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Structural and functional interrogation of single amino acid residues in fluorescent proteins

Description

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties…

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.

ContributorsWatkins, Jennifer L (Author) / Wachter, Rebekka M. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)

Created2012

Fiber dosage effects in asphalt binders and hot mix asphalt mixtures

Description

The application of fibers and other materials in asphalt mixes has been studied and applied over the past five decades in order to improve pavement performance around the world. This thesis highlights the characteristics and performance properties of modified asphalt mixes using a blend of polypropylene and aramid fibers, The…

The application of fibers and other materials in asphalt mixes has been studied and applied over the past five decades in order to improve pavement performance around the world. This thesis highlights the characteristics and performance properties of modified asphalt mixes using a blend of polypropylene and aramid fibers, The main objective of this study was to evaluate the effect of adding different fiber dosages on the laboratory performance of both asphalt binder and mixture. The laboratory study was conducted on sixteen different dosages and blends of the fibers, with various combinations of polypropylene and aramid, using binder tests as well as hot mix asphalt tests. The binder tests included: penetration, softing point, and Brookfield viscosity tests. The asphalt mixture tests included the dynamic modulus, and indirect tensile strength. The binder test results indicated that the best viscosity - temperature susceptibility performance would be from the blend of three dosages of polypropylene and one dosage of aramid, the dynamic modulus test results also confirmed this finding. Overall, in almost every case, the addition of fibers resulted in an increase in mixture stiffness regardless of fiber content. From the indirect tensile strength results, the polypropylene fibers had less of an effect on post peak failure than the aramid fibers. Overall, the aramid fibers yielded better results than the polypropylene fibers. This study has important implications for the future of pavement design and the prospect of using optimal dosages of polypropylene and aramid fibers in further research to further determine their long-term performance and characteristics used in real world applications.

ContributorsAlrajhi, Ashraf (Author) / Kaloush, Kamil (Thesis advisor) / Mamlouk, Michael (Committee member) / Witzcak, Matthew (Committee member) / Arizona State University (Publisher)

Created2012

Integrated model of the urban continuum with dynamic time-dependent activity-travel microsimulation: framework, prototype, and implementation

Description

The development of microsimulation approaches to urban systems modeling has occurred largely in three parallel streams of research, namely, land use, travel demand and traffic assignment. However, there are important dependencies and inter-relationships between the model systems which need to be accounted to accurately and comprehensively model the urban system.…

The development of microsimulation approaches to urban systems modeling has occurred largely in three parallel streams of research, namely, land use, travel demand and traffic assignment. However, there are important dependencies and inter-relationships between the model systems which need to be accounted to accurately and comprehensively model the urban system. Location choices affect household activity-travel behavior, household activity-travel behavior affects network level of service (performance), and network level of service, in turn, affects land use and activity-travel behavior. The development of conceptual designs and operational frameworks that represent such complex inter-relationships in a consistent fashion across behavioral units, geographical entities, and temporal scales has proven to be a formidable challenge. In this research, an integrated microsimulation modeling framework called SimTRAVEL (Simulator of Transport, Routes, Activities, Vehicles, Emissions, and Land) that integrates the component model systems in a behaviorally consistent fashion, is presented. The model system is designed such that the activity-travel behavior model and the dynamic traffic assignment model are able to communicate with one another along continuous time with a view to simulate emergent activity-travel patterns in response to dynamically changing network conditions. The dissertation describes the operational framework, presents the modeling methodologies, and offers an extensive discussion on the advantages that such a framework may provide for analyzing the impacts of severe network disruptions on activity-travel choices. A prototype of the model system is developed and implemented for a portion of the Greater Phoenix metropolitan area in Arizona to demonstrate the capabilities of the model system.

ContributorsKonduri, Karthik Charan (Author) / Pendyala, Ram M. (Thesis advisor) / Ahn, Soyoung (Committee member) / Kuby, Michael (Committee member) / Kaloush, Kamil (Committee member) / Arizona State University (Publisher)

Created2012

Development of enhanced cylindrical specimen thermal conductivity testing procedure

Description

The current method of measuring thermal conductivity requires flat plates. For most common civil engineering materials, creating or extracting such samples is difficult. A prototype thermal conductivity experiment had been developed at Arizona State University (ASU) to test cylindrical specimens but proved difficult for repeated testing. In this study, enhancements…

The current method of measuring thermal conductivity requires flat plates. For most common civil engineering materials, creating or extracting such samples is difficult. A prototype thermal conductivity experiment had been developed at Arizona State University (ASU) to test cylindrical specimens but proved difficult for repeated testing. In this study, enhancements to both testing methods were made. Additionally, test results of cylindrical testing were correlated with the results from identical materials tested by the Guarded Hot&ndashPlate; method, which uses flat plate specimens. In validating the enhancements made to the Guarded Hot&ndashPlate; and Cylindrical Specimen methods, 23 tests were ran on five different materials. The percent difference shown for the Guarded Hot&ndashPlate; method was less than 1%. This gives strong evidence that the enhanced Guarded Hot-Plate apparatus in itself is now more accurate for measuring thermal conductivity. The correlation between the thermal conductivity values of the Guarded Hot&ndashPlate; to those of the enhanced Cylindrical Specimen method was excellent. The conventional concrete mixture, due to much higher thermal conductivity values compared to the other mixtures, yielded a P&ndashvalue; of 0.600 which provided confidence in the performance of the enhanced Cylindrical Specimen Apparatus. Several recommendations were made for the future implementation of both test methods. The work in this study fulfills the research community and industry desire for a more streamlined, cost effective, and inexpensive means to determine the thermal conductivity of various civil engineering materials.

ContributorsMorris, Derek (Author) / Kaloush, Kamil (Thesis advisor) / Mobasher, Barzin (Committee member) / Phelan, Patrick E (Committee member) / Arizona State University (Publisher)

Created2011

Mass spectrometric and molecular analyses of biological agents in environmental compartments

Description

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as…

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as bioterrorism agents. The first chapter introduces the hurdles in detecting in diverse environmental compartments in which they could be found, a select list of single-celled pathogens representing known or potential bioterrorism agents. These hurdles take the form of substances that interfere either directly or indirectly with the detection method. In the case of mass spectrometry-based detection, many of these substances (interferences) can be removed via effective sample pretreatment. Chapters 2 through 4 highlight specific methods developed to detect bioremediation or bioterrorism agents in environmental matrices. These methods are qualitative mass spectrometry, quantitative PCR, and quantitative mass spectrometry, respectively. The targeted organisms in these methods include several bioremediation agents, e.g. Pseudomonas putida F1 and Sphingomonas wittichii RW1, and bioterrorism agents, e.g. norovirus and Cryptosporidium parvum. In Chapter 2, I identify using qualitative mass spectrometry, biomarkers for three bacterial species involved in bioremediation. In Chapter 3, I report on a new quantitative PCR method suitable for monitoring of a key gene in yet another bioremediation agent, Sphingomonas wittichii RW1; furthermore, I apply this method to track the efficacy of bioremediation in bioaugmented environmental microcosms. In Chapter 4, I report on the development of new quantitative mass spectrometry methods for two organisms, S. wittichii RW1 and Cryptosporidium parvum, and evaluate two previously published methods for their applicability to the analysis of complex environmental samples. In Chapter 5, I review state-of-the-art methods for the detection of emerging biological contaminants, specifically viruses, in environmental samples. While this summary deals exclusively with viral pathogens, the advantages and remaining challenges identified are also applicable to all single-celled organisms in environmental settings. The suggestions I make at the end of this chapter are expected to be valid not only for future needs for emerging viruses but also for bacteria, eukaryotic pathogens, and prions. In general, it is advisable to continue the trend towards quantification and to standardize methods to facilitate comparison of results between studies.

ContributorsHartmann, Erica Marie (Author) / Halden, Rolf U. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Nelson, Randall W. (Committee member) / Arizona State University (Publisher)

Created2012

The investigation and characterization of the group 3 [NiFe]-Hydrogenases using protein film electrochemistry

Description

Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and…

Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. First, HoxEFUYH is shown to be biased towards hydrogen production. Upon exposure to oxygen, HoxEFUYH inactivates to two states, both of which can be reactivated on the timescale of seconds. Second, we show that PfSHI is the first example of an oxygen tolerant [NiFe]-hydrogenase that produces two inactive states upon exposure to oxygen. Both inactive states are analogous to those characterized for HoxEFUYH, but oxygen exposed PfSHI produces a greater fraction that reactivates at high potentials, enabling hydrogen oxidation in the presence of oxygen. Third, it is shown that removing the NAD(P)-reducing subunits from PfSHI leads to a decrease in bias towards hydrogen oxidation and renders the enzyme oxygen sensitive. Both traits are likely due to impaired intramolecular electron transfer. Mechanistic hypotheseses for these functional differences are considered.

ContributorsMcIntosh, Chelsea Lee (Author) / Jones, Anne K (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)

Created2012

Endurance limit for HMA based on healing phenomenon using viscoelastic continuum damage analysis

Description

Perpetual Pavements, if properly designed and rehabilitated, it can last longer than 50 years without major structural rehabilitation. Fatigue endurance limit is a key parameter for designing perpetual pavements to mitigate bottom-up fatigue cracking. The endurance limit has not been implemented in the Mechanistic Empirical Pavement Design Guide software, currently…

Perpetual Pavements, if properly designed and rehabilitated, it can last longer than 50 years without major structural rehabilitation. Fatigue endurance limit is a key parameter for designing perpetual pavements to mitigate bottom-up fatigue cracking. The endurance limit has not been implemented in the Mechanistic Empirical Pavement Design Guide software, currently known as DARWin-ME. This study was conducted as part of the National Cooperative Highway Research Program (NCHRP) Project 9-44A to develop a framework and mathematical methodology to determine the fatigue endurance limit using the uniaxial fatigue test. In this procedure, the endurance limit is defined as the allowable tensile strains at which a balance takes place between the fatigue damage during loading, and the healing during the rest periods between loading pulses. The viscoelastic continuum damage model was used to isolate time dependent damage and healing in hot mix asphalt from that due to fatigue. This study also included the development of a uniaxial fatigue test method and the associated data acquisition computer programs to conduct the test with and without rest period. Five factors that affect the fatigue and healing behavior of asphalt mixtures were evaluated: asphalt content, air voids, temperature, rest period and tensile strain. Based on the test results, two Pseudo Stiffness Ratio (PSR) regression models were developed. In the first model, the PSR was a function of the five factors and the number of loading cycles. In the second model, air voids, asphalt content, and temperature were replaced by the initial stiffness of the mix. In both models, the endurance limit was defined when PSR is equal to 1.0 (net damage is equal to zero). The results of the first model were compared to the results of a stiffness ratio model developed based on a parallel study using beam fatigue test (part of the same NCHRP 9-44A). The endurance limit values determined from uniaxial and beam fatigue tests showed very good correlation. A methodology was described on how to incorporate the second PSR model into fatigue analysis and damage using the DARWin-ME software. This would provide an effective and efficient methodology to design perpetual flexible pavements.

ContributorsZeiada, Waleed (Author) / Kaloush, Kamil (Thesis advisor) / Witczak, Matthew W. (Thesis advisor) / Zapata, Claudia (Committee member) / Mamlouk, Michael (Committee member) / Arizona State University (Publisher)

Created2012

Reliability associated with the estimation of soil resilient modulus at different hierarchical levels of pavement design

Description

Deterministic solutions are available to estimate the resilient modulus of unbound materials, which are difficult to interpret because they do not incorporate the variability associated with the inherent soil heterogeneity and that associated with environmental conditions. This thesis presents the stochastic evaluation of the Enhanced Integrated Climatic Model (EICM), which…

Deterministic solutions are available to estimate the resilient modulus of unbound materials, which are difficult to interpret because they do not incorporate the variability associated with the inherent soil heterogeneity and that associated with environmental conditions. This thesis presents the stochastic evaluation of the Enhanced Integrated Climatic Model (EICM), which is a model used in the Mechanistic-Empirical Pavement Design Guide to estimate the soil long-term equilibrium resilient modulus. The stochastic evaluation is accomplished by taking the deterministic equations in the EICM and applying stochastic procedures to obtain a mean and variance associated with the final design parameter, the resilient modulus at equilibrium condition. In addition to the stochastic evaluation, different statistical analyses were applied to determine that the uses of hierarchical levels are valid in the unbound pavement material design and the climatic region has an impact on the final design resilient moduli at equilibrium. After determining that the climatic regions and the hierarchical levels are valid, reliability was applied to the resilient moduli at equilibrium. Finally, the American Association of State Highway and Transportation Officials (AASHTO) design concept based on the Structural Number (SN) was applied in order to illustrate the true implications the hierarchical levels of design and the variability associated with environmental effects and soil properties have in the design of pavement structures. The stochastic solutions developed as part of this thesis work together with the SN design concept were applied to five soils with different resilient moduli at optimum compaction condition in order to evaluate the variability associated with the resilient moduli at equilibrium condition. These soils were evaluated in five different climatic regions ranging from arid to extremely wet conditions. The analysis showed that by using the most accurate input parameters obtained from laboratory testing (hierarchical Level 1) instead of Level 3 analysis could potentially save the State Department of Transportation up to 10.12 inches of asphalt in arid and semi-arid regions.

ContributorsRosenbalm, Daniel (Author) / Zapata, Claudia (Thesis advisor) / Witczak, Matthew (Committee member) / Kaloush, Kamil (Committee member) / Arizona State University (Publisher)

Created2011

Relative benefit of chip seal application in different climatic conditions based on initial pavement roughness

Description

Pavement preservation is the practice of selecting and applying maintenance activities in order to extend pavement life, enhance performance, and ensure cost effectiveness. Pavement preservation methods should be applied before pavements display significant amounts of environmental distress. The long-term effectiveness of different pavement preservation techniques can be measured in terms…

Pavement preservation is the practice of selecting and applying maintenance activities in order to extend pavement life, enhance performance, and ensure cost effectiveness. Pavement preservation methods should be applied before pavements display significant amounts of environmental distress. The long-term effectiveness of different pavement preservation techniques can be measured in terms of life extension, relative benefit, and benefit-cost ratio. Optimal timing of pavement preservation means that the given maintenance treatment is applied so that it will extend the life of the roadway for the longest possible period with the minimum cost. This document examines the effectiveness of chip seal treatment in four climatic zones in the United States. The Long-Term Pavement Performance database was used to extract roughness and traffic data, as well as the maintenance and rehabilitation histories of treated and untreated sections. The sections were categorized into smooth, medium, and rough pavements, based upon initial condition as indicated by the International Roughness Index. Pavement performance of treated and untreated sections was collectively modeled using exponential regression analysis. Effectiveness was evaluated in terms of life extension, relative benefit, and benefit-cost ratio. The results of the study verified the assumption that treated sections performed better than untreated sections. The results also showed that the life extension, relative benefit, and benefit cost ratio are highest for sections whose initial condition is smooth at the time of chip seal treatment. These same measures of effectiveness are lowest for pavements whose condition is rough at the time of treatment. Chip seal treatment effectiveness showed no correlation to climatic conditions or to traffic levels.

ContributorsDosa, Matild (Author) / Mamlouk, Michael S. (Thesis advisor) / Kaloush, Kamil (Committee member) / Zapata, Claudia E (Committee member) / Arizona State University (Publisher)

Created2012

Impact of Material Properties and Urban Geometry on Urban Heat Island Effect

Description

Utilizing an urban canopy model (UCM) developed by Zhihua Wang, Ph.D. for a research study conducted for the National Asphalt Pavement Association (NAPA), several scenarios were run in order to determine the impact on the mitigation of the urban heat island (UHI) effect. These scenarios included various roof albedo, wall…

Utilizing an urban canopy model (UCM) developed by Zhihua Wang, Ph.D. for a research study conducted for the National Asphalt Pavement Association (NAPA), several scenarios were run in order to determine the impact on the mitigation of the urban heat island (UHI) effect. These scenarios included various roof albedo, wall albedo, ground albedo, a combination of all three albedos, roof emissivity, wall emissivity, ground emissivity, a combination of all three emissivities, and normalized building height as independent variables. Dependent variables included canyon air temperature, effective ground temperature, effective roof temperature, effective wall temperature, and sensible heat flux. It was found that emissivity does play a part in reducing the different dependent variables; however, typically emissivity values are already within a preferred range that not much can be done with them. Normalized building height has a minor impact but the impact that it does have upon the different variables is lessened with lower values of the normalized building height. Increasing the wall albedo decreased the canyon air temperature and the effective wall temperature the most compared to the other variables when considering expenses. An increase in roof albedo reduced effective roof temperature and sensible heat flux the most when taking into consideration the cost of changing the albedo of the surface. Larger values of ground albedo helped to reduce the effective ground temperature more than the other variables considered when a budget is necessary.

ContributorsHousenga, Hannah Eileen (Author) / Kaloush, Kamil (Thesis director) / Wang, Zhihua (Committee member) / Civil, Environmental and Sustainable Engineering Programs (Contributor) / Barrett, The Honors College (Contributor)

Created2015-05