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During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses of the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C–O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr -OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr -OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the PDB has established that Tyr -OH electron density loss is not generally a dominant damagepathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposedto account for instances of observable Tyr -OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to disulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported.

Journal URL: http://www.proteinscience.org/; http://www3.interscience.wiley.com/journal/121502357/home

The Amino acid/Polyamine/Cationic transporter superfamily is among the largest of the sec-ondary active transporters. APC family members use either a proton or sodium gradient to drive substrate translocation across the membrane. Members of the APC superfamily are responsible for the uptake of amino acids and are found in all kingdoms of life. Amino acids, and their derivatives, are crucial for both prokaryotic and eukaryotic cell biology, being the building blocks of a variety of macromolecules and play important roles in metabolism and cell signalling. Therefore, the systems responsible for their transport are important targets of further study, in particular to gain greater insight into how these proteins recognise and discriminate between different types of amino acids. The transport mechanism of several APC members has been elucidated using a variety of structural, biophysical and functional approaches. However, these studies have focussed on sodium coupled transporters due to their function as neurotransmitter transporters. Here, the crystal structure of the first pro-ton-coupled amino acid transporter of the APC superfamily, from Geobacillus kaustophilus, GkApcT, is presented. GkApcT closely related to the mammalian cationic amino acid trans-porters (CATs) of the SLC7 family, responsible for the transport arginine and lysine into the cell. The mechanism of transport was further investigated using liposome-based functional assays to characterise the substrate specificity and kinetics. The crystal structure was used to guide a detailed site-directed mutagenesis study, which identified several conserved res-idues involved in substrate binding and proton-coupling. The current structure serves as a template to further understand proton coupled amino acid transport in bacteria and to un-derstand mammalian amino acid transport via the SLC7 and SLC36 families.

Quantitative studies of global radiation damage are presented for two different types of experiments in structural biology: macromolecular crystallography (MX) and small angle X-ray scattering (SAXS) MX is the most common technique to elucidate the atomic resolution structures of biological macromolecules. However, these molecules undergo radiation induced changes during the experiment that undesirably affect the data. Global radiation damage, which is characterised by an overall loss in the diffracted intensity of Bragg reflections, limits the amount of useful data that can be collected from a single crystal in an experiment. Furthermore, for experimental phasing experiments, the radiation induced intensity changes can be so significant that the phasing signal becomes undetectable, thereby hindering successful structure determination. This thesis investigates methods to track and correct the diffraction data that are affected as a result of global radiation damage. First, extensions to the diffraction weighted dose (DWD) metric are investigated for the ability of DWD to track the overall intensity decay of reflections. This metric then is combined with a new mathematical model of intensity decay to perform zero-dose extrapolation. An additional probabilistic extrapolation approach is incorporated into the traditional regression based approach to allow extrapolation of low multiplicity reflections. As an alternative approach, a new hidden Markov model representation of the data collection experiment is developed that allows the time-resolved calculation of structure factor amplitudes, with error estimates calculated explicitly. This method gives comparable refinement statistics to that obtained from data processed with the current data reduction pipeline, and improvements to the algorithm are proposed. SAXS, on the other hand, is a complementary structural technique that results in low resolution information about macromolecules. However it still requires the probing of the macromolecules with ionising radiation, so radiation induced changes are still a problem. Unfortunately the tools for assessing radiation damage in SAXS experiments are not mature enough for them to be used routinely. This thesis presents extensions to RADDOSE-3D to perform dose calculations for SAXS samples. Additionally, a free, open source Python library has been developed to allow the exploration and visualisation of the results of a similarity analysis of frames within a dataset. These tools are then used to determine the efficacy of various radioprotectant compounds at different concentrations to mitigate radiation damage effects.

Two research questions are investigated here: the first, major, section addresses the problem of uneven distributions of dose (absorbed energy per unit mass) in crystals used for macromolecular crystallography (MX), and the second presents the develop- ment of a high-throughput metalloprotein characterisation technique, HT microPIXE. In MX, the advent of X-ray microbeam data collection has led to uneven distributions of dose within the crystal volume becoming increasingly common. In these cases, the rotation method creates a highly damaged central region of crystal that stays within the beam throughout exposure, and less damaged outer regions, which are introduced during rotation. This thesis presents a new software program, raddose-3d, which performs a full 3D simulation of the profile of absorbed energy (the dose state) within a crystal during X-ray exposure. In order to utilise this time resolved, 3D picture of the dose state of the crystal, a new metric – Diffraction Weighted Dose – is proposed. This metric is then experimentally validated, and is found to summarise the dose state into a single dose value, which reflects the damage state of the crystal. Simulations are performed using raddose-3d and Diffraction Weighted Dose to compare possible dose spreading strategies, and generalised recommendations for MX experimentalists are offered. Uniquely identifying the species and stoichiometry of bound metals in protein sam- ples is a significant challenge for biophysical characterisation. Low throughput mi- crobeam Proton Induced X-ray Emission (microPIXE) provides an unambiguous anal- ysis of these properties, but has a limited throughput of ∼10 samples per day. As a consequence, its applicability has been restricted to niche cases. This thesis presents significant progress, including proof of principle experiments, on developing sample preparation methods, data acquisition systems, and data analysis protocols to increase this throughput by an order of magnitude, opening up major new applications for the technique.

The main objective of this study was to determine the relationship between household characteristics and the expenditure shares allocated among various categories of women's clothing for U.S. households belonging to different ethnic groups. The study also estimated unobserved latent quality variables based on household characteristics, and examined the effects of the latent quality variables on the expenditure shares for the various apparel categories. A Multiple Indicator-Multiple Cause Model, which is a special case of the general Structural Equations Model, was used to estimate separate Engel equations for 15 expenditure shares for women's clothing categories, for four different ethnic groups. The results of the study showed that household characteristics had a significant impact on the latent quality variables associated with different categories of women's clothing, and the latent quality variables themselves impacted the clothing expenditure shares. Also, for different ethnic groups, household characteristics had differing effects on women's clothing expenditure shares. Of all the characteristics examined, annual total household expenditures and numbers of children and adults in the household had significant effects on the largest numbers of latent quality variables associated with the clothing categories for the four ethnic groups. The socio-economic variables also significantly affected several clothing expenditure shares for the four ethnic groups. These results imply that socio-economic variables impact consumers' quality choices, and presumably prices paid, for women's clothing. The results support the conclusions of Paulin (1998), and Wagner and Soberon-Ferrer (1990), in that different ethnic groups have distinct expenditure patterns possibly due to differences in socio-economic characteristics; such characteristics may signify resources and constraints faced by a household. The distinct expenditure patterns and tastes of the four ethnic groups are reflected in the significantly different effects of annual total expenditures on the expenditure shares for each category of women's clothing, as well as in the significantly different effects of the latent quality variables on several expenditure shares, for the four ethnic groups. Ph. D.

The purpose of this study was to examine a sample of employed individuals who participated in credit counseling through a non-profit consumer credit counseling agency in the Mid-Atlantic. Using data collected at two points in time, this sample was examined to measure changes in personal financial variables, health status, and work outcomes. The sample respondents were also examined to determine the extent to which they instituted positive financial behaviors following participation in credit counseling. In addition, this research assessed differences in the demographics among the clients. Also studied was the extent to which individual and family characteristics, health status, financial concerns and related stress, and financial wellness accounted for the variance in work outcomes of productivity, presenteeism, and worktime used for personal financial matters. Significant changes in personal financial outcomes, health status, and work outcomes were found between the initial and follow-up study. One year following credit counseling, respondents had decreased levels of financial concerns and financial stress, experienced fewer workloss days, and spent less time using work hours to handle personal financial matters. They also indicated improvements in their level of financial wellness, health status, and job productivity. Respondents had instituted a number of positive financial behaviors since receiving credit counseling one year earlier. Most had reduced some of their personal debts and cut down on living expenses. A model of work and personal financial outcomes was presented in this study. Hierarchical regression analyses using both data sets revealed that health status and financial concerns explained a significant amount of the variance in four work outcomes: (1) productivity, (2) presenteeism, (3) work time used for personal financial matters, and (4) workloss days. Adding financial wellness as the final step in the analysis, did not explain any additional variance in each of the work outcomes. This research assessed only the demographic and personal financial variables explanatory relationships to work outcomes. Therefore, life events beyond these variables may offer additional explanation of the work outcomes. Of importance is that this research provides documentation of positive changes in personal finances and work outcomes of employed individuals who participated in credit counseling one year earlier. In addition, the research presented a model of personal financial and work outcomes that can be advanced through further research. Ph. D.

The purpose of this research was to examine the effects of workplace financial education on workers' personal finances and work outcomes and determine relationships among financial management (attitudes, knowledge and behaviors), financial well-being, personal finance-work conflict, and work outcomes with data of white-collar workers in an insurance company in mid-western states. Research questions were (1) What are the profiles of financial attitudes, financial knowledge, financial behaviors, financial well-being, personal finance-work conflict, productivity, absenteeism, work time use, organizational commitment, pay satisfaction, loyalty, and intention to leave?, (2) Do the profiles of financial attitudes, financial knowledge, financial behaviors, and financial well-being differ by the individual characteristics?, (3) Do the profiles of personal finance-work conflict, productivity, absenteeism, work time use, organizational commitment, and pay satisfaction differ by the individual characteristics?, (4) What are the relationships among financial attitudes, financial knowledge, financial behaviors, financial well-being, and work outcomes (productivity, absenteeism, organizational commitment, and pay satisfaction)?, (5) What are the effects of workplace financial education on financial attitudes, financial knowledge, financial behaviors, financial well-being, and work outcomes?, and (6) What are the individual profiles of workplace financial education including participation, value of workplace financial education, reasons for participation and non-participation, desire for financial check-up, and desired topics of workplace financial education? The research design was a pre- and post-assessment survey. A pre-assessment survey was conducted in February and March 1999 before workplace financial education was provided during March 1999. One-and one-half hour workplace financial education workshops were provided at no cost to employer or employees in March 1999. Three months after the workplace financial education was provided, a post-assessment survey was conducted from June through August 1999. A pre-assessment questionnaire was mailed to all 476 workers (five were undeliverable) and 262 responses were utilized for data analysis. In the post-assessment, 482 questionnaires were mailed to workers and five were undeliverable. Usable return rates for the data analysis were 56.0% in the pre-assessment (262/471) and 40.0% in the post-assessment (189/477). Overall, the respondents in this study were somewhat positive toward financial management, were not knowledgeable on financial matters, and were practicing their financial behaviors fairly well. Objective financial well-being measures showed that workers were in fairly good financial condition but the levels of subjective financial well-being were about the mid-point on a scale, when each score was converted into a percentage. The workers reported that they were very productive, did not miss work days frequently, were highly committed to their organization, and they showed fairly high levels of pay satisfaction. Workers were very hesitant to admit to direct questions asking about whether or not their financial concerns interfered with their responsibilities at the workplace while they were not always able to do normal work even though they were present in the office and spent some work time handling financial matters. Some of individual characteristics influenced financial attitude, financial knowledge, financial behavior, financial well-being, personal finance-work outcomes, and work outcomes. The tests of the structural equation model showed that worker's personal finances had direct and indirect effects on work outcomes. The financial well-being had a negative effect on personal finance-work conflict. The financial well-being had direct effects on negative work time use and pay satisfaction. The financial well-being had indirect effects through personal finance-work conflict on absenteeism, negative work time use, and organizational commitment. The t-test results did not show the significant effects of workplace financial education on personal finances and work outcomes between the pre- and the post-assessment. Ph. D.

The problem that was examined in this research was to develop and test a conceptual model that describes the relationship between personal financial wellness and worker job productivity. The research questions were (1) what is the personal financial wellness profile?; (2) how does the personal financial wellness profile differ by the demographic characteristics?; (3) what is the relationship between financial stressors and personal financial wellness profile?; (4) what is the relationship between personal financial wellness and financial stress level?; (5) what is the worker job productivity profile?; (6) what is the relationship between personal financial wellness and worker job productivity?; (7) what is the relationship between financial stress and worker job productivity?; and (8) what financial education programs do employees want in the future? In order to test a part of the conceptual model, a survey research design was undertaken. A questionnaire was developed and pre-tested. A mail survey (N=474) of white-collar clerical workers of a large employer located in mid-eastern state was conducted during January, February, and March of 1998. From a random sample of 447 (27 out of original 474 were undeliverable), 288 questionnaires were returned (64.4%). Seventeen questionnaires were determined unusable resulting in a 60.4% usable return rate (271/447). In terms of subjective perception, as a group, the respondents were not financially well. In the behavioral assessment, the respondents reported above a mid-point score. On overall financial wellness scales, the respondents were not satisfied with their financial situation. Personal financial wellness was influenced by some of the demographic characteristics and financial stressors. The lower levels of personal financial wellness were related to the financial stress level. Those who have high levels of personal financial wellness reported better performance ratings, less absenteeism, and less work time used for personal financial matters. Workers are interested in comprehensive financial education programs which include retirement education, better use of employee benefits, money management, credit management, and consumer protection. Some workers are not financially well because they have financial problems. If employers can improve personal financial wellness of workers, such as through financial education, it may increase productivity, because personal financial wellness is related to worker productivity. Ph. D.

The main objective of this study was to evaluate the effects of the changes in total nondurables expenditures, prices, and demographics on the U.S. aggregate demand for clothing categories and shoes. In particular, this study focused on identifying and parameterizing the effects of such changes. To this purpose, a demand system for two clothing categories, shoes, and other nondurable commodities for the U.S. was estimated using aggregate time-series data sets (1929-1994), and a second-stage budgeting model was developed and estimated. The basis for the demand model was the Almost Ideal Demand System model, which was modified to account for the demographic effects. Demographic variables included in the final model were age distribution of the U.S. population (median age and variance), proportion of non-White population in the total U.S. population, and labor force participation rate of U.S. women. The main data sources were documents published by the Bureau of the Census, Bureau of Labor Statistics, and Bureau of Economic Analysis in the U.S. Department of Commerce. The results indicate that the total nondurables expenditures is a significant variable in determining consumers' nondurables expenditure allocation on clothing categories and shoes. The estimated total expenditure elasticities suggest that the clothing categories and shoes are expenditure elastic, ranging from 1.1019 to 1.4944. Most own and cross prices appear to be significant variables in determining the consumer budget allocations for clothing categories and shoes. The median age and non-White population variables evidence as significant variables that affect the U.S. aggregate nondurables expenditure allocation on men’s and boys’ clothing and on shoes. Women’s labor force participation rate evidences as a significant variable that affects the U.S. aggregate nondurables expenditure allocation on women’s and children’s clothing. The estimated own-price elasticities of demand for clothing categories and shoes indicate that all the clothing categories and shoes are inelastic (i.e., -0.3908 to -0.9175). The estimated crossprice elasticities of clothing categories, shoes and other nondurable goods show substitution and complementary relationships between the categories. The demand elasticities with respect to the demographic variables were also estimated. Ph. D.

The study was designed to ascertain the level of knowledge, opinions, and degree of preparedness of a group of Virginia Polytechnic Institute and State University faculty and staff concerning selected practices and regulations of the funeral industry. Questionnaires were returned by 226 respondents; this represented 75.00% of the sample. The Consumer Funeral Test was developed for this study and included a knowledge, opinion, and preparedness section. Mean achievement score on the knowledge section was 4.00, representing 57.10% correct. Kuder-Richardson formula twenty reliability estimate equaled 0.14; an expected low figure given the small number of questions (seven). The opinions expressed indicated that the respondents were "conventional" in their feelings; that is, there was a general tendency toward agreement with selected practices and regulations of the funeral industry, that was anticipated according to previous studies and responses by consumers. "Unconventional" would indicate a general tendency toward agreement with the status quo of selected practices and regulations of the funeral industry. The preparedness scores reported by respondents indicated that the greater majority are unprepared for their own funerals. Of the five preparedness questions asked, four received a negative response by 79.80% or more of the respondents. No statistically significant relationship existed between the knowledge, opinions, and preparedness scores. Also, age, education, income, sex, and religion were not related to the opinions and preparedness scores reported. However, a significant relationship existed between the knowledge of selected practices and regulations of the funeral industry and the age, education, and sex of the respondents. Those who were older scored higher; the males scored significantly higher than the females on the knowledge section; and those with more education scored higher. Master of Science