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La membrane cellulaire est principalement une bicouche phospholipidique constituant une barrière qui régule les échanges entre la cellule et son environnement. Son intérieur hydrophobe empêche le passage d’espèces hydrophiles, chargées, de grande masse moléculaire et polaires, qui sont généralement transportées par des protéines à travers la bicouche. Dans certains cas de systèmes défectueux (e.g. les canalopathies), l’équilibre des concentrations en ions à l’intérieur et à l’extérieur des cellules est perturbé et les cellules sont compromises. C’est pourquoi le développement de transporteurs transmembranaires synthétiques est nécessaire. De nombreux travaux ont été faits dans le développement de transporteurs synthétiques d’anions (particulièrement du chlorure). Dans cette thèse, nous présentons nos travaux sur un nouveau transporteur d’anion appelé axe parapluie, capable de changer de conformation dépendamment de la polarité de son environnement. Dans un premier temps, nous avons conçu le design, puis synthétisé ces axes parapluie qui montrent une importante activité en tant que transporteur de chlorures. Ces composés réunissent deux concepts : - Le parapluie, constitué d’acides biliaires amphiphiles (une face hydrophile, une face hydrophobe). La flexibilité des articulations combinée à la grande surface des acides choliques permettent d’empêcher les interactions défavorables entre les parties hydrophiles et hydrophobes, ce qui facilite l’insertion dans la bicouche. - Un site ammonium secondaire en tant que site de reconnaissance, capable de former des ponts hydrogène avec des ions chlorure. De plus, l’axe peut complexer une roue de type éther couronne pour former un pseudo-rotaxane ou rotaxane parapluie ce qui résulte en l’inhibition partielle de leurs propriétés de transport. Ceci nous mène au second objectif de cette thèse, le développement d’un nouveau moyen de transport pour les médicaments cycliques. Certains macrocycles polaires et biologiquement actifs tels que les nactines ont besoin d’atteindre leur objectif à l’intérieur de la cellule pour jouer leur rôle. La membrane cellulaire est alors un obstacle. Nous avons imaginé tirer profit de notre axe parapluie pour transporter un médicament cyclique (en tant que roue d’un rotaxane parapluie). Les assemblages des rotaxanes parapluie furent accomplis par la méthode de clipage. Le comportement de l’axe et du rotaxane parapluie fut étudié par RMN et fluorimétrie. Le mouvement du parapluie passant d’une conformation fermée à exposée dépendamment du milieu fut observé pour le rotaxane parapluie. Il en fut de même pour les interactions entre le rotaxane parapluie et des vésicules constituées de phospholipides. Finalement, la capacité du rotaxane parapluie à franchir la bicouche lipidique pour transporter la roue à l’intérieur de la vésicule fut démontrée à l’aide de liposomes contenant de la α-chymotrypsine. Cette dernière pu cliver certains liens amide de l’axe parapluie afin de relarguer la roue. / The cell membrane is a phospholipid bilayer barrier that controls the exchanges between the cell and its environment. Its hydrophobic core prevents the entrance of hydrophilic, charged or large polar species that are transported through the bilayer by proteins. In some dysfunctional systems e.g. channelopathies), the balance of ion concentrations between the interior and exterior of the cell is no longer insured and the cell’s health is compromised. That is why the synthesis of synthetic transmembrane transporters is needed. There have been many synthetic anion carriers (especially chloride) developed in this area using different strategies. In this thesis we present our work on a new anion transporter, an umbrella thread. First, we designed and synthesized umbrella threads that showed significant chloride transport activity. These compounds combine two concepts: - the umbrella moiety, made from facial amphiphilic bile acids. The flexibility and large surface of the cholic acids can shield disfavored interactions between hydrophilic and hydrophobic elements that should ease their insertion into the bilayer. - a secondary ammonium recognition site on the thread that can form hydrogen bonds with chloride ions. Furthermore, the thread moiety is able to complex a crown-ether like wheel to form an umbrella pseudo-rotaxane or rotaxane that showed partially inhibited properties for chloride transport. This leads us to the second goal of this thesis, i.e. the development of a new vehicle for drug delivery. Some biologically active polar macrocycles (e.g. nactins) need to reach their target inside the cell to be efficient. The cell membrane also represents an obstacle here. In our work, we imagined using an umbrella thread as the vehicle for the cyclic drug as the wheel of the umbrella rotaxane). The umbrella rotaxanes were successfully assembled by the clipping method. The behavior of both the umbrella thread and umbrella rotaxane was extensively studied by NMR and fluorimetry. The umbrella motion from a shield conformation to an exposed one depending on the environment was observed for the rotaxane. Interactions between the umbrella rotaxane and phospholipid vesicles were also noticed. Finally, its ability to cross the lipid bilayer to deliver the wheel inside the vesicle was shown with α-chymotrypsin-filled liposome assays. This enzyme was able to cleave amide bonds on the umbrella thread to release the wheel.

[pt] A simulação da adsorção da prednisolona em um modelo de surfactante pulmonar foi realizada com sucesso usando dinâmica molecular coarse grained a uma temperatura de 310 K. O modelo coarse grained da prednisolona foi parametrizado usando o modelo do colesterol e validado utilizando cálculos de coeficientes de partição octanol-água e coeficientes de difusão lateral. O coeficiente de partição octanol-água calculado para prednisolona a 298 K é 3,9 mais ou menos 1,6 que possui um acordo razoável com o valor experimental. O coeficiente de difusão lateral da prednisolona na monocamada mista de DPPC/POPC é estimado ser (6 mais ou menos 4) x10(-7) cm(2) s(-1) a 20 mN m(-1), o que está de acordo com o encontrado para o colesterol. A monocamada mista de DPPC/POPC foi utilizada como modelo de surfactante pulmonar onde moléculas de prednisolona foram adsorvidas formando nanoagregados. Os nanoagregados de prednisolona foram transferidos dentro da monocamada mista DPPC/POPC sendo espalhados na tensão superficial de 20 mN m(-1). A 0 e 10 mN m(-1) os nanoagregados de prednisolona induzem o colapso da monocamada mista DPPC/POPC formando bicamadas. A implicação deste trabalho é que a prednisolona pode apenas ser administrada com surfactante pulmonar utilizando baixas frações em massa de prednisolona por lipídio (menor que 10 por cento). Com frações elevadas, o colapso inativa as propriedades do surfactante pulmonar pela formação de uma bicamada. Os resultados desta pesquisa podem ser utilizados para o desenvolvimento de novos tratamentos clínicos de doenças como a síndrome da angústia respiratória do recém-nascido, asma e doença pulmonar obstrutiva crônica. / [en] The simulation of prednisolone adsorption on a lung surfactant model was successfully performed using coarse grained molecular dynamics at 310 K (dynamics first performed). The coarse grained model for prednisolone was parameterized using a well-established cholesterol model and validated by using calculations of octanol–water partition coefficients and lateral diffusion coefficients. The calculated octanol–water partition coefficient of prednisolone at 298 K is 3.9 more or less 1.6, which is in reasonable agreement with experiment. The lateral diffusion coefficient of prednisolone in the DPPC/POPC mixed monolayer is estimated to be (6 more or less 4) x10(-7) cm(2) s(-1) at 20 mN m(-1), which is in agreement with that found for cholesterol. The DPPC/POPC mixed monolayer was used as lung surfactant model where prednisolone molecules were adsorbed forming nanoaggregates. The nanoaggregates of prednisolone were transferred into the DPPC/POPC mixed monolayer being spread at the surface tension of 20 mN m(-1). At 0 and 10 mN m(-1) , the prednisolone nanoaggregates induce the collapse of the DPPC/POPC mixed monolayer forming a bilayer. The implications of this work are that prednisolone may only be administered with lung surfactant by using low mass fractions of prednisolone per lipid (less than 10 percent). And, with high fractions, the collapse inactivates the properties of the lung surfactant by forming a bilayer. The results of this research can be used to develop new clinical treatments for diseases such as respiratory distress syndrome of the newborn, asthma and chronic obstructive pulmonary disease.

Les fluorocarbures ont un fort potentiel en médecine. Cependant, et en dépit du fait que certaines formulations employant des fluorocarbures sont utilisées en clinique, il n’existe que relativement peu d’études visant à déterminer les interactions entre un fluorocarbure et une membrane de phospholipides. Notre étude concentre à l’interface fluorocarbure/phospholipide, qui représente d’une part un modèle simplifié du surfactant pulmonaire natif dont le composant majoritaire est la dipalmitoylphosphatiylcholine (DPPC), et d’autre part la paroi de microbulles développées comme nouveaux agents théranostiques.Tout d’abord, nous montrons que les fluorocarbures abaissent considérablement la tension interfaciale d’équilibre d’une série de phospholipides et accélèrent fortement leur adsorption. Nous montrons que des oscillations périodiques appliquées à la bulle induisent une transition du film de DPPC vers un état d’organisation plus dense. L’application d’oscillations périodiques permet aussi à la DPPC d’expulser du film interfacial une protéine, l’albumine, dont la présence est souvent liée aux troubles dus au mauvais fonctionnement du surfactant pulmonaire. L’effet des fluorocarbures, qui accélère considérablement l’expulsion de l’albumine par la DPPC, est également étudié. D’autre part, nous avons obtenu des microbulles exceptionnellement stables grâce à une série homologue de phosphates perfluoroalkylés. Nous avons également réussi à former des microbulles couvertes par des nanoparticules magnétiques, tout en gardant les propriétés échogènes des bulles. De telles microbulles offrent un potentiel comme des agents de contraste bimodaux pour l’IRM et l’échosonographie. / Fluorocarbons have a great potential in medicine. However, and despite the fact that some formulations using fluorocarbons are used clinically, only a few studies are reported that aim to determining the interactions between a fluorocarbon and a membrane of phospholipids. Our work concentrated on the fluorocarbon/phospholipid interface, which represents, on one hand, a simplified model of the lung surfactant, the major component of which is dipalmitoylphosphatiylcholine (DPPC), and on the other hand, the shell of microbubbles developed as new theranostic agents. In a first part, we show that fluorocarbons significantly reduce the equilibrium interfacial tension of a series of phospholipids and greatly accelerate their adsorption rate. We also show that periodical oscillations applied to the bubble induce a transition of DPPC film to state with a denser organization. The application of periodical oscillations also allows DPPC to expel from the interfacial film a protein, albumin, whose presence is often associated with disorders caused by dysfunction of the lung surfactant. The impact of fluorocarbons, which considerably accelerate the expulsion from the interfacial film of albumin, is also studied. In a second part, we have obtained exceptionally stable microbubbles with a homologous series of perfluoroalkylated phosphates. We were also able to form microbubbles covered by magnetic nanoparticles, while preserving the echogenicity of the bubbles. Such microbubbles offer a potential as bimodal contrast agents for MRI and echography.

L'étude des membranes biologiques nécessite l'examen des différentes propriétés de ses composantes principales: les lipides et les protéines. Dans ce manuscrit, l'interaction lipide- lipide et lipide-protéine ont été suivies par spectroscopie vibrationnelle (Raman, Infrarouge). Nous sommes intéressés en premier lieu à l'étude de la structure et l'organisation des phospholipides dans leur phase gel et leur phase cristalline liquide en utilisant la spectroscopie moyen infrarouge. En outre, l'effet de la composition du groupement hydrophiles des lipides sur le comportement de la liaison hydrogène des mélanges lipidiques a été sondé en utilisant la spectroscopie lointain infrarouge. Dans la seconde partie, l'interaction de la protéine NADH ubiquinone oxydoréductase et du mutant NuoL (D563N) avec le zinc ont été étudiés par spectroscopie différentielle et les changements conformationnels induits par la liaison du zinc avec les protéines ont été examinés. Enfin, les vibrations métal-ligand des groupements fer-soufre dans le mutant de NuoB (C64A G100C) à différents pH ont été analysées par spectroscopie Raman. / The study of biological membranes involves the examination of the different properties of its main components: as lipids and proteins. In this manuscript, the lipid-lipid interaction and the lipid-protein interaction were monitored by vibrational spectroscopy (Raman and Infrared). We have been interested in the first part in studying the structure and organization of phospholipids in the gel phase and the liquid crystalline phase using mid infrared spectroscopy. In addition, the effect of the head group composition on the hydrogen bonding behaviour of lipid mixtures was probed using far infrared spectroscopy. In the second part, the interaction of the NADH ubiquinone oxidoreductase protein and NuoL mutant (D563N) with zinc was investigated through FTIR difference spectroscopy where the conformational changes upon zinc binding were monitored. Finally, the metal-ligand vibrations of the iron- sulfur clusters in NuoB mutants (C64A G100C) at different pH were analysed using Raman spectroscopy.

This thesis deals with the non-equilibrium phenomena under shear observed mainly in bilayer forming liquid crystalline phases of mixed surfactant systems, anisotropic colloidal dispersions as well as Langmuir monolayers of membrane peptides. To correlate the structural transitions under shear with the mechanical properties or flow behaviour, the rheological measurements are combined with different techniques such as optical imaging (bright field, polarizing or confocal), small angle light scattering as well as small angle x-ray scattering (Rheo-SAXS) measurements. The bilayer forming phases that have been studied consist of mixed surfactant system formed by a mixture of ionic amphiphiles with strong binding organic counter ions. The propensity of the hydrophobic counterion to modify the spontaneous curvature at the micelle-water interface gives rise to a rich equilibrium phase behaviour consisting of different bilayer forming liquid crystalline mesophases in between the hexagonal and lamellar phases. The liquid crystalline mesophases presently examined under shear are the weakly swollen isotropic and lamellar phases as well as the random and rhombohedral mesh phases. The main motivation of the thesis was to examine the stability of these phases under shear since all the existing studies so far on shear induced structural transitions are mainly confined to highly swollen isotropic sponge phase of interconnecting bilayers that can transform to a lamellar phase consisting of a stack of bilayers with 1D quasi long range order or a dilute lamellar phase is shear transformed to a collapsed surfactant rich lamellar phase coexisting with excess solvent at Peclet Number greater than 1. The present study revealed for the first time a shear reversible crystallization above the equilibrium crystallization temperature in the weakly swollen isotropic and lamellar phases formed in the SDS-PTHC-water system where the structural transition is feasible through a shear induced segregation/microphase separation of the hydrophobic counterions to tune the curvature of the bilayer-water interface. These results incited us to examine the role of shear on another class of mesophases that are structurally similar to lamellar phase but with a non-uniform interfacial curvature of the bilayers identified as the intermediate mesh phases. Mesh phases are formed by a 1D stack of perforated bilayers with quasi-long range order where the water filled pores or curvature defects can have a liquid-like ordering in the plane of the bilayers as in a random mesh phase or the pores can have a square or hexagonal ordering locking into a three dimensional lattice with either tetragonal or rhombohedral symmetry to form Tα or R3m ordered mesh phases. Two characteristic features of the mesh phases that is noteworthy are i) the non-uniform mean curvature for the bilayers formed by 3-coordinated hexagonal mesh or the 4-coordinated square mesh; ii) the elasticity of the bilayers forming the hexagonal or square ordered mesh in R3m or Tα phases as opposed to the fluid-like bilayers with zero surface shear modulus in the random mesh or classical lamellar phases (Lα). Hence the structural similarity as well as differences of the mesh phases with the lamellar phase raises some pertinent questions regarding the stability of surfactant mesh phases under shear. Two striking consequences of shear flow on the random and ordered mesh phases of a cationic-anionic mixed surfactant system were revealed: a shear-induced 3D ordering of the curvature defects in LDα phase as well as a hydrodynamic instability wherein a sequence of structural rearrangements leading to buckling instability gives rise to unstable flows in the R3m phase. These studies on shear induced structural transitions on partially ordered mesophases is juxtaposed with the study on another class of systems that were examined under shear comprising dispersion of anisotropic colloidal rods. We demonstrate that these suspensions shear thicken at low concentrations (≥ 25 %) and origin of shear thickening is formation of stress bearing hydrodynamic clusters rather than order-disorder transition. Finally we also examine the rheological properties of a monolayer of membrane peptide Alamethicin where the coexistence of solid-like domains in a backround of liquid expanded phase at high surface concetrations gives rise to a dense anisotropic suspension in 2D. The rheological properties of these jammed fluid/fluid interfaces formed by membrane proteins is well explained by the soft glassy rheology model proposed earlier in the context of 3D soft glasses, comprising emulsions, foams, colloidal glasses and gels. Chapter 1 starts with a general introduction of soft condensed matter systems and then we proceed to describe surfactant systems, their phase behaviour and self assembly. The formation of liquid crystalline phases in pure surfactant systems and in presence of additives such as salt or counterions are discussed. A brief introduction to colloids is explained further. This is followed by the discussion on the inter-macromolecular forces governing soft matter systems such as van der Waals interaction, the screened Coulomb repulsion, hydrogen bond, depletion, peristaltic, hydrophobic and hydration forces and steric repulsion. We further explain the systems studied and their phase behaviour at different concentrations formed by SDS-PTHC-water, CTAB-SHN-water system in detail. In the next section we describe the characterization of different liquid crystalline phases viz. nematic, hexagonal, isotropic, lamellar, intermediate mesh and ribbon phases using cross polarizing optical microscopy, small angle x-ray and rheology. Then, a theoretical background of linear and nonlinear rheology, optical/confocal microscopy and x-ray scattering techniques are given. This is followed by discussion on flow properties of colloidal suspensions in dilute and semi-dilute regime and finally shear thickening phenomena observed in concentrated suspensions. We discuss shear thickening phenomena observed in anisotropic precipitated calcium carbonate (PCC) colloids. We have also discussed shear induced phase and structural transitions observed in different liquid crystalline phases. Chapter 2 discusses the experimental apparatus and techniques used in our studies. We have discussed the different components of the MCR-300 and 101 stress-controlled rheometer (Paar Physica, Germany). The cross polarizing optical microscopy in transmission and reflection mode using a home-made shear cell and in built set-up respectively, and small angle light scattering set-up are discussed. Next, we discuss in-situ small angle x-ray rheology setup, a home made Couette cell installed at RRI Bangalore, Couette installed at SWING beamline Soleil, Paris, France and parallel plate setup at PETRA III, Germany. This is followed by discussion on sample preparation and synthesis technique of silica colloidal rods and modification of surface potential using a thermo-responsive polymer. Further, we explain the algorithm to track rods and analysis of SAXS 2D diffraction pattern. Flow behaviour of different phases formed in SDS-PTHC-water system are described in Chapter 3. This chapter has been divided into four sections. In section I, we describe the phase behaviour and rheology of micellar solution at different surfactant concentrations (ϕ) and molar ratios (α = [PTHC]/[SDS]) of two components. At ϕ = 0.3, a transition from viscous to visco-elastic behaviour is observed with increasing α from 0 to 0.3. Zero shear viscosity shows a non-monotonic behaviour with increasing α and reveals a maxima at α = 0.15. At low α, we observe a Newtonian behaviour which changes to shear thinning behaviour with increasing α and finally again retains the Newtonian behaviour. Dynamic light scattering studies in conjunction with presence of nematic phase made up of disks (confirmed by cross-polarizing optical microscopy) at higher α > 0.325, suggest that the drop in zero shear viscosity is due to decrease in length of the micelles from rods to disks. A similar behaviour is observed with increasing ϕ at constant α = 0.2, 0.25, 0.6. A change in the morphology of micellar aggregates with increase in α is expected in mixed surfactant systems with strongly binding counterions. However the change in morphology of micellar aggregates with surfactant content in surprising which is witnessed for the first time in mixed surfactant systems. In section II of this chapter we discuss the phase behaviour and rehological properties of different liquid crystalline phases formed in SDS-PTHC-water system at ϕ = 0.4, and varying α from 0 to 0.4. Using deutrium nuclear magnetic resonance (NMR) studies we show that the transition from hexagonal phase at α = 0, to lamellar phase at α = 0.4 occurs through a nemtaic phase of rods at α = 0.05 and nemtaic phase of disks at α = 0.2 through an isotropic phase of rods at α = 0.15. NMR studies reveal a decrease in variation of the quadrupole splitting across the transition from NC to ND. The visco-elastic and flow behaviour of the different phases were examined. A decrease in the steady shear viscosity across the different phases with increasing α suggests a decrease in the aspect ratio of the micellar aggregates. From the transient shear stress response of the NC and ND nematic phases in step shear experiments, they were characterized to be tumbling and flow aligning, respectively. Our studies reveal that by tuning the morphology of the surfactant micelles, strongly binding counterions modify the phase behaviour and rheological properties of concentrated surfactant solutions. In section III, we discuss shear induced phase transition in SDS-PTHC-water system using in-situ rheo-optical imaging and in-situ rheo-SAXS. Bilayer forming liquid crystalline phases namely isotropic (Li - optically isotropic) and lamellar (Lα - optically birefringence) are formed at α = 1.5, ϕ = 0.4 and α = 1, ϕ = 0.5. Both phases co-exist with excess solvent and remain fully swollen at temperature T > 50 oC. We have constructed a dynamic phase diagram in the parameter space of shear rate and temperature which demonstrate a novel shear induced phase transition to a crystalline phase (Lc) above a critical shear rate. At constant shear rate, the increase in viscosity is accompanied by presence of birefringent texture of Lα phase after a waiting time (t) which decreases with increasing shear rate. The Lc phase is stable under shear and melts back to equilibrium Li phase once shear is stopped. At higher temperature a transition from Li → Lα is observed. In-situ small angle x-ray scattering reveals an evolution of additional peaks in small as well as wide angle region which does not evolve any further once the viscosity reaches a maxima. The Lc phase obtained under shear at different shear rates can be indexed to a triclinic lattice with the lattice parameters depending on shear rates. We propose that the possible origin of phase transition is re-distribution of counterions under shear which results in counterion-rich and counterion-poor region. This counterion rich region results in crystalline Lc phase. In addition to revealing a unique class of non-equilibrium phase transition, the present study urges a unique approach toward understanding shear-induced phenomena in concentrated meso-phases of mixed amphiphilic systems In section IV we propose a shear induced nucleation and growth of crystalline phase in metastable bilayer forming Li and Lα phases. Nucleation and growth of crystalline phase ac-celerated by shear exhibits a power law dependence on time. The time of nucleation strongly depends on the shear rate with different exponents for different phase compositions. The crystalline phase formed under the influence of shear is stable and irreversible for tempera-ture < 28 oC. The crystal structure obtained under shear can be indexed to a triclinic unit cell with different lattice parameters depending on the shear rate and concentration probed. In Chapter 4, we discuss shear induced transitions observed in mesh phases formed in cationic surfactant system CTAB in the presence of strongly binding counterions SHN formed at different surfactant concentration (ϕ) and molar ratio (α). Random mesh phase (LDα) formed at ϕ = 0.3, 0.4, 0.5 and α = 1 are identified as stack of bilayers having curvature defects in form of water filled pores in the plane of bilayers . These pores do not have any long range correlation either in-plane or across the plane. A 3D ordered mesh phase (R3m) is formed at α = 1 and ϕ = 0.6, where these pores have in-plane and out of plane positional correlation and locked into a 3D lattice with rhombohedral symmetry. These phases are easily identified from small angle x-ray scattering studies wherein LDα a diffuse peak corresponding to in-plane defect spacing (ddef ) is observed along with lamellar d-spacing (d). However several additional peaks along with lamellar peak are observed for R3m phase revealing a long range correlation of pores. By shearing different LDα phases formed at different ϕ′s, we D phases formed probe the effect of shear far and near to the R3m phase boundary. When Lα at ϕ = 0.3 and 0.4 are sheared at constant shear rate, we observe a structural transition to an onion phase which is accompanied by increase in viscosity at the onset of the transition. D When Lα phase formed near R3m phase at ϕ = 0.5 is sheared, we observe a decrease in viscosity which is accompanied by the presence of a sharp peak near the diffuse peak corresponding to ddef along with several other small as well as wide angle peaks. All these D phase. We propose that peaks can be indexed to R3m phase co-existing with equilibrium Lα the locking of the defects into a 3D lattice occurs when the in-plane correlation length (ddef ) is larger than the bilayer periodicity (d). Prior to appearance of sharp peak near ddef , we observe an a-orientation of lamellae i.e. bilayers align along the shear-gradient plane where shear is likely to increase the length of cylindrical arrays or rods. A shear driven increase in the length of the rods implies a larger radius of this in-plane circle forming the pores, leading to a lower curvature and consequently a lower curvature energy. This increase in average size of the pores under shear favored by the lower curvature energy is expected to increase the in-plane as well as the trans-bilayer correlation length of the defects. The Lα → R3m phase transition is also observed in another system cetylpyridinium chloride (CPCl)-SHN-water. Thus this type of transition is general feature of random mesh phases when sheared near R3m phase in the equilibrium phase diagram. A thixotropic behaviour with yield stress (σy = 500 Pa, is observed when equilibrium R3m phase is sheared. When the shear stress crosses a threshold value of 1000 Pa, we observe an avalanche behaviour with drop in viscosity of more than 4 orders of magnitude. This drop is accompanied by appearance of several sharp peaks which can be indexed to two or three R3m phases. The similar transition is observed under shear in R3m phase formed in CPCl-SHN-water system. We propose that shearing a 3D ordered lattice of defects as in the R3m phase leads to additional structural transitions, though the rhombohedral symmentry is retained. In Chapter 5, we discuss shear thickening observed in colloidal rods. Using rheology combined with microscopy, we demonstrate that origin of shear thickening in colloidal rods is the formation of hydroclusters and not order-disorder transition. We observe continuous (CST) as well as discontinuous shear thickening (DST) at volume fractions of colloidal sus-pension at 25 % and > 30 % respectively. In DST, in controlled stress measurements, flow curve exhibits an S-shaped flow curve (stress vs. shear rate) where we observe a negative slope in shear thickening regime. By combining fast confocal microscopy with rheometer (parallel plate geometry), we investigate the possible mechanism for shear thickening in our suspension and rule out order-disorder transition. This indicates that the shear thickening might be a consequence of formation of hydroclusters which is confirmed by modifying sur-face properties of these colloids where a thermo responsive microgel PNIPAM was used as a shell to the silica core. The advantage of using PNIPAM is that the polymer brush remains fully swollen below the lower critical solution temperature (LCST) and shrinks above the LCST (34 oC) acting as hard particles. Thus by controlling the temperature, the interparticle separation can be tuned. We observe a pure shear thinning and shear thickening behaviour below and above LCST respectively. We show that by changing the interparticle separation we can avoid hydrocluster formation arsing due to the hydrodynamic lubrication forces re-sponsible for the shear thickening. The calculation the order parameter and measurements on core-shell particles illustrate that microscopic origin of shear thickening is the formation of hydroclusters and not order-disorder transition. Chapter 6 deals with the 2D interfacial rheology of antibiotic alamethecin film at air-water interface. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfa-cial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behaviour was investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P>4. Our studies also confirm that the visco-elasticity of alame-thicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interac-tions at the air-water interface.

Présentement, la désorption ionisation laser assistée par la matrice (MALDI) est la méthode d’ionisation préférentielle pour étudier les lipides par l’imagerie par spectrométrie de masse (IMS). Bien qu’il existe les matrices spécifiques aux lipides, tel que la 1,5-DAN pour les phospholipides et la 2,5-DHB pour les triacylglycérols, il est toujours nécessaire d’augmenter la sensibilité de cette technique pour les échantillons atypiques ou certaines classes de lipides. Dans la première étude, nous avons amélioré la sensitivité pour les phospholipides sur les tubes de Malpighi de mouches prélevés par microdissection dans un tampon physiologique à base de sodium et potassium. Un protocole de lavage à deux étapes a était trouvé favorable : un premier rinçage dans le glycérol suivi d’un second rinçage dans l’acétate d’ammonium. Ce protocole permet de réduire au maximum la présence de sels sans délocalisation notoire des phospholipides. La détection et l’imagerie des phospholipides en ionisation négative et positive ont suggéré une distribution uniforme sur toute la longueur des tubes. Ces résultats ont été comparés à ceux obtenus sur des sections tissulaires minces de mouche entière acquis avec les deux polarités. Néanmoins, la structure tridimensionnelle complexe des tubes rénaux suggère que la microdissection est l’approche la plus favorable pour en étudier leur lipidome. Dans la deuxième étude, nous avons déterminé que l’addition de formate d’ammonium (AF) peut améliorer la détection des gangliosides par IMS dans le cerveau. Curieusement, il est nécessaire de rincer l’échantillon dans une solution d’AF avant l’addition de ce même sel suivit d’une conservation de l’échantillon dans un congélateur pendant 24 heures après la déposition de la matrice afin d’obtenir la meilleure augmentation de sensibilité. En moyenne, cette approche a permis d’augmenter l’intensité d’un facteur dix avec trois fois plus d’espèces de gangliosides détectées. De plus, malgré l’étape de lavage, nous n’avons pas observé la délocalisation des gangliosides puisqu’il est toujours possible d’obtenir les résultats d’IMS de qualité avec une résolution spatiale de 20 µm. Finalement, nous avons établi que le nitrate d’argent permet l’analyse des oléfines par IMS, en particulier du cholestérol. En optimisant le protocole de déposition par nébulisation, il est possible de générer une couche mince et homogène de nitrate d’argent ce qui rend la possibilité d’effectuer l’IMS à haute résolution spatiale, jusqu’à 10 µm, sans perte de qualité comparativement aux autres approches publiées. L’ensemble de ce travail démontre l’effet du sel sur la sélectivité et la sensibilité pour cibler les familles de lipides désirées, ce qui nécessite les études ultérieures sur le rôle de ces sels lors du processus de la désorption-ionisation. / Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is currently the ionization method of choice for elucidating the spatial distribution of lipids on thin tissue sections. Despite the discovery of lipid friendly matrices such as 1,5-DAN for phospholipids and 2,5-DHB for triacylglycerols, there is a continued need to improve sensitivity. In the first study, we improved the overall sensitivity for phospholipids of entire fly Malpighian tubules microdissected in PBS with a two-step wash in glycerol followed by ammonium acetate that removed the bulk of the salt with minimal species delocalization and tubule displacement. We were able to detect phospholipids in both positive and negative ion modes and revealed an even distribution of most phospholipids along the length of this organ. We compared the method to the results from whole body fly sections acquired in dual-polarity mode at the same spatial resolution and found it to be more suitable for studying the tubules because of the complex three-dimensional structure of this organ within the fly. In the second study, we observed a marked improvement in ganglioside signals on mouse brain tissue sections with ammonium salt addition. Specifically, when the sample was first desalted in a low concentration ammonium formate solution, spray-coated with the same salt, coated with matrix and finally left in the freezer overnight before data acquisition, we observed an average overall improvement in ganglioside signal intensity by ten-fold and the number of species detected by three-fold. This method also did not affect the spatial distribution of the gangliosides, as high spatial resolution IMS results acquired at 20 µm showed no species delocalization. Finally, we sought to determine if salts could be employed directly as matrices. In this work, we tested silver-based metal salts and discovered that spray depositing silver nitrate alone is a viable method for the IMS detection of olefins, particularly cholesterol. With the optimized dry spray parameter, the overall deposition is homogeneous and composed of microscopic salt crystals that allow for high spatial resolution IMS down to 10 µm while maintaining acceptable overall signal quality comparable to that of previously published protocols. Overall, this thesis demonstrates we can manipulate the local salt distribution to influence the sensitivity and selectivity to target specific lipid subfamilies, opening the door for future research to understanding the role salts play during the laser desorption/ionization process.

Simulation of processes in cellular membranes Abstract Many important processes in cells involve ions, e.g., fusion of synaptic vesi- cles with neuronal cell membranes is controlled by a divalent cation Ca2+ ; and the exchange of Na+ and K+ drives the the fast electrical signal transmis- sion in neurons. We have investigated model phospholipid membranes and their interactions with these biologically relevant ions. Using state-of-the-art molecular dynamics simulations, we accurately quantified their respective affinites towards neutral and negatively charged phospholipid bilayers. In order to achieve that, we developed a new model of phospholipids termed ECC-lipids, which accounts for the electronic polarization via the electronic continuum correction implemented as charge rescaling. Our simulations with this new force field reach for the first time a quantitative agreement with the experimental lipid electrometer concept for POPC as well as for POPS with all the studied cations. We have also examined the effects of transmembrane voltage on phospholipid bilayers. The electric field induced by the voltage exists exclusively in the hydrophobic region of the membrane, where it has an almost constant strength. This field affects the structure of nearby water molecules highlighting its importance in electroporation. 1

The current study investigates the effect of strain state, strain rate, and membrane planar area on phospholipid bilayer mechanoporation and failure. Using molecular dynamics, a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer was deformed biaxially to represent injury-induced neuronal membrane mechanoporation and failure. For all studies, water forming a bridge through both phospholipid bilayer leaflets was used as a failure metric. To examine the effect of strain state, 72 phospholipid structures were subjected to equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial tensile deformations at the von Mises strain rate of 5.45 × 108 s-1. The stress magnitude, failure strain, headgroup clustering, and damage behavior were strain state dependent. The strain state order of detrimentality in descending order was equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial with failure von Mises strains of 0.46, 0.47, 0.53, 0.77, and 1.67, respectively. Additionally, pore nucleation, growth, and failure were used to create a Membrane Failure Limit Diagram (MFLD) to demonstrate safe and unsafe membrane deformation regions. This MFLD allowed representative equations to be derived to predict membrane failure from in-plane strains. To examine the effect of strain rate, the equibiaxial and strip biaxial strain states were repeated at multiple strain rates. Additionally, a 144 phospholipid structure, which was twice the size of the 72 phospholipid structure in the x dimension, was subjected to strip biaxial tensile deformations to examine planar area effect. The applied strain rates, planar area, and cross-sectional area had no effect on the von Mises strains at which pores greater than 0.1 nm2 were detected (0.509 plus/minus 7.8%) or the von Mises strain at failure (0.68 plus/minus 4.8%). Additionally, changes in bilayer planar and cross-sectional areas did not affect the stress response. However, a strain rate increase from 1.4 × 108 to 6.8 × 108 s-1 resulted in a yield stress increase of 44.1 MPa and a yield strain increase of 0.17. Additionally, a stress and mechanoporation behavioral transition was determined to occur at a strain rate of ~1.4 × 108 s-1. These results provide the basis to implement a more accurate mechano-physiological internal state variable continuum model that captures lower-length scale damage.

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