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.

Electrostatic Contributions to the Thermodynamics of Ribonuclease P Protein Folding

Electrostatic interactions are of fundamental importance in determining the structure and stability of macromolecules. For example, charge-charge interactions modulate the folding and binding of proteins and influence protein solubility. Electrostatic interactions are highly variable and can be both favorable and unfavorable. The ability to quantify these interactions is challenging but vital to understanding the detailed balance and major roles that they have in different proteins and biological processes. Measuring pKa values of ionizable groups provides a sensitive method for experimentally probing the electrostatic properties of a protein.

pKa values report the free energy of site-specific proton binding and provide a direct means of studying protein folding and pH-dependent stability. Using a combination of NMR, circular dichroism, and fluorescence spectroscopy along with singular value decomposition, we investigated the contributions of electrostatic interactions to the thermodynamic stability and folding of the protein subunit of Bacillus subtilis ribonuclease P, P protein. Taken together, the results suggest that unfavorable electrostatics alone do not account for the fact that P protein is intrinsically unfolded in the absence of ligand because the pKa differences observed between the folded and unfolded state are small. Presumably, multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

(Table 1) AMS 14C dates from sediment cores RC12-10 and GYRE97-6PV-20

Proxy records from two piston cores in the Gulf of Mexico (GOM) provide a detailed (50-100 year resolution) record of climate variability over the last 14,000 years. Long-term (millennial-scale) trends and changes are related to the transition from glacial to interglacial conditions and movement of the average position of the Intertropical Convergence Zone (ITCZ) related to orbital forcing. The d18O of the surface-dwelling planktic foraminifer Globigerinoides ruber show negative excursions between 14 and 10.2 ka (radiocarbon years) that reflect influx of meltwater into the western GOM during melting of the Laurentide Ice Sheet. The relative abundance of the planktic foraminifer Globigerinoides sacculifer is related to transport of Caribbean water into the GOM. Maximum transport of Caribbean surface waters and moisture into the GOM associated with a northward migration of the average position of the ITCZ occurs between about 6.5 and 4.5 ka. In addition, abundance variations of G. sacculifer show century-scale variability throughout most of the Holocene. The GOM record is consistent with records from other areas, suggesting that century-scale variability is a pervasive feature of Holocene climate. The frequency of several cycles in the climate records is similar to cycles identified in proxy records of solar variability, indicating that at least some of the century-scale climate variability during the Holocene is due to external (solar) forcing.

Stable isotopic composition of Poritres lutea from New Caledonia

A 335 year stable isotope record from a New Caledonia coral (22°S, 166°E) helps fill a large gap in historical climate reconstructions. Although the long-term coral s18O-based sea surface temperature (SST) trend is one of warming, there are notable decadal fluctuations, especially in the early 18th and early 19th centuries. Mean annual SSTs between 1658 and 1900 are estimated to be ~0.3°C lower than the 20th century average, with interdecadal excursions of 0.5°-0.8°C. Time series analyses of the coral isotope record reveals significant concentrations of variance in the El Niño band; an inderdecadal spectral peak is present, but its robustness requires additional statistical evaluation. A secular but irregular decrease in coral d13C values begins in the mid-1800s and may reflect the anthropogenic perturbation of the carbon reservoir. These and other results indicate that the New Caledonia coral isotope record is a valuable source of information on southwest Pacific climate history.

Information from Paleoclimate Archives

Compilation of figure recipes for all figures of Chapter 5 of IPCC Working Group I, Fifth Assessment Report. In addition to figure captions, figure recipes are supposed to serve as detailed figure creation info. If not publicly available elsewhere, processed data underlying the respective figures are also provided here.

Cultural Nationalism

Nationalism may involve the combination of culture and politics, but for many of its most prominent students, the former is subordinate to the latter. In this view, nationalist appeals to culture are a means to a political end; that is, the achievement of statehood. Hence, for Ernest Gellner (2006 [1983]: 124), culture is but an epiphenomenon, a ‘false-consciousness … hardly worth analyzing …’. For their part, Eric Hobsbawm and Terrence Ranger (1983) suggest that national traditions are ‘invented’ by elites concerned with the legitimization of state power. Similarly, John Breuilly (2006 [1982]: 11) defines national movements as ‘political movements … which seek to gain or exercise state power and justify their objectives in terms of nationalist doctrine’. A broadly similar characterization of nationalism can be found in the writings of many other esteemed scholars (Giddens, 1985; Laitin, 2007; Mann, 1995; Tilly, 1975). The privileging of politics over culture remains the dominant approach to understanding nationalism, but it is not without criticism. There is now a vast and rapidly growing body of literature insisting that the role of culture should be made more prominent. In opposition to the argument that nationalist appeals to culture are but an exercise in legitimation, this body of literature suggests that they can be ends unto themselves. This latter phenomenon, generally referred to as cultural nationalism, is the subject of this chapter. The chapter proceeds as follows. I begin with the definition and history of cultural nationalism before discussing several key themes in its study. To conclude, I briefly outline several lines of research that I believe hold particular potential for developing the field. In the light of the huge array of literature on cultural nationalism, the review is focused on seminal contributions.

The 1998 BFA Graduating Class: Department of Visual Arts

The exhibition of works by Sir Wilfred Grenfell College's graduating Class in visual arts constitutes the culmination of four years of intensive study and practice. our students are trained in the fundamentals of their craft and exposed tio a variety of media and styles ranging from the traditional to the avant garde. But their education has more to do with the exploration and development of their creativity, the enlargement of their capacity to see, and their mastery of the vocabulary of sight, enabling them to discover, uncover and represent. Each of the students ha sustained and nurtured an individual and unique quest over the four years of this rigorous programme.

Heritage Language Maintenance and Biliteracy Development for Immigrants' Children: A Study of Chinese Immigrants' Family Language Policy and Biliteracy Practices

This study reports on research that examines the family language policy (FLP) and biliteracy practices of middle-class Chinese immigrant families in a metropolitan area in the southwest of the U.S. by exploring language practices pattern among family members, language and literacy environment at home, parents’ language management, parents’ language attitudes and ideologies, and biliteracy practices. In this study, I employed mixed methods, including survey and interviews, to investigate Chinese immigrant parents’ FLP, biliteracy practices, their life stories, and their experience of raising and nurturing children in an English-dominant society. Survey questionnaires were distributed to 55 Chinese immigrant parents and interviews were conducted with five families, including mothers and children. One finding from this study is that the language practices pattern at home shows the trend of language shift among the Chinese immigrants’ children. Children prefer speaking English with parents, siblings, and peers, and home literacy environment for children manifests an English-dominant trend. Chinese immigrant parents’ language attitudes and ideologies are largely influenced by English-only ideology. The priority for learning English surpasses the importance of Chinese learning, which is demonstrated by the English-dominant home literacy practices and an English-dominant language policy. Parents invest more in English literacy activities and materials for children, and very few parents implement Chinese-only policy for their children. A second finding from this study is that a multitude of factors from different sources shape and influence Chinese immigrants’ FLP and biliteracy practices. The factors consist of family-related factors, social factors, linguistic factors, and individual factors. A third finding from this study is that a wide variety of strategies are adopted by Chinese immigrant families, which have raised quite balanced bilingual children, to help children maintain Chinese heritage language (HL) and develop both English and Chinese literacy. The close examination and comparison of different families with English monolingual children, with children who have limited knowledge of HL, and with quite balanced bilingual children, this study discovers that immigrant parents, especially mothers, play a fundamental and irreplaceable role in their children’s HL maintenance and biliteracy development and it recommends to immigrant parents in how to implement the findings of this study to nurture their children to become bilingual and biliterate. Due to the limited number and restricted area and group of participant sampling, the results of this study may not be generalized to other groups in different contexts.