Quitosana é um polissacarídeo usado em diversas aplicações biológicas, por exemplo, em liberação controlada de drogas, transfecção, aceleração da cicatrização de feridas e como agente bactericida, entre outras. Em todas essas aplicações, o polímero interage com tecidos e células. Entretanto, embora sua ação seja comprovada, os mecanismos de ação e a interação do polímero com células e biomembranas no nível molecular ainda não são conhecidos. Nesta tese de doutorado, filmes de Langmuir e Langmuir-Blodgett (LB) de lipídios foram usados como modelos de membrana celular para estudar em nanoescala a interação e os efeitos causados pela quitosana. Primeiramente, observou-se que a quitosana, um polieletrólito solúvel em pH ácidos, possui atividade superficial induzida na presença de um filme interfacial de lipídio, demonstrando que o polímero possui interação favorável com membranas. Após adsorver sobre as monocamadas, a quitosana expande as mesmas, o que ocorre apenas até uma determinada concentração de polímero, denominada concentração de saturação. A magnitude dessa expansão é menor para filmes compactos, o que sugere que a quitosana é parcialmente expulsa da interface, localizando-se na subsuperfície. Isso foi comprovado com o uso de filmes LB, que mostraram que filmes mistos com quitosana têm rugosidade cerca de 10 vezes a de filmes puros de ácido dimiristoil fosfatídico (DMPA). Foi possível confirmar que a quitosana penetra na monocamada, formando agregados com até 150 nm de altura. Além disso, a maior orientação das moléculas de fosfolipídios, sugerida por isotermas de potencial de superfície (V-A) para filmes de Langmuir, também foi comprovada para os filmes LB por medidas de espectroscopia de geração de soma de freqüências (SFG). Filmes mistos de DMPA e colesterol também foram estudados, sendo que o colesterol provoca condensação nos filmes de DMPA a baixas pressões, mas expande as monocamadas em altos estágios de compactação. Quando a quitosana interage com os filmes mistos, ela provoca a mesma expansão para todas as monocamadas independentemente da proporção de colesterol na mistura. Embora esse comportamento possa sugerir um papel inerte do colesterol, ele é explicado pela modulação da penetração da quitosana nos filmes pelo colesterol. Isso ocorre porque há um número fixo de pontos de interações eletrostáticas entre os grupos NH3+ da quitosana e PO2- do DMPA, o que foi comprovado por medidas de espectroscopia de reflexão-absorção na região do infravermelho com modulação da polarização (PM-IRRAS). Com esta técnica para filmes de Langmuir, e espectroscopia SFG para filmes LB, pôde ser traçado um panorama dos efeitos da inserção de colesterol na membrana de DMPA, seguido da interação da quitosana com a membrana mista. A adição do colesterol ao filme de fosfolipídio acarreta em diminuição da ordem das cadeias de DMPA, detectado por variações nas bandas de s(CH2) e ass(PO2-) do fosfolipídio no espectro de PM-IRRAS, e pela razão s(CH3)/s(CH2) nos espectros de SFG. Por outro lado, a interação da quitosana com esse filme misto causa recuperação da orientação das caudas polares do fosfolipídio, verificada pela análise das mesmas bandas de PM-IRRAS e pela razão s(CH3)/s(CH2), que diminui de 6,62 para 4,58 com a adição de colesterol, mas volta a 5,97 após a interação com o polímero. De forma geral, a ação da quitosana sobre biomembranas é governada principalmente por interações eletrostáticas com lipídios carregados negativamente, na superfície externa das mesmas. Dentre os principais efeitos causados pelo polímero, destaca-se a diminuição da elasticidade da membrana e o aumento da orientação das moléculas de lipídio, que podem ter importantes implicações biológicas. A observação de uma concentração de saturação dos efeitos, na maioria dos casos, sugere que a dosagem e a estrutura química da quitosana devem ser bem controladas para alcançar o efeito biológico desejado. / Chitosan is a polyssaccharide with many biological applications, as in drug delivery, transfection, wound healing and as bactericidal agent, for instance. In all these applications the polymer interacts with tissues and cells. The efficacy of chitosan has been proven, but the mechanisms of action and the interactions with cells and biomembranes are still unknown. In this thesis, Langmuir and Langmuir-Blodgett (LB) films made of lipids were employed as cell membrane models, in order to investigate the interactions and modulations caused by chitosan at the molecular level. Firstly, the soluble polyelectrolyte chitosan was found to induce surface activity when a lipid monolayer is at the air/water interface, demonstrating that the interaction of chitosan with membranes is favorable. Upon chitosan adsorption, the monolayers were increasingly expanded with increasing chitosan concentration in the subphase up to a saturation concentration. The extension of this expansion was lower for highly packed films, suggesting that chitosan was partially expelled from the interface after the compression, being located at the sub-monolayer region. This was confirmed by the 10-fold increase in film roughness observed for the areas without aggregates in LB films. Also, we could observe aggregates as high as 150 nm on the film surface, thus confirming chitosan penetration in the dimyristoyl phosphatidic acid (DMPA) monolayer. Mixed DMPA-cholesterol Langmuir monolayers were also produced, with cholesterol inducing condensation of the DMPA films at low pressures, and film expansion at high pressures. Regardless of the cholesterol proportion in the film, chitosan always induced the same degree of expansion on the DMPA mixed monolayers as for a neat DMPA monolayer. Although this behaviour may suggest an inert role for cholesterol, it can only be explained if the sterol is assumed to regulate the extension of chitosan penetration into the monolayer. This occurs because there is a fixed number of sites for electrostatic interactions between NH3+ groups from chitosan and PO2- from DMPA, probed by infrared reflection-absorption spectroscopy (PM-IRRAS) measurements. Indeed, with PM-IRRAS measurements for Langmuir monolayers and sum-frequency generation spectroscopy (SFG) measurements for LB films, we could establish an overview of the effects from cholesterol on DMPA films upon interaction with chitosan. The addition of cholesterol to the DMPA monolayer caused a decrease in the chain order, which was detected by changes in the s(CH2) and ass(PO2-) bands from the phospholipid in the PM-IRRAS spectrum, and by the s(CH3)/s(CH2) intensity ratio in SFG measurements. On the other hand, the interaction of chitosan with these mixed monolayers restored chain order, as observed from the analysis of PM-IRRAS bands and the s(CH3)/s(CH2) in SFG. The latter dropped from 6.62 to 4.58 with cholesterol addition, but further increased to 5.97 with the chitosan interaction. Overall, the chitosan action on biomembranes is mainly governed by electrostatic interactions with negatively charged lipids at the external leaflet of the membrane. The main effects from chitosan to the membrane models are the decrease in membrane elasticity and the increase in molecular ordering, which can lead to important biological implications. Moreover, the existence of the so-called concentration of saturation for most systems suggests that the dosage and chemical structure of chitosan must be well controlled to obtain the desired biological effect.

L’objectif de mon travail de thèse a été de mettre au point une nouvelle technique de nanoscopie de fluorescence par excitation non radiative pour l’étude des interactions membrane/substrat. Cette technique repose sur la modification d’une lamelle de verre par une monocouche de boîtes quantiques (QDs). Les QDs joueront alors le rôle de donneurs lors du transfert d’énergie non radiatif. Afin d’obtenir un transfert d’énergie entre cette surface et une membrane (vésicule géante unilamellaire ou cellule vivante), cette dernière est marquée par un fluorophore amphiphile jouant le rôle d’accepteur. Notre étude s’est principalement portée sur l’étude de l’adhésion des vésicules (système modèle de cellule) sur une surface de QDs recouverte de poly-L-lysine. Une attraction électrostatique forte est alors induite, conduisant à l’adhésion des vésicules sur la surface En ajoutant un sel en solution, nous avons pu contrôler finement la force de l’interaction et donc modifier la distance d’équilibre entre la surface et la membrane. A partir de mesure quantitative du quenching des QDs et de la fluorescence émise par le transfert non radiatif, nous avons pu calculer les distances d’équilibre et obtenir une cartographie de ces distances avec une résolution optique nanométrique. Nous avons également utilisé cette technique pour étudier l’adhésion membranaire des cellules U87MG sur différentes surfaces afin d’observer leur points focaux / The objective of my thesis work was to develop a new technique of Non-radiative Excitation Fluorescence Microscopy to study the interactions membrane/substrate.This technique is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer.To prevent dewetting of the donor film, we have implemented a silanization process to covalently bond the QDs on the substrate. A monolayer of QDs was then deposited on only one side of the coverslips. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed together a quenching of QDs emission and emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs. By changing salt concentration in the solution, we have been able to finely control the force of the interaction and thus modify the equilibrium distance between the surface and the membrane. From quantitative measurements of quenching of QDs and fluorescence emitted by non-radiative transfer, we calculate the equilibrium distances and obtain a mapping of these distances with a nanometric optical resolution. Based on this study, our functionalization technique is also used to observe the adhesion of living cells, U87MG on different surfaces in order to observe their focal points

Indiana University-Purdue University Indianapolis (IUPUI) / Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a <italic>trans</italic> or <italic>cis</italic> double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its <italic>cis</italic> counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.

In order for organisms to co-exist with nonliving matter, envelop to protect their delicate internal functions must be present. There are other reasons for this boundary to exist, e.g. to limit the volume occupied by the organism and to compartmentalize the contents of the organism so that certain critical concentrations may be easily maintained. Such envelopes are, of course, the cellular membranes. Membranes differ greatly between species, as well as within species. Given the complexity to which organisms have evolved, membranes have developed with a myriad of functions and components. It is easy to see differences between plant cellular membranes and animal cell membranes as well as to see differences between mitochondrial and nuclear membranes within a single cell.

No / Review article. No abstract.

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied.

Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied.

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.