Student Number : 9306227A - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for -helices. N-capping motifs have been identified within hGST A1-1 at the N-terminal ends of -helix 9 and helix 6. The conservational role of these two motifs in protein stability, folding and function was investigated. -Helix 9 is a unique structural feature to class Alpha GSTs that is important for its catalytic functioning. This amphipathic helix is highly dynamic, where upon ligand binding at the active-site, the delocalised C-terminal region becomes immobilised to form a structured helix forming a “lid” over the active-site. The specific role of the Asp N-cap motif toward the stability and dynamics of helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding and urea-induced activity studies showed that by removing the N-cap motif of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic binding site compared to that in the wild-type. The helical content of the peptide, corresponding to helix 9 in the C-terminal region of hGST A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif. The explanation for the conservation of the Asp N-cap residue can be found in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising role was however less apparent in context of the protein compared to that in the peptide. Majority of the atomic contacts owing to the stability of helix 9 appear to be governed by non-local tertiary interactions rather than local interactions, such as the N-cap motif. These tertiary interactions are likely to include short and long range contacts between residues on the surface of the protein that are already known to contribute towards the stability of the C-terminal region. In this study, the ligand displacement-studies and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1. The N-capping motif of helix 6 identified in class Alpha GSTs is located within the core of domain 2. This motif is a common feature found amongst almost all GST-like proteins and is thought to be the folding nucleation site (Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser- 154) and N3 (Asp-157) residues were each substituted with an Ala in hGST A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both substitutions resulted in thermal sensitive mutants compared to that of the wild-type. The N3 substitution (D157A) was however too disruptive, where the yields of this mutant were insufficient for any further studies to be carried out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a significantly destabilised core in domain 2 compared to that of the wild-type. The kinetic folding studies monitored by fluorescence spectroscopy, revealed that the N-cap motif contributes to the efficient folding and dimerisation of the subunits, and to a far lesser extent towards the final tight packing and reorganisation of tertiary interactions in hGST A1-1. Since no changes in the burst-phase of S154A was evident compared to that of the wild-type, it seems unlikely that this motif is a folding nucleation site in hGST A1-1. These results do not exclude the possibility that this motif contributes to the rapid formation secondary structure during the burst-phase of folding. Due to the highly conserved region surrounding helix 6 , the role of this motif contributing to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike proteins. In this study, further insight into the mechanism of folding for hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6 occurred as a late folding event, that is during the final packing and reorganisation of tertiary interactions of the protein. The N-cap motif is an important structural feature for the fast folding of domain 2. This N-cap motif is a unique structural feature important for the efficient folding of the monomers, which is exclusive to its role in stabilising helix 6 in hGST A1-1.

This thesis focuses on the performace of pipeline converters and their integration on mixed signal processes. With this in mind, a 12-b 50MHz pipeline ADC has been realized in a 0.6um digital CMOS process. The architecture is based on a 1.5-b per stage structure utilizing digital correction for the first six stages. A differeintial switched capacitor circuit consisting of a cascode gm-c op-amp with 250MHz of bandwidth is used for sampling and amplification in each stage. Comparators with an internal offset voltage are used to implement the decision levels required for the 1.5-b per stage structure. Correction of the pipeline is accomplished by measuring the offset and gain of each of the first six stages using subsequent stages. The measured values are used to calculate digtal values the compensate for the inaccuracies of the analog pipeline. Corrected digital values for each stage are stored in the pipeline and used to create corrected output codes. Errors caused by measuring the first six stages using uncalibrated stages are minimized by using extra switching circuitry during calibration.

This thesis describes several molecular dynamics studies of polymers, proteins, and active colloids. These diverse systems fall under the purview of soft matter physics and in Part I, I explain what is soft matter and describe some of its essential features. In Part II, I introduce some basic polymer physics and show how confined polymers can be described using blob theory. I also discuss how phase separation of polymer mixtures can occur. These concepts are applied to systems of mixed polymer brushes on spheroids, objects that have surfaces with non-uniform curvature. I show how the interplay of phase separation and surface curvature give rise to striped patterns, and how an extension of blob theory can give analytical expressions for the free energy. Finally, I show how phase separation of miscible polymers can occur, driven solely by surface curvature. In Part III, I present an overview of self-assembly and describe how active, or self-propelled colloids can be used to assemble new materials. I show how two large colloids immersed in a bath of smaller active colloids exhibit an effective short-ranged repulsion and long-ranged attraction, which stands in contrast to the standard short-ranged depletion attraction. I also explore how self-propulsion changes clustering by focusing on a system with short-ranged attractive and long-ranged repulsive particles, which under equilibrium, exhibit finite-sized clusters. I show that for certain parameters, spheres can form a fluid of living crystals, and dumbbells can form a crystal of rotors. In Part IV, I give a brief introduction to protein folding and describe how molecular chaperones combat misfolding in the human body. Then, taking inspiration from the chaperones, I show that a polymer-grafted “soft” nanopore can be used to unfold misfolded proteins and destroy undesired aggregates. I also show preliminary results for a hydrophobic “smart” nanopore that can selectively capture and unfold misfolded proteins.

Oxidative folding, the process by which folding and disulfide oxidation occur in concert, is a critical step in the production of many extracellular proteins and is therefore centrally linked to a vast multitude of important physiological functions. The primary focus of this dissertation is the remarkable disulfide oxidoreductase DsbA, the sole catalyst of oxidative folding in Escherichia coli. DsbA was the first oxidative folding catalyst to be discovered, and remains the strongest known oxidant among the thioredoxin superfamily of disulfide oxidoreductases due to unique biochemical and biophysical properties. Through the activity of its substrate repertoire, which includes adhesion structures and toxins, DsbA is an essential component of many pathogenic processes and therefore is an active target for the development of novel antibiotics. Though DsbA has been analyzed through a host of biochemical, genetic, and cellular experiments over the quarter-century since its identification, the elucidation of certain mechanistic details of its catalytic process have proven elusive to conventional techniques. This primarily results from the experimental difficulties in independently monitoring the progress of folding and oxidation during oxidative folding that arise with conventional, ensemble-averaged approaches. In this work, single molecule force spectroscopy methods are applied to investigate the process of oxidative folding as catalyzed by DsbA. Through observing single substrate molecules as they undergo DsbA-catalyzed oxidative folding, a precise kinetic analysis of the enzyme is constructed. DsbA is demonstrated to be a highly effective catalyst of oxidative folding, outperforming its eukaryotic counterpart by substantial margins in every metric considered. This efficacy complements the strong preference for simpler disulfide connectivity patterns in the Escherichia coli proteome, which in conjunction likely represent a strategy for navigating the physiological demands that are imposed by the inherent speed of prokaryotic life, in which a generation can be as short as twenty minutes.

Mechanical forces regulate biological processes in unique and unexpected ways, but many biochemical methods are unable to reproduce the vectorial stretching experienced by proteins in cells. Force spectroscopy techniques remedy these shortcomings by utilizing microscopic force probes to stretch and relax single protein, DNA, and RNA molecules. The central focus of this thesis is the development and implementation of a custom-built protein magnetic tweezers for unfolding and refolding Ig domains from titin, a critical filament of the sarcomere, and the longest continuous peptide in the human body. Suspended from the Z-disc to the tip of the thick filament, titin sustains the brunt of intracellular forces during muscle elongation. Since the discovery of titin, it has been widely debated whether Ig domain unfolding contributes to muscle mechanics. A combination of single quantum dot tracking in myofibrils extracted from rabbit muscle and single molecule magnetic tweezers experiments on recombinant titin fragments confirms, for the first time, the presence of titin Ig domain unfolding and refolding at physiological sarcomere lengths and stretching forces. The magnetic tweezers experiments show the surprising ability of titin Ig domains to generate piconewton level forces during folding, and we advance the hypothesis that titin folding is an important source of energy during muscle contraction. Muscle elongation recruits Ig domains to the unfolded state, whereby folding is initiated through reduction of force on titin upon actomyosin crossbridges formation. Magnetic tweezers measurements demonstrate that titin Ig folding generates peak work, velocity, and power output of 64 zeptojoules, 1.9 microns per second, and 6,000 zeptowatts, matching or exceeding the equivalent single molecule measurements from single molecule myosin II powerstrokes. The forces generated by protein folding are therefore likely to be an integral part of the contractile process of animal muscle.

This is a study of Japanese folding screens decorated with a variety of cartographic imagery of European origin. The central argument of this work is that Japanese cartographic namban screens made during the period considered in this dissertation can assist us to further understand the marked Christian eschatological character of the pictorial programmes decorating these screens, reflecting European contemporary hopes about the messianic coming of a universal Christian King, and about the Christian future of Japan at the onset of Shogun Tokugawa Hidetada's ban against Christianity (1614). By taking into account the use of folding screens as diplomatic gifts, this research seeks to argue that the hybridity of namban cartographic screens reveals as much about the expectation of Jesuit missionaries towards the evangelization of the Japanese archipelago as they did about how Japanese artists and observers understood European cartographic knowledge within a pre-existing local ritual use of maps and cartography. This dissertation is composed of four chapters. In chapter one I describe the material qualities of folding screens, the architectural environments in which they were displayed, and how the practice of donating folding screens as diplomatic gifts was eventually co-opted by the Jesuit missionaries operating in Japan. Chapter two is a discussion on the organization and the passage of the first Japanese diplomatic mission in Europe and the role that European cartography and geographical allegories played in this event. In chapter three I will examine the dissemination of Christian sacred images in Japan and the establishment of a Jesuit school to train Japanese artists in western-style painting. Chapter four unpacks the discussion developed in the preceding chapters and focuses on two specific pairs of namban cartographic screens - the Map of the World and Twenty-Eight Cities (today at the Imperial Household Agency in Tokyo) and the Battle of Lepanto and World Map (today at the Kosetsu Museum in Kobe) - for which I propose a new interpretation.

O presente trabalho tem como objetivo mostrar a influência do recozimento na recuperação e recristalização de tiras de aço baixo carbono dobradas por deformação a frio. Muitas indústrias que produzem peças a partir de dobramento a frio, buscam constantemente garantir a qualidade de seus produtos. Isto se torna um desafio a partir do momento que se considera a diversidade do formato das dobras exigidas. Através de observações práticas, o que se vê é que, com o objetivo de facilitar o processo, muitos profissionais são induzidos a acreditar que simplesmente aquecendo o material, o trabalho será facilitado e garantirá a qualidade do produto final. Assim, independente do tipo do aço e/ou nível de encruamento, os parâmetros de temperatura utilizados são determinados, na sua grande maioria, de forma empírica, sem critérios estabelecidos, ou seja, em muitos casos, os valores de temperatura tendem a ser os mesmos. Como a grande maioria de peças produzidas na indústria submetida a deformação é em aço baixo carbono, este foi escolhido como material para a fabricação das amostras utilizadas no experimento. Estas amostras foram submetidas a diversos graus de dobramento a frio e aplicação de recozimento para recristalização com variação controlada dos parâmetros. Através da intercomparação das amostras e com aplicação de diversos ensaios foram caracterizados a dureza, estrutura metalográfica, tamanho médio do precipitado, bem como a correlação entre as condições de recozimento versus o nível de recuperação do encruamento. Através dos dados obtidos, observou-se a importância de definir de forma científica os parâmetros de aquecimento para a recristalização, sob pena de prejudicar as características das peças. / This work aims to show the influence of annealing on the recovery and recrystallization of low carbon steel strips bent by cold forming. Many industries that produce parts from cold bending, constantly seek to ensure the quality of their products. This becomes a challenge from the moment that one considers the diversity of the shape of folds required. Through practical observation, we can see that, in order to facilitate the process, many professionals are led to believe that simply heating the material, the work will be facilitated and ensure final product quality. Thus, regardless of the type of steel and / or level of work hardening, the parameters used in temperature are determined, mostly, empirically, without established criteria, ie, in most cases, the temperature values tend to be same. As the vast majority of parts produced in the industry is subjected to deformation in steel low carbon, this was chosen as material for the manufacture of the samples used in the experiment. These samples were subjected to various degrees of cold bending and applying for recrystallization annealing with controlled variation of parameters. By intercomparison of samples and application of various tests were characterized hardness, metallographic structure, average size of the precipitate, and the correlation between the annealing conditions versus the level of recovery of work hardening. Through the data obtained it observed the importance of defining the parameters in a scientific way of heating for recrystallization, failing to affect the characteristics of components.

Polyglutamine (polyQ) diseases are progressive fatal neurodegenerative movement disorders. Although many cellular processes are perturbed in polyQ disease, recent studies highlight the importance of protein misfolding as a central event in polyQ toxicity. Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a particularly interesting polyQ disease because of the special qualities of the disease protein ataxin-3, which normally participates in cellular protein quality control. Here I use multiple mouse models of disease to explore toxic protein species and the role of protein homeostasis in SCA3 pathogenesis. In Chapter 1, I review the key features of polyQ disease, and outline the background and rationale behind our strategy for identifying toxic protein species in SCA3. In Chapter 2, I examine the role of the protein quality control ubiquitin ligase, CHIP (C-terminus of Hsp70 interacting protein), in regulating the toxicity of expanded ataxin-3 in vivo. Genetic reduction or removal of CHIP increases formation of detergent-resistant ataxin-3 microaggregates specifically in the brain. Concomitant with this, reduction or removal of CHIP exacerbates the phenotype of SCA3 mice, revealing a correlation between high levels of microaggregates and phenotypic severity. Additional cell-based studies confirm that CHIP may not directly mediate ataxin-3 degradation, suggesting that CHIP reduces expanded ataxin-3 toxicity in the brain primarily by enhancing ataxin-3 solubility. In Chapter 3, I use various biochemical techniques to reveal the presence of brain-specific ataxin-3 microaggregates in two genetically distinct mouse models of SCA3. Selective neuropathological evaluation of SCA3 mice reveals that major neuronal loss and reactive glial proliferation are not shared features of phenotypically-manifesting SCA3 mice. Additional studies fail to provide evidence for loss-of-function of endogenous ataxin-3 in SCA3 mice. Our results suggest that neuronal dysfunction in SCA3 is mediated through a toxic gain-of-function mechanism by ataxin-3 microaggregates in the CNS. In Chapter 4, I discuss important areas for future research in polyQ disease. I describe studies that would help elucidate the structural nature of toxic soluble microaggregates, and their effects on other cellular proteins. I also consider how the results described in this thesis inform potential treatment strategies.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

The design of a pop-up book or card has hitherto been labour intensive with tasks of trials and errors. The constructions of collapsible pop-up structures can be demanding and inefficient without adequate knowledge of their geometric properties. This paper examines the properties of creases in 90° pop-up structures. A 90° pop-up structure is one that erects fully when two adjacent base pages, on which it sits, are opened to a right angle. In particular, we define a boundary region for creating 90° pop-ups. Similarly, paper folds are able to achieve pop-up effects and can be integrated with 90° pop-up constructions. The development of these pop-up structures can be represented graphically. Through this study, a fundamental foundation for pop-up topology and geometry is built. This foundation would be vital for understanding the applications of pop-up making techniques. The mathematical relationships devised would be useful for developing computer-enhanced pop-up design. / Singapore-MIT Alliance (SMA)