The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

Incorporation of monoacetylferrocene to the sulfonated polystyrene ionomers imparted some changes in the properties of sulfonated polystyrene. Sulfonation was carried out by acetic anhydride and concentrated sulphuric acid. The sulfonation reaction and the degree of sulfonation were determined by analytical titration and adiabatic bomb calorimeter . For this purpose, sulfonated polystyrene (SPS) samples with varying percentages of sulfonation were prepared between 0.85% and 6.51%. Monoacetyl ferrocene was used in equivalent amount of sulfonation through addition procedure. FTIR Spectroscopy was one of the major techniques used to support the successful addition of AcFe to the SPS samples. Altering the sulfonation degree did not change the characteristic peak positions, but increased the peak intensities with increasing the degrees of sulfonation. Mechanical properties of resultant polymers were investigated. As a result, elastic modulus of polymers decreased by the amount of monoacetylferrocene. Thermal characteristic were found by Differential Scanning Calorimeter (DSC). Thermal analysis revealed that sulfonated polystyrene samples after addition of monoacetylferrocene displayed lower values of Tg. Microscopic analysis were made by Scanning Electron Microscopy (SEM) and single phase for each sample was observed. Besides, energy dispersed micro analysis showed an increase in the intensity of the iron (II) peaks that is related to the amount of monoacetylferrocene added to the SPS samples. Flame retardancy for each polymer was also examined and found that addition of monoacetylferrocene to sulfonated polystyrene does not change the Limiting Oxygen Index value (LOI)(17). However, LOI value for polystyrene is 18.

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity. / QC 20100924

ANEEL; FINEP; CAPES; CNPQ; Fundação Araucária / Neste trabalho é apresentada uma aplicação de sensores à fibra ótica baseados em redes de Bragg encapsulados em compósito de resina polimérica com reforço de fibra de vidro. Foram avaliadas três resinas epóxi comerciais. O objetivo do trabalho é caracterizar os compósitos e investigar a viabilidade de embeber sensores a fibra ótica, baseados em redes de Bragg em fibras óticas em compósito epóxi. Na caracterização das amostras, foram realizados: Ensaios para avaliar a tensão residual após a cura das amostras. Nos ensaios, foram utilizados sensores FBGs incrustados no compósito. Ensaios para avaliar a temperatura de transição vítrea através da técnica Calorimetria Exploratória Diferencial (DSC). Ensaios de tração axial e flexão simples utilizando máquina de teste universal e ensaios para avaliar o comportamento do compósito quando sujeito a uma carga fixa e temperatura variando de 20 °C até a temperatura limite da transição vítrea do compósito. Os resultados mostram um elevado grau de integração das FBGs no compósito epóxi. Um dos resultados é promissor para aplicações em um gerador de alta potência e em ambientes hostis com temperatura de trabalho até 127 °C. / This work presents an application of optical fiber sensors based on Bragg gratings encapsulated in polymeric composite resin with glass fiber reinforcement. Three commercial epoxy resins were evaluated. The main objective of the study is to characterize the composites and investigate the feasibility of embedding the optical fiber sensors based on Bragg gratings in epoxy composite. In the characterization of the samples tensile tests were performed to evaluate the residual stress after the curing process. The residual stress was investigated by mains of a FBG sensor embedded in the composite. Additionally, tests were conducted to evaluate the glass transition temperature by DSC technique. The values of the axial tensile and simple flexural stress were investigated using a universal testing machine. In addition, tests were performed for evaluating the composite behavior when subjected to a fixed load and variable temperature ranging from 20 °C to the temperature limit of the glass transition of the composite. The results show a high level of integration of the FBGs with the epoxy composite. One of the results is promising for applications in a high power generator and in hostile environments working at temperatures up to 127 °C. / 5000

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This paper describes a study of reaction parameters in a ball mill in the synthesis of 1H-pyrazole acid catalyzed. The studied parameters were: amount of reactants, frequency, reaction time, amount of catalyst, number of milling balls, different catalysts, diameter and material of the milling balls. Another study was conducted to study the mechanism of solid-solid reaction involved in the cyclocondensation between enaminones and hydrazine hydrochloride acid catalyzed. These mixtures were studied by thermal analysis (TGA and DSC), where eight enaminones were evaluated and four eutectic mixtures were identified. / Este trabalho descreve o estudo de parâmetros reacionais em moinho de bolas na síntese de 1H-pirazol catalisada por ácido. Os parâmetros estudados foram: quantidade de reagentes, frequência, tempo, quantidade de catalisador, número de esferas, diferentes catalisadores, diâmetro e material das esferas de moagem. Outro trabalho realizado foi o estudo do mecanismo de reação sólido-sólido envolvido na reação de ciclocondensação entre enaminonas e cloridrato de hidrazina catalisada por ácido. Estas misturas foram estudadas através de análises térmicas (TGA e DSC), sendo que de oito enaminonas avaliadas, 4 misturas eutéticas binárias foram identificadas.

Os métodos termo-analíticos são ferramentas úteis na avaliação da compatibilidade entre fármacos e adjuvantes, com destaque à calorimetria exploratória diferencial. Neste trabalho foram avaliados a compatibilidade e o comportamento térmico entre a isoniazida e adjuvantes tecnológicos primários usualmente empregados em formas farmacêuticas sólidas. A compatibilidade foi examinada por meio da preparação de misturas físicas binárias do tipo fármaco/adjuvante. Foi investigada também a influência da granulação por via úmida e do processo de compactação para as misturas de isoniazida e adjuvantes com função de material de enchimento e carga e deslizante. A isoniazida apresentou um comportamento térmico não encontrado na literatura. Os adjuvantes avaliados foram: ácido esteárico, amido, celulose microcristalina, crospovidona, croscarmelose sódica, dióxido de silício coloidal estearato de magnésio, glicolato de amido sódico, hipromelose, lactose, manitol, polidona e talco. Para as misturas físicas, a maioria dos adjuvantes mostrou-se compatível com o fármaco em questão. Foram verificadas interações com o ácido esteárico, o glicolato de amido sódico, a lactose, o manitol e a povidona. A isoniazida mostrou a formação de uma mistura eutética com o manitol e de interação química com a lactose. A agregação por via úmida e o processo de compactação não mostraram influências adicionais na compatibilidade das misturas avaliadas. Os resultados observados foram confirmados por métodos não-térmicos como difratometria de raios X, espectroscopia de infravermelho e ressonância nuclear magnética. / Thermo-analytical methods, and specially Differential Scanning Calorimetry, are useful support for the evaluation of compatibility between drug substances and pharmaceutical excipients. In this work were studied the compatibility and the thermal behavior of isoniazid and pharmaceutical excipients, commonly used for the formulation of solid dosage forms. Colloidal silicon dioxide, corn starch, crospovidone, hypromellose, lactose, magnesium stearate, mannitol, microcrystalline cellulose, povidone, sodium croscarmellose, sodium starch glycolate, stearic acid and talc were the excipients employed in these experiments. The compatibility was analyzed testing binary physical drug/excipient admixtures. The effect of wet granulation and compression was also investigated, in this case especially between isoniazid, fillers and lubricant. For almost all excipients no incompatibilities with isoniazid were observed. Interactions were detected when the drug substance was added to stearic acid, sodium starch glycolate, lactose, mannitol and povidone. Isoniazid formed a euthetic mixture with mannitol, whereas a possible chemical reaction occurred between isoniazid and lactose. Wet granulation and compaction of the tested admixtures did not affect the results observed above. These observations were confirmed by non-thermal techniques, such as X-Ray diffractometry, infrared spectroscopy and nuclear magnetic resonance.

Introdução: O gasto energético (GE) dos indivíduos pode ser determinado por diversos métodos, dentre os quais estão a calorimetria indireta (CI) e as equações de predição. Objetivo: Comparar o gasto energético de pacientes com doença pulmonar obstrutiva crônica (DPOC) medido através da CI com o estimado pela equação de Harris-Benedict (HB). Métodos: Estudo transversal incluindo 30 indivíduos com diagnóstico médico de DPOC, segundo critérios GOLD, atendidos no Centro de Reabilitação Pulmonar do Pavilhão Pereira Filho e do ambulatório de Pneumologia, ambos do Complexo Hospitalar Santa Casa de Porto Alegre, no período de fevereiro à setembro de 2010. O gasto energético foi mensurado pela CI usando monitor específico, assim como predito pela equação de HB. Os participantes também foram submetidos à avaliação antropométrica, através dos parâmetros de peso, altura, índice de massa corporal (IMC), dobra cutânea tricipital (DCT), circunferência do braço (CB) e circunferência muscular do braço (CMB), além de aplicação da avaliação nutricional subjetiva global (ANSG), bem como verificação do consumo alimentar. Os valores encontrados foram analisados através do teste t de Student, do teste qui-quadrado de McNemar e pelo método de Bland-Altman, e expressos pela média ± desvio-padrão, com nível de significância estatística p 0,05. Resultados: Do total de 30 portadores de DPOC, 70% eram do sexo masculino com idades de 62,5 ± 11,5 anos e IMC médio de 24,2 ± 4,2kg/m². O gasto energético em repouso (GER) medido pela CI foi de 1.568 ± 234,8kcal e o estimado pela equação de HB foi de 1.312 ± 120,5kcal, com diferença estatisticamente significativa entre os dois métodos (p<0,001). Quanto ao gasto energético total (GET), o valor medido pela CI foi de 2.038 ± 305,23kcal e o predito pela equação de HB foi de 2.047 ± 188kcal, sem apresentar diferença estatística significativa (p=0,853) e demonstrando uma concordância de 96,7% entre os dois métodos. Relativo ao diagnóstico nutricional, ao considerarmos apenas o IMC, 3,3% dos participantes apresentavam desnutrição, 63,3% eutrofia, 23,3% sobrepeso e 10% obesidade enquanto que pelo agrupamento de parâmetros (IMC, DCT, CB, CMB e ANSG), 53,3% dos pacientes apresentaram desnutrição, 33,3% eutrofia, 10% sobrepeso e 3,3% obesidade. Conclusão: O GER foi subestimado pela equação de HB, não apresentando boa concordância com o medido pela CI. Quanto ao GET, os resultados foram significativamente semelhantes demonstrando boa concordância entre os dois métodos. Em relação ao estado nutricional, talvez o IMC não seja suficiente para avaliar a real condição de pacientes com DPOC. / Introduction: The energy expenditure (EE) of individuals can be determined by various methods, among which are the indirect calorimetry (IC) and the prediction equations. Objective: To compare the energy expenditure of patients with chronic obstructive pulmonary disease (COPD) measured by the IC estimate by the Harris-Benedict equation (HB). Methods: Cross sectional study including 30 individuals diagnosed with COPD according to GOLD criteria, seen in the Pulmonary Rehabilitation Center of the Pereira Filho and outpatient pulmonology, both of Santa Casa Hospital Complex of Porto Alegre in the period from February to September 2010. Energy expenditure was measured by IC using a specific monitor, as predicted by the HB equation. Participants also underwent anthropometric assessment, through the parameters of weight, height, body mass index (BMI), triceps skinfold thickness (TSF), mid-arm circumference (MAC) and mid-arm muscle circumference (MAMC), and application subjective global nutritional assessment (SGA) and to determine food consumption. The values were analyzed using the Student t test, chi-square, McNemar and the Bland-Altman and expressed as mean + standart deviation, with statistical significance level p 0.05. Results: Of 30 patients with COPD, 70% were male, aged 62.5 ± 11.5 years and average BMI of 24.2 ± 4.2kg/m². The resting energy expenditure (REE) measured by IC was 1568 ± 234.8kcal and estimated by the HB equation was 1312 ± 120.5kcal, with a statistically significant difference between the two methods (p<0.001). As for the total energy expenditure (TEE), the value measured by ICwo methods (p <0.001). As for the total energy expenditure (TEE), the value measured by IC was 2038 ± 305.23kcal and foretold the HB equation was 2047 ± 188kcal, no statistical significant difference (p=0.853) and showed a concordance of 96,7% between the two methods. Concerning the nutritional diagnosis, we consider only the BMI, 3.3% of participants had malnutrition, 63.3% were eutrophic, 23.3% overweight and 10% were obese while the grouping of parameters (BMI, TSF, CB, CMB and SGA), 53.3% of patients suffered from malnutrion, 33.3% were eutrophic, 10% overweight and 3.3% obese. Conclusion: REE was underestimated by the HB equation, not a good agreement with that measured by IC. As for the GET, the results were significantly similar showing good agreement between the two methods. In relation to nutritional status, BMI may not be sufficient to evaluate the actual condition of patients with COPD.

Neste trabalho foram avaliados o comportamento do ácido gálico e de adjuvantes tecnológicos frequentemente empregados em formas farmacêuticas sólida, e das suas misturas físicas, através de métodos termoanalíticos e por espectroscopia de infravermelho. Foi investigada também a influência da compactação sobre as misturas físicas equiponderais. Os adjuvantes avaliados foram amidoglicolato de sódio, celulose microcristalina, croscarmelose sódica, crospovidona, dióxido de silício coloidal, estearato de magnésio e polimetacrilato. O ácido gálico apresentou um comportamento térmico diferenciado nas misturas, assumindo, provavelmente, uma forma instável com menor ponto de fusão. Os resultados obtidos por DSC demonstraram interação de natureza física com mudança de entalpia para misturas do ácido gálico com celulose microcristalina, crospovidona, estearato de magnésio e polimetacrilato. A interação não pode ser confirmada por espectroscopia de infravermelho para a crospovidona e polimetacrilato, devido à sobreposição das bandas com o ácido gálico. Os demais adjuvantes também apresentaram interação física, porém, sem alteração da entalpia, confirmada por espectroscopia de infravermelho, relacionada ao estabelecimento de ligações de hidrogênio entre os componentes da mistura. A compactação demonstrou particular influência sobre a interação com celulose microcristalina, croscarmelose sódica e crospovidona. / In this work were evaluated the behavior of the gallic acid and technological excipients used in sold dosage forms and their physical powder mixtures, by Differential Scanning Calorimetry (DSC) and Thermogravimety (TGA) and infrared spectroscopy (IR). The influence of the compression force on the 1:1 (w/w) physical mixtures was also investigated. The excipients evaluated were sodium starch glycolate, microcrystalline cellulose, croscarmellose sodium, crospovidone, colloidal silicon dioxide, magnesium stearate and polymethacrylate. Gallic acid presented a different thermal behavior in the mixtures, assuming, probably, an unstable form with a lower melting point. The results obtained by (DSC) demonstrated the occurrence of physical interactions with enthalpy changes for the mixtures of gallic acid with microcrystalline cellulose, crospovidone, magnesium stearate and polymethacrylate. The interaction could not be confirmed by infrared spectroscopy for crospovidone and polymethacrylate, due to overlapping of the gallic acid IR bands. The other excipients also presented physical interaction, however, without alteration of the enthalpy, confirmed by IR, which could be correlated to the establishment of hydrogen bonds between the components of the mixture. The compression of the powder mixtures demonstrated a particular influence of the interaction of gallic acid with microcrystalline cellulose, croscarmellose sodium and crospovidone.

Neste trabalho foram avaliados o comportamento do ácido gálico e de adjuvantes tecnológicos frequentemente empregados em formas farmacêuticas sólida, e das suas misturas físicas, através de métodos termoanalíticos e por espectroscopia de infravermelho. Foi investigada também a influência da compactação sobre as misturas físicas equiponderais. Os adjuvantes avaliados foram amidoglicolato de sódio, celulose microcristalina, croscarmelose sódica, crospovidona, dióxido de silício coloidal, estearato de magnésio e polimetacrilato. O ácido gálico apresentou um comportamento térmico diferenciado nas misturas, assumindo, provavelmente, uma forma instável com menor ponto de fusão. Os resultados obtidos por DSC demonstraram interação de natureza física com mudança de entalpia para misturas do ácido gálico com celulose microcristalina, crospovidona, estearato de magnésio e polimetacrilato. A interação não pode ser confirmada por espectroscopia de infravermelho para a crospovidona e polimetacrilato, devido à sobreposição das bandas com o ácido gálico. Os demais adjuvantes também apresentaram interação física, porém, sem alteração da entalpia, confirmada por espectroscopia de infravermelho, relacionada ao estabelecimento de ligações de hidrogênio entre os componentes da mistura. A compactação demonstrou particular influência sobre a interação com celulose microcristalina, croscarmelose sódica e crospovidona. / In this work were evaluated the behavior of the gallic acid and technological excipients used in sold dosage forms and their physical powder mixtures, by Differential Scanning Calorimetry (DSC) and Thermogravimety (TGA) and infrared spectroscopy (IR). The influence of the compression force on the 1:1 (w/w) physical mixtures was also investigated. The excipients evaluated were sodium starch glycolate, microcrystalline cellulose, croscarmellose sodium, crospovidone, colloidal silicon dioxide, magnesium stearate and polymethacrylate. Gallic acid presented a different thermal behavior in the mixtures, assuming, probably, an unstable form with a lower melting point. The results obtained by (DSC) demonstrated the occurrence of physical interactions with enthalpy changes for the mixtures of gallic acid with microcrystalline cellulose, crospovidone, magnesium stearate and polymethacrylate. The interaction could not be confirmed by infrared spectroscopy for crospovidone and polymethacrylate, due to overlapping of the gallic acid IR bands. The other excipients also presented physical interaction, however, without alteration of the enthalpy, confirmed by IR, which could be correlated to the establishment of hydrogen bonds between the components of the mixture. The compression of the powder mixtures demonstrated a particular influence of the interaction of gallic acid with microcrystalline cellulose, croscarmellose sodium and crospovidone.