<p>The mechanical behaviour of coated papers during folding has been investigated. This problem has been studied with experimental techniques and numerical analyses in order to give a better understanding of the folding properties of coated papers pertinent to the mechanical behaviour in general, and particularly cracking along the fold. </p><p>A microscopy investigation has been performed. The surface of the folded paper has been carefully examined to study the event of fracture and related issues. The influence of the grammage on the cracking event has been studied and it was shown that the coating material would not fail if the paper sample was sufficiently thin. It was found that a stress or strain based criterion is sufficient to describe the cracking of the coating layers and that the anisotropy of paper should be taken into account when studying the folding process. </p><p>The finite element method has been used for the numerical analyses remembering that the geometry of the problem is rather complicated, excluding a solution in analytical form. Using different constitutive models for the base stock, it has been shown that the deformation of the coated paper during folding is much governed by the paper substrate. The numerical results also suggested that particular forms of plastic anisotropy can substantially reduce the maximum strain levels in the coating. Furthermore, it has also been shown that delamination buckling, in the present circumstances, has a very small influence on the strain levels in the coating layer subjected to high tensile loading. </p><p>Dynamic effects have also been studied and it has been shown that a quasi-static analysis of the problem is sufficient in order to describe many of the important features related to cracking. An attempt to model strong anisotropy of paper has been presented and the results indicate that the large anisotropy in the thickness direction of coated papers needs to be taken into account in order to fully understand the mechanics of folding. </p><p>Finally, an experimental investigation has been presented in order to study if important mechanical properties of the coating material could be determined by microindentation techniques. The results presented indicate that microindentation can be a powerful tool for characterization of these materials, but only if careful efforts are made in order to account for the influence from plasticity as well as from boundary effects. </p><p><b>KEYWORDS:</b> folding, coated papers, finite element method, cracking, indentation, anisotropy, plasticity.</p>

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 202-216).

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 93-101).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 141-161).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-86).

The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail. / text

Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis.

Many functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures. / text

Group I introns are structured RNAs that have been used extensively as model systems for RNA folding because they are experimentally tractable, yet complex enough to have folding challenges associated with larger RNAs. The Tetrahymena group I intron consists of a set of conserved core helices and a set of peripheral elements. Peripheral elements surround the core helices and form long range tertiary contacts between each other and to the core. Interestingly, a long-lived misfolded state is populated that has the same long range tertiary contacts as the native state but differs locally within the core. Our lab showed that the intact periphery is necessary to specify the correct core structure, as mutating tertiary contacts or removing the P5abc peripheral element dramatically destabilized the native ribozyme relative to the misfolded form. However, we also showed that the thermodynamic benefit peripheral structure provided is accompanied by kinetic liability in folding, apparently because native tertiary contacts formed by peripheral elements around the misfolded core must come apart to allow refolding of the misfolded RNA to the native state. In addition to peripheral elements, proteins also play a role in stabilizing the native structures of many group I introns. The CYT-18 protein, which occupies the same binding site as P5abc, stabilizes the functional structures of certain group I introns by using a set of insertions that are absent in other related bacterial and mitochondrial aminoacyl tRNA synthetases. Using the P5abc deletion variant of the Tetrahymena ribozyme, I sought to further define CYT-18 roles in RNA folding by probing its thermodynamic and kinetic effects on the native state formation relative to the misfolded state. I demonstrated that CYT-18, like P5abc, provided thermodynamic stability to the native state. However, unlike P5abc, CYT-18 had no apparent effect on the refolding kinetics, suggesting that a protein co-factor can stabilize the functional structure without acquiring the associated costs in RNA folding kinetics. Furthermore, I found that the mechanism of CYT-18 action appears to be distinct from P5abc. Disruption of the long-range contact P14, which is formed between P5c and L2 and is part of the network of peripheral contacts, dramatically weakened P5abc binding to the native ribozyme core by ~10⁸ fold. Interestingly, CYT-18 maintained specific and tight binding to these mutants, which suggests that CYT-18 does not rely on a circular network of contacts to specifically stabilize the native state. Instead, the specificity may arise from a more direct and intimate contact of CYT-18 with the ribozyme core. This study gives insight into an evolutionary advantage of protein co-factors in RNA folding; proteins may offer thermodynamic assistance without inhibiting folding kinetics. / text