No description available.

No description available.

This work focuses on understanding deformation mechanisms and responsiveness associated with the wrinkling, folding, and snapping of thin polymer films. We demonstrated the use of elastic instabilities in confined regimes, such as the crumpling and snapping of surface attached sheets. We gained fundamental insight into a thin film's ability to localize strain. By taking advantage of geometric strain localization we were able to develop new strategies for responsive surfaces that will have a broad impact on adhesive, optical, and patterning applications. Using the rapid closure of the Venus flytrap's leafets as dictated by the onset of a snap instability as motivation, we created surfaces with patterned structures to transition through a snap instability at a prescribed stress state. This mechanism causes surface topography to change over large lateral length scales and very short timescales. Changes in the stress state can be related to triggers such as chemical swelling, light-induced architecture transitions, mechanical pressure, or voltage. The primary advantages of the snap transition are that the magnitude of change, the rate of change, and the sensitivity to change can be dictated by a balance of materials properties and geometry. The patterned structures that exhibit these dynamics are elastomeric shells that geometrically localize strain and can snap between concave and convex curvatures. We have demonstrated the control of the microlens shell geometry and that the transition time follows scaling relationships presented for the Venus flytrap. Furthermore, the microlens arrays have been demonstrated as surfaces that can alter wettability. Using a similar novel processing technique, microarrays of freestanding elastomeric plates were placed in equibiaxial compression to fabricate crumpled morphologies with strain localized regions that are difficult to attain through traditional patterning techniques. The microstructures that form can be initially described using classical plate buckling theory for circular plates under an applied compressive strain. Upon the application of increasing compressive strain, axisymmetric microstructures undergo a secondary bifurcation into highly curved, nonaxisymmetric structures. The inherent interplay between geometry and strain in these systems provides a mechanism for generating responsiveness in the structures. By swelling the elastomeric plates with a compatible solvent, we demonstrated the microstructures ability to reversibly switch between axisymmetric and nonaxisymmetric geometries. To further explore the localization of strain in materials, we have fabricated sharply folded films of glassy, homogenous polymers directly on rigid substrates. The films were uniaxially compressed and buckle after delaminating from the substrate. As the applied strain is increased, we observed strain localization at the center of the delaminated features. We found that normally brittle, polystyrene films can accommodate excessive compressive strains without fracture by undergoing these strain localizing fold events. This technique provided a unique way to examine the curvature and stability of folded features, but was not adequate for understanding the onset of folding. By taking thin films, either glassy or elastomeric, and simply lifting them from the surface of water, we observed and quantified the wrinkle-to-fold transition in an axisymmetric geometry. The films initially wrinkle as they are lifted with a wavelength that is determined by the film thickness and material properties. The wrinkle-to-fold transition is analogous to the transition observed in uniaxially compressed films, but the axisymmetric geometry caused the fold to act as a disclination that increased the radial stress in the film, thereby decreasing the wavelength of the remaining wrinkles. Further straining the films caused the remaining wrinkles to collapse into a discrete number of folds that is independent of film thickness and material properties.

No description available.

Understanding architecture through a contemporary context of the transformations of art and technology was the springboard for this thesis. Identifying folding as a basic transformation became the focus for developing an Old town, Alexandria, Virginia law firm building. Folding is conceptually used in the spatial and inhabitable forms of the building as well as the materials, textures, and finishes of the walls, ceilings, and floors. Folding is structurally investigated by taking once planer and flimsy elements and creating folded, rigid, and load-bearing elements. Architectural concepts of day lighting, shading, rain runoff, partitioning, vertical circulation, horizontal circulation, library stacks, file storage, solar energy collection, gardening, building services, furnishings, reading, and inhabitation are all thought of in terms of folding. Designing a law firm for Old town, Alexandria, Virginia was chosen from a random number generating process cross referenced with the Alexandria, Virginia phone book. I interviewed a local law firm and based the programmatic spaces on their office needs and relationships. / Master of Architecture

Yes / It is important for the formulators of biopharmaceuticals to predict the folding–unfolding transition of proteins. This enables them to process proteins under predetermined conditions, without denaturation. Depending on the apparent denaturation temperature (Tm) of lysozyme, we have derived an equation describing its folding–unfolding transition diagram. According to the water content and temperature, this diagram was divided into three different areas, namely, the area of the water-folded lysozyme phase, the area of the water-folded lysozyme phase and the bulk water phase, and the area of the denatured lysozyme phase. The water content controlled the appearance and intensity of the Raman band at ∼1787 cm–1 when lysozyme powders were thermally denatured at temperatures higher than Tm. / MAM gratefully acknowledges CARA (Stephen Wordsworth and Ryan Mundy) and University of Bradford for providing an academic fellowship.

Protein sequences may evolve to avoid highly hydrophobic local regions of sequence, in part because such sequences promote nonnative aggregation. Hydrophobic local sequences are avoided in proteins even in buried regions, where native structure requirements tend to favor them. In this dissertation, I describe three explorations of this hydrophobic suppression. In Chapter 2, I examine the occurrence of hydrophobic and polar residues in completely buried β-strand elements, and find evidence for hydrophobic suppression that decreases as a β-strand becomes more exposed. In Chapter 3, I present a generalized study of the tendency of local sequences to deviate from the hydropathy (hydrophobicity) expected based on their solvent exposure. First, I examined the hydropathy of local and nonlocal sequence groups over a large range of solvent exposures, within folded protein domains in the ASTRAL Compendium database; second, I calculated the tendency of residues within 10 positions of a nonpolar or polar reference residue to deviate from the hydropathy expected based on their structural environment. Both analyses suggested that protein sequences exhibit 'local hydropathic balance' across a range of 6-7 residues, meaning that polar and nonpolar residues are more dispersed in the sequence than expected based on solvent exposure patterns. This balance occurs in all major fold classes, domain sizes and protein functions. An unexpected finding was that it partly arises from a tendency of buried or exposed residues to be flanked by polar or nonpolar residues, respectively. This relationship may result from evolutionary selection for folding efficiency, which might be enhanced by reduced local competition for buried or exposed sites during folding. Finally, in Chapter 4, I present several exploratory analyses, including a decision-tree approach, to visualize the influence of a large number of sequence-structure properties on residue hydrophobicity. Overall, the work in this dissertation confirms that hydrophobic suppression and local hydropathic balance in general are intrinsic properties of folded proteins. I speculate that local hydropathic balance results from selection for reduced aggregation propensity, increased folding efficiency and increased native state specificity. The concept of local hydropathic balance might be used to improve the properties of designed and engineered proteins.

Proteínas são heteropolímeros lineares essenciais à vida, responsáveis pela estruturação dos organismos e pela maioria dos processos bioquímicos que os mantêm vivos e permitem sua reprodução. Essa variedade de funções é refletida na diversidade estrutural encontrada no universo das proteínas, já que sua função é intrinsecamente ligada à sua rigorosa conformação espacial. A partir dos experimentos de Anfinsen (1973), ficou demonstrado que o enovelamento dessas moléculas (folding) se dá essencialmente por meio de um processo físico-químico guiado pela interação entre os aminoácidos da cadeia protéica e entre estes e o meio solvente, quando sob condições fisiológicas (temperatura, pressão, pH). O completo entendimento do mecanismo de folding tem também importância médica, pois várias doenças como mal de Alzheimer, diabetes tipo II, encefalite bovina espongiforme e várias formas de câncer estão relacionadas com falhas estruturais das proteínas. Neste trabalho, por meio de experimentação computacional por dinâmica molecular (DM) em diferentes condições térmicas, estudamos inicialmente o papel das pontes dissulfeto (S-S) e das ligações de hidrogênio (LH) na estabilidade da proteína. Em seguida, adotando exclusivamente o regime de alta temperatura (T = 448K) em combinação com simulações de longa duração (até ~100ns), no intuito de expandir a exploração do espaço configuracional, verificamos a premissa de que as forças entrópicas, geradas pelo efeito hidrofóbico, seriam dominantes no processo de busca pela estrutura nativa. Neste trabalho foi utilizada como um protótipo de proteína pequena e com pontes S-S, a toxina Ts Kappa (MM=3,8 Kda; pdb id: 1tsk), que é dotada de três pontes S-S. A estabilidade conformacional foi analisada por meio de uma série de simulações de DM em temperaturas crescentes e em duas situações: com e sem os cross-links S-S. Nossos resultados indicam que para incrementos nas temperaturas significativamente elevadas, como 50K acima da temperatura em que a estrutura nativa foi determinada por NMR (283K), a remoção das S-S não compromete a estabilidade conformacional da proteína. De fato, a ausência dos cross-links elimina certas restrições geométricas permitindo agora que diferentes combinações de LH sejam feitas, inclusive entre resíduos adjacentes à cisteína, os quais de certa forma substituem as pontes S-S em seus papeis conformacionais pois a estrutura nativa é essencialmente mantida. No segundo experimento o espaço configuracional foi varrido extensamente durante 100ns e à temperatura de 398K. No caso da Ts Kappa com suas pontes dissulfeto intactas, a desestruturação da proteína é limitada pelas fortes pontes covalentes S-S, mas com a remoção delas, a proteína se desnaturou completamente ao longo dos primeiros 50ns. Contudo, a partir deste ponto a cadeia desnaturada passou a seguir, de forma espontânea e sistemática, uma rota de re-estruturação em direção à nativa, com o reestabelecimento de todas suas estruturas secundárias. Ao redor de 100ns a cadeia atingiu um estado de grande identidade estrutural com sua correspondente estrutura nativa. Em conclusão, os presentes resultados corroboram as premissas de que o folding de proteínas ocorre por meio de um processo em duas etapas, temporalmente separadas: no início, as forças entrópicas são dominantes e são as que induzem a cadeia para a conformação nativa. Então, uma vez na vizinhança da estrutura nativa, as pontes de hidrogênio (agora protegidas da competição com o meio solvente), juntamente com um mais eficiente empacotamento estrutural das cadeias laterais devido às complementaridade estéricas das mesmas (e assim otimizando as interações de van der Waals), iniciam a etapa de estabilização energética da proteína. / Proteins are linear heteropolymers essential for life; they are responsible for many distinct functions as the structural components of organism, and for most of the biochemical processes to maintain a reproductive life. Such diversity of functions is correlated with the extremely large accessible conformational space, since function and spatial structure are interdependent. After Anfinsen experiments (1973), it becomes clear that the protein folding is essentially a physical-chemical process guided by interactions among the chain constituents (amino acid sequence) and interactions between the chain and the solvent, under physiological conditions (temperature, pressure, pH). Because miss-folded proteins are related with diseases (Alzheimer, type II diabetes, several forms of cancer, etc.) the full understanding of the folding mechanism has also significant medical interest. In this work, by means of molecular dynamics (MD) simulations under distinct thermal conditions, we first consider the role of disulfide cross-links (S-S) and hydrogen bonds (HB) with respect to the protein thermal stability. Then, using exclusively high temperature regime (T = 448K) combined with extended time simulations (up to ~100ns), in order to fully span of configurational space, we analyzed the hypothesis that the entropic forces, generated by the hydrophobic effect, are dominant in the search process for the native structure. The protein Ts Kappa was used a prototype for small proteins having S-S bridges (MM=3,8Kda; 3 S-S - pdb id: 1tsk). The thermal conformational stability was analyzed from a series of MD simulations under growing temperatures, using two distinct cases: with and without cross-links S-S. Our results suggest that for significant temperature increments, such as 50K above the temperature used in the Ts Kappa structure determination (by NMR at 283K), the thermal conformational stability of the proteins is not affected if the S-S bridges are removed. Indeed, cutting of the cross-links eliminates certain geometrical constraints, what permits the formation of new combinations of HB, which in some way take the place of the S-S bridges on its conformational role since the native structure is essentially maintained. In the second computational experiment, the configurational space was extensively swapped during 100ns at a fixed temperature T=398K. In the case with preserved S-S bridges, the structural unpacking is limited by the three covalent cross-links, but without the S-S bridges the protein denaturation was complete after 50ns. However, after this point the chain started spontaneous and systematically a configurational rote that finally, after about 100ns, reached a conformation very similar with the native (RMSD » 0.5nm), reestablishing all its secondary structure. Concluding, the present results corroborate the hypothesis that the protein folding is a process in two stages temporally separated: first, entropic forces are dominant and guide the chain into the native structure, and then, once in the native neighborhood, the HB (now protected from competition with the solvent), altogether with a more efficient structural specificity of the side chains (optimizing the van de Walls interactions), start the energetic stabilization of the protein.

In the study of alphaA-crystallin (CRYAA), G98R CRYAA was cloned into a mammalian expression vector pcDNA6-His/myc version B and the sequence was confirmed by direct sequencing. Following lipophilic transfection to lens epithelial B3 cells, the recombinant mutated CRYAA protein was highly insoluble upon 0.5% Triton X-100 (Tx) extraction. It was retained and formed aggregation, and distributed in the endoplasmic reticulum (ER) along with the ER resident protein (protein disulfide isomerise). The wild-type (WT) CRYAA was found to be soluble and diffusely distributed in the cytoplasm. The accumulation of G98R mutant induced ER stress, and the affected cells were prone to apoptosis. After treatment with a small chemical molecule, the natural osmolyte trimethylamine N oxide (TMAO), the Tx insolubility of mutant protein was reduced in dose- and time-dependent manners. It was also prone to be degraded via ubiquitin proteasome pathway (UPP). In mutant-expressing cells, the mutant protein aggregation was decreased after treatment. The ER stress and the rate of apoptosis were also alleviated, probably mediated by heat shock response, as demonstrated by the effect of TMAO on heat shock protein 70 expression. / The third eye gene model was myocilin (MYOC ), the first identified gene responsible for primary open angle glaucoma. The aim of this study was to investigate if glaucoma-causing MYOC variants, including D384N MYOC, could be correctable. D384N MYOC was identified in a Chinese family diagnosed with high tension juvenile-onset primary open-angle glaucoma. Disease causing mutations in MYOC (R82C, C245Y, Q368X P370L, T377M, D380A, D384N, R422C, R422H, C433R, Y437H, I477N, I477S and N480K) were cloned into mammalian expression vector p3XFLAG-myc-CMV"-25 and the sequences confirmed by direct sequencing. Following lipophilic transfection to human trabecular meshwork (HTM) cells, the Tx solubility and secretion of MYOC and cell apoptosis were examined in the presence or not with small chemical treatments. 4-PBA, TMAO and deuterium oxide (D2O), reduced the portion of insoluble fractions to various extents in the mutant proteins. The osmolytes TMAO and D2O were more effective than 4-PBA in improving MYOC solubility. TMAO was further shown to improve the secretion and ER-Golgi trafficking of D384N MYOC, thereby reducing the ER stress and rescuing cells from apoptosis. (Abstract shortened by UMI.) / The truncated G165fsX8 gammaD-crystallin ( CRYGD) variant was studied to further examine the effects of small chemical-assisted protein rescue of a CRYGD mutant that causes congenital cataract. G165fsX8 CRYGD was identified in a Chinese family with nuclear type of congenital cataract. The mutation was cloned into a mammalian expression vector p3XFLAG-myc-CMV"-25 and sequence was confirmed by direct sequencing. Following lipophilic transfection to COS-7 cells, the G165fsX8 CRYGD mutant protein was significantly insoluble upon 0.5% Tx extraction and was mistrafficked to the nuclear envelope with co-localization with nuclear lamins, whereas WT protein was Tx soluble and nuclear located. Treatment with small chemical sodium 4-phenylbutyrate (4-PBA) substantially reduced the Tx insolubility and reversed the mutant protein to nuclear localization. This correction has resulted in better cell survival, probably via a heat-shock response, as demonstrated by heat-shock protein 70 up-regulation. / To date, many genes and mutations are identified to cause various ocular diseases. Some of them result in a disruption of protein folding, an important cause of disease pathogenesis and progression. In my laboratory, novel mutations of crystallins and myocilin have been identified to segregate with congenital cataract and primary open-angle glaucoma, respectively. In this thesis, I reported molecular investigations of the resultant protein variants and their altered cellular functions in relation to the clinical phenotypes that contributed to new understanding of the roles of these genes in ocular tissues. / Gong, Bo. / Adviser: Chi-Pui Pang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 163-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Proteínas são heteropolímeros lineares essenciais à vida, responsáveis pela estruturação dos organismos e pela maioria dos processos bioquímicos que os mantêm vivos e permitem sua reprodução. Essa variedade de funções é refletida na diversidade estrutural encontrada no universo das proteínas, já que sua função é intrinsecamente ligada à sua rigorosa conformação espacial. A partir dos experimentos de Anfinsen (1973), ficou demonstrado que o enovelamento dessas moléculas (folding) se dá essencialmente por meio de um processo físico-químico guiado pela interação entre os aminoácidos da cadeia protéica e entre estes e o meio solvente, quando sob condições fisiológicas (temperatura, pressão, pH). O completo entendimento do mecanismo de folding tem também importância médica, pois várias doenças como mal de Alzheimer, diabetes tipo II, encefalite bovina espongiforme e várias formas de câncer estão relacionadas com falhas estruturais das proteínas. Neste trabalho, por meio de experimentação computacional por dinâmica molecular (DM) em diferentes condições térmicas, estudamos inicialmente o papel das pontes dissulfeto (S-S) e das ligações de hidrogênio (LH) na estabilidade da proteína. Em seguida, adotando exclusivamente o regime de alta temperatura (T = 448K) em combinação com simulações de longa duração (até ~100ns), no intuito de expandir a exploração do espaço configuracional, verificamos a premissa de que as forças entrópicas, geradas pelo efeito hidrofóbico, seriam dominantes no processo de busca pela estrutura nativa. Neste trabalho foi utilizada como um protótipo de proteína pequena e com pontes S-S, a toxina Ts Kappa (MM=3,8 Kda; pdb id: 1tsk), que é dotada de três pontes S-S. A estabilidade conformacional foi analisada por meio de uma série de simulações de DM em temperaturas crescentes e em duas situações: com e sem os cross-links S-S. Nossos resultados indicam que para incrementos nas temperaturas significativamente elevadas, como 50K acima da temperatura em que a estrutura nativa foi determinada por NMR (283K), a remoção das S-S não compromete a estabilidade conformacional da proteína. De fato, a ausência dos cross-links elimina certas restrições geométricas permitindo agora que diferentes combinações de LH sejam feitas, inclusive entre resíduos adjacentes à cisteína, os quais de certa forma substituem as pontes S-S em seus papeis conformacionais pois a estrutura nativa é essencialmente mantida. No segundo experimento o espaço configuracional foi varrido extensamente durante 100ns e à temperatura de 398K. No caso da Ts Kappa com suas pontes dissulfeto intactas, a desestruturação da proteína é limitada pelas fortes pontes covalentes S-S, mas com a remoção delas, a proteína se desnaturou completamente ao longo dos primeiros 50ns. Contudo, a partir deste ponto a cadeia desnaturada passou a seguir, de forma espontânea e sistemática, uma rota de re-estruturação em direção à nativa, com o reestabelecimento de todas suas estruturas secundárias. Ao redor de 100ns a cadeia atingiu um estado de grande identidade estrutural com sua correspondente estrutura nativa. Em conclusão, os presentes resultados corroboram as premissas de que o folding de proteínas ocorre por meio de um processo em duas etapas, temporalmente separadas: no início, as forças entrópicas são dominantes e são as que induzem a cadeia para a conformação nativa. Então, uma vez na vizinhança da estrutura nativa, as pontes de hidrogênio (agora protegidas da competição com o meio solvente), juntamente com um mais eficiente empacotamento estrutural das cadeias laterais devido às complementaridade estéricas das mesmas (e assim otimizando as interações de van der Waals), iniciam a etapa de estabilização energética da proteína. / Proteins are linear heteropolymers essential for life; they are responsible for many distinct functions as the structural components of organism, and for most of the biochemical processes to maintain a reproductive life. Such diversity of functions is correlated with the extremely large accessible conformational space, since function and spatial structure are interdependent. After Anfinsen experiments (1973), it becomes clear that the protein folding is essentially a physical-chemical process guided by interactions among the chain constituents (amino acid sequence) and interactions between the chain and the solvent, under physiological conditions (temperature, pressure, pH). Because miss-folded proteins are related with diseases (Alzheimer, type II diabetes, several forms of cancer, etc.) the full understanding of the folding mechanism has also significant medical interest. In this work, by means of molecular dynamics (MD) simulations under distinct thermal conditions, we first consider the role of disulfide cross-links (S-S) and hydrogen bonds (HB) with respect to the protein thermal stability. Then, using exclusively high temperature regime (T = 448K) combined with extended time simulations (up to ~100ns), in order to fully span of configurational space, we analyzed the hypothesis that the entropic forces, generated by the hydrophobic effect, are dominant in the search process for the native structure. The protein Ts Kappa was used a prototype for small proteins having S-S bridges (MM=3,8Kda; 3 S-S - pdb id: 1tsk). The thermal conformational stability was analyzed from a series of MD simulations under growing temperatures, using two distinct cases: with and without cross-links S-S. Our results suggest that for significant temperature increments, such as 50K above the temperature used in the Ts Kappa structure determination (by NMR at 283K), the thermal conformational stability of the proteins is not affected if the S-S bridges are removed. Indeed, cutting of the cross-links eliminates certain geometrical constraints, what permits the formation of new combinations of HB, which in some way take the place of the S-S bridges on its conformational role since the native structure is essentially maintained. In the second computational experiment, the configurational space was extensively swapped during 100ns at a fixed temperature T=398K. In the case with preserved S-S bridges, the structural unpacking is limited by the three covalent cross-links, but without the S-S bridges the protein denaturation was complete after 50ns. However, after this point the chain started spontaneous and systematically a configurational rote that finally, after about 100ns, reached a conformation very similar with the native (RMSD » 0.5nm), reestablishing all its secondary structure. Concluding, the present results corroborate the hypothesis that the protein folding is a process in two stages temporally separated: first, entropic forces are dominant and guide the chain into the native structure, and then, once in the native neighborhood, the HB (now protected from competition with the solvent), altogether with a more efficient structural specificity of the side chains (optimizing the van de Walls interactions), start the energetic stabilization of the protein.