include semi-flexible, anionic linear polyelectrolyte (LPE) molecules in our grafted dendrimer SCFT framework to model the interactions between dendrimers and negatively charged genetic materials. Specifically, we examine how neutral dendrimer grafts, LPE stiffness, and solution pOH affect the interactions between dendrimers and LPEs. We then use our SCFT potential fields as input into Monte Carlo simulations in order to determine the dendrimer-LPE potentials of mean force and the resulting loop and tail statistics of the dendrimer-adsorbed LPE chains.We incorporate a negatively charged bilayer into our grafted dendrimer SCFT framework to model dendrimer interactions with a cellular membrane. We specifically examine the role of dendrimer grafting length, solution pH, and membrane tension on such interactions. By comparing our results with SCFT calculations of fixed dendrimer conformations and hard sphere nanoparticles in the presence of membranes, we delineate the role of dendrimer flexibility and porosity on the interactions between dendrimers and anionic bilayers. / text

No description available.

The soft matter field considers a wide class of objects such as liquids, polymers, gels, colloids, liquid crystals and biological macromolecules, which have complex internal structure and conformational flexibility leading to phenomena and properties having multiple spacial and time scales. Existing computer simulation methods are able to cover these scales, but with different resolutions, and ability to link them together performing a multiscale simulation is highly desirable. The present work addresses systematic multiscaling approach for soft matter studies, using structure-based coarse-graining (CG) methods such as iterative Boltzmann inversion and inverse Monte Carlo. A new software package MagiC implementing these methods is introduced. The software developed for the purpose of effective CG potential derivation is applied for ionic water solution and for water solution of DMPC lipids. A thermodynamic transferability of the obtained potentials is studied. The effective inter-ionic solvent mediated potentials derived for NaCl successfully reproduce structural properties obtained in explicit solvent simulation, which indicates the perspectives of using the structure-based coarse-graining for studies of ion-DNA and other polyelectrolytes systems. The potentials have temperature dependence, dominated mostly by the electrostatic long-range part which can be described by temperature dependent effective dielectric permittivity, leaving the short-range part of the potential thermodynamically transferable. For CG simulations of lipids a 10-bead water-free model of dimyristoylphosphatidylcholine is introduced. Four atomistic reference systems, having different lipid/water ratio are used to derive the effective bead-bead potentials, which are used for subsequent coarse-grained simulations of lipid bilayer. A significant influence of lipid/water ratio in the reference system on the properties of the simulated bilayers is noted, however it can be softened by additional angle-bending interactions. At the same time the obtained bilayers have stable structure with correct density profiles. The model provides acceptable agreement between properties of coarse-grained and atomistic bilayer, liquid crystal - gel phase transition with temperature change, as well as realistic self-aggregation behavior, which results in formation of bilayer, bicell or vesicle from a dispersed lipid solution in a large-scale simulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted. </p><p> </p>

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Membrane-bound proteins and charged lipids are separated based on their charge-to-size ratio by electrophoretic-electroosmotic focusing (EEF) method on supported lipid bilayers (SLBs). EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands from an initially homogeneous mixture. Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations and it overcame diffusive peak broadening problem. A "SLB differentiation" post-separation SLB treatment method was also developed by using magnetic particles to rapidly slice the whole SLB into many small patches after electrophoretic separation, while keeping the majority of materials on surface and avoiding the use of chemical reactions. Label-free detection techniques were also developed based on EEF on SLBs. First, a new separation based label-free detection method was developed based on the change of focusing position of fluorescently labeled ligands. This technique is capable of simultaneous detecting multiple protein competitive binding on the same ligand on SLBs. Low concentration protein can be detected in the presence of interfering proteins and high concentration of BSA. The fluorescent ligands were moved to different focusing positions in a charged SLB patch by different binding proteins. Both free ligand and protein bound ligand concentrations were obtained. Therefore, both protein identity and quantity information were obtained simultaneously. Second, the focusing position of fluorescent biomarkers on SLB was used to monitor the phospholipase D catalyzed hydrolysis of phosphatidylcholine (PC) to form phosphatidic acid (PA), which is involved with the change of charge on the phospholipids. The focusing position of fluorescent membrane-bound biomarker in the EEF experiment is directly determined by the negative charge density on SLB. Other enzyme reactions involved with the change of phospholipids charge can be monitored in a label-free fashion in a similar way.

No description available.

Une des clés pour comprendre les mécanismes d’action biologiques des molécules naturelles et thérapeutiques est leur faculté à incorporer ou traverser les membranes lipidiques. Parce que les méthodes expérimentales sont parfois couteuses et répondent partiellement aux questions posés par les interactions composé-membrane, la modélisation moléculaire est devenue une sérieuse alternative. Les simulations de dynamique moléculaire ont ouvert de nombreuses perspectives ces dernières années en offrant la possibilité de décrire ces interactions intermoléculaires au niveau atomique. À l’aide de ces simulations, nous avons évalué la capacité de plusieurs composés (polyphénols, vitamines E et C, plantazolicine et carprofènes) à s’incorporer dans les membranes. Ces molécules ont été choisies pour leurs activités biologiques diverses, à savoir (i) activité antioxydante, précisément inhibition de la peroxydation lipidique, (ii) activité antibiotique et possibilité de former un pore transmembranaire, et (iii) inhibition d’enzymes impliquées dans la maladie d’Alzheimer. Leurs positions et orientations ainsi que leur capacité à s’accumuler ou à traverser les membranes ont été évaluées pour comprendre leurs mécanismes d’action.Dans le but d’utiliser les simulations de dynamique moléculaire en drug design, l’accent a été mis sur la précision des calculs, qui dépend de la qualité sous-jacente du modèle utilisé. En corrélant données expérimentales et théoriques, la méthodologie de nos modèles a été systématiquement revisitée. Le choix du champ de force, les paramètres des composés étudiés ainsi que la composition de la membrane sont en particulier apparus comme d’importants facteurs dans la description des interactions entre les molécules naturelles et thérapeutiques et les membranes. Des mélanges de lipides contenant du cholestérol ont notamment été utilisés et ont montré un impact significatif sur les résultats obtenus. / One of the key lockers to understand mechanisms of biological action of drugs and natural compounds is their capacity to incorporate/cross lipid bilayer membranes. In the light of demanding experimental techniques, in silico molecular modelling has become a powerful alternative to tackle these issues. In the past few years, molecular dynamics (MD) has opened many perspectives, providing an atomistic description of the related intermolecular interactions. Using MD simulations, we have explored the capacity of several compounds (polyphenols, vitamins E and C, plantazolicin, carprofens) to incorporate lipid bilayer membranes. The different compounds were chosen according to their different biological functions, namely (i) antioxidant activity against lipid peroxidation, (ii) antimicrobial activity with the possibility of trans-membrane pore formation, and (iii) inhibition of enzymes involved in Alzheimer’s disease. In order to rationalize their mechanisms of action, their position and orientation in membranes as well as their capacity to accumulate or permeate lipid bilayers were assessed. Having in mind a predictive purpose in drug design for MD simulations, the accuracy of the results relies on the quality of the in silico membrane models. By ensuring relationships between experimental and theoretical data, methodological improvements have been proposed. In particular, force field selection, xenobiotic parameterization and bilayer constitution emerged as crucial factors to appropriately depict drug-membrane interactions. For the latter issue, lipid mixtures e.g., including cholesterol have been developed.