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The occurrence of thrombosis in several pathophysiological conditions creates a huge need for anticoagulation therapy. Thrombin and factor Xa have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. This work investigates chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids (DHPs) as potential anticoagulants. Oligomers were prepared through peroxidase-catalyzed oxidative coupling of 4-hydroxycinnamic acids. The products resulting from this reaction are called CDs, FDs and SDs. Structurally, these sulfated DHPs are unique and do not resemble any of the anticoagulants known in the literature.DHP oligomers were found to increase clotting times at concentrations comparable to heparin. Studies in blood and plasma show that DHPs possess an anticoagulation profile similar to enoxaparin. To understand the mechanism of action of DHPs, we studied the inhibition of thrombin, FXa, FIXa, and FVIIa in the presence and absence of antithrombin. CDs and FDs display a preference for direct inhibition of thrombin and FXa, and exhibit a high level of specificity over FIXa and FVIIa. In the presence of AT, CDs and FDs displayed weaker inhibition of FXa and thrombin suggesting that binding to AT is a competitive side reaction. SDs exhibited potent inhibition of FXa and thrombin in the absence of antithrombin, but was inactive against FIXa and FVIIa representing the best selectivity among the DHPs. For SDs, inhibition of all the pro-coagulant enzymes favored the antithrombin dependent pathway. Binding studies were performed to determine how CDs directly inhibits thrombin. Competitive binding studies suggest that CDs interacts with exosite II and disrupts the catalytic triad of thrombin. These results indicate that the preferred mechanism of CDs action is exosite II mediated allosteric disruption of thrombin. CDs appears to be the first exosite II mediated DTI and this represents a novel mechanism of inhibitor function. The inhibition characteristics of DHPs are unique and radically different in structure from all the current clinically used anticoagulants. To the best of our knowledge this dual mechanism of anticoagulation and unique binding mode has not been described as yet in literature and represents a novel strategy that our laboratory has discovered.

Filmes de Langmuir e Langmuir-Blodgett (LB) de ligninas extraídas do bagaço de cana-de-açúcar, via processo organossolve, foram fabricados. As isotermas de pressão revelaram uma área para a seção média da lignina em torno de 100 &#197 para a monocamada condensada, enquanto a modelagem a partir dos espectros elipsométricos indicou uma espessura por camada de cerca 60 &#197 para os filmes LB. Porém a deposição não se mostrou homogênea sobre toda a superfície do substrato como observado pelas medidas de microscopia de força atômica (AFM), de forma que filmes mais homogêneos foram obtidos a partir de uma solução mista de Iignina com estearato de cádmio. A área por molécula, calculada com base no número de moléculas de ácido esteárico sobre a subfase, aumenta com a quantidade de lignina na solução mista, indicando a presença de ambos os componentes também na monocamada. Os espectros de infravermelho com transformada de Fourier (FTIR) mostraram que tanto a lignina quanto o estearato de cádmio são transferidos e os espectros de ultravioleta visível (UV-vis) indicaram que esta deposição é homogênea até a 20&#170 camada. Os difratogramas de raios-X e as medidas de AFM mostram que a deposição de lignina e estearato de cádmio ocorre em domínios separados. As medidas de AFM revelaram ainda um aumento da rugosidade do filme com o aumento do número de camadas depositadas e com a maior irregularidade do substrato utilizado. Vale destacar que os filmes de lignina pura mostraram-se mais homogêneos que os filmes mistos, apesar da maior estabilidade destes sobre a subfase. A uniformidade macroscópica dos filmes LB foram confirmadas por medidas de potencial de superfície. Um estudo comparativo de filmes de Langmuir de ligninas extraídas por diferentes processos e de diferentes fontes (madeira mole, madeira dura e cana-de- açúcar) foi realizado. Destacou-se a importância dos grupos funcionais nas medidas de pressão e potencial de superfície, sendo que a área molecular média mostrou-se aumentar com a massa molar das ligninas em questão. A observação de uma inversão nos valores do potencial de superfície para filmes LB de lignina de cana e de pinus abriu caminho para uma investigação mais detalhada dos fatores que contribuem para o potencial de superfície dos filmes LB. Além da Iignina, filmes LB de polianilina e estearato de cádmio também foram estudados. Os resultados revelaram que os valores do potencial de superfície para estes filmes podem ser melhor compreendidos levando-se em consideração modelos teóricos que tratam tanto da contribuição dipolar das moléculas que constituem o filme como da contribuição da interface filme/substrato, a qual geralmente é negativa e se dá pela injeção de cargas. No caso dos filmes LB de estearato de cádmio o potencial é positivo para um número ímpar de camadas e negativo para um número par / Langmuir monolayers and Langmuir-Blodgett (LB) films were produced from lignins extracted from sugar cane bagasse, via the organosolv process The surface pressure isotherms revealed an area per molecule of 100 &#197 for a condensed monolayer, and the modelling of ellipsometric data led to a thickness of 60 &#197 per layer for the LB films. The latter were not homogeneous, as demonstrated by atomic force microscopy (AFM) studies, and therefore mixed monolayers of Iignin and cadmium stearate were transferred with the aim of achieving more homogeneous films. The area per molecule, based on the number of stearic acid molecules on the airlwater interface, increased with the lignin contents in the mixed monolayers, thus confirming the presence of both components in the Langmuir monolayer. Fourier transform infra-red (FTIR) spectroscopy of LB films indicated that cadmium stearate as well as lignin were transferred onto the substrate. An equal amount of material was transferred in each deposition step, as demonstrated by the linear increase in UV-vis absorbance with the number of deposited layers up to the 20&#170 layer. Lignin and cadmium stearate are transferred in separate domains, which was indicated in X-ray diffraction measurements and AFM images. The latter also reveal that the roughness increases with the number of layers and may depend upon the substrate. Interestingly, films of pure lignin were more homogeneous than mixed films with cadmium searate, in spite of the higher stability and transterability of the mixed monolayers. Surface potential measurements showed a unitorm profile when the probe was scanned across an LB film, which may be taken as demonstration of uniformity at least at the macroscopic levei, though the film comprises domains and is usually rough for a molecular film. A comparative study was made ot monolayer characteristics for lignins obtained trom different sources and using different extracting procedures. The importance of functional groups was highlighted in these measurements, in which the area per molecule increased with the molecular weight ot the material. The observation that LB films from pinus and sugar cane bagasse exhibit surface potentials ot inverted sign has prompted us to investigate the possible contributions to such surface potentials. In addition to Iignins, in this study cadmium stearate and polyaniline LB films were also used. The overall conclusion was that the surface potential depends on the dipole moment of the monolayer-torming molecules, but is also affected substantially by the contribution from the substrate/film interface. The latter arises from charge injection from the electrode and is generally negative. For simple cadmium stearate LB films, the surface potential is positive for an odd number of layers, but negative for even numbers, since the dipole contribution is cancelled out

Filmes de Langmuir e Langmuir-Blodgett (LB) foram fabricados a partir de ligninas de bagaço de cana-de-açúcar e da Pinus caribaea hondurensis, extraídas via processo organossolve. Obteve-se nove tipos de lignina de pinus, sendo que cada uma foi extraída com um sol vente diferente, e seis tipos de lignina de cana, as quais passaram por um fracionamento, diminuindo sua polidispersividade. Os filmes de Langmuir foram fabricados sobre subfases de água ultrapura e caracterizados por medidas de pressão e potencial de superfície. Em todos os casos constatou-se a formação de agregados não monomoleculares quando o filme é comprimido além do ponto de colapso, o que é notado pela grande histerese na isoterma pressãoárea. Para ligninas de baixa massa molecular, estruturas estáveis não monomoleculares são formadas antes do colapso. No caso das ligninas menos polidispersas (cana), a massa molecular aumenta linearmente com a área molecular média da lignina. Monocamadas das ligninas de pinus e cana puderam ser transferidas para substratos de vidro, constituindo-se nos primeiros filmes de Langmuir-Blodgett destes materiais. Através do monitoramento da velocidade de imersão e retirada do substrato da subfase pode-se fabricar diferentes tipos de filmes LB, os quais podem ser do tipo Y com a deposição ocorrendo na imersão e retirada do substrato e também dos tipos X e Z se a deposição ocorre preferencialmente na imersão ou retirada do substrato, respectivamente. Os filmes LB foram caracterizados por medidas do potencial de superfície e elipsometria. O potencial de superfície para os filmes de pinus são positivos, enquanto que para os filmes de cana são negativos. Esta inversão de sinal foi surpreendente, pois para as monocamadas os valores de potencial são sempre positivos, tanto para as ligninas de cana como para as de pinus, embora sejam maiores no caso da pinus. A razão para esta inversão é uma contribuição negativa da interface filme/substrato que suplanta a contribuição positiva dos dipolos do filme de lignina de cana. Os dados elipsométricos revelaram que a lignina apresenta um arranjo tridimensional, com muitos espaços vazios e uma espessura em torno de 60 \'angstrom\' por camada. / Langmuir monolayers and Langmuir-Blodgett (LB) films were fabricated from lignins extracted from Pinus caribaea hondurensis and sugar cane bagasse using the organosolv processo Nine types of pinus lignins were obtained by employing different solvents. The lignins of sugar cane bagasse were extracted using only one solvent but the material was fractioned into six fractions according to the molecular sizes. The resulting lignins were then less polydisperse than the pinus lignins. Langmuir monolayers were spread onto ultrapure water subphases and characterized by surface pressure and surface potential measurements. In all cases, non-monomolecular aggregates are formed when the monolayer is compressed beyond the collapse pressure, which is denoted by large hysteresis in pressurearea isotherms. For the low molecular weight lignins, stable multilayer structures are formed even before collapse. In the less polydisperse bagasse lignins, the average area per molecule increases linearly with the molecular weight. Monolayers from both pinus and bagasse lignins could be transferred onto glass substrates, thus forming the first ever reported Langmuir-Blodgett (LB) films of these materials. By controlling the dipping speed one can build-up different types of LB film which can be y-type with deposition occurring in both upstrokes and downstrokes and also Z or x-type if transfer occurs only in the upstrokes or downstrokes, respectively. The deposited LB films were characterized by surface potential and ellipsometric measurements. The surface potential of pinus films is positive whereas that of bagasse lignins is negative. This sign inversion was surprising since the monolayer surface potentials were always positive for all materials, even though they were higher for the Pinus lignins. The reason for the inversion is the negative contribution of the film/substrate interface which surpass the small, positive contribution from the dipole moments in the bagasse lignins. The ellipsometric data showed that the lignin molecules assume a three-dimensional arrangement, even within a single layer. The LB film appears to be a highly porous structure, with a thickness of 60 Á per layer.

Filmes de Langmuir e Langmuir-Blodgett (LB) foram fabricados a partir de ligninas de bagaço de cana-de-açúcar e da Pinus caribaea hondurensis, extraídas via processo organossolve. Obteve-se nove tipos de lignina de pinus, sendo que cada uma foi extraída com um sol vente diferente, e seis tipos de lignina de cana, as quais passaram por um fracionamento, diminuindo sua polidispersividade. Os filmes de Langmuir foram fabricados sobre subfases de água ultrapura e caracterizados por medidas de pressão e potencial de superfície. Em todos os casos constatou-se a formação de agregados não monomoleculares quando o filme é comprimido além do ponto de colapso, o que é notado pela grande histerese na isoterma pressãoárea. Para ligninas de baixa massa molecular, estruturas estáveis não monomoleculares são formadas antes do colapso. No caso das ligninas menos polidispersas (cana), a massa molecular aumenta linearmente com a área molecular média da lignina. Monocamadas das ligninas de pinus e cana puderam ser transferidas para substratos de vidro, constituindo-se nos primeiros filmes de Langmuir-Blodgett destes materiais. Através do monitoramento da velocidade de imersão e retirada do substrato da subfase pode-se fabricar diferentes tipos de filmes LB, os quais podem ser do tipo Y com a deposição ocorrendo na imersão e retirada do substrato e também dos tipos X e Z se a deposição ocorre preferencialmente na imersão ou retirada do substrato, respectivamente. Os filmes LB foram caracterizados por medidas do potencial de superfície e elipsometria. O potencial de superfície para os filmes de pinus são positivos, enquanto que para os filmes de cana são negativos. Esta inversão de sinal foi surpreendente, pois para as monocamadas os valores de potencial são sempre positivos, tanto para as ligninas de cana como para as de pinus, embora sejam maiores no caso da pinus. A razão para esta inversão é uma contribuição negativa da interface filme/substrato que suplanta a contribuição positiva dos dipolos do filme de lignina de cana. Os dados elipsométricos revelaram que a lignina apresenta um arranjo tridimensional, com muitos espaços vazios e uma espessura em torno de 60 \'angstrom\' por camada. / Langmuir monolayers and Langmuir-Blodgett (LB) films were fabricated from lignins extracted from Pinus caribaea hondurensis and sugar cane bagasse using the organosolv processo Nine types of pinus lignins were obtained by employing different solvents. The lignins of sugar cane bagasse were extracted using only one solvent but the material was fractioned into six fractions according to the molecular sizes. The resulting lignins were then less polydisperse than the pinus lignins. Langmuir monolayers were spread onto ultrapure water subphases and characterized by surface pressure and surface potential measurements. In all cases, non-monomolecular aggregates are formed when the monolayer is compressed beyond the collapse pressure, which is denoted by large hysteresis in pressurearea isotherms. For the low molecular weight lignins, stable multilayer structures are formed even before collapse. In the less polydisperse bagasse lignins, the average area per molecule increases linearly with the molecular weight. Monolayers from both pinus and bagasse lignins could be transferred onto glass substrates, thus forming the first ever reported Langmuir-Blodgett (LB) films of these materials. By controlling the dipping speed one can build-up different types of LB film which can be y-type with deposition occurring in both upstrokes and downstrokes and also Z or x-type if transfer occurs only in the upstrokes or downstrokes, respectively. The deposited LB films were characterized by surface potential and ellipsometric measurements. The surface potential of pinus films is positive whereas that of bagasse lignins is negative. This sign inversion was surprising since the monolayer surface potentials were always positive for all materials, even though they were higher for the Pinus lignins. The reason for the inversion is the negative contribution of the film/substrate interface which surpass the small, positive contribution from the dipole moments in the bagasse lignins. The ellipsometric data showed that the lignin molecules assume a three-dimensional arrangement, even within a single layer. The LB film appears to be a highly porous structure, with a thickness of 60 Á per layer.

Abstract Lignins have polyphenolic structures, making them candidates to replace phenols and polyphenols in polymers. Lignins are highly recalcitrant, making their refining challenging, requiring harsh temperatures and pressures. Lignins could be partially modified under milder conditions for their use in biopolymers. The main purpose of this research was to upgrade Indulin AT, a kraft pine lignin, to enhance its properties. The first part of this thesis dealt with formic acid aided pressurized hot water extraction (FAPHWE) of hemicelluloses from birch hardwood as the first step in separating the components of a lignocellulosic feedstock (LCF). More than half of the hemicelluloses were extracted as hydrolysis products, while keeping the cellulose hydrolysis products in the extract under 5% and the lignin under 3%. In the second part of this work, a method to determine the amount of phenolic hydroxyl groups (OHph) in lignins was assessed. The Δε IDUS method was found to be useful for comparing the OHph of pine kraft and birch milox lignins, albeit not as precise as carbon-13 nuclear magnetic resonance spectroscopy (13C-NMR). The third part of this thesis explored the tuning of the molar mass (MM) and OHph of Indulin AT by aqueous ethanol fractionation. The results showed that a higher water content favored the extraction of fractions with low MM and low OHph. A high ethanol content favored the extraction of fractions with medium MM and high OHph. A 50–60 wt% ethanol content allowed for near complete solubilization of Indulin AT, which could be beneficial for a single-phase chemical reaction. The fourth part of this research dealt with the depolymerization of Indulin AT in an ethanol-water solvent with formic acid as hydrogen donor. The properties of interest were MM, polydispersity (PDI), OHph, and formaldehyde uptake capability (FUC). The results of the reaction were affected predominantly by temperature. Higher temperatures led to lower MM and PDI, and higher OHph and FUC. The results of this thesis suggest that, in a biorefinery, the first step before delignification of an LCF could be FAPHWE. It was found that the properties of Indulin AT (OHph, FUC, and MM) could be enhanced by chemical depolymerization and physical fractionation. Modified lignins with higher OHph and FUC could be utilized in biopolymer applications such as phenolic resins and polyurethanes. / Tiivistelmä Ligniini on rakenteeltaan polyfenoli, mikä tekee siitä mahdollisen fenolien korvaajan polyfenolien valmistuksessa. Ligniinin rakenne on hyvin kestävä, mikä tekee sen jalostuksesta haastavaa vaatien usein korkean lämpötilan ja paineen käyttöä. Tästä huolimatta ligniiniä voidaan tietyssä määrin muokata miedommissa olosuhteissa, mikä lisää sen käyttökelpoisuutta biopolymeerien raaka-aineena. Tämän tutkimuksen tarkoitus oli jalostaa Indulin AT -kraft ligniiniä siten, että sen ominaisuudet paranevat. Aluksi väitöstyössä tarkasteltiin puun hemiselluloosan muurahaishappokatalysoidun kuumavesiuutton soveltuvuutta lignoselluloosaraaka-aineiden fraktioinnin ensimmäiseksi vaiheeksi. Yli puolet hemiselluloosasta voitiin uuttaa monosakkarideiksi samalla, kun selluloosasta uuttui alle 5 % ja ligniinistä alle 3 %. Seuraavaksi arvioitiin fenolisten hydroksyyliryhmien määritysmenetelmää. Δε IDUS metodin havaittiin olevan hyödyllinen ainakin sulfaattimenetelmän havupuuligniinien ja Milox-prosessin koivuligniininäytteiden vertailussa, vaikkakaan se ei ole yhtä tarkka kuin ydinmagneettiseen resonanssispektroskopiaan (NMR) perustuva analyysi. Tämän jälkeen tutkittiin mahdollisuuksia tuottaa etanoli-vesiliuosfraktioinnilla jakeita, joissa ligniinillä on haluttu molekyylikoko ja fenolisten hydroksyyliryhmien pitoisuus. Tulokset näyttivät, että korkea vesipitoisuus suosii pienen molekyylikoon ja matalan OHph -pitoisuuden sisältäviä jakeiden uuttumista. Korkea etanolipitoisuus suosii keskikokoisen molekyylikoon jaetta, jossa ligniinillä on korkea OHph -pitoisuus. 50–60 m-% etanolipitoisuudessa Indulin AT liukenee lähes täydellisesti, mikä voi olla edullista kemiallisten reaktioiden toteuttamiseen yhdessä faasissa. Lopuksi tutkimuksessa tarkasteltiin ligniinin osittaista depolymerisointia etanoli-vesiliottimessa muurahaishapon toimiessa vetylähteenä. Tarkasteltavat tuotteen ominaisuudet olivat molekyylikoko, polydispersiteetti, fenolisten hydroksyyliryhmien määrä ja formaldehydin sitomiskapasiteetti. Lämpötilan havaittiin olevan merkittävin depolymerisointireaktioon vaikuttava tekijä. Korkea lämpötila johtaa pienempään molekyylikokoon ja kapeampaan molekyylikokojakaumaan sekä suurempaan hydroksyyliryhmien määrään ja formaldehydin sitomiskykyyn. Tämän työn tulokset viittaavat siihen, että hemiselluloosan vesiuutto happamissa olosuhteissa voi olla biojalostamon ensimmäinen vaihe, ennen delignifiointia. Lisäksi havaittiin, että ligniinin ominaisuuksia voidaan muokata kemiallisella depolymerisoinnilla ja fysikaalisella fraktioinnilla. Korkeamman hydroksyyliryhmäpitoisuuden ja formaldehydin sitomiskyvyn muokattuja ligniinejä voidaan hyödyntää biopolymeerisovellutuksissa, kuten fenolisissa hartseissa ja polyuretaaneissa.

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