Structural characterization and immunogenicity in wildtype and immune tolerant mice of degraded recombinant human interferon alpha2b

Purpose - To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods - RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance. Results - M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions - RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon Alpha2b

Purpose: This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods: RhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. Results: M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions: RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Structural and optical studies of Au doped titanium oxide films

Thin films of TiO2 were doped with Au by ion implantation and in situ during the deposition. The films were grown by reactive magnetron sputtering and deposited in silicon and glass substrates at a temperature around 150 degrees C. The undoped films were implanted with Au fiuences in the range of 5 x 10(15) Au/cm(2)-1 x 10(17) Au/cm(2) with a energy of 150 keV. At a fluence of 5 x 10(16) Au/cm(2) the formation of Au nanoclusters in the films is observed during the implantation at room temperature. The clustering process starts to occur during the implantation where XRD estimates the presence of 3-5 nm precipitates. After annealing in a reducing atmosphere, the small precipitates coalesce into larger ones following an Ostwald ripening mechanism. In situ XRD studies reveal that Au atoms start to coalesce at 350 degrees C, reaching the precipitates dimensions larger than 40 nm at 600 degrees C. Annealing above 700 degrees C promotes drastic changes in the Au profile of in situ doped films with the formation of two Au rich regions at the interface and surface respectively. The optical properties reveal the presence of a broad band centered at 550 nm related to the plasmon resonance of gold particles visible in AFM maps. (C) 2011 Elsevier B.V. All rights reserved.

Nanotech thin film photovoltaics

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia de Electrónica e Telecomunicações

Automatic assignment of absolute configuration from 1D NMR data

Opposite enantiomers exhibit different NMR properties in the presence of an external common chiral element, and a chiral molecule exhibits different NMR properties in the presence of external enantiomeric chiral elements. Automatic prediction of such differences, and comparison with experimental values, leads to the assignment of the absolute configuration. Here two cases are reported, one using a dataset of 80 chiral secondary alcohols esterified with (R)-MTPA and the corresponding 1H NMR chemical shifts and the other with 94 13C NMR chemical shifts of chiral secondary alcohols in two enantiomeric chiral solvents. For the first application, counterpropagation neural networks were trained to predict the sign of the difference between chemical shifts of opposite stereoisomers. The neural networks were trained to process the chirality code of the alcohol as the input, and to give the NMR property as the output. In the second application, similar neural networks were employed, but the property to predict was the difference of chemical shifts in the two enantiomeric solvents. For independent test sets of 20 objects, 100% correct predictions were obtained in both applications concerning the sign of the chemical shifts differences. Additionally, with the second dataset, the difference of chemical shifts in the two enantiomeric solvents was quantitatively predicted, yielding r2 0.936 for the test set between the predicted and experimental values.

Espetroscopia (1H) por ressonância magnética do disco intervertebral lombar no adulto e sua aplicação na rotina imagiológica

Objetivos – Demonstrar o potencial da espetroscopia (1H) por ressonância magnética na doença degenerativa discal lombar e defender a integração desta técnica na rotina clínico‑imagiológica para a precisa classificação da involução vs degenerescência dos discos L4‑L5 e L5‑S1 em doentes com lombalgia não relacionável com causa mecânica. Material e métodos – O estudo incluiu 102 discos intervertebrais lombares de 123 doentes. Foram estudados 61 discos de L4‑L5, 41 discos de L5‑S1 e 34 discos de D12‑L1. Utilizou‑se um sistema de ressonância magnética de 1,5 T e técnica monovoxel. Obtiveram‑se os rácios [Lac/Nacetyl] e [Nacetyl/(Lac+Lípidos)] e aplicou‑se a ressonância de lípidos para avaliar a bioquímica do disco com o fim de conhecer o estado de involução vs degenerescência que o suscetibilizam para a instabilidade e sobrecarga. Avaliou‑se o comportamento dos rácios e do teor lipídico dos discos L4‑L5‑S1 e as diferenças apresentadas em relação a D12‑L1. Foi também realizada a comparação entre os discos L4‑L5, L5‑S1 e D12‑L1 na ponderação T2 (T2W), segundo a classificação ajustada (1‑4) de Pfirrmann. Resultados – Verificou‑se que os rácios e o valor dos lípidos dos discos L4‑L5‑S1 apresentaram diferenças estatisticamente significativas quando relacionados com os discos D12‑L1. O rácio [Lac/Nacetyl] em L4‑L5‑S1 mostrou‑se aumentado em relação a D12‑L1 (p=0,033 para os discos com grau de involução [1+2] e p=0,004 para os discos com grau [3+4]). Estes resultados sugerem que a involução vs degenerescência dos discos nos graus mais elevados condiciona um decréscimo do pico do Lactato. O rácio [Nacetyl/(Lac+Lip)] discrimina os graus de involução [1+2] do [3+4] no nível L4‑L5, apresentando os valores dos rácios (média 0,65 e 0,5 respetivamente com p=0,04). O rácio médio de [Nacetyl/(Lac+Lip)] dos discos L4‑L5 foi 1,8 vezes mais elevado do que em D12‑L1. O espetro lipídico em L4‑L5‑S1 nos graus mais elevados não mostrou ter uma prevalência constante quanto às frequências de ressonância. Conclusão – A espetroscopia (1H) dos discos intervertebrais poderá ter aplicação na discriminação dos graus de involução vs degenerescência e representar um contributo semiológico importante em suplemento à ponderação T2 convencional. As ressonâncias de lípidos dos discos L4‑L5 e L5‑S1, involuídos ou degenerados, devem ser avaliadas em relação a D12‑L1, utilizando este valor como referência, pois este último é o nível considerado estável e com baixa probabilidade de degenerescência.

Visible range plasmonic effect produced by aluminium nanoparticles embedded in amorphous silicon

We present results, obtained by means of an analytic study and a numerical simulation, about the resonant condition necessary to produce a Localized Surface Plasmonic Resonance (LSPR) effect at the surface of metal nanospheres embedded in an amorphous silicon matrix. The study is based on a Lorentz dispersive model for a-Si:H permittivity and a Drude model for the metals. Considering the absorption spectra of a-Si:H, the best choice for the metal nanoparticles appears to be aluminium, indium or magnesium. No difference has been observed when considering a-SiC:H. Finite-difference time-domain (FDTD) simulation of an Al nanosphere embedded into an amorphous silicon matrix shows an increased scattering radius and the presence of LSPR induced by the metal/semiconductor interaction under green light (560 nm) illumination. Further results include the effect of the nanoparticles shape (nano-ellipsoids) in controlling the wavelength suitable to produce LSPR. It has been shown that is possible to produce LSPR in the red part of the visible spectrum (the most critical for a-Si:H solar cells applications in terms of light absorption enhancement) with aluminium nano-ellipsoids. As an additional results we may conclude that the double Lorentz-Lorenz model for the optical functions of a-Si:H is numerically stable in 3D simulations and can be used safely in the FDTD algorithm. A further simulation study is directed to determine an optimal spatial distribution of Al nanoparticles, with variable shapes, capable to enhance light absorption in the red part of the visible spectrum, exploiting light trapping and plasmonic effects. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solubilities of C‑Tetraalkylcalix[4]resorcinarenes in SCCO2: experimental measurements, characterization, and correlation

The solubilities of two C-tetraalkylcalix[4]resorcinarenes, namely C-tetramethylcalix[4]resorcinarene and C-tetrapentylcalix[4]resorcinarene, in supercritical carbon dioxide (SCCO2) were measured in a flow-type apparatus at a temperature range from (313.2 to 333.2) K and at pressures from (12.0 to 35.0) MPa. The C-tetraalkylcalix[4]resorcinarenes were synthesized applying our optimized procedure and fully characterized by means of gel permeation chromatography, infrared and nuclear magnetic resonance spectroscopy. The solubilities of the C-tetraalkylcalix[4]resorcinarenes in SCCO2 were determined by analysis of the extracts obtained by HPLC with ultraviolet (UV) detection methodology adapted by our team. Four semiempirical density-based models, and the SoaveRedlichKwong cubic equation of state (SRK CEoS) with classical mixing rules, were applied to correlate the solubility of the calix[4]resorcinarenes in the SC CO2. The physical properties required for the modeling were estimated and reported.