This Thesis presents studies of molecular association in aqueous solution and at the liquid/solid interface. The investigated molecular systems range from self-aggregating surfactants to hydration water in contact with micelles or individual molecules. In most studies, combinations of various NMR methods were applied. These vary from simple chemical shift and intensity measurements to more elaborate self-diffusion and intermolecular cross-relaxation experiments. Non-ideal mixed micelles of fluorinated and hydrogenated surfactants were studied by an experimental procedure that allows an analysis in terms of micellar structure, using a minimal number of initial assumptions. Quantitative conclusions about micro-phase separation within mixed micelles were obtained within the framework of the regular solution theory. Additionally, NMR was introduced and developed as a powerful method for studying adsorption of surfactants at solid interfaces. Adsorption isotherms for pure and mixed surfactant systems and non-ideal mixing behavior of fluorinated and hydrogenated surfactants at solid surfaces were quantified. Fluorosurfactant-protein association was investigated using the methods described. Intermolecular cross-relaxation rates between solute and solvent molecules were recorded at several different magnetic fields. The results reveal strong frequency dependence for both small molecules and micelles. This finding demonstrates that intermolecular cross-relaxation is not solely controlled by fast local motions, but also by long-range translational dynamics. Data analysis in terms of recently developed relaxation models provides information about the hydrophobic hydration and micellar structure. / QC 20100914

Líquidos iônicos (LIs) são sais que se encontram no estado líquido em temperaturas menores que 100ºC e que vêm ganhando protagonismo na área chamada química verde, prometendo: substituir solventes nocivos ao meio ambiente, aprimorar componentes eletrônicos, favorecer biocatálises dentre outros. Sua alta estabilidade e baixa toxicidade são frequentemente afirmadas, porém, devem ainda ser melhor investigadas. Com o objetivo de implementar o entendimento da interação dos líquidos iônicos com sistemas de relevância biológica, realizamos um estudo sistemático acerca da interação de 3 diferentes líquidos iônicos anfifílicos de mesma cabeça polar e diferentes caudas carbônicas ([C10mim][Cl], [C12mim][Cl] e [C14mim][Cl]) com 3 diferentes proteínas modelo, através das técnicas de absorção óptica, fluorescência, dicroísmo circular (CD) e espalhamento de raios-X a baixos ângulos (SAXS). Para Tanto, utilizamos as proteínas BSA e HSA (Albuminas de Soro Bovino e Humano, respectivamente) além da lisozima. Observamos a supressão da fluorescência das proteínas em todos os casos analisados, onde a diminuição da intensidade correspondeu a, para as proteínas BSA, HSA e lisozima, respectivamente, (55±3)%, (16.1±0.8)% e (4.1±0.2)%, em presença de 0.6mM de [C14mim][Cl], (38±2)%, (13.2±0.7)% e (0.6±0.1)% em presença de 0.6mM de [C12mim][Cl] e (11.0±0.5)%, (9.2±0.5)% e (0.0±0.1)% em presença de 0.6mM de [C10mim][Cl]. Os espectros de absorbância e fluorescência de todos os sistemas nos indicam uma interação de contato entre as proteínas e os líquidos iônicos. Constatamos também o deslocamento do pico de fluorescência, das proteínas BSA e HSA, para menores comprimentos de onda (blue-shift), na medida em que a concentração de LI era aumentada. O máximo deslocamento () alcançado correspondeu a (21±1)nm para ambas albuminas, enquanto que a lisozima não apresentou deslocamento significativo. O blue-shift pode ser explicado pela aproximação das cadeias carbônicas e formações de pontes de hidrogênio nas proximidades dos triptofanos. De acordo com a técnica de SAXS, evidenciamos o aumento do raio de giro das proteínas, na medida em que adicionamos LIs. O raio de giro da BSA, da HSA e lisozima em ausência de LI são (29±1)Å, (30±1)Å e (15±1)Å, respectivamente, e passam para (46±1)Å, (44±1)Å e (20±1)Å respectivamente, em presença de 0.6mM de [C14mim][Cl]. As curvas de SAXS também apresentaram o indício da formação de estruturas micelares a partir de uma dada concentração. Além da alteração em sua estrutura terciária, os dados de CD indicam uma leve perda de estrutura secundária de ambas as albuminas (BSA e HSA), passando de 80 para 65% de -hélice em ausência e presença de 0.6mM de [C14mim][Cl], respectivamente. Sugerimos que as interações das proteínas com os líquidos iônicos, embora inicialmente movidas por forças eletroestática, possuem como principal fator o efeito hidrofóbico, portanto quanto maior a cadeia carbônica do LI maior é sua interação com a proteína. Tal interação causa o desenovelamento das proteínas e formação de um complexo e estruturas micelares a altas concentrações de LI. Acreditamos que este trabalho traz novas informações acerca da interação dos LIs com proteínas modelo, indicando sua capacidade de alterar a conformação das mesmas. / Ionic liquids (ILs) are salts that are liquid at temperatures smaller than 100 ° C and are gaining prominence in the so-called green chemistry, promising: replace harmful solvents to the environment, improve electronic components, and favor biocatalysis, among others. Its high stability and low toxicity are often asserted; nevertheless, they are ascribed to ILs due to its small volatility. With the aim of improving the understanding of the interaction of ILs with biological relevant systems, we conducted a systematic study of the interaction of three different ionic liquids of the same polar head and different paraffinic tails ([C10mim][Cl], [C12mim][Cl] and [C14mim][Cl]) with three different model proteins, through the techniques of optical absorption, fluorescence, circular dicrhoism (CD) and small angle X-ray scattering (SAXS). To do so, we use BSA and HSA proteins (Bovine Serum Albumin and the Human Serum Albumin, respectively) and lysozyme. We observed fluorescence quenching, of all studied proteins, where the decrease in the fluorescence was (for BSA, HAS and lysozyme, respectively): (55 ± 3)%, (16.1 ± 0.8)% to (4.1 ± 0.2 )% in the presence of 0.6mm [C14mim][Cl], (38 ± 2)%, (13.2 ± 0.7)% to (0.6 ± 0.1)% in the presence of 0.6mm [C12mim][Cl] and ( 11.0 ± 0.5)% (9.2 ± 0.5)% and (0.0 ± 0.1)% in the presence of 0.6mm [C10mim][Cl]. UV-vis absorbance spectra and fluorescence indicate all systems in a contact interaction between proteins and ionic liquids. We also note the shift of the fluorescent peak of BSA and HSA proteins for shorter wavelengths (blue-shift), as the IL content was increased. The maximum shift () achieved corresponded to (21 ± 1) nm for both albumins, whereas no significant displacement was observed for lysozyme. The blue-shift can be explained by the approach of carbon chains and formation of hydrogen bonds in the vicinity of tryptophan. SAXS data indicate an increasing in the proteins radius of gyration value as ILs was added in the solution. The turning radius of BSA, HSA and lysozyme in the absence of IL are (29 ± 1) Å, (30 ± 1) Å and (15 ± 1) Å, respectively, and go to (46 ± 1) Å, ( 44 ± 1) Å and (20 ± 1) Å, respectively, in the presence of 0.6mm [C14mim][Cl]. The SAXS curves also show evidence of the formation of micellar structures from a given concentration. Besides the change in its tertiary structure, the CD data indicates a slight loss of secondary structure of both albumins (BSA and HSA), from 80 to 65% of -helix in the absence and presence of 0.6mm [C14mim][Cl], respectively. We suggest that the interactions of the protein with the ionic liquid, although initially driven by electrostatic forces, have a major factor hydrophobic effect and thus the higher the carbon chain of greater IL is its interaction with the protein. This interaction causes unfolding of the protein and formation of a micellar structures at high concentrations of IL. We believe this work provides new information about the interaction of ILs with model proteins, indicating its ability to alter the conformation of the same.

Abstract Pulmonary surfactant is a lipid-protein mixture that lines the inner surface of the lung. The main function of surfactant is to reduce surface tension at the air-liquid interface, thus preventing alveolar collapse at the end of expiration. Lack of surfactant is the main cause of respiratory distress syndrome (RDS) in preterm infants. Very preterm babies are at risk of developing a lung disease called bronchopulmonary dysplasia (BPD). The surfactant proteins SP-A, -B, -C and -D have important functions in surfactant structure, homeostasis and innate immunity of the lung. The genes of these proteins have been studied as candidates for several multifactorial lung diseases both in adults and in children. The aim of the present study was to examine the genetic variation in SP genes and to evaluate the role of SP gene polymorphism in the etiology of severe pulmonary infantile diseases, including RDS, BPD and severe respiratory syncytial virus (RSV) infection among the Finnish population. Conventional allelic association methods in combination with multiparameter analysis and family-based transmission disequilibrium test (TDT) were used. The SP-D Met11 allele was associated with a risk for severe RSV bronchiolitis in a matched case-control setting of 84 infants with severe RSV infection and 93 control infants. The variants of the SP-C gene had no detectable association with BPD. However, a modest association of SP-C Asn138 and Asn186 alleles with RDS was found. A length variation in the SP-B gene was associated with BPD among very preterm infants born before 32 weeks of gestation. The SP-B intron 4 deletion variant allele increased the risk for BPD especially in very low birth weight infants. The association was confounded by birth order, being evident only among presenting infants, who are more prone to ascending infections during a preterm birth process. The present study provides new evidence about the significance of SP gene polymorphisms in the etiology of complex infantile pulmonary diseases, including RDS, BPD and severe RSV bronchiolitis. The results help us to understand the molecular mechanisms underlying these diseases and may, in the long run, enable better treatment of these life-threatening diseases. / Tiivistelmä Keuhkosurfaktantti on keuhkon sisäpintaa peittävä kalvomainen rasva-proteiinikompleksi, jonka tärkein ominaisuus on pintajännityksen vähentäminen keuhkorakkuloissa. Surfaktantin puutos ennenaikaisesti syntyneillä lapsilla aiheuttaa hengitysvaikeusoireyhtymän, RDS-taudin (respiratory distress syndrome). Alle 30 raskausviikon iässä syntyneistä, useimmiten RDS-taudin saaneista keskosista n. 30 % sairastuu vakavaan krooniseen keuhkotautiin, BPD-tautiin (bronchopulmonary dysplasia). Surfaktanttiproteiineilla SP-A, -B, -C ja -D on osoitettu olevan tärkeä tehtävä surfaktantin toiminnassa ja keuhkon synnynnäisessä immuniteetissa. Tämän tutkimuksen tavoitteena oli selvittää surfaktanttiproteiineissa esiintyvän geneettisen muuntelun määrää ja merkitystä keskosten RDS- ja BPD-taudeissa sekä pienten lasten vakavassa respiratory syncytial -viruksen (RSV) aiheuttamassa keuhkotulehduksessa. Tutkimuksen laajin osa keskittyi tutkimaan keskosten BPD-tautia ja surfaktanttiproteiinien geenien osuutta siinä. Geneettisen muuntelun merkitystä tarkasteltiin populaatiogeneettisin keinoin tapaus-verrokkiasetelmissa ja perheaineistojen avulla. Yhteensä analysoitiin noin tuhannen lapsen ja yli kahdensadan vanhemman DNA-näytteet. Tutkimuksessa havaittiin SP-D-geenissä olevan metioniini11-geenimuodon liittyvän pienten lasten vakavaan RSV-infektioon. Lisäksi saatiin uutta tietoa SP-C-geenin populaatiotason yleisestä muuntelusta ja todettiin SP-C:n asparagiini138 ja asparagiini186 -geenimuotojen yhteys keskosten RDS-taudin esiintymiseen. Merkittävin löydös oli SP-B-geenissä olevan deleetiovariantin kytkeytyminen alle 32-viikkoisina syntyneiden keskosten BPD-tautiin. Geneettisen altistuksen lisäksi BPD-tautiin sairastumiseen vaikuttivat lukuisat keskosuudelle ominaiset seikat, kuten alhainen syntymäpaino, RDS-tauti ja syntymähetkellä todettu hapenpuute. Geneettisen tekijän vaikutus oli voimakkain erittäin pienipainoisilla keskosilla. Tutkimuksen tulokset ovat tuoneet arvokasta lisätietoa surfaktanttiproteiinien geenien osuudesta keskosten RDS- ja BPD-taudeissa sekä pienten lasten vakavassa RSV-infektiossa. Ne auttavat ymmärtämään näiden molekyylibiologisia syntymekanismeja ja voivat ajan mittaan olla edistämässä uusien hoitomuotojen kehittämistä.

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