Mesoscale simulation of grain boundary diffusion creep in the presence of grain growth

Grain-boundary (GB) diffusion creep (Coble creep) is the dominant deformation mechanism for the fine-grained materials under low stress and at elevated temperature. During creep deformation the grains become elongated in the tensile direction because of atoms diffusion along GBs from places in compression to those in tension. Consequently, the GB diffusion rate depends on the normal stress gradient along the boundaries. It is widely accepted that the GB migration generally plays two important roles during Coble creep: one leading to the decrease of the creep rate due to the increase of the grain size by GB migration mediated grain growth and the other one leading to the relaxation of the stress concentrations along the GBs and at the triple junctions. In this study we use mesoscopic simulations to investigate the influence of the external stress and grain-boundary migration (static grain growth) on creep deformation of polycrystalline materials. Our simulation methodology is based on the variational principle of dissipated power and the simulation results reveal that the grains comprising the microstructure remain almost equiaxed during grain-boundary diffusion creep with accommodation by GB migration. In addition, the average grain size of the evolving microstructure is controlled by the interplay between the static and dynamic grain growth and depends strongly on both the externally applied stress and the strain

Alpha-tocopherol: extraction from rice bran by microwave-assisted method, and entrapment and release from polymeric nanoparticles

The purpose of this study was two-fold, 1) to extract and quantify vitamin E components from rice bran using microwave extraction, to determine the antioxidant activity of the rice bran oil, and the effect of solvent and temperature on vitamin E components and oil yield, and 2) to entrap alpha-tocopherol into polymeric nanoparticles, to characterize the nanoparticles in terms of morphology, size and size distribution, zeta potential, entrapment efficiency, and amount of residual PVA associated with the nanoparticles, as well as to study the release of alpha-tocopherol from PLGA nanoparticles. Microwave-assisted extraction was an efficient method for the extraction of oil and vitamin E components from rice bran. Hexane was a better solvent for rice bran oil extraction as compared to isopropanol at 40ºC. At higher temperature, isopropanol was a better solvent for oil extraction. Hexane extracted large amount of α-tocotrienol at 120ºC while the increase in temperature for isopropanol was more beneficial for the extraction of γ-tocopherol. No significant differences in the oil yield, total vitamin E, and antioxidant activity of rice bran oil was noticed between the conventional solvent and microwave-assisted extractions, at 40ºC. For the second part of the study, emulsion evaporation method was used to synthesize spherical PLGA(αT) nanoparticles with SDS and PVA as surfactants. For SDS nanoparticles, the size of the nanoparticles decreased significantly with the entrapment of α-tocopherol in the PLGA matrix, while the size of PVA nanoparticles remained unchanged. The PDI after synthesis was under 0.100 for PVA nanoparticles and around 0.150 for SDS nanoparticles. Zeta potential was negative for all PVA nanoparticles. The entrapment efficiency of α-tocopherol in the polymeric matrix was approximately 89% and 95% for nanoparticles with 8% and 16% α-tocopherol theoretical loading. The residual PVA associated to the nanoparticles after purification was approximately 6% (w/w relative to the nanoparticles). The release profile showed an initial burst followed by a slower release of the α-tocopherol entrapped inside the PLGA matrix. The release for nanoparticles with 8% α-tocopherol theoretical loading (86% released/first hour) was faster than the release for the nanoparticles with 16% α-tocopherol theoretical loading (34% released/first hour)

α-Synuclein Interaction with Membranes

The association of α-synuclein with membranes appears to be an important factor in Parkinson's disease. My dissertation research was focused on understanding the interactions of the protein with artificial membranes, the extracellular plasma membrane, and intracellular membranes. Fluorescence and nuclear magnetic resonance spectroscopy were used to monitor these interactions. Large unillamelar vesicles with a composition similar to mitochondrial membranes were studied. Cardiolipin, present mainly in the inner mitochondrial membrane, is key for protein binding, and reducing the amount of cardiolipin decreases binding. The nature of cardiolipin's acyl chains is also important; cardiolipin with chains containing one double bond interact more strongly than those with chains having two double bonds or saturated acyl chains. This finding is physiologically relevant for Parkinson's disease because cardiolipin containing fatty acids with one double bond are the most abundant phospholipid in the brain. The affinity of α-synuclein for plasma membrane was tested, and only N-terminal region of the protein binds. Also, 19F NMR proved useful for monitoring the interactions of proteins and fused peptide-proteins with the plasma membrane. Information on protein interactions with intracellular membranes reveals that the N-terminal region may be cleaved in cells, but further studies are needed to confirm this idea.Doctor of Philosoph

Interaction of α-Synuclein and a Cell Penetrating Fusion Peptide with Higher Eukaryotic Cell Membranes Assessed by 19 F NMR

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a 19F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of 19F resonances of 3-fluoro-L-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

Abstractα-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and 19F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The 19F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes

Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids

Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis

Synthetic peptides incorporating well-folded β-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG β-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the β-hairpin-conjugated substrate and its PKCα-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity

α-Synuclein Conformation Affects Its Tyrosine-Dependent Oxidative Aggregation †

Oxidative stress and aggregation of the protein α-synuclein are thought to be key factors in Parkinson’s disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including α-synuclein. Here, we examine the role of each of α-synuclein’s four tyrosine residues and how the protein’s conformation affects covalent oxidative aggregation. When α-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when α-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson’s disease

α-Synuclein Conformation Affects Its Tyrosine-Dependent Oxidative Aggregation †

Oxidative stress and aggregation of the protein α-synuclein are thought to be key factors in Parkinson’s disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including α-synuclein. Here, we examine the role of each of α-synuclein’s four tyrosine residues and how the protein’s conformation affects covalent oxidative aggregation. When α-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when α-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson’s disease