Hidrogéis são membranas formadas pela reticulação de cadeias poliméricas, empregados na área farmacêutica como produtos biomédicos. Dentre os principais polímeros selecionados para a síntese de hidrogéis, destaca-se a poliacrilamida (PAAM) devido às suas propriedades como hidrofilicidade e alto grau de intumescimento. Proteínas terapêuticas e enzimas são veiculadas em hidrogéis como carreadores de fármaco ou como dispositivos para tratamento de feridas e escaras na pele. Este trabalho teve como objetivo a síntese de uma membrana à base de PAAM favorável para veiculação de proteínas. As proteínas empregadas foram papaína e albumina de soro bovino (BSA) e as etapas do processo englobaram síntese da membrana, adição das proteínas no sistema, irradiação em condições específicas e caracterização da membrana. Ao utilizar temperaturas criogênicas na síntese e na irradiação das amostras, houve predomínio de reticulação da cadeia polimérica, fato que não ocorria em temperatura ambiente. As membranas foram obtidas com incorporação dos ativos na concentração de 0,2 a 1% (p/p), obtendo-se concentração de PAAM entre 4% a 10% (p/p), as quais receberam irradiação com raios gama provenientes de uma fonte 60Co, na dose de 25 kGy. Nas condições realizadas, as membranas não apresentaram citotoxicidade nem adesão celular, o perfil de liberação das proteínas foi adequado, a papaína manteve sua bioatividade preservada apesar do decaimento biológico e, segundo estudos de carga das moléculas, a membrana possui maior afinidade com a papaína, liberando-a mais lentamente. Desta forma, o método proposto e as membranas obtidas foram apropriados para a obtenção de um biomaterial. / The use of hydrogels for biomedical purposes has been extensively investigated. Polyacrylamide (PAAM) is widely used due to properties such as hydrophilicity and swelling degree. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release using papain and bovine serum albumin (BSA) as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using gamma radiation at 25 kGy under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density, fluid handling capacity, determination pH at point of polymer zero charge and evaluated according to protein release, bioactivity, cytotoxicity and cell adhesion. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-10% polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties of polymer and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 hours), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay and cell adhesion it was also adequate for biomedical purposes and this method was able to generate a matrix to protein release.

Les floculants à base de polyacrylamide (PAM) sont produits à partir du monomère toxique : l’acrylamide (AMD) et peuvent en contenir des quantités résiduelles (jusqu’à 0,1% en Europe). Après utilisation pour faciliter la séparation solide/liquide des eaux de procédés dans les industries de granulat, ces floculants sont stockés avec les boues de décantation dans des lagunes à partir desquelles une dissémination de l’AMD et du PAM vers les eaux de surface ou les eaux souterraines peut avoir lieu. Dans ces travaux de thèse, les interactions du PAM et de l’AMD avec des particules de boue et des phases argileuses (kaolinite et illite, utilisées pour étanchéifier les lagunes de décantation) ont été étudiées. Pour pouvoir quantifier correctement l’AMD, une méthode d’analyse basée sur la HPLC/MS/MS en injection directe a été développée. Cette méthode a été validée avec les normes Afnor NF T 90-210 et NF T 90-220 avec une limite de quantification égale à 1 µg/L. L’étude de l’interaction de l’AMD avec des particules de boue d’un site de granulat et deux argiles (kaolinite et illite) a mis en évidence une faible adsorption de l’AMD sur ces phases solides (<10%), indépendante du temps, de la concentration en AMD et du pH. Inversement, le PAM s’adsorbe fortement et irréversiblement sur la boue, la kaolinite et l’illite avec une cinétique rapide de 1er ordre. Les isothermes d’adsorption sont bien corrélées avec les modèles de Langmuir et de Freundlich. Les quantités d’adsorption du PAM sont indépendantes du pH des suspensions mais fortement impactées par la force ionique qui influence les interactions électrostatiques entre le PAM et les surfaces solides. / Polyacrylamide (PAM) based floculants are produced from the highly toxic acrylamide (AMD) monomer and can contain residual amounts (up to 0.1% in Europe) of AMD. After they are used to facilitate liquid/solid separation of process water in aggregate quarries, PAM floculants are stored, with the sewage sludge, in decantation lagoons. Dissemination of AMD and PAM to groundwater and surface water from these lagoons can occur. In this work, we aimed to study the interactions of AMD and PAM with sludge particles and clays (kaolinite and illite used for decantation lagoon sealing) from aggregate quarry. To correctly quantify the AMD, analytical method based on HPLC/MS/MS with direct injection was developed. This method was validated according to the Afnor guidelines (NF T 90-210 and NF T 90-220) with a limit of quantification of 1 µg/L. Results of AMD adsorption experiments showed a low adsorption of AMD to sludge and clay (kaolinite and illite) particles, which is independent of time, AMD concentration and pH. Inversely, PAM was found to adsorb strongly and irreversibly to sludge, kaolinite and illite with a rapid kinetic of adsorption which consists of first order kinetic. Adsorption isotherms are well correlated with Langmuir and Freundlich models. PAM adsorption quantities are independent on the pH of suspensions, but are strongly impacted by the ionic strength which affects electrostatic interactions between PAM and solid surfaces.

The objectives of this study were to determine the effects of two forms of polyacrylamide (PAM) conditioners (Cross-linked and Non-cross-linked PAM) on evaporation, saturated hydraulic conductivity, water retention, crust and crack formation of soils, seed germination, and seedling and tubeling growth. The two PAM conditioners, 0.2% concentration by weight, were mixed with seven soils of different textures (sandy loam, silt, silty clay loam, silt loam, fine sand, medium sand, and coarse sand) to investigate the effects on evaporation, saturated hydraulic conductivity, and water retention. Soil samples of different textures were brought to field capacity and placed in a growth chamber for two weeks to measure evaporation under a controlled environment. A second experiment was carried out in the field to determine the effects of the two PAM conditioners on seedling emergence of crested wheatgrass, Agropyron desertorum, as well as on soil cracking, penetrometer resistance, and soil moisture. The two PAMs were mixed with a silt loam Xerollic Calciorthid at 0.2% concentration by weight. Seedling emergence was monitored directly for two weeks. Soil moisture was measured by TDR. Cracking was described by photographic means. Penetrometer resistance was measured by a hand-held. penetrometer. The third experiment was also carried out in the field, using the same soil texture as in experiment 2, to investigate the effects of the two PAMs on soil moisture at depths between 25 to 45 cm and on sagebrush (Artemisia tridentata) growth. Evaporation was found to be significantly lower in the fine-textured controls than under the two PAM treatments. The sandy loam and sandy soils experienced significantly higher evaporation from the controls. The two PAM conditioners significantly reduced saturated hydraulic conductivity on all soil textures. Water retention increased in the PAM-treated textures at the matric potential range used (0.0, 0.05, 0.1, 1.5 MPa) • The PAM application also did not improve grass seedling emergence or improve soil moisture, and did not have any significant affects on sagebrush growth. Larger cracks were found in the two plots treated with PAM than the controls. Lower penetrometer resistance occurred in the two PAM treatments compared to the untreated control. From this study, it can be concluded that the application of PAM conditioners, at relatively high concentrations used, could be more viable on sandy textures. Other researchers are advised to try lower application rates than used here, particularly with finer textured soils.

[pt] Os copolímeros de poliacrilamida desempenham um papel importante na recuperação avançada de petróleo, cuja utilização visa o aumento da produção de óleo. A avaliação do polímero na água produzida é, portanto, necessária para monitorar a operação do campo petrolífero e para questões ambientais. Entretanto, tal avaliação é desafiadora devido à complexidade da matriz e à possível degradação da macromolécula durante o processo. Entre os diferentes métodos para determinação de polímero, o carbono orgânico total (TOC, do inglês total organic carbon) é vantajoso em comparação com outros métodos reportados na literatura devido ao seu baixo custo, fácil operação e curto tempo de análise. Devido a isso, um método de análise por TOC foi desenvolvido para a determinação do teor de poliacrilamida, e a influência da salinidade, massa molar e dois tipos de degradação química (hidrólise e por Fe2+/O2) na quantificação do polímero por este método foram exploradas neste trabalho. Medidas reológicas, análises de ressonância magnética nuclear (RMN) e de espectroscopia de infravermelho (ATR-FTIR, do inglês Fourier-transform infrared with attenuated total reflectance) também foram realizadas com o intuito de avaliar a degradação. A quantificação do polímero na presença de diferentes teores de sal não foi afetada. De modo similar, as diferentes massas molares dos polímeros não afetaram os resultados das análises por TOC. O grau de hidrólise dos polímeros foi confirmado por 13C RMN e ATR-FTIR, enquanto medidas reológicas confirmaram a degradação por Fe2+/O2. Notavelmente, ambos os mecanismos de degradação investigados não impactaram a determinação de poliacrilamida. Para todos os fatores avaliados, os erros não ultrapassaram 8 por cento. Assim, os resultados obtidos destacam o método TOC como adequado e confiável para a determinação de poliacrilamida com boa exatidão, conferindo-lhe um potencial de aplicação para análises de amostras reais. / [en] Polyacrylamide copolymers play an important role in enhanced oil recovery (EOR), whose widespread use aims to improve oil production. Assessment of polymer in the produced water is thus required for monitoring the oilfield operation and for environmental concerns. However, this task is challenging due to the matrix complexity and the possible degradation of the macromolecule during the process. Among the different methods for polymer determination, total organic carbon (TOC) is advantageous in comparison to others reported in the literature because of its low cost, easy operation and short analysis time. Due to this, a TOC method was developed for the determination of polyacrylamide content, and the influence of salinity, molar mass, and two types of chemical degradation (by hydrolysis and Fe2+/O2) on the polymer quantification through this method were explored in this work. Rheological measurements, NMR and ATR-FTIR analyses were also carried out to evaluate the extent of degradation. The polymer quantification was not affected in the presence of different salt contents. Similarly, analyses of samples with different molar masses did not affect TOC results. The hydrolysis degree of the polymers was confirmed by 13C NMR and ATR-FTIR, while rheological measurements confirmed degradation by Fe2+/O2. Noticeably, both degradation mechanisms investigated did not impair polyacrylamide determination. For all factors evaluated, the errors were never greater than 8 percent. The results obtained highlight the TOC as a suitable and reliable method for the determination of polyacrylamide with good accuracy, showing its potential application in the analysis of real samples.

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The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.

Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated using traditional methods of enzyme activity measurements and recent proteomic approaches. The findings of the study can be used to guide future studies on the proteomic responses of vertebrates to environmental stress. Chapter 2 focuses on measurement of the temporal effects of oxygen treatments on the maximal specific activities of nine glycolytic enzymes in liver and skeletal muscle during chronic exposure (28d) of Fundulus heteroclitus. The fish was exposed to four different oxygen treatments: hyperoxia, normoxia, moderate hypoxia, and severe hypoxia. The time course of changes in maximal glycolytic enzyme specific activities was assessed at 0, 8, 14 and 28 d. The results demonstrate that chronic hypoxia alters the capacity for carbohydrate metabolism in F. heteroclitus, with the important observation that the responses are both tissue- and enzyme-specific. Chapter 3 studies the effect of tissue storage on protein profile of tissues of F. grandis. The technique of one dimensional gel electrophoresis (1D-SDS-PAGE) was used to assess the effects of tissue sampling, flash frozen in liquid nitrogen versus immersion of fresh tissue in RNA later, for five tissues, liver, skeletal muscle, brain, gill, and heart, followed by LC-MS/MS to identify protein bands that were differentially stabilized in gill and liver. The study shows that, in F. grandis, the preferred method of preservation was tissue specific. xi Chapter 4 focuses on the use of advanced 2DE-MS/MS to characterize the proteome of multiple tissues in F. grandis. Database searching resulted in the identification of 253 non-redundant proteins in five tissues: liver, muscle, brain, gill, and heart. Identifications include enzymes of energy metabolism, heat shock proteins, and structural proteins. The protein identification rate was approximately 50 % of the protein spots analyzed. This identification rate for a species without a sequenced genome demonstrates the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.

Este trabalho descreve a síntese, caracterização e aplicação de sistemas poliméricos baseados em polímeros condutores em sistemas de liberação controlada de drogas. Esta tese pode ser dividida em duas partes: na primeira se apresentam os resultados da aplicação de filmes de polianilina e polipirrol na liberação de drogasmodelo como a dopamina protonada e o ácido salicílico. Na liberação de salicilato utilizou-se um filme polianilina eletrosintetizado e dopado com íons cloreto. Já para a liberação de dopamina protonada (um cátion) a liberação foi conduzida a partir de um sistema bicamadas, com um filme de polianilina recoberta com uma camada de Náfion. É mostrada a liberação controlada nos dois casos, porém também se discutem limitaçãoes deste tipo de sistema que levaram ao estudo de uma forma alternativa de controle eletroquímico utilizando polímeros condutores. A segunda parte do trabalho mostra então esta nova metodologia que se baseia em compósitos de poianilina eletropolimerizada no interior de hidrogéis de poliacrilamida. É mostrado que este novo material é eletroativo e mantém as características de intumescimento dos hidrogéis, tanto necessárias ao desenvolvimento destes sistemas de liberação controlada. Mecanismos para o crescimento e distribuição da polianilina na matriz isolante e para a atuação do compósito no controle eletroquímico da liberação são propostos com base nos dados de microscopia de força atômica, Raman e eletrônica de varredura, além de testes de liberação controlada com moléculas de diferentes cargas. / This work describes the synthesis, characterization and application of polimeric systems based on contucting polymers for electrochemical release devices. The thesis is divided into two parts: frrst, results conceming the application of polyaniline and polypyrrole films in the controlled release of dopamine and salicilyc acid, are showed. An electrosyntetized polyaniline film doped with chloride ions was used in the salicilate release. On the other hand, for the controlled release of protonated dopamine, a bilayer system consisting of a polyaniline film recovered with Nafion was employed. The release control was reached in both cases, but this kind of actuator show serious limitations that lead to the conclusion that a new type of electrochemical control based in conducting polymers must be developped. The second part shows the new methodology based on composites of polyaniline grown inside polyacrylamide hydrogels. It\'s showed that this new material is electroactive and still maintains the hydrogel\'s swelling properties, which makes it an interesting and suitable material for drug delivery devices. Raman Microscopy, Scaning Electron Microscopy and Atomic Force Microscopy, and controlled release tests of different charged molecules were tools for the elucidation of polyaniline\'s growth and distribuition inside the hydrogels and for the mecanism of actuation in the controlled release.

A avaliação das modificações no proteinograma do leite tem sido utilizada como meio de diagnóstico das mamites dos bovinos, pois, em decorrência dos processos inflamatórios na glândula mamária, ocorrem alterações, tanto na concentração dos componentes protéicos do leite, como também, surgimento de compostos protéicos não elaborados no processo de secreção láctea. A técnica de fracionamento protéico por eletroforese em gel de poliacrilamida mostrou-se eficiente na detecção de pequenas quantidades de proteínas em fluídos orgânicos. As características das proteínas do leite de vacas sadias, e a avaliação de possíveis fatores de variabilidade foram determinadas em 139 amostras de leite de vacas. Os animais foram, inicialmente, submetidos a exame clínico geral e do úbere, complementado por exames físico-químicos, celulares e microbiológicos do leite, complementares ao diagnóstico das mamites. Do conjunto das amostras, 97 eram de vacas sadias, em plena lactação, sendo utilizadas para estabelecer valores padrões dos constituintes do leite de vacas sadias e avaliar a influência racial - Jersey e Gir; e do número de lactações - animais em primeira lactação, duas ou três lactações e, quatro ou mais lactações sobre o proteinograma lácteo. Além do mais a amostragem serviu de base para a avaliação da fase da lactação sobre o quadro protéico e, permitiram a formação de grupos experimentais para avaliação da influência do número de células somáticas, do isolamento bacteriano e do estado de saúde do úbere no proteinograma lácteo, obtido por eletroforese em gel de poliacrilamida. Os resultados demonstraram influência significativa de fatores raciais no teor de proteína total do soro lácteo, e de suas frações imunoglobulínica, &alpha;1-antitripsina, &beta;-lactoglobulina, bem como de um conjunto de proteínas do soro lácteo separadas, mas não identificadas; influência do número de lactações no teor de albumina de origem plasmática, imunoglobulinas, &beta;-lactoglobulina e &alpha;-lactoalbumina. A fase da lactação influenciou de forma significativa os teores de proteína total do leite, assim como nas frações protéicas do soro lácteo de vacas sadias, variações evidentes na fase colostral da lactação. O número de células somáticas influenciou o proteinograma do leite, apenas nas amostras com mais de 1.500.000 células somáticas/ml. Não foi demonstrada a influência significativa do isolamento bacteriano no proteinograma lácteo de vacas. Entretanto, observou-se significativa influência da mamite no proteinograma do leite de vacas, agindo, principalmente, nas proteínas não sintetizadas no ciclo fisiológico de secreção da glândula mamária. / The evaluation of the modifications in milk proteinogram is used as a diagnostic tool for bovine mastitis, once the inflammation of the mammary glands leads to changes in the concentration of milk proteic components, as well as the appearance of proteic components that are not elaborated in the milk secretion process. The technique of protein fractioning by electrophoresis in polyacrylamide gel is effective in detecting small amounts of proteins in organic fluids. The features of milk proteins of healthy cows and the evaluation of possible variability factors were determined in 139 cow milk samples. Animals were first submitted to general clinical examination and udder examination, and to physical-chemical, cellular and microbiological milk tests, complementary to mastitis diagnosis. Of the total samples, 97 were from healthy cows in full lactation, used to establish reference values for the components of healthy cow milk and to evaluate the influence of breed - Jersey and Gir, and number of lactations - first, two or three lactations, and four or more lactations on milk proteinogram. Additionally, sampling was useful as a base for the formation of experimental groups for evaluation of the influence of the number of somatic cells, bacterial isolation and health condition of the udder on milk proteinogram obtained by electrophoresis in polyacrylamide gel. Results showed a significant influence of breed related factors on the level of total protein and its fractions immunoglobulinic, &alpha;1-antitripsin, &beta;-lactoglobulin in whey, as well as on the level of a group of separated but not identified whey proteins; influence of the number of lactations on the level of albumin of plasmatic origin, immunoglobulins, &beta;-lactoglobulin and &alpha;-lactoalbumin. The lactation phase influenced the levels of milk serum protein significantly, as well as the proteic fractions of healthy cow milk serum, variation evident in the colostrum phase of lactation. The number of somatic cells influenced milk proteinogram only in samples with more than 1,500,000 somatic cells. No significant influence of bacterial isolation on milk proteinogram was demonstrated. However, a significant influence of mastitis on the proteinogram of cow milk was observed, affecting mostly those proteins not synthetized in the physiologic cycle of mammary gland secretion.