H-1 NMR study of the states of water in equilibrium Poly(HEMA-co-THFMA) hydrogels

The spin-spin relaxation times, T-2, of hydrated samples of poly(hydroxymethyl methacrylate), PHEMA, poly(tetrahydrofurfuryl methacrylate),PTHFMA, and the,corresponding HEMA-THFMA copolymers have been examined to probe the states of,the imbibed water in these polymers. The decay in the transverse magnetization of water. in fully hydrated samples of PHEMA, PTHFMA, and copolymers of HEMA and THFMA was described by a multiexponential function. The short component of T-2 was interpreted as water molecules that were strongly interacting with the polymer chains. The intermediate component of T-2 was assigned to water residing in the porous structure of the samples. The long component of T-2 was believed to arise from water residing in the remnants of cracks formed in the polymer network during water sorption.

PFG-NMR measurements of the self-diffusion coefficients of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The self-diffusion coefficients for water in a series of copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA, swollen with water to their equilibrium states have been studied at 310 K using PFG-NMR. The self-diffusion coefficients calculated from the Stejskal-Tanner equation, D-obs, for all of the hydrated polymers were found to be dependent on the NMR storage time, as a result of spin exchange between the proton reservoirs of the water and the polymers, reaching an equilibrium plateau value at long storage times. The true values of the diffusion coefficients were calculated from the values of D-obs, in the plateau regions by applying a correction for the fraction of water protons present, obtained from the equilibrium water contents of the gels. The true self-diffusion coefficient for water in polyHEMA obtained at 310 K by this method was 5.5 x 10(-10) m(2) s(-1). For the copolymers containing 20% HEMA or more a single value of the self-diffusion coefficient was found, which was somewhat larger than the corresponding values obtained for the macroscopic diffusion coefficient from sorption measurements. For polyTHFMA and copolymers containing less than 20% HEMA, the PFG-NMR stimulated echo attenuation decay curves and the log-attenuation plots were characteristic of the presence of two diffusing water species. The self-diffusion coefficients of water in the equilibrium-hydrated copolymers were found to be dependent on the copolymer composition, decreasing with increasing THFMA content.

Spectroscopic study of the penetration depth of grafted polystyrene onto poly (tetrafluoroethylene-co-perfluoropropylvinylether) substrates. 1. Effect of grafting conditions

This study concerns the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinylether) (PFA) substrates and the penetration depth of the graft. Grafting was obtained by the simultaneous irradiation method, and the spectroscopic analysis was made with the micro-Raman technique. Effects of grafting conditions such as the type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol, respectively. A mixture of methanol and dichloromethane used as a solvent for styrene achieved a higher degree of grafting and concentration of grafted polystyrene onto the surface of PFA substrates than solutions of the monomer in the separate solvents. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate. (C) 2002 Wiley Periodicals, Inc.

The copolymerization of N-vinyl-2-pyrrolidone with 2-hydroxyethyl methacrylate

The bulk free radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N-vinyl-2-pyrrolidone (VP) was carried out to low conversions at 50 degreesC, using benzoyl peroxide (BPO) as initiator. The compositions of the copolymers; were determined using C-13 NMR spectroscopy. The conversion of monomers to polymers was studied using FT-NIR spectroscopy in order to predict the extent of conversion of monomer to polymer. From model fits to the composition data, a statistical F-test revealed that die penultimate model describes die copolymerization better than die terminal model. Reactivity ratios were calculated by using a non-linear least squares analysis (NLLS) and r(H) = 8.18 and r(V) = 0.097 were found to be the best fit values of the reactivity ratios for the terminal model and r(HH) = 12.0, r(VH) = 2.20, r(VV) = 0.12 and r(HV) = 0.03 for the penultimate model. Predictions were made for changes in compositions as a function of conversion based upon the terminal and penultimate models.

Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle. (C) 2002 Wiley Periodicals, Inc.

Water sorption into poly[(2-hydroxyethyl methacrylate)-co-(1-vinyl-2-pyrrolidone]at 310 K

Poly(2-hydroxyethyl methacrylate) and copolymers of 2-hydroxyethyl methacrylate (HEMA) and 1-vinyl-2-pyrrolidone (VP) in the form of cylindrical samples (approximate to8mm x 20mm) have been prepared and the sorption of water into these cylinders has been studied by the mass-uptake methods and by magnetic-resonance imaging. The equilibrium water contents for the cylinders were found to vary systematically with the copolymer composition. Diffusion of water into the cylinders was found to follow Fickian behaviour for cylinders with high HEMA contents, with the diffusion coefficients obtained from mass-uptake studies dependent on the copolymer composition, varying from 1.7 x 10(-11) m(2) s(-1) for poly(HEMA) to 2.0 x 10(-11) m(2) s(-1) for poly(HEMA-co-VP) with a composition of 1:1. However, NMR-imaging studies showed that, while the profiles of the water diffusion fronts for cylinders with high HEMA contents were Fickian, that for the 1:1 copolymer was not and indicated that the mechanism was Case III. The polymers which were rich in VP were characterized by a water-sorption process which follows Case-III behaviour. (C) 2003 Society of Chemical Industry.

Radical formation on gamma-radiolysis of poly [(methacrylic acid)-co-acrylonitri

The radicals formed on gamma-radiolysis of a series of copolymers of methacrylic acid and acrylonitrile have been investigated by ESR spectroscopy. This series of copolymers spanned the full composition range and the study was carried out at 77 K and ambient temperature. The radicals formed in the copolymers at 77 and 303 K were found to be similar to those found in the two homopolymers, but in the intermediate composition range the presence of acrylonitrile propagation radicals was also detected. These radicals were not observed to be formed in significant quantities on the radiolysis of polyacrylonitrile. They are believed to result from a scission of the main chain at methacrylic acid/acrylonitrile diad sequences following loss of the methacrylic acid carboxyl group. At 77 K, the copolymers with high methacrylic acid contents were found to be more sensitive to radical formation than the methacrylic acid homopolymer, but this enhanced sensitivity was not evident at ambient temperature, where the G-values for radical formation for the copolymers were slightly less than the values for the homopolymers. (C) 2003 Society of Chemical Industry.

A study of the radiation chemistry of poly(chlorotrifluoroethylene) by ESR spectroscopy

The ESR spectra of poly(chlorotrifluoroethylene) were recorded following gamma-radiolysis under vacuum at room temperature and 77 K. The very broad spectrum at 77 K revealed little fine structure with which to identity the radicals formed upon irradiation, but subsequent photobleaching and annealing studies, together with radiolytic studies at higher temperatures, afforded scope for making radical assignments. Both main-chain radicals and a range of chain-end radicals have been identified. The G-values for radical formation were 1.55, 0.36 and 0.32 at 77 K, 273 K and room temperature, respectively. (C) 2003 Elsevier Science Ltd. All rights reserved.

An investigation of the thermal and tensile properties of PFA following γ-radiolysis

For some applications for fluoropolymers they must be subjected to high-energy radiation, e.g., when they are grafted with styrene using an irradiation method to produce fuel cell membranes or matrix supports for combinatorial chemistry. In some of these applications they may be subjected to mechanical stress or elevated temperature, so it is important to elucidate the effects of the radiolysis on these properties. In the present work the effect of gamma-radiolysis on the glass transition, melting behavior, and thermal stability of PFA has been studied as well as the effect of the radiolysis on the tensile properties of the polymer.

PHB-PEO electrospun fiber membranes containing chlorhexidine for drug delivery applications

Fiber meshes of poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate)/ poly(ethylene oxide) (PHB/PEO) with different concentrations of chlorhexidine (CHX) were prepared by electrospinning, for assessment as a polymer based drug delivery system. The electrospun fibers were characterized at morphological, molecular and mechanical levels. The bactericidal potential of PHB and PHB/PEO electrospun fibers with and without CHX was investigated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by disk diffusion susceptibility tests. Electrospun fibers containing CHX exhibited bactericidal activity. PHB/PEO-1%CHX displayed higher CHX release levels and equivalent antibacterial activity when compared to PHB/PEO with 5 and 10 wt% CHX. Bactericidal performance of samples with 1 wt% CHX was assessed by Colony Forming Units (CFU), where a reduction of 100 % and 99.69 % against E. coli and S. aureus were achieved, respectively.

Synthesis, structure, and optical properties of an alternating calix[4]arene-based meta-linked phenylene ethynylene copolymer

Novel alternating copolymers comprising biscalix[4]arene-p-phenylene ethynylene and m-phenylene ethynylene units (CALIX-m-PPE) were synthesized using the Sonogashira-Hagihara cross-coupling polymerization. Good isolated yields (60-80%) were achieved for the polymers that show M-n ranging from 1.4 x 10(4) to 5.1 x 10(4) gmol(-1) (gel permeation chromatography analysis), depending on specific polymerization conditions. The structural analysis of CALIX-m-PPE was performed by H-1, C-13, C-13-H-1 heteronuclear single quantum correlation (HSQC), C-13-H-1 heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), and nuclear overhauser effect spectroscopy (NOESY) in addition to Fourier transform-Infrared spectroscopy and microanalysis allowing its full characterization. Depending on the reaction setup, variable amounts (16-45%) of diyne units were found in polymers although their photophysical properties are essentially the same. It is demonstrated that CALIX-m-PPE does not form ground-or excited-state interchain interactions owing to the highly crowded environment of the main-chain imparted by both calix[4]arene side units which behave as insulators inhibiting main-chain pi-pi staking. It was also found that the luminescent properties of CALIX-m-PPE are markedly different from those of an all-p-linked phenylene ethynylene copolymer (CALIX-p-PPE) previously reported. The unexpected appearance of a low-energy emission band at 426 nm, in addition to the locally excited-state emission (365 nm), together with a quite low fluorescence quantum yield (Phi = 0.02) and a double-exponential decay dynamics led to the formulation of an intramolecular exciplex as the new emissive species.

Demixing instability in coil-rod blends undergoing polycondensation reactions

The authors extend their earlier work on the stability of a reacting binary polymer blend with respect to demixing [D. J. Read, Macromolecules 31, 899 (1998); P. I. C. Teixeira , Macromolecules 33, 387 (2000)] to the case where one of the polymers is rod-like and may order nematically. As before, the authors combine the random phase approximation for the free energy with a Markov chain model for the chemistry to obtain the spinodal as a function of the relevant degrees of reaction. These are then calculated by assuming a simple second-order chemical kinetics. Results are presented, for linear systems, which illustrate the effects of varying the proportion of coils and rods, their relative sizes, and the strength of the nematic interaction between the rods. (c) 2007 American Institute of Physics.

Terapêuticas antigénio-especificas no tratamento das doenças desmielinizantes: estudos sobre a vacinação com ADN e a descoberta de um novo alvo antigénico

RESUMO A Esclerose Múltipla (EM) é uma doença desmielinizante crónica do Sistema Nervoso Central (SNC), provocada, em grande parte, por um ataque imuno-mediado contra diversos elementos da bainha de mielina. Dentro dos alvos antigénicos desta resposta autoimune, vários componentes proteicos e lipídicos da mielina têm vindo a ser identificados ao longo dos anos, entre os quais se destacam a proteína básica de mielina(MBP), glicoproteína ligodendrocitária da mielina (MOG), proteína proteolipídica (PLP) e glicoproteína associada à mielina (MAG). Com o desenvolvimento do modelo animal de Encefalomielite Autoimune Experimental (EAE), diversas terapias antigénio-específicas foram desenhadas, baseadas na modificação benéfica da resposta autoimune contra a mielina, tais como a administração de mielina ou seus componentes, os copolímeros terapêuticos, os ligandos peptídeos alterados e, recentemente, a vacinação com ácido desoxirribonucleico (ADN) codificador de proteínas de mielina, integrado em plasmídeos e purificado para administração parentérica. Neste trabalho, apresentamos os resultados de um extenso conjunto de experiências, subordinadas a dois temas fundamentais: 1) avaliação do potencial terapêutico, e dos mecanismos de acção, da vacinação tolerizadora com ADN codificador de proteínas de mielina (MBP, MOG, PLP, MAG) na EAE, e da associação desta vacinação com a administração de ADN de citocinas Th2, ou de oligonucleótidos imunomoduladores; 2) identificação e caracterização da resposta imune contra um novo componente da mielina com potencial antigénico, a proteína inibidora do recrescimento axonal, Nogo-A. No que respeita à vacinação com ADN, os nossos resultados comprovam a eficácia desta terapêutica antigénio-específica na prevenção e tratamento da EAE. Os seus mecanismos de acção incluem, entre outros, a supressão anérgica da proliferação antigénioespecífica dos linfócitos T anti-mielina (no modo de prevenção da doença), o enviesamento Th2 da resposta imune (quando co-administrada com a vacina de ADN codificadora da citocina IL-4, funcionando como terapia génica local), e a redução da diversificação de epítopos da resposta humoral anti-mielina, avaliada através de myelin spotted arrays. A associação das vacinas de ADN com oligonucleótidos imunomoduladores GpG, desenvolvidos para contrariar as sequências CpG imunoestimuladoras presentes no vector de vacinação, levou à melhoria da sua eficácia terapêutica, devida, provavelmente, ao efeito estimulador preferencial dos oligonucleótidos GpG sobre linfócitos Th2 e sobre células reguladoras NK-T. Com base nestes resultados a vacinação com ADN foi desenvolvida para o tratamento da EM em humanos, com ensaios clínicos a decorrerem neste momento. Em relação à proteína Nogo-A, estudos de estrutura primária e de previsão de antigenicidade identificaram a região Nogo-66 como alvo antigénico potencial para a EAE. Nas estirpes de ratinho SJL/J e C57BL/6, fomos capazes de induzir sinais clínicos e histológicos de EAE após imunização com os epítopos encefalitogénicos Nogo1-22, Nogo23- 44 e Nogo45-66, utilizando protocolos de quebra de tolerância imune. Ao mesmo tempo, identificámos e caracterizámos uma resposta linfocitária T específica contra os antigénios contidos na região Nogo-66, e uma resposta linfocitária B com diversificação intra e intermolecular a vários determinantes presentes noutras proteínas da mielina. A transferência adoptiva de linhas celulares Th2 anti-Nogo45-66, levou à melhoria clínica e histológica da EAE em animais recipientes induzidos com outros antigénios de mielina, após migração destas células para o SNC. Estes dados comprovam a importância da Nogo-66 como antigénio na EAE, e a eficácia de terapias antigénio-específicas nela baseadas. No seu conjunto, os nossos resultados confirmam o potencial terapêutico das vacinas de ADN codificadoras de proteínas de mielina, bem como a importância dos encefalitogénios contidos na proteína Nogo-A para a fisiopatologia da EAE e da EM, com eventual relevância para o desenvolvimento de novas terapias antigénio-específicas. O aperfeiçoamento futuro destas terapias poderá levar, eventualmente, a uma capacidade de manipulação da resposta imune que permita o tratamento eficaz das doenças inflamatórias desmielinizantes, como a Esclerose Múltipla. ABSTRACT Multiple Sclerosis (MS) is a chronic demyelinating disease of the Central Nervous System (CNS), caused, mainly, by an immune-mediated attack against several elements of the myelin sheath. Among the antigenic targets for this autoimmune response, several proteic and lipidic myelin components have been identified throughout the years, of which myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipidic protein (PLP), and myelin associated glycoprotein (MAG) are the best characterized. With the development of the animal model for MS, Experimental Autoimmune Encephalomyelitis (EAE), several antigen-specific therapies have been designed, based on beneficial modifications of the autoimmune response against myelin. These have included myelin and myelin component administration, therapeutic copolymers, altered peptide ligands and, more recently, vaccination with myelin-protein encoding deoxyribonucleic acid (DNA), integrated into plasmids and purified for parenteral administration. In this work we present the results of an extensive series of experiments, subordinate to two fundamental areas: 1) evaluating the therapeutic potential, and mechanisms of action, of tolerizing myelin protein (MBP, MOG, PLP, MAG) DNA vaccination in EAE, alone and in association with Th2 cytokine DNA administration, or immunomodulatory oligonucleotides; 2) identifying and characterizing the immuneresponse against a new myelin component with antigenic potential, the axonal regrowth inhibitor Nogo-A. Regarding DNA vaccination, our results prove the efficacy of this antigen-specific therapy for the prevention and treatment of EAE. Its mechanisms of action include, among others, anergic suppression of antigen-specific T-cell proliferation against myelin (in prevention mode), Th2 biasing of the immune response (when co-administered with the IL- 4 codifying DNA vaccine, acting as local gene therapy), and reduction of epitope spreading of the anti-myelin antibody response, assessed by myelin spotted arrays. The combination of myelin DNA vaccination with the administration of GpG immunomodulatory oligonucleotides, designed to counteract immunostimulatory CpG motifs present in the vaccination vector, led to an improvement in therapeutic efficacy, probably due to the preferential stimulatory effect of GpG oligonucleotides on Th2 lymphocytes and on regulatory NK-T cells. Based on these results, tolerizing DNA vaccination is being developed for human use, with ongoing clinical trials. As concerns the Nogo-A protein, based on studies of primary structure and prediction of antigenicity, we identified the Nogo-66 region (responsible for the most of the inhibitory capacity of this protein) as a potential antigenic target for EAE. In the SJL/Jand C57BL/6 mouse strains, we were able to induce clinical and histological signs of EAE,after immunization with the encefalitogenic epitopes Nogo1-22, Nogo23-44 and Nogo45-66,using a tolerance breakdown protocol. Concomitantly, we identified and characterized a specific T cell response against these antigens, together with a B cell response which showed extensive intra and intermolecular epitope spread to several determinants present in other myelin proteins. Adoptive transfer of nti-Nogo45-66 Th2 cell lines resulted in clinical and histological improvement of EAE in recipient animals induced with other myelin antigens, after intraparenchymal CNS migration of anti-Nogo cells. These data confirm the relevance of Nogo-66 as an antigen in EAE, as well as the efficacy of antigenspecific therapies based on the response against this protein.In conclusion, our results substantiate the therapeutic potential of myelin-encoding DNA vaccination, as well as the importance of encefalitogenic epitopes present in the Nogo-A protein for the pathophysiology of EAE and MS, with potential relevance for the creation of new antigen specific-therapies. The future development of these therapies may eventually lead to a degree of manipulation of the immune response that allows the effective treatment of autoimmune, inflammatory, demyelinating diseases, such as Multiple Sclerosis.

Development of 2-oxazoline-based hydrogels and porous polyester microparticles in scCO2

Thesis submitted to Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa in partial fulfillment of the requirements for the obtention of the degree of Master of Science in Biotechnology

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology