Compensatory Education: Approaching the Problem from the Wrong Direction

*Designated as an exemplary master's project for 2015-16*

Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Nanofabrication towards biophotonics

This thesis explores methods for fabrication of nanohole arrays, and their integration into a benchtop system for use as sensors or anti-counterfeit labels. Chapter 1 gives an introduction to plasmonics and more specifically nanohole arrays and how they have potential as label free sensors compared to the current biosensors on the market. Various fabrication methods are explored, including Focused Ion Beam, Electron Beam Lithography, Nanoimprint lithography, Template stripping and Phase Shift Lithography. Focused Ion Beam was chosen to fabricate the nanohole arrays due to its suitability for rapid prototyping and it’s relatively low cost. In chapter 2 the fabrication of nanohole arrays using FIB is described, and the samples characterised. The fabricated nanohole arrays are tested as bulk refractive index sensors, before a bioassay using whole molecule human IgG antibodies and antigen is developed and performed on the senor. In chapter 3 the fabricated sensors are integrated into a custom built system, capable of real time, multiplexed detection of biomolecules. Here, scFv antibodies of two biomolecules relevant to the detection of pancreatic cancer (C1q and C3) are attached to the nanohole arrays, and detection of their complementary proteins is demonstrated both in buffer (10 nM detection of C1q Ag) and human serum. Chapter 4 explores arrays of anisotropic (elliptical) nanoholes and shows how the shape anisotropy induces polarisation sensitive transmission spectra, in both simulations and fabricated arrays. The potential use of such samples as visible and NIR tag for anti-counterfeiting applications is demonstrated. Finally, chapter 5 gives a summary of the work completed and discusses potential future work in this area.

Socialization of Early Prosocial Behavior: Parents' Talk about Emotions is Associated with Sharing and Helping in Toddlers.

What role does socialization play in the origins of prosocial behavior? We examined one potential socialization mechanism, parents' discourse about others' emotions with very young children in whom prosocial behavior is still nascent. Two studies are reported, one of sharing in 18- and 24-month-olds (n = 29), and one of instrumental and empathy-based helping in 18- and 30-month-olds (n = 62). In both studies, parents read age-appropriate picture books to their children and the content and structure of their emotion-related and internal state discourse were coded. Results showed that children who helped and shared more quickly and more often, especially in tasks that required more complex emotion understanding, had parents who more often asked them to label and explain the emotions depicted in the books. Moreover, it was parents' elicitation of children's talk about emotions rather than parents' own production of emotion labels and explanations that explained children's prosocial behavior, even after controlling for age. Thus, it is the quality, not the quantity, of parents' talk about emotions with their toddlers that matters for early prosocial behavior.

Mediterranean cold-water coral measurements

This study presents aggradation rates supplemented for the first time by carbonate accumulation rates from Mediterranean cold-water coral sites considering three different regional and geomorphological settings: (i) a cold-water coral ridge (eastern Melilla coral province, Alboran Sea), (ii) a cold-water coral rubble talus deposit at the base of a submarine cliff (Urania Bank, Strait of Sicily) and (iii) a cold-water coral deposit rooted on a predefined topographic high overgrown by cold-water corals (Santa Maria di Leuca coral province, Ionian Sea). The mean aggradation rates of the respective cold-water coral deposits vary between 10 and 530 cm kyr?1 and the mean carbonate accumulation rates range between 8 and 396 g cm?2 kyr?1 with a maximum of 503 g cm?2 kyr?1 reached in the eastern Melilla coral province. Compared to other deep-water depositional environments the Mediterranean cold-water coral sites reveal significantly higher carbonate accumulation rates that were even in the range of the highest productive shallow-water Mediterranean carbonate factories (e.g. Cladocora caespitosa coral reefs). Focusing exclusively on cold-water coral occurrences, the carbonate accumulation rates of the Mediterranean cold-water coral sites are in the lower range of those obtained for the prolific Norwegian coral occurrences, but exhibit much higher rates than the cold-water coral mounds off Ireland. This study clearly indicates that cold-water corals have the potential to act as important carbonate factories and regional carbonate sinks within the Mediterranean Sea. Moreover, the data highlight the potential of cold-water corals to store carbonate with rates in the range of tropical shallow-water reefs. In order to evaluate the contribution of the cold-water coral carbonate factory to the regional or global carbonate/carbon cycle, an improved understanding of the temporal and spatial variability in aggradation and carbonate accumulation rates and areal estimates of the respective regions is needed.

Synthesis, Modification, and Application of Siloxane Materials for Environmental Sensing

As human populations and resource consumption increase, it is increasingly important to monitor the quality of our environment. While laboratory instruments offer useful information, portable, easy to use sensors would allow environmental analysis to occur on-site, at lower cost, and with minimal operator training. We explore the synthesis, modification, and applications of modified polysiloxane in environmental sensing. Multiple methods of producing modified siloxanes were investigated. Oligomers were formed by using functionalized monomers, producing siloxane materials containing silicon hydride, methyl, and phenyl side chains. Silicon hydride-functionalized oligomers were further modified by hydrosilylation to incorporate methyl ester and naphthyl side chains. Modifications to the siloxane materials were also carried out using post-curing treatments. Methyl ester-functionalized siloxane was incorporated into the surface of a cured poly(dimethylsiloxane) film by siloxane equilibration. The materials containing methyl esters were hydrolyzed to reveal carboxylic acids, which could later be used for covalent protein immobilization. Finally, the siloxane surfaces were modified to incorporate antibodies by covalent, affinity, and adsorption-based attachment. These modifications were characterized by a variety of methods, including contact angle, attenuated total reflectance Fourier transform infrared spectroscopy, dye labels, and 1H nuclear magnetic resonance spectroscopy. The modified siloxane materials were employed in a variety of sensing schemes. Volatile organic compounds were detected using methyl, phenyl, and naphthyl-functionalized materials on a Fabry-Perot interferometer and a refractometer. The Fabry-Perot interferometer was found to detect the analytes upon siloxane extraction by deformation of the Bragg reflectors. The refractometer was used to determine that naphthyl-functionalized siloxanes had elevated refractive indices, rendering these materials more sensitive to some analytes. Antibody-modified siloxanes were used to detect biological analytes through a solid phase microextraction-mediated enzyme linked immunosorbent assay (SPME ELISA). The SPME ELISA was found to have higher analyte sensitivity compared to a conventional ELISA system. The detection scheme was used to detect Escherichia coli at 8500 CFU/mL. These results demonstrate the variety of methods that can be used to modify siloxanes and the wide range of applications of modified siloxanes has been demonstrated through chemical and biological sensing schemes.

QUADRUPOLE CENTRAL TRANSITION NMR SPECTROSCOPY OF QUADRUPOLAR NUCLEI IN SOLUTION

This thesis reports on 17O (I = 5/2) and 59Co (I = 7/2) quadrupole central transition (QCT) NMR studies of three classes of biologically important molecules: glucose, nicotinamide and Vitamin B12 derivatives. Extensive QCT NMR experiments were performed over a wide range of molecular motion by changing solvent viscosity and temperature. 17O-labels were introduced at the 5- and 6-positions respectively: D-[5-17O]-glucose and D-[6-17O]-glucose following the literature method. QCT NMR greatly increased the molecular size limit obtained by ordinary solution NMR. It requires much lower temperatures to get the optimal spectral resolution, which are preferable for biological molecules. In addition, quadrupolar product parameter (PQ) and shielding anisotropy product parameter (PSA) were obtained for hydroxide group and amide group for the first time. For conventional NMR studies of quadrupolar nuclei, only PQ is accessible while QCT NMR obtained both PQ and PSA simultaneously. Our experiments also suggest the resolution of QCT NMR can be even better than that obtained by conventional NMR. We observed for the first time that the second-order quadrupolar interaction becomes a dominant relaxation mechanism under ultraslow motion. All these observations suggest that QCT NMR can become a standard technique for studying quadrupolar nuclei in solution.

A look into diversity in the radio and music industry in Spain

UNESCO’s approval of the Convention on the Protection and Promotion of the Diversity of Cultural Expressions (UNESCO, 2005) has been an important element in catalyzing any attempt to measure the diversity of cultural industries (UIS, 2011). Within this framework, this article analyzes the relations between the music and radio industries in Spain from a critical perspective through the analysis of available data on recorded music offer and consumption (sales lists, radio-formula lists, the characteristics of the phonographic and radio markets) in different key moments due to the emergence of new formats and devices (CDS, Mp3, Internet).The main goal of this work is to study the evolution of the Spanish record market in terms of diversity from the end of the 1970s to the present, through the study of radio music hits lists and, the business structure of the phonographic and radio sectors, and phonograms top sales

Combining Taxonomies using Word2vec

Taxonomies have gained a broad usage in a variety of fields due to their extensibility, as well as their use for classification and knowledge organization. Of particular interest is the digital document management domain in which their hierarchical structure can be effectively employed in order to organize documents into content-specific categories. Common or standard taxonomies (e.g., the ACM Computing Classification System) contain concepts that are too general for conceptualizing specific knowledge domains. In this paper we introduce a novel automated approach that combines sub-trees from general taxonomies with specialized seed taxonomies by using specific Natural Language Processing techniques. We provide an extensible and generalizable model for combining taxonomies in the practical context of two very large European research projects. Because the manual combination of taxonomies by domain experts is a highly time consuming task, our model measures the semantic relatedness between concept labels in CBOW or skip-gram Word2vec vector spaces. A preliminary quantitative evaluation of the resulting taxonomies is performed after applying a greedy algorithm with incremental thresholds used for matching and combining topic labels.

English product names and descriptions in two Swedish supermarkets : A quantitative and qualitative analysis

This study aimed to investigate what proportion of two household staples, soap and crispbread products, in two Swedish supermarkets had English product names or descriptions, and attempted a qualitative analysis of the English language used. Out of the Swedish brands, 54-62% of the soap products had names and/or product descriptions containing English, compared to 13-15% of the crispbread; these differences were in line with previous research, suggesting English is used more to market certain product groups than other ones. Earlier studies have also proposed that English could be considered an ‘elite’ language in Sweden, and it might thus be more commonly found on more exclusive/expensive products, or in the supermarket primarily aiming at higher-income customers. However, the differences between the two supermarkets, and between the more and less expensive products, were not great enough for any firm conclusions. When products had a mixture of languages on the label, English was most often used for product names or part of names, not so often for product descriptions. Further studies with a larger amount of data would be required for more reliable conclusions, especially for the qualitative analyses. It would also be interesting to investigate customers’ attitudes towards the use of English on product labels.

Designing and Developing Digital and Blended Learning Solutions

Chapter 6 concerns ‘Designing and developing digital and blended learning solutions’, however, despite its title, it is not aimed at developing L&D professionals to be technologists (in so much as how Chapter 3 is not aimed at developing L&D professionals to be accounting and financial experts). Chapter 6 is about developing L&D professionals to be technology savvy. In doing so, I adopt a culinary analogy in presenting this chapter, where the most important factors in creating a dish (e.g. blended learning), are the ingredients and the flavour each of it brings. The chapter first explores the typical technologies and technology products that are available for learning and development i.e. the ingredients. I then introduce the data Format, Interactivity/ Immersion, Timing, Content (creation and curation), Connectivity and Administration (FITCCA) framework, that helps L&D professionals to look beyond the labels of technologies in identifying what the technology offers, its functions and features, which is analogous to the ‘flavours’ of the ingredients. The next section discusses some multimedia principles that are important for L&D professionals to consider in designing and developing digital learning solutions. Finally, whilst there are innumerable permutations of blended learning, this section focuses on the typical emphasis in blended learning and how technology may support such blends.

Optics-Based Quantum Information and Sensing Platforms Utilizing the Nitrogen-Vacancy Center in Diamond

Thesis (Ph.D.)--University of Washington, 2016-08

Enjeux de la cueca chilienne : vocalité et représentations sociales

Cette recherche aborde le chant de la cueca chilienne à travers le spectre de la voix dans la construction de représentations sociales, et ce, sur la base d’une méthodologie mixte qui combine la recherche bibliographique, l’observation participante, les entrevues et l’analyse musicale. Comme point de départ, on remarque la pratique d’une cueca surnommée urbana, brava ou chilenera, caractérisée par un timbre vocal singulier associé au milieu « populaire » des chanteurs. Remontant aux premières traces de la cueca, au Chili, le chapitre 2 aborde les descriptions de la voix de la zamacueca au XIXe siècle publié dans des récits de voyage. L’analyse du contexte dans lequel ces récits se publient permet de constater que l’idée du caractère nasal de la cueca s’associe à la constitution d’altérité. Le chapitre 3 aborde la façon dont une théorie spécifique sur l’origine de la cueca chilienne contribue aux conceptions de la voix dans le genre. Le sujet de la nasalité apparaît encore, cette fois-ci imbriqué dans l’imagination de l’origine arabo-andalouse de la cueca. S’intéressant à la représentation du sujet populaire, le chapitre 4 expose deux figures centrales de la culture chilienne : le huaso et le roto, représentants du paysan et du sujet urbain, tous deux entremêlés avec des discours nationalistes. Le « parler populaire » apparaît représenté dans divers styles de cueca, en produisant des vocalités affectées par l’imagination de la classe sociale, et ce dans le contexte de débats sur l’authenticité. Le chapitre 5 aborde finalement l’expérience vécue par de jeunes chanteurs actifs sur une scène de revitalisation. Leurs dynamiques de chant en groupe soulignent l’impact de la compétition sur le déploiement de la voix. La pratique structurée selon le chant en ronde - chant à la rueda – montrera que la production d’un « bon pito », soit d’une voix adéquate à la cueca, révèle la nécessité d’adapter les voix aux besoins du groupe. La conclusion met en lumière que le rapport entre voix et style se présente comme une correlation cruciale pour comprendre non seulement la diversité des variantes de cuecas existantes à un moment donné, mais également leurs transformations au cours du temps par l’entremise d’un processus de stylisation. Au-delà du style, pourtant, la voix paraît exprimer quelque chose du sujet qui la fait résonner. Par ce biais, les diverses étiquettes accompagnant le terme cueca nous informent sur les caractéristiques ethniques, du genre et de classe des chanteurs, y compris les différentes voix d’un même sujet qui chante.

Information Presentation Features and Comprehensibility of Hospital Report Cards: Design Analysis and Online Survey Among Users

Background: Improving the transparency of information about the quality of health care providers is one way to improve health care quality. It is assumed that Internet information steers patients toward better-performing health care providers and will motivate providers to improve quality. However, the effect of public reporting on hospital quality is still small. One of the reasons is that users find it difficult to understand the formats in which information is presented. Objective: We analyzed the presentation of risk-adjusted mortality rate (RAMR) for coronary angiography in the 10 most commonly used German public report cards to analyze the impact of information presentation features on their comprehensibility. We wanted to determine which information presentation features were utilized, were preferred by users, led to better comprehension, and had similar effects to those reported in evidence-based recommendations described in the literature. Methods: The study consisted of 5 steps: (1) identification of best-practice evidence about the presentation of information on hospital report cards; (2) selection of a single risk-adjusted quality indicator; (3) selection of a sample of designs adopted by German public report cards; (4) identification of the information presentation elements used in public reporting initiatives in Germany; and (5) an online panel completed an online questionnaire that was conducted to determine if respondents were able to identify the hospital with the lowest RAMR and if respondents’ hospital choices were associated with particular information design elements. Results: Evidence-based recommendations were made relating to the following information presentation features relevant to report cards: evaluative table with symbols, tables without symbols, bar charts, bar charts without symbols, bar charts with symbols, symbols, evaluative word labels, highlighting, order of providers, high values to indicate good performance, explicit statements of whether high or low values indicate good performance, and incomplete data (“N/A” as a value). When investigating the RAMR in a sample of 10 hospitals’ report cards, 7 of these information presentation features were identified. Of these, 5 information presentation features improved comprehensibility in a manner reported previously in literature. Conclusions: To our knowledge, this is the first study to systematically analyze the most commonly used public reporting card designs used in Germany. Best-practice evidence identified in international literature was in agreement with 5 findings about German report card designs: (1) avoid tables without symbols, (2) include bar charts with symbols, (3) state explicitly whether high or low values indicate good performance or provide a “good quality” range, (4) avoid incomplete data (N/A given as a value), and (5) rank hospitals by performance. However, these findings are preliminary and should be subject of further evaluation. The implementation of 4 of these recommendations should not present insurmountable obstacles. However, ranking hospitals by performance may present substantial difficulties.