908 resultados para poly(Ethylene Glycol) (PEG)
Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting
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Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.
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Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG+PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties.
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Superparamagnetic iron oxide nanoparticles for biomedical applications are usually coated with organic molecules to form a steric barrier against agglomeration. The stability of these coatings is well established in the synthesis medium but is more difficult to assess in physiological environment. To obtain a first theoretical estimate of their stability in such an environment, we perform density functional theory calculations of the adsorption of water, polyvinyl alcohol (PVA) and polyethylene glycol (PEG) coating molecules, as well as the monomer and dimer of glycine as a prototype short peptide, on the (110) surface of magnetite (Fe3O4) in vacuo. Our results show that PVA binds significantly stronger to the surface than both PEG and glycine, while the difference between the latter two is quite small. Depending on the coverage, the wateradsorption strength is intermediate between PVA and glycine. Due to its strongly interacting OH side groups, PVA is likely to remain bound to the surface in the presence of short peptides. This stability will have to be further assessed by molecular dynamics in the solvated state for which the present work forms the basis.
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Context. The ESA Rosetta spacecraft (S/C) is tracking comet 67P/Churyumov-Gerasimenko in close vicinity. This prolonged encounter enables studying the evolution of the volatile coma composition. Aims. Our work aims at comparing the diversity of the coma of 67P/Churyumov-Gerasimenko at large heliocentric distance to study the evolution of the comet during its passage around the Sun and at trying to classify it relative to other comets. Methods. We used the Double Focussing Mass Spectrometer (DFMS) of the ROSINA experiment on ESA’s Rosetta mission to determine relative abundances of major and minor volatile species. This study is restricted to species that have previously been detected elsewhere. Results. We detect almost all species currently known to be present in cometary coma with ROSINA DFMS. As DFMS measured the composition locally, we cannot derive a global abundance, but we compare measurements from the summer and the winter hemisphere with known abundances from other comets. Differences between relative abundances between summer and winter hemispheres are large, which points to a possible evolution of the cometary surface. This comet appears to be very rich in CO2 and ethane. Heavy oxygenated compounds such as ethylene glycol are underabundant at 3 AU, probably due to their high sublimation temperatures, but nevertheless, their presence proves that Kuiper belt comets also contain complex organic molecules.
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The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^
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The efficacy of waste stabilization lagoons for the treatment of five priority pollutants and two widely used commercial compounds was evaluated in laboratory model ponds. Three ponds were designed to simulate a primary anaerobic lagoon, a secondary facultative lagoon, and a tertiary aerobic lagoon. Biodegradation, volatilization, and sorption losses were quantified for bis(2-chloroethyl) ether, benzene, toluene, naphthalene, phenanthrene, ethylene glycol, and ethylene glycol monoethyl ether. A statistical model using a log normal transformation indicated biodegradation of bis(2-chloroethyl) ether followed first-order kinetics. Additionally, multiple regression analysis indicated biochemical oxygen demand was the water quality variable most highly correlated with bis(2-chloroethyl) ether effluent concentration. ^
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Objetivos: reducir pérdidas durante la conservación frigorífica, emplear atmósfera modificada como método suplementario a la refrigeración, alargar el período de aptitud comercial. Metodología: se trabajó con fruta acondicionada a 0±1 °C y 90±5 % HR, según las siguientes variantes: 1. testigo: 20 kg fruta a granel sin seleccionar en caja plástica; 2. granel + film PVC: 10 kg de fruta a granel en bandejas de madera más cartón corrugado recubierta con film de PVC; 3. celpack: bandejas de madera recubiertas de cartón corrugado con dos celpack de 23 frutos cada uno; 4. celpack + atmósfera modificada: ídem anterior pero cada celpack en bolsa de polietileno de baja densidad de 20 μ. A partir de los 30 días de conservación se extrajo semanalmente, durante 9 semanas, una muestra de 46 frutos, de los cuales 23 fueron analizados al momento de ser extraídos y los 23 restantes luego de 48 horas de comercialización simulada (sc). Para la evaluación estadística se aplicó análisis de la varianza con el programa SAS (Statistical Analysis System) y se determinaron las diferencias entre tratamientos con el test de Duncan. Para sabor, en cambio, se aplicó una prueba de homogeneidad de P2. La evaluación de sabor se realizó mediante degustación con panel de 5 catadores entrenados. Resultados: Los frutos tenían las siguientes características al inicio de conservación: calibre 61.4 mm, peso 117.8 g, firmeza de pulpa 3.1 kgf, sabor agridulce, contenido de sólidos solubles 17.5 °Bx, acidez 0.78 g ác. málico%g, % cubrimiento 83.69 %. Luego de la conservación frigorífica (97días): % de color de cobertura 95 %. La firmeza de la pulpa en el tratamiento celpack + bolsa se diferencia con valores más altos, media de 2.8 kgf , el resto con media 2.6 kgf. En sc la firmeza es inferior y esta disminución es menor en celpack + bolsa. Sólidos solubles, media 17.21 °Bx, en sc valores con media de un 0.3 % más. Acidez titulable: disminución progresiva, de 0.68 a 0.47 g%g al fin de conservación. Sabor: a partir de los 59 días aumentan los frutos insípidos y desagradables excepto en celpack + bolsa. Síntomas de deshidratación: a partir de los 79 días la única variante que no presenta síntomas es celpack + bolsa. Conclusiones: El acondicionamiento en celpack redujo la incidencia de ataque por mohos (fue el único tratamiento sin ataque durante 94 días); tampoco presentó sabores desagradables y su limitación en conservación se debió a la deshidratación evidente a partir de 74 días. La fruta embalada en celpack + bolsa tuvo mayores valores de resistencia a la presión y 100 % de frutos sin deshidratación a los 94 días de conservación; a partir de 80 días es evidente el ataque de mohos y frutos con sabores desagradables. Las variantes granel y granel + film presentan deterioro por deshidratación a partir de 74 días. La conservación no debería superar 80 días. Celpack + bolsa muestra mejores resultados, con mayores valores de resistencia a la presión que los otros tratamientos; con respecto al sabor, mantiene una mayor proporción de sabor dulce.
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El contenido de materia orgánica (MO) del suelo se relaciona positivamente, en algunos casos, con la superficie específica (SE) del mismo, pero grandes cantidades de MO pueden resultar en una SE menor, cuando se la determina a través de la adsorción de nitrógeno a bajas temperaturas. Tradicionalmente se asocia a la MO una elevada SE, del orden de los 500 a 800 m2 g-1. Estos valores, obtenidos mediante técnicas que emplean solventes polares, por ejemplo, etilenglicol, hicieron suponer que la misma contribuye en gran medida a la SE del suelo. No obstante, hay referencias que señalan una sobrestimación de los resultados obtenidos, atribuida a la utilización de estos solventes. Mediciones que emplean el método con adsorción de N2 a 77 K, cuya interpretación se basa en el modelo de Brunauer, Emmett y Teller (BET), considerado internacionalmente como método patrón, arrojan valores de SE para la MO que son menores que los primeros, en dos órdenes de magnitud. Con el objetivo de aportar conocimiento que contribuyera a una mejor comprensión del papel de la MO en procesos edáficos, se realizaron determinaciones de SE en muestras de un Argiudol típico, con diferentes contenidos de MO y contenido de arcilla constante (20%). Las medidas se llevaron a cabo interpretando las isotermas de adsorción de N2 a 77 K, mediante el modelo BET. Los resultados obtenidos mostraron una relación no lineal negativa entre la SE y el contenido de MO. La eliminación de la MO mediante H2O2 incrementó la SE de las muestras en todos los casos. La variación de SE por unidad de MO eliminada sugiere que la misma no se adsorbe de manera uniforme y continua sobre la superficie de la fase mineral.
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Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C—C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.
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The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux–response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of ≈1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux–response coefficient, in one case to values of ≈1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux–response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.
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Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca2+ or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca2+ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca2+-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca2+. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca2+. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca2+ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca2+ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca2+ from both intracellular and extracellular sources.
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Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.
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O trabalho visa o desenvolvimento do sistema para medidas de distribuição de corrente e ampliação de escala (50 cm²) buscando aperfeiçoar as condições de preparação do conjunto eletrodo membrana (MEA) quanto às condições de operação da célula e avaliar a melhor geometria. Foram realizados estudos de síntese de catalisadores de Pt-M e avaliação do desempenho desses materias e das rotas de síntese utilizadas com objetivo de aplicar estes materias em sistemas de maior escala. A insuficiência do desempenho e estabilidade dos catalisadores são fatores que ainda inviabilizam o uso em larga escala das células a combustível de eletrólito polimérico sólido, destacando-se as perdas associadas ao desempenho do cátodo. Os catalisadores preparados foram nanopartículas bimetálicas PtM/C (M = Fe, Co e Ni) suportadas em carbono de elevada área superficial, por duas rotas sintéticas. Foram utilizadas as rotas: ácido fórmico e etilenoglicol modificado (EG). Em ambas as rotas se buscou catalisadores com alto grau de incorporação do segundo metal, tamanho de partícula pequeno e bom desempenho catalítico do cátodo. Observou-se que pela rota do ácido fórmico com modificações no processo de síntese é possível obter a incorporação nominal do segundo metal no catalisador, porém há desvantagem de o tamanho de partícula ser elevado. Pela rota do EG obteve-se catalisadores com pequeno tamanho de partícula, porém a incorporação do segundo metal mostrou-se ineficiente. Os estudos de ampliação de escala foram realizados em células de 50 cm2 variando-se as condições de operação; i) diferentes placas de distribuição de gás, e ii) diferentes valores de fluxo dos gases reagentes. Foi observado que a baixos fluxos de gases a quantidade de reagente é insuficiente para ser difundida por todo eletrodo, o que ocasiona reação apenas na região de entrada de gases no sistema, ocasionando uma rápida limitação em obter-se densidades de corrente alta. Pode-se observar que a diferença de desempenho entre as placas é pequena, porém a placa serpentina 6 apresentou melhor desempenho. O desempenho dos cátodos preparados com catalisadores comerciais e os sintetizados no laboratório nas células de 50 cm² mostrou sofrer bastante influência das condições de operação comparada com as células de 4,6 cm².
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As diversas aplicações tecnológicas de nanopartículas magnéticas (NPM) vêm intensificando o interesse por materiais com propriedades magnéticas diferenciadas, como magnetização de saturação (MS) intensificada e comportamento superparamagnético. Embora MNP metálicas de Fe, Co e bimetálicas de FeCo e FePt possuam altos valores de MS, sua baixa estabilidade química dificulta aplicações em escala nanométrica. Neste trabalho foram sintetizadas NPM de Fe, Co, FeCo e FePt com alta estabilidade química e rigoroso controle morfológico. NPM de óxido metálicos (Fe e Co) também foram obtidas. Dois métodos de síntese foram empregados. Usando método baseado em sistemas nanoheterogêneos (sistemas micelares ou de microemulsão inversa), foram sintetizadas NPM de Fe3O4 e Co metálico. Foram empregados surfactantes cátion-substituídos: dodecil sulfato de ferro(III) (FeDS) e dodecil sulfato de cobalto(II) (CoDS). Para a síntese das NPM, foram estudados e determinados a concentração micelar crítica do FeDS em 1-octanol (cmc = 0,90 mmol L-1) e o diagrama de fases pseudoternário para o sistema n-heptano/CoDS/n-butanol/H2O. NPM esferoidais de magnetita com3,4 nm de diâmetro e comportamento quase-paramagnético foram obtidas usando sistemas micelares de FeDS em 1-octanol. Já as NPM de Co obtidas via microemulsão inversa, apesar da larga distribuição de tamanho e baixa MS, são quimicamente estáveis e superparamagnéticas. O segundo método é baseado na decomposição térmica de complexos metálicos, pelo qual foram preparadas NPM esféricas de FePt e de óxidos metálicos (Fe3O4, FeXO1-X, (Co,Fe)XO1-X e CoFe2O4) com morfologia controlada e estabilidade química. O método não mostrou a mesma efetividade na síntese de NPM de FeAg e FeCo: a liga FeAg não foi obtida enquanto que NPM de FeCo com estabilidade química foram obtidas sem controle morfológico. NPM de Fe e FeCo foram preparadas a partir da redução térmica de NPM de Fe3O4 e CoFe2O4, as quais foram previamente recobertas com sílica. A sílica previne a sinterização inter-partículas, além de proporcionar caráter hidrofílico e biocompatibilidade ao material. As amostras reduzidas apresentaram aumento dos valores de MS (entre 21,3 e 163,9%), o qual é diretamente proporcional às dimensões das NPM. O recobrimento com sílica foi realizado via hidrólise de tetraetilortosilicato (TEOS) em sistema de microemulsão inversa. A espessura da camada de sílica foi controlada variando-se o tempo de reação e as concentrações de TEOS e de NPM, sendo então proposto um mecanismo do processo de recobrimento. Algumas amostras receberam um recobrimento adicional de TiO2 na fase anatase, para o qual foi empregado etilenoglicol como solvente e ligante para formação de glicolato de Ti como precursor. A espessura da camada de TiO2 (2-12 nm) é controlada variando as quantidades relativas entre NPM e o precursor de Ti. Ensaios de hipertermia magnética foram realizados para as amostras recobertas com sílica. Ensaios de hipertermia magnéticas mostram grande aumento da taxa de aquecimento das amostras após a redução térmica, mesmo para dispersões diluídas de NPM (0,6 a 4,5 mg mL-1). Taxas de aquecimento entre 0,3 e 3,0oC min-1 e SAR entre 37,2 e 96,3 W g-1. foram obtidos. A atividade fotocatalítica das amostras recobertas foram próximas à da fase anatase pura, com a vantagem de possuir um núcleo magnético que permite a recuperação do catalisador pela simples aplicação de campos magnéticos externos. Os resultados preliminares dos ensaios de hipertermia magnética e fotocatálise indicam um forte potencial dos materiais aqui relatados para aplicações em biomedicina e em fotocatálise.
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The transitions and reactions involved in the thermal processing of binary mixtures of polyethylene and poly(ethylene-co-vinyl acetate) copolymers with different concentrations of a foaming agent (azodicarbonamide) were studied using differential scanning calorimetry (DSC). The effect of ZnO as a kicker also was discussed. The temperature at the maximum rate and the heat evolved were measured for all the processes—melting, transitions, and reactions—all the mixtures prepared were measured and compared. Azodicarbonamide decomposed differently depending on the polymeric matrix. These data can be very useful for the plastic processing industry.