Amyloid-proteins are among the most actively researched biological topics today, because they have been associated with many serious human diseases, such as Alzheimer's disease and type II diabetes. In particular the deposition of protein aggregates on cell membranes has been suspected as the causes of the diseases, although the proof is still elusive. Studying the interactions of amyloid-forming peptides with lipid-bilayer membranes may clarify the pathway of the β-aggregate formation and provide new insights into the amyloid hypothesis of diseases. In this thesis, I investigate how three peptides, penetratin, amylin, and LL-37, interact with lipid membranes by using several techniques well-developed in our lab. In the study of penetratin interacting with lipid membranes, we were able to clarify the energy pathway of amyloid formation mediated by membrane-binding. This provides the sole experimental proof for the Jarrett-Lansbury theory of β- amyloid formation. Our investigation on amylin-membrane interaction clarifies how amylin in different forms damage bilayer membranes. Between penetratin and amylin we have clarified the complicated pattern of interactions between amyloid-forming peptides and lipid bilayers. The third peptide LL-37 studied in my thesis turned out to a pore forming peptide. I found the mistake made by previous investigators in several different laboratories that made them erroneously conclude that LL-37 was not a pore forming peptide. The results of these three peptides show that methods we used are a comprehensive set of tools that can reveal a broad range of peptide properties. Both the formation of amyloid aggregates and formation of membrane pores can be explained by a two-state model proposed by Huang describing peptide-membrane interactions. For LL-37, the second state is a pore in membrane. But for penetratin and amylin the second state is an aggregation in the β form. We found that β-aggregates have low affinity within a lipid bilayer, and therefore exit from the bilayer structure. However, this exit process extracts lipid molecules from the bilayer and incorporates them in the peptide aggregates. We suggest that this is the molecular process of how amylin might damage of the membranes of β-cells.

No description available.

<p>One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. </p><p>We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, &#945;-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.</p><p>Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.</p> / Dissertation

Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors. Part II aims to encapsulate in model lipid bilayers, metallic nanoparticles, which are used as probes in surface enhanced Raman spectroscopy. Three strategies of encapsulating particles, and incorporating Raman-active dyes are demonstrated, each using a different dye: malachite green, rhodamine-PE, and Tryptophan. Dye incorporation is verified by SERS and the bilayer is visualized and measured by TEM, with support from DLS and UV-Vis spectroscopy. In both parts, lipid-coated sensors are successfully fabricated and characterized. These results represent important and novel solutions to the functionalization of plasmonic surfaces with biologically relevant cell membrane mimics.

No description available.

Ácidos graxos livres (FFA) são importantes mediadores do transporte de prótons através de membranas. Porém, pouco se sabe sobre a influência estrutural tanto dos FFA como do ambiente lipídico na translocação de prótons através de membranas. Tanto os efeitos do comprimento da cadeia e número de insaturações dos FFA como a composição da membrana foram analisados por medidas elétricas em bicamadas lipídicas planas. Condutância a prótons (GH+) e condutância de vazamento (Gleak) foram calculadas a partir de medidas de voltagem em circuito aberto e de corrente de curto-circuito obtidas através de um eletrômetro ou um amplificador de patch-clamp (modo de voltage-clamp). Nossos resultados mostram que FFA com cis-insaturações causam um efeito mais pronunciado no transporte de próton quando comparados com FFA saturados ou trans-insaturação. Colesterol e cardiolipina diminuem Gleak de membranas. Cardiolipina também diminui GH+. Esses efeitos indicam uma dupla modulação do transporte de prótons: pelo mecanismo de flip-flop dos FFA e por uma via difusional simples adicional. / Free fatty acids (FFA) are important mediatiors of proton transport across membranes. However, little is known about the structural influence of both FFA and the membrane environment have in proton translocation across phospholipid membranes and by which means this influence is brought about. Both the effects of FFA chain length and insaturation and membrane composition on proton transport have been addressed in this study by electrical measurements in planar lipid bilayers. Proton conductance (GH+) and leak conductance (Gleak) were calculated from open-circuit voltage and short-circuit current measurements obtained using either an electrometer or a patch-clamp amplifier (voltage-clamp mode). We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to saturated or trans-unsaturated FFA. Cholesterol and cardiolipin decreased Gleak. Cardiolipin also decreased GH+. These effects indicate a dual modulation of protein-independent proton transport by FFA through flip-flop and by an additional simple diffusional pathway.

La sonoporation, c'est-à-dire l'utilisation d'ultrasons pour augmenter la perméabilité de la membrane cellulaire et permettre le transfert de molécules dans la cellule, est une méthode de transfection alternative intéressante. Cependant, même s'il est généralement admis que la cavitation acoustique joue un rôle important dans la sonoporation, les mécanismes physiques sous-jacents à ce phénomène ne sont pas totalement compris. Pour obtenir des informations sur les interactions entre les bulles, les cellules et le milieu environnant, nous avons développé un système de sonoporation adapté à la visualisation en temps-réel sous microscope et dédié aux cellules adhérentes. Le champ acoustique dans le puits cellulaire est composé d'ondes stationnaires et possède donc des positions d'équilibres pour les bulles au fond du puits, c'est-à-dire à proximité des cellules. Après avoir confirmé que les effets biologiques sont liés à la cavitation inertielle, un système de régulation de la cavitation inertielle a été implémenté pour augmenter la reproductibilité de la sonoporation. L'utilisation de cette régulation permet de s'affranchir des problèmes d'initiation et de maintien de l'activité de cavitation ainsi que de travailler sans ajout d'agents de contraste ultrasonore. Ce système permet de sonoporer des cellules adhérentes et ceci de manière plus reproductible lors de l'utilisation de la régulation qu'à intensité acoustique fixée. L'utilisation de la régulation permet également de s'affranchir de la température du milieu (à 24 ou 37 °C). De plus, la sonoporation des cellules ne semblent pas induire d'effets négatifs sur la reprise de croissance des cellules. L'utilisation de membrane modèle (des bicouches lipidiques fluorescentes) permet l'observation des interactions bulles-membrane, principalement sous la forme de dégâts de type fissures ou impacts présents à la fois sur les ventres et nœuds de pression acoustique. Ces positions particulières sont également le siège du détachement cellulaire et de la sonoporation / Sonoporation, the use of ultrasound to increase cell membrane permeability and allow the transfer of molecules into cells, is an interesting alternative method of transfection. However, even if it is generally admitted that acoustic cavitation plays an important role in sonoporation, the physical mechanisms acting during sonication are not fully understood. To obtain information on the interaction between bubbles, cells and the _owing medium during sonication, we designed a sonoporation device adapted to real-time microscope visualization and dedicated to adherent cells. The acoustic field in the well is composed by standing waves and provides cavitation bubble equilibrium positions at the bottom of the well, near cells. After biological effects have been confirmed to be linked to inertial cavitation, a regulation device on inertial cavitation has been implemented in order to improve reproducibility of sonoporation. This regulation allows overcoming problems linked to initiation and time stability of cavitation activity without adding ultrasound contrast agents in the medium. The sonoporation device allows the sonoporation of adherent cells, and this, with more reproducible results when using regulation instead of a fixed acoustic intensity. The cavitation control allows also to obtain the same biological effects at 24 and 37 °C. Furthermore, cell sonoporation does not apparently induce negative effects on cell growth. The use of a membrane model (fluorescent lipid bilayer) allows the observation of bubbles-membrane interactions, principally in the form of damages as cracks or impacts present at both nodal and anti-nodal positions of the acoustic field. Cell detachment and sonoporation appear also at these particular locations

One of the most important aspects for all life on this planet is theact to keep their cellular processes in a state where they do notreach equilibrium. One part in the upholding of this imbalanced stateis the barrier between the cells and their surroundings, created bythe cell membrane. In addition to experiments, the investigation ofprocesses occuring in the cell membrane can be performed by usingmolecular dynamics simulations. Through this method we can obtain anatomistic description of the dynamics associated with events that arenot accessible to experimental setups. Molecular dynamics relies onthe integration of Newton's equations of motion in order to sample therelevant parts of phase-space for the system, and therefore it isdependent on a correct description of the interactions between all thesimulated particles. In this thesis I first present an improved methodfor the calculation of long-range interactions in molecular dynamicssimulations, followed by a study of cholesterol's impact on thepermeation of small solutes across a lipid bilayer. The first paper presents a previously derived modification to theparticle-mesh Ewald method, which makes it possible to apply thisto long-range Lennard-Jones interactions. Old implementations of themethod have been haunted by an extreme performance degradation andhere I propose a solution to this problem by applying a modifiedinteraction potential. I further show that the historical treatmentof long-range interactions in simulations of lipid bilayers hasnon-negligible effects on their structural properties.In the second paper, this modification is improved such that the smallerrors introduced by the modified interaction potential becomenegligible. Furthermore, I demonstrate that I have also improved theimplementation of the method so that it now only incurs a performanceloss of roughly 15% compared to conventional simulations using theGromacs simulation package.The third paper presents a simulation study of cholesterol's effect onthe permeation of six different solutes across a variety of lipidbilayers. I analyze the effect of different head groups, tail lengths,and tail saturation by performing simulations of the solutes in fourdifferent bilayers, with cholesterol contents between 0% and50%. Analysis of the simulations shows that the impact of the surfacearea per lipid on the partitioning of the solute could be lower thanpreviously thought. Furthermore, a model with a laterallyinhomogeneous permeability in cholesterol-containing membranes isproposed, which could explain the large differences betweenpermeabilities from experiments and calculated partition coefficientsin simulations. / <p>QC 20140609</p>

No description available.

Inspired by the roles of hair cells in nature, this study aims to develop and characterize two new sets of novel flow sensors. One set of sensors developed and studied in this work are flow sensors fabricated using carbon nanomaterials. These sensors are made by embedding carbon nanotubes (CNT) and carbon nanohorns (CNH) into a polymeric substrate and then tested by flowing a conductive aqueous solution over the surface of the exposed CNT and CNH. In response, a flow-dependent voltage is generated. The surface coverage and the electrical relationship between the sensor and water is investigated and the voltage measurements of sensors with different levels of resistance were tested in varying fluid velocities. In response to these fluid velocities, the least resistive sensor showed small, but detectable changes in voltages, while higher resistance sensors showed less response. In addition, plasma treatment of the carbon nanomaterial/PDMS films were conducted in order to render the PDMS on the surface hydrophilic and in turn to pull more fluid towards the carbon material. This showed to improve the sensitivity of the flow sensors. This work also builds on previous research by investigating the flow dependent electrical response of a "skin"-encapsulated artificial hair cell in an aqueous flow. An artificial cell membrane is housed in a flexible polyurethane substrate and serves as the transduction element for the artificial hair cell. Flow experiments are conducted by placing the bio-inspired sensor in a flow chamber and subjecting it to pulse-like flows. This study demonstrates that the encapsulated artificial hair cell flow sensor is capable of sensing changes in flow through a mechanoelectrical response and that its sensing capabilities may be altered by varying its surface morphology. Furthermore, the sensor's response and dynamics as a function of its surface morphology and structural properties are investigated through synchronized motion tracking of the hair with a laser vibrometer and current measurements across the artificial cell membrane. / Master of Science