Um sistema diferencial foi desenvolvido para resolver as principais dificuldades na medida do aumento da condução protônica na interface filme de Langmuir-água, a saber: a) pequeno aumento da condução devido ao filme comparado com a condução da água; b) diminuição da área imersa dos eletrodos para tensões superficiais diferentes da tensão da água; c) obtenção de uma subfase isenta de impurezas. Com o novo sistema mediu-se a condutância de filmes de Langmuir tradicionais (moléculas anfipáticas com cadeias de hidrocarbonos longas), de filmes mistos de fosfolipídio-fármacos e de um polímero condutor. Realizaram-se, também, medidas de pressão de superfície e potencial de superfície. Os resultados da condutância lateral e do potencial de superfície obtidos com os filmes tradicionais indicam que a área crítica (área em que a condutância do filme aparece e o potencial de superfície aumenta abruptamente) é área em que se forma uma rede de pontes de hidrogênio. Cálculos de área crítica realizados para os ácidos graxos, utilizando um modelo unidimensional para a transferência de prótons, reproduzem os resultados experimentais. Resultados obtidos com o polímero condutor mostram que é possível medir a contribuição eletrônica para a condutância com o novo sistema diferencial. Um estudo de filmes mistos de fosfolípídios-anestésicos locais (tetracaína e dibucaína) foi realizado utilizando a técnica de coespalhamento. Está técnica tem a vantagem, sobre as de adsorção de Langmuir usada tradicionalmente, de se ter certeza de que todas as mudanças nas propriedades do filme sejam causadas pelos compostos adicionados na monocamada. Ambos os anestésicos expandiram a matriz de fosfolípidios. Os resultados mostraram que a tetracaína possui ligações hidrofóbicas mais fortes que a dibucaína, fazendo com que a tetracaína penetrasse mais profundamente na matriz de fosfolipídio / A different system has been developed for overcoming the main difficulties associated with the measurement of an enhanced protonic conductance at the Langmuir film/water interface. These difficulties are: a) small increase in conductance due to the film as compared to the bulk water conductance; b) decrease in the electrode immersed area for surface tensions lower than that of the subphase water; c) obtaining an impurity-free subphase. With the new system the lateral conductance of traditional Langmuir films (possessing amphiphilic molecules with long hydrocarbon tails), of mixed films madeup of phospholipid and pharmaceutical drugs and of a conducting polymer. Measurements were also performed of monolayer surface pressure and surface potential. The conductance and surface potential results obtained with the traditional compounds have indicated that the critical area (at which the conductance appears and the surface potential increase abruptly) is the area for which a H-bonded network is formed. The critical area for fatty acids was calculated , employing a unidimensional proton transfer model, which is consistent with the measured value. Results obtained using the differential system with a conducting polymer also show that it is a possible to detect an electronic contribution to the lateral conductance. A study of mixed films from phospholipid and local anesthetics (tetracaine and dibucaine) was conducted using the co-spreading technique. This method offers the advantage, compared to the traditionally employed Langmuir adsorption method, of making sure that all changes in the film properties are caused by incorporation of the drugs added to the phospholipid monolayer. Both anesthetics caused the phospholipid matrix to expand. The results showed that tetracaine has stronger hydrophobic interaction than dibucaine, and therefore tetracaine penetrates deeper into the phospholipid matrix

As quitosanas são polissacarídeos usados em medicina, farmácia, odontologia e na inibição do crescimento de microrganismos, como agente bactericida. Nessas aplicações sua ação deve depender da interação com membranas celulares, o que é difícil de verificar uma vez que não se isola uma membrana facilmente. Uma alternativa é investigar a interação com modelos de membrana, como um filme de Langmuir de fosfolipídios, a partir do qual é possível obter informações no nível molecular. Nesta dissertação, é avaliada a influência do conteúdo médio de unidades N-acetilglucosamina (GlcNAc) de quitosanas e da massa molecular na interação com filmes de Langmuir do ácido fosfatídico de dipalmitoíla (DMPA). Quitosanas com diferentes graus médios de acetilação e de baixa massa molecular foram produzidas com auxílio de ultrassom de alta intensidade. As quitosanas afetam as isotermas de pressão e potencial de superfície em grandes áreas por molécula, em virtude de interações eletrostáticas e hidrofóbicas com o DMPA. Nos filmes condensados, localizam-se na subsuperfície, com pouco efeito nas isotermas. A quitosana com menos grupos GlcNAc induziu alterações maiores nas isotermas de pressão de superfície e na elasticidade dos filmes, provavelmente devido à maior interação eletrostática com um número maior de grupos amina na quitosana interagindo com as cabeças polares do DMPA. A quitosana com baixa massa molecular foi a mais eficaz para alterar as propriedades dos filmes de DMPA, o que pode ser atribuído à facilidade na adsorção. Um tamanho mínimo de cadeia parece ser essencial, entretanto, pois misturas das unidades repetitivas N-acetilglucosamina (GlcNAc) e glucosamina (GlcN) praticamente não alteraram as isotermas de pressão e a elasticidade dos filmes de DMPA, pela ausência de interações hidrofóbicas. Concluímos que quitosanas com grau de acetilação e massa molecular baixos têm efeitos maiores sobre um modelo de membrana e devem ser mais adequadas em aplicações biológicas que dependam dessa interação. / Chitosans are polysaccharides used in medicine, pharmacy, dentistry and in the inhibition of microorganisms growth (eg. as bactericidal agent). In these applications their action should depend on the interaction with cell membranes, which is difficult to verify because isolating a membrane is not easy. An alternative is to investigate the interaction with membrane models, such as a Langmuir film of phospholipids, from which information on the molecular level can be obtained. This dissertation evaluates the influence of the average content of N-acetylglucosamine units (GlcNAc) of chitosan and molecular interaction with Langmuir films of dipalmitoyl phosphatidic acid (DMPA). Chitosans with different average degrees of acetylation and low molecular weight were produced with the high-intensity ultrasound procedure. Chitosans affect the surface pressure and surface potential isotherms at large areas per molecule due to electrostatic and hydrophobic interactions with DMPA. In condensed films, they are located in the subsurface with little effect on the isotherms. The chitosan with fewer GLcNAc groups induced larger changes in the isotherms and in the film elasticity, probably due to stronger electrostatic interaction owing to a larger number of amine groups in chitosan interacting with the polar heads of DMPA. The most effective sample to induce changes in the DMPA monolayers was the low molecular weight chitosan, which can be attributed to the ease of adsorption. A minimum size chain seems essential, however, for mixtures of repeating units N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) did not change the surface pressure isotherms and the elasticity of the DMPA films, owing to the absence of hydrophobic interactions. We conclude that the chitosan with better prospects for biological applications relying on the cell membrane interaction should have a low degree of acetylation and low molecular weight.

Polyhedral oligomeric silsesquioxanes (POSS) have attracted substantial academic interest for many years as hybrid materials and nanofillers for controlling thermal and mechanical properties, and providing thermal and chemical resistance while retaining ease of processing. A natural extension of these studies has been POSS-based amphiphiles and thin film coatings. Studies at the air/water (A/W) interface have shown that trisilanol-POSS derivatives are amphiphilic and form uniform Langmuir films, whereas closed-cage POSS derivatives are hydrophobic and aggregate. In previous work, a triester (POSS-triester) and a triacid (POSS-triacid) were synthesized from PSS-(3-hydroxypropyl)-heptaisobutyl (POSS-OH) and Weisocyanate and fully characterized by surface pressure – area per molecule (Π-A) isotherm and Brewster angle microscopy (BAM) studies at the A/W interface. The results indicated that POSS-triester is surface active forming a liquid expanded (LE) monolayer, whereas POSS-triacid forms a liquid condensed (LC) monolayer that is only weakly affected by pH. A face-on conformation was proposed and examined to understand the packing of POSS-based amphiphilic molecules at the A/W interface. The face-on/vertex-on comparison is rarely discussed for Langmuir monolayers at the A/W interface. In this thesis, three other POSS-based esters were synthesized from POSS-OH and aminopropylisobutyl-POSS (POSS-NH₂) using Weisocyanate and a similar isocyanate containing two tert-butyl protected carboxylic acids. The synthesized materials are characterized by Π-A isotherm and BAM. For POSS-OH based diester (PAlDE) and POSS-NH2 based diester (PAmDE), LE/LC phase transitions were observed in Π-A isotherms over part of the experimentally accessible temperature range and were attributed to a change from a vertex-on to face-on conformation. Apparent BAM images confirmed LC islands coexisted with the LE phase. The experimentally observed dynamic estimates of the critical temperatures (Tc) were estimated from a two-dimensional Clausius-Clapeyron analysis and were consistent with the temperature dependence of the Π-A isotherms. These LE/LC phase transitions are the first observed for POSS amphiphiles. / Ph. D.

Polyhedral oligomeric silsesquioxanes (POSS) have attracted substantial academic interest for many years as hybrid materials and nanofillers for controlling thermal and mechanical properties, and for providing thermal and chemical resistance while retaining ease of processing. A natural extension of these studies has been working on POSS-based amphiphiles and thin film coatings. Studies at the air/water (A/W) interface have shown that trisilanol-POSS derivatives are amphiphilic and form uniform Langmuir films, whereas closed-cage POSS derivatives are hydrophobic and aggregate. In this study, two novel POSS derivatives were synthesized from PSS-(3-hydroxypropyl)-heptaisobutyl substituted (POSS-OH) and completely characterized. Weisocyanate reacted with POSS-OH, and a POSS-based triester (POSS-triester) was obtained. Trifluoroacidolysis of the POSS-triester at room temperature afforded the corresponding triacid (POSS-triacid). Purified POSS-OH, POSS-triester, and POSS-triacid were studied by using surface pressure - area per molecule (? -A) isotherms as well as Brewster angle microscopy (BAM) at the air/water (A/W) interface. Compared with previous work on trisilanol-POSS derivatives, the results indicated that POSS-triester was surface active and formed a liquid-expanded (LE) monolayer. In contrast, POSS-triacid monolayers were more condensed (LC) and were not dramatically affected by changes in pH. Results for the lift-off areas (Alift-off), limiting areas (A0), collapse areas (Ac), and collapse pressures (? c) of POSS-OH, POSS-triester, and POSS-triacid were compared to trisilanolisobutyl-POSS (TiBP) and were interpreted in terms of possible molecular conformations. Whereas, TiBP has been hypothesized to exist in a vertex-on conformation, POSS-OH and POSS-triacid packing at the A/W interface was consistent with face-on conformations. For POSS-triester, the isotherm was consistent with a vertex-on conformation at low ? , but a face-on conformation at high ? . / Master of Science

Les liquides ioniques et le graphène sont intensivement étudiés, respectivement en tant qu’électrolyte et électrode, pour le développement des supercondensateurs. Dans ce cadre, il est primordial de caractériser l’interface entre les deux espèces. Pour ce faire, nous avons réalisé ce type d’interface par la procédure des films de Langmuir que nous avons observés à différentes échelles via des mesures d’isothermes, de microscopies à angle de Brewster et à force atomique ainsi que par diffusion des rayons X de surface. Nous avons étudié des films formés par des liquides ioniques, de l’oxyde de graphène et enfin d’un mélange de ces deux espèces. L’étude sur les liquides ioniques purs montre que le cation joue un rôle non négligeable sur l’organisation des films à l’interface air-eau, aussi bien en monocouche que lors du passage en phase tridimensionnelle. Par ailleurs, nous avons montré que les films d’oxyde de graphène forment spontanément une bicouche de feuillets à l’interface eau-air même pour de faibles densités superficielles. Enfin concernant les films mixtes, nous avons observé une ségrégation verticale des espèces quand la pression de surface devient suffisamment élevée. Le film est alors composé d’une première couche en contact avec l'eau, majoritairement composée de feuillets d’oxyde de graphène parallèles à l’interface, sur laquelle se superpose une seconde couche formée des domaines de liquide ionique désorganisé. / Graphene and ionic liquids are intensively studied, respectively as electrolyte and as electrode materials, for the development of supercapacitors. In this framework, the characterization between the two species is essential. We realized such kind of interfaces through the Langmuir film procedure and characterized them at different scales, using isotherm measurements, Brewster Angle and Atomic Force Microscopies, and surface X-ray scattering. We studied films formed by different ionic liquids, by graphene oxide and finally by a mixture of the two species. The study on the pure ionic liquids evidences the role of the cation on the film organization at the air-water interface, for the monolayer as well as for the tridimensional phase. Moreover, we showed that the graphene oxide films are composed of a bilayer of sheets à the interface, even at low surface densities. Finally, regarding the mixed film, we observed a vertical segregation of the species for high enough surface pressures. The film is formed by a first layer in contact with the water surface, mostly composed of graphene oxide sheets parallel to the interface, on which a second layer is superimposed, composed of disorganized ionic liquid domains.

Made available in DSpace on 2017-07-20T12:40:14Z (GMT). No. of bitstreams: 1 Bianca Sandrino.pdf: 3429333 bytes, checksum: e320b1605b6c2c81bf2ecd35d8aa88f5 (MD5) Previous issue date: 2014-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-RuCl3(dppb)(VPy)] (dppb = 1,4-bis (diphenylphosphine) butane and VPy = 4-vinylpyridine),RuVPy) was analyzed. Results showed that this compound led to a mortality rate of 50% of human laryngeal carcinoma HEp-2 cell with 120 ±10 mol L-1, indicating its high toxicity. Toward a better understanding if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylserine (DPPS) phospholipids. Results of thermodynamic parameters indicated miscibility between the components is not ideal mixed monolayers. Preferably attractive and repulsive interactions between RuVPy and zwitterionic or anionic phospholipids, respectively, are observed with mixed monolayer of DPPS/RuVPy energetically unfavorable. These data were confirmed polarization - modulated infrared reflection-absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air-water interface. Proof of interaction was confirmed by electrochemical results of Langmuir-Blodgett films of the phospholipid/RuVPy mixture. The presence of the RuVPy on the conductor substrate, which presents higher electron density, form "defects" in the monolayer of phospholipids increasing accumulation of electrons in the electrode/solution interface making it more permeable material. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy. / Um dos grandes desafios na concepção de medicamentos é a identificação de compostos com potencial toxicidade para as células-alvo e a compreensão do seu modo de ação. Nesta tese, foi analisada a propriedade antitumoral do complexo de rutênio mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis (difenilfosfina)butano e VPy = 4-vinilpiridina) (RuVPy), e os resultados mostraram que este composto levou a uma taxa de mortalidade de 50% de células de câncer de laringe (HEp-2) com 120 ± 10 μmol L-1, indicando sua alta toxicidade. Para a compreensão do modo de ação em nível molecular deste complexo, associada à sua interação com membranas celulares, monocamadas de Langmuir foram utilizadas como um modelo simples de membrana. O RuVPy apresentou um forte efeito sobre as isotermas de pressão de superfície, especialmente sobre as propriedades elásticas do zwitteriônico dipalmitoilfosfatidilcolina (DPPC) e dos fosfolipídios carregados negativamente dipalmitoilfosfatidilglicerol (DPPG) e dipalmitoilfosfatidilserina (DPPS). Resultados dos parâmetros termodinâmicos indicaram que há miscibilidade entre os componentes das monocamadas mistas não ideais. Interações preferencialmente atrativas e repulsivas foram constatadas entre o RuVPy e os fosfolipídios zwitteriônico e aniônicos, respectivamente, sendo a monocamada mista de DPPS/RuVPy energeticamente desfavorável. A interação entre o grupo de maior densidade eletrônica do RuVPy, obtido por cálculo de teoria funcional da densidade (DFT), e o grupo fosfato dos fosfolipídios foi confirmada por espectroscopia de infravermelho de reflexão e absorção de modulo polarizado (PM-IRRAS) realizada na interface ar-água. Prova desta interação foi constatada por resultados eletroquímicos dos filmes Langmuir-Blodgett da mistura fosfolipídio/RuVPy. A presença do complexo no substrato condutor, por ter maior densidade eletrônica, forma “defeitos” na monocamada dos fosfolipídios aumentando o acúmulo de elétrons na interface eletrodo/solução tornando o material mais permeável. Desta forma, é evidente que além de eventuais contribuições de outros receptores ou componentes celulares não poderem ser descartadas, os resultados aqui apresentados trazem os efeitos significativos nas membranas celulares que provavelmente estão associados à alta toxicidade do RuVPy.

Pesquisa em novas moléculas e estratégias antimicrobianas é crucial devido ao aumento de resistência a antibióticos pelos microrganismos. Polímeros antimicrobianos tem várias vantagens quando comparados a outros biocidas pequenos: maiores tempo de vida, potência, especificidade e baixa toxicidade residual. Logo, outras aplicações tecnológicas como recobrimentos, embalagens ou produtos têxteis antimicrobianos poderem ser exploradas. Em particular, derivados hidrossolúveis de quitosana, como os oligômeros de quitosana (OQ), são biopolímeros catiônicos extraídos de fontes renováveis que são candidatos promissores a serem agentes antimicrobianos de amplo espectro (fungos, bactérias gram-positivas e bactérias gram-negativas). Diferentemente da quitosana, que é sobretudo bioativa em pHs ácidos, OQ permanece catiônico – e portanto ativo – em pH fisiológico. Não obstante, o mecanismo exato pelo qual o polímero age nas membranas celulares permanece desconhecido em nível molecular. Este trabalho visa investigar o mecanismo de interação entre os OQ e modelos de membrana biomiméticos (Filmes de Langmuir). Para comparação, outro polieletrólito catiônico sintético com propriedades antibacterianas, o PAH – poli(hidrocloreto de alilamina) – foi investigado. Nós realizamos a Espectroscopia por Geração de Soma de Frequência (SFG) em Filmes de Langmuir de fosfolipídeos em água pura e em subfases contendo antimicrobianos. A Espectroscopia SFG nos permite obter o espectro vibracional de moléculas interfaciais (filme lipídico e moléculas que estão interagindo com ele: água e antimicrobianos) sem nenhuma contribuição de moléculas do interior do volume e é muito sensível às conformações lipídicas da membrana. Um fosfolipídeo zwitteriônico (DPPC) foi usado para modelar membranas tipo-humana, enquanto outro carregado negativamente (DPPG) modelava a tipo-bacteriana. Isotermas em subfases contendo antimicrobianos mostraram que ambos PAH e OQ causam uma pequena expansão das monocamadas de DPPC. Entretanto, para as monocamadas de DPPG ambos os polieletrólitos geraram uma expansão significativa. Entre eles, os OQ causaram um efeito mais drástico. Espectros SFG dos estiramentos CH mostraram que a conformação lipídica permaneceu bem empacotada em todos os casos (ligeiramente menos ordenada com PAH) apesar das expansões da membrana. Isto indica que os OQ foram inseridos formaram ilhas de OQ dentro do filme lipídico. Mudanças na forma de linha dos estiramentos da água interfacial indicaram que a adsorção de PAH em ambos os filmes foram capazes de compensar as cargas negativas, gerando uma inversão de cargas na superfície. Os espectros SFG dos grupos fosfato também indicaram que, em água pura, as cabeças polares de DPPC estão com uma orientação mais ordenada do que no caso do DPPG. Contudo, quando interagindo com os polieletrólitos catiônicos, as cabeças dos DPPGs se ordenam, ficando preferencialmente perpendicular à interface. Experimentos com antimicrobianos injetados na subfase enquanto os filmes de Langmuir já estavam condensados indicaram que os OQ foram capazes de penetrar na monocamada, embora causando uma expansão no filme menor. Esta comparação evidencia que a escolha da metodologia experimental afeta o resultado, mas ambas podem ser complementares, visto que podem representar diferentes fases do ciclo celular das biomembranas. A visão detalhada provida aqui para as interações moleculares desses polieletrólitos com filmes lipídicos podem os elucidar mecanismos de atividade biocida deles e auxiliar no planejamento racional de novos polímeros antimicrobianos. / Research on new antimicrobial molecules and strategies is crucial due to the increasing microorganism resistance to antibiotics. Antimicrobial Polymers have many advantages when compared to other small biocides: increased lifetimes, potency, specificity and lower residual toxicity. Therefore, they have great potential for technological applications, such as antimicrobial coatings, packages, or textile products. In particular, water-soluble derivatives of chitosan, such as chitosan oligomers (CO), are cationic biopolymers obtained from renewable sources that are promising candidates to a wide-spectrum antimicrobial agent (fungi, gram positive and gram negative bacteria). Unlike chitosan, which is mainly bioactive at acidic pH, CO remain cationic - and therefore active - at physiological pH. Nevertheless, the exact mechanism by which this polymer acts on the cell membranes remains unknown at the molecular level. This work aims at investigating the molecular interaction between CO and a biomimetic cell membrane model (Langmuir Film). For comparison, another synthetic cationic polylelectrolyte with antibacterial properties, PAH – poly(alylamine hydrochloride), has been investigated. We have carried out Sum-Frequency Generation (SFG) Spectroscopy on Langmuir Films of phospholipids on pure water and on antimicrobial containing subphases. SFG Spectroscopy allows obtaining the vibrational spectrum of interfacial molecules (lipid Langmuir Film and molecules interacting with it – water and antimicrobials), without any contribution from the bulk molecules, and is quite sensitive to the conformation of membrane lipids. A zwitterionic phospholipid (DPPC) was used to model human-like membranes, while a negatively charged phospholipid (DPPG) modeled bacterial-like membranes. Surface pressure-area isotherms on antimicrobial-containing subphases showed that both PAH and CO led to a small expansion of DPPC monolayers. However, for DPPG monolayers both polyelectrolytes led to significant expansion, with CO causing a more dramatic effect. SFG spectra in the CH stretch range showed that the lipid chain conformation remained always well ordered in all cases (slightly less ordered upon interacting with PAH), despite membrane expansion. This indicates that CO were inserted in the monolayer, forming islands of CO within the lipid film. Changes in the SFG spectral lineshape of OH stretches for the interfacial water molecules indicated that PAH adsorption on both DPPC and DPPG films was able to overcompensate the lipid negative charge and led to an overall surface charge reversal. The SFG spectra of the phosphate groups also indicated that in pure water the DPPC headgroups had a more ordered orientation than in the case of DPPG. Nevertheless, upon interaction with the cationic polyelectrolytes, the DPPG headgroups also become ordered, with a preferential orientation towards the subphase. Experiments with the antimicrobials injected in the subphase under a condensed Langmuir film indicated that CO were also capable of monolayer penetration, albeit causing a reduced film expansion. This comparison indicates that the choice of experimental methodology affects the outcome, but both may be complementary, as they may represent different phases of a biomembrane lifecycle. The detailed view provided here for the molecular interaction of these polyelectrolytes with lipid films may shed light on the mechanism of their biocidal activity and aid on a rational design of new antimicrobial polymers.

The Langmuir – Blodgett (LB) technique is about forming insoluble monolayer on the surface of aqueous solution and recently, it has emerged as one of the best method to study floating monolayer at the air – water interface. It has gained popularity after the use of monolayer with chemical complexes as well as biological species, and recently it has been used for the formation of biosensors. Langmuir monolayer arrays the amphiphilic molecules in a fashion where the hydrophobic part points towards the air and the hydrophilic group remains in contact with the aqueous subphase. Due to this property of Langmuir monolayer to orient the molecules at the air – water interface in a particular fashion, it can successfully serve as a template for two – dimensional reactions with restricted freedom. Hence, Langmuir monolayer has been extensively employed to study chemical and biological reactions at the air – water interface. To understand the behavior of Langmuir monolayer, surface pressure – molecular area (P – A) isotherms are studied as these P – A isotherms illustrate general conditions regarding the phase behavior of the two-dimensional Langmuir monolayer. Any change occurring due to the alignment of aliphatic molecules forming the monolayer is reflected by the change in P – A isotherms, which is known as phase transition. The phase transition is the most important element of the P – A isotherms with a characteristic signature of a plateau region in the isotherms. This phase transition point changes with the change of certain external parameters such as temperature, pH, and ionic strength, and as a result gives general information regarding the phase transition behavior. Therefore, with the little change of external parameters, the arrangement of the molecules in the monolayer also changes, which is reflected in the change in the nature of the isotherms. Thus, the system can, in principle, be used to define several physical parameters associated with it. On account of the property of Langmuir monolayer to orient the molecules at the air – water interface with restricted mobility and due to their condensed nature known as solid like phase, it closely mimics the situation inside a biological cell. Hence, we wanted to test whether an artificial nucleus can be generated at LB films. This can be achieved by immobilizing DNA or protein at the air – water interface and then by promoting their biological properties through macromolecular recognition. Here, immobilization of a macromolecule of biological relevance, its interaction with another component of a cell and extracting the thermodynamic parameters utilizing the LB technique will be of significance. This thesis embodies the immobilization of some biologically important proteins then follows their activity as well as DNA recognition properties at the air – water interface. A set of equations are derived here for the two dimensional Langmuir monolayer, which are used to calculate the thermodynamics of the system under study. Chapter 1 outlines the information about Langmuir monolayer and LB films. It sketches the historical background of the Langmuir monolayer and also elucidates the theory behind the same. This chapter cites the technical details of formation of Langmuir monolayer and LB films viś – a – viś other methods available for the fabrication of monomolecular films. It adequately discusses the functional LB films and their utilization for various different purposes. Finally, the role of metal ions in the LB films and in immobilizing biological macromolecules is discussed. Chapter 2 discusses the different techniques employed to perform the experiments described in this thesis. It includes the purification methods for the different proteins and DNA; the details of formation of Langmuir monolayer and fabrication of LB films. This chapter also describes the various techniques used for the characterization of the LB films, i.e Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) spectroscopy. In Chapter 3, immobilization and imaging of protein molecules and protein DNA complexes on a LB substrate have been explored. Firstly, we describe the preparation of a Ni (II) – arachidate (NiA) monolayer and its characterization through P – A isotherm on a LB trough. Then, recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the NiA monolayer through a Ni (II) – histidine interaction. A single molecule of RNA polymerase (RNAP) could be seen through intermittent-contact AFM. Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. In Chapter 4, the interaction between NiA and histidine tagged RNAP (HisRNAP), and RNAP and DNA were studied. LB films of Arachidic acid – NiA, NiA -HisRNAP and NiA – HisRNAP – DNA with different mole fractions were fabricated systematically. P -A isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for FTIR spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the AA monolayer with the change in pH. The increase in mole fraction of RNAP and DNA in the NiA and NiA – RNAP monolayer, respectively, with their increasing concentration in the subphase are also noticed. The system developed here is robust and can be utilized to follow macromolecular interactions. In chapter 5, the Langmuir monolayer has been utilized to array a protein, Dps, specific for Fe (II) and non-specific for DNA. Dps from Mycobacterium smegmatis is known to have a cage like structure, exists in two oligomeric states, trimer and dodecamer, and can accommodate Fe (II) ions in its internal cavity. In addition, it converts Fe (II) to Fe (III), both in trimeric and dodecameric form, whereas the latter species is specific for non-specific DNA binding. We demonstrate here that, histidine tagged Dps in both oligomeric states can be immobilized on NiA LB films, where both ferroxidation and DNA binding ability remained unaffected in the ordered protein assembly. Interestingly, when Fe (II) – arachidate was used to generate a LB layer instead of NiA, Dps protein not only recognizes Fe (II) ion in the monolayer, it also converts it to Fe (III) ion in a time dependent fashion. However, once Fe (III) – Dps complex is formed and arrayed on LB monolayers, it remains very stable.

Pesquisa em novas moléculas e estratégias antimicrobianas é crucial devido ao aumento de resistência a antibióticos pelos microrganismos. Polímeros antimicrobianos tem várias vantagens quando comparados a outros biocidas pequenos: maiores tempo de vida, potência, especificidade e baixa toxicidade residual. Logo, outras aplicações tecnológicas como recobrimentos, embalagens ou produtos têxteis antimicrobianos poderem ser exploradas. Em particular, derivados hidrossolúveis de quitosana, como os oligômeros de quitosana (OQ), são biopolímeros catiônicos extraídos de fontes renováveis que são candidatos promissores a serem agentes antimicrobianos de amplo espectro (fungos, bactérias gram-positivas e bactérias gram-negativas). Diferentemente da quitosana, que é sobretudo bioativa em pHs ácidos, OQ permanece catiônico – e portanto ativo – em pH fisiológico. Não obstante, o mecanismo exato pelo qual o polímero age nas membranas celulares permanece desconhecido em nível molecular. Este trabalho visa investigar o mecanismo de interação entre os OQ e modelos de membrana biomiméticos (Filmes de Langmuir). Para comparação, outro polieletrólito catiônico sintético com propriedades antibacterianas, o PAH – poli(hidrocloreto de alilamina) – foi investigado. Nós realizamos a Espectroscopia por Geração de Soma de Frequência (SFG) em Filmes de Langmuir de fosfolipídeos em água pura e em subfases contendo antimicrobianos. A Espectroscopia SFG nos permite obter o espectro vibracional de moléculas interfaciais (filme lipídico e moléculas que estão interagindo com ele: água e antimicrobianos) sem nenhuma contribuição de moléculas do interior do volume e é muito sensível às conformações lipídicas da membrana. Um fosfolipídeo zwitteriônico (DPPC) foi usado para modelar membranas tipo-humana, enquanto outro carregado negativamente (DPPG) modelava a tipo-bacteriana. Isotermas em subfases contendo antimicrobianos mostraram que ambos PAH e OQ causam uma pequena expansão das monocamadas de DPPC. Entretanto, para as monocamadas de DPPG ambos os polieletrólitos geraram uma expansão significativa. Entre eles, os OQ causaram um efeito mais drástico. Espectros SFG dos estiramentos CH mostraram que a conformação lipídica permaneceu bem empacotada em todos os casos (ligeiramente menos ordenada com PAH) apesar das expansões da membrana. Isto indica que os OQ foram inseridos formaram ilhas de OQ dentro do filme lipídico. Mudanças na forma de linha dos estiramentos da água interfacial indicaram que a adsorção de PAH em ambos os filmes foram capazes de compensar as cargas negativas, gerando uma inversão de cargas na superfície. Os espectros SFG dos grupos fosfato também indicaram que, em água pura, as cabeças polares de DPPC estão com uma orientação mais ordenada do que no caso do DPPG. Contudo, quando interagindo com os polieletrólitos catiônicos, as cabeças dos DPPGs se ordenam, ficando preferencialmente perpendicular à interface. Experimentos com antimicrobianos injetados na subfase enquanto os filmes de Langmuir já estavam condensados indicaram que os OQ foram capazes de penetrar na monocamada, embora causando uma expansão no filme menor. Esta comparação evidencia que a escolha da metodologia experimental afeta o resultado, mas ambas podem ser complementares, visto que podem representar diferentes fases do ciclo celular das biomembranas. A visão detalhada provida aqui para as interações moleculares desses polieletrólitos com filmes lipídicos podem os elucidar mecanismos de atividade biocida deles e auxiliar no planejamento racional de novos polímeros antimicrobianos. / Research on new antimicrobial molecules and strategies is crucial due to the increasing microorganism resistance to antibiotics. Antimicrobial Polymers have many advantages when compared to other small biocides: increased lifetimes, potency, specificity and lower residual toxicity. Therefore, they have great potential for technological applications, such as antimicrobial coatings, packages, or textile products. In particular, water-soluble derivatives of chitosan, such as chitosan oligomers (CO), are cationic biopolymers obtained from renewable sources that are promising candidates to a wide-spectrum antimicrobial agent (fungi, gram positive and gram negative bacteria). Unlike chitosan, which is mainly bioactive at acidic pH, CO remain cationic - and therefore active - at physiological pH. Nevertheless, the exact mechanism by which this polymer acts on the cell membranes remains unknown at the molecular level. This work aims at investigating the molecular interaction between CO and a biomimetic cell membrane model (Langmuir Film). For comparison, another synthetic cationic polylelectrolyte with antibacterial properties, PAH – poly(alylamine hydrochloride), has been investigated. We have carried out Sum-Frequency Generation (SFG) Spectroscopy on Langmuir Films of phospholipids on pure water and on antimicrobial containing subphases. SFG Spectroscopy allows obtaining the vibrational spectrum of interfacial molecules (lipid Langmuir Film and molecules interacting with it – water and antimicrobials), without any contribution from the bulk molecules, and is quite sensitive to the conformation of membrane lipids. A zwitterionic phospholipid (DPPC) was used to model human-like membranes, while a negatively charged phospholipid (DPPG) modeled bacterial-like membranes. Surface pressure-area isotherms on antimicrobial-containing subphases showed that both PAH and CO led to a small expansion of DPPC monolayers. However, for DPPG monolayers both polyelectrolytes led to significant expansion, with CO causing a more dramatic effect. SFG spectra in the CH stretch range showed that the lipid chain conformation remained always well ordered in all cases (slightly less ordered upon interacting with PAH), despite membrane expansion. This indicates that CO were inserted in the monolayer, forming islands of CO within the lipid film. Changes in the SFG spectral lineshape of OH stretches for the interfacial water molecules indicated that PAH adsorption on both DPPC and DPPG films was able to overcompensate the lipid negative charge and led to an overall surface charge reversal. The SFG spectra of the phosphate groups also indicated that in pure water the DPPC headgroups had a more ordered orientation than in the case of DPPG. Nevertheless, upon interaction with the cationic polyelectrolytes, the DPPG headgroups also become ordered, with a preferential orientation towards the subphase. Experiments with the antimicrobials injected in the subphase under a condensed Langmuir film indicated that CO were also capable of monolayer penetration, albeit causing a reduced film expansion. This comparison indicates that the choice of experimental methodology affects the outcome, but both may be complementary, as they may represent different phases of a biomembrane lifecycle. The detailed view provided here for the molecular interaction of these polyelectrolytes with lipid films may shed light on the mechanism of their biocidal activity and aid on a rational design of new antimicrobial polymers.

Nous avons étudié les films de Langmuir de copolymères PS-b-PAA et la formation de nanostructures inorganiques par réduction d’ions Ag+ à leur voisinage. D’une part, nous avons déterminé précisément la structure de ces copolymères à l’interface air-eau pour différents pH, à l’aide de techniques complémentaires (isothermes π-A, réflectivité de neutrons, diffusion de rayons x rasants). Cette étude a démontré que l’interprétation usuelle de ces isothermes devait être reconsidérée et en particulier nous avons mis en évidence le rôle majeur du solvant d’étalement. Ce dernier reste partiellement adsorbé à la surface après le dépôt, la monocouche mixte copolymère-solvant est alors formée de nanostructures de surface. Ces nanostructures ont de taille caractéristique bien. Lorsque les chaînes de PAA sont neutres, le pseudo-plateau observé sur les isothermes correspond à l’expulsion réversible du solvant vers la sous-phase ce qui induit une réorganisation des nanostructures. Dans le cas de chaînes de PAA chargées, l’isotherme ne présente plus ce pseudo-plateau. Ceci est dû aux répulsions électrostatiques entre ces chaînes. La compression du film entraîne le «plongeon» d’une partie de la monocouche, copolymère et solvant, vers le volume. D’autre part, nous avons étudié le comportement des films de copolymères chargés en présence d’ions Ag+ dans la sous-phase avant et après leur réduction. Nous avons montré que la présence d’ions Ag+ induit le collapse des chaînes PAA à l’interface. La réduction des ions par la radiolyse de surface via les rayons x, produit des colloïdes d’argent agrégés selon une structure 2D «bicontinue» de taille caractéristique marquée. En revanche, la réduction par photochimie ne semble pas produire de couche homogène et dense de colloïdes. / We studied the copolymer PS-b-PAA Langmuir films and the formation of non-organic nanostructures by reduction of silver ions Ag+ in their neighborhood. On the one hand, we have precisely determined the structure of these copolymers at the air-water interface for different pH with many complementary techniques (isothermal, ?-A, neutron reflectivity, grazing-incidence x-ray scattering). This study proved that the classic interpretation of these isotherms should be reconsidered and we emphasized the major role played by the spreading solvent. This (solvent) is partially absorbed by the surface after the deposit and the mixed copolymer-solvent monolayer is created by the surface nanostructure. These nanostructures have a well-defined typical size. When the PAA chains are neutral, the pseudo-surface observed on the isotherms correspond to the reversible expulsion of the solvent to a subphase which induces a reorganization of the nanostructures. In the case of charged PPA chains, the isotherm does not show the flat surface. This is due to the electrostatic repulsion between these chains. The compression of the film induces the ?dive? of a part of the monolayer ? copolymer and solvent ? towards the volume. On the other hand, we studied the behavior of these charged copolymer films in the presence of silver ions Ag+ in the before and after reduction. We showed that the presence of ions Ag+ ions induces the collapse of the PAA chains at the interface. The ions reduction by surface x-rays radiolysis produces silver colloids following a ?bi-continuous? 2D structure with a typical length scale. However, the photo-chemical reduction does not seem to produce homogenous and dense layers of colloids.