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Description
In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle.
ContributorsChakravadhanula, Madhavi (Author) / Capco, David G. (Thesis advisor) / Chandler, Douglas (Committee member) / Clark-Curtiss, Josephine (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2012
Description
DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.
ContributorsLiu, Minghui (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2013
Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
For some time it has been recognized amongst researchers that individual and collective change should be the goal in educating for sustainability, unfortunately education has generally been ineffective in developing pro-environmental behaviors among students. Still, many scholars and practitioners are counting on education to lead us towards sustainability but suggest that in order to do so we must transition away from current information-intensive education methods. In order to develop and test novel sustainability education techniques, this research integrates pedagogical methods with psychological knowledge to target well-established sustainable behaviors. Through integrating education, behavior change, and sustainability research, I aim to answer: How can we motivate sustainable behavioral change through education programs? More specifically: How do diverse knowledge domains (declarative, procedural, effectiveness, and social) influence sustainable behaviors, both in general as well as before and after a sustainability education program? And: What are barriers hindering education approaches to changing behaviors? In answering these questions, this research involved three distinct stages: (1) Developing a theoretical framework for educating for sustainability and transformative change; (2) Implementing a food and waste focused sustainability educational program with K-12 students and teachers while intensively assessing participants' change over the course of one year; (3) Developing and implementing an extensive survey that examines the quantitative relationships between diverse domains of knowledge and behavior among a large sample of K-12 educators. The results from the education program demonstrated that significant changes in knowledge and behaviors were achieved but social knowledge in terms of food was more resistant to change as compared to that of waste. The survey results demonstrated that K-12 educators have high levels of declarative (factual or technical) knowledge regarding anthropocentric impacts on the environment; however, declarative knowledge does not predict their participation in sustainable behaviors. Rather, procedural and social knowledge significantly influence participation in sustainable food behaviors, where as procedural, effectiveness, and social knowledge impact participation in sustainable waste behaviors. Overall, the findings from this research imply that in order to effectively educate for sustainability, we must move away from nature-centric approaches that focus on declarative knowledge and embrace different domains of knowledge (procedural, effectiveness, and social) that emphasis the social implications of change.
ContributorsRedman, Erin (Author) / Larson, Kelli (Thesis advisor) / Eakin, Hallie (Committee member) / Spielmann, Katherine (Committee member) / Arizona State University (Publisher)
Created2013
Description
Over the last two decades programs and mandates to encourage and foster sustainable urban development have arisen throughout the world, as cities have emerged as key opportunity sites for sustainable development due to the compactness and localization of services and resources. In order to recognize this potential, scholars and practitioners have turned to the practice of visioning as a way to motivate actions and decision making toward a sustainable future. A "vision" is defined as desirable state in the future and scholars believe that the creation of a shared, motivational vision is the best starting point to catalyze positive and sustainable change. However, recent studies on city visions indicate that they do not offer substantive sustainability content, and methods or processes to evaluate the sustainability content of the resulting vision (sustainability appraisal or assessment) are often absent from the visioning process. Thus, this paper explores methods for sustainability appraisal and their potential contributions to (and in) visioning. The goal is to uncover the elements of a robust sustainability appraisal and integrate them into the visioning process. I propose an integrated sustainability appraisal procedure based on sustainability criteria, indicators, and targets as part of a visioning methodology that was developed by a team of researchers at Arizona State University (ASU) of which I was a part. I demonstrate the applicability of the appraisal method in a case study of visioning in Phoenix, Arizona. The proposed method allows for early and frequent consideration and evaluation of sustainability objectives for urban development throughout the visioning process and will result in more sustainability-oriented visions. Further, it can allow for better measurement and monitoring of progress towards sustainability goals, which can make the goals more tangible and lead to more accountability for making progress towards the development of more sustainable cities in the future.
ContributorsMinowitz, Amy (Author) / Wiek, Arnim (Thesis advisor) / Golub, Aaron (Committee member) / Pfeiffer, Deirdre (Committee member) / Arizona State University (Publisher)
Created2013
Description
Sustainability visioning (i.e. the construction of sustainable future states) is considered an important component of sustainability research, for instance, in transformational sustainability science or in planning for urban sustainability. Visioning frees sustainability research from the dominant focus on analyzing problem constellations and opens it towards positive contributions to social innovation and transformation. Calls are repeatedly made for visions that can guide us towards sustainable futures. Scattered across a broad range of fields (i.e. business, non-government organization, land-use management, natural resource management, sustainability science, urban and regional planning) are an abundance of visioning studies. However, among the few evaluative studies in the literature there are apparent deficits in both the research and practice of visioning that curtails our expectations and prospects of realizing process-based and product-derived outcomes. These deficits suggests that calls instead should focus on the development of applied and theoretical understanding of crafting sustainability visions, enhancing the rigor and robustness of visioning methodology, and on integrating practice, research, and education for collaborative sustainability visioning. From an analysis of prominent visioning and sustainability visioning studies in the literature, this dissertation articulates what is sustainability visioning and synthesizes a conceptual framework for criteria-based design and evaluation of sustainability visioning studies. While current visioning methodologies comply with some of these guidelines, none adhere to all of them. From this research, a novel sustainability visioning methodology is designed to address this gap to craft visions that are shared, systemic, principles-based, action-oriented, relevant, and creative (i.e. SPARC visioning methodology) and evaluated across all quality criteria. Empirical studies were conducted to test and apply the conceptual and methodological frameworks -- with an emphasis on enhancing the rigor and robustness in real world visioning processes for urban planning and teaching sustainability competencies. In-depth descriptions of the collaborative visioning studies demonstrate tangible outcomes for: (a) implementing the above sustainability visioning methodology, including evaluative procedures; (b) adopting meaningful interactive engagement procedures; (c) integrating advanced analytical modeling, sustainability appraisal, and creativity enhancing procedures; and (d) developing perspective and methodological capacity for long-range sustainability planning.
ContributorsIwaniec, David (Author) / Wiek, Arnim (Thesis advisor) / Childers, Daniel L. (Committee member) / Lant, Timothy (Committee member) / Arizona State University (Publisher)
Created2013
Description
A methodology is developed that integrates institutional analysis with Life Cycle Assessment (LCA) to identify and overcome barriers to sustainability transitions and to bridge the gap between environmental practitioners and decisionmakers. LCA results are rarely joined with analyses of the social systems that control or influence decisionmaking and policies. As a result, LCA conclusions generally lack information about who or what controls different parts of the system, where and when the processes' environmental decisionmaking happens, and what aspects of the system (i.e. a policy or regulatory requirement) would have to change to enable lower environmental impact futures. The value of the combined institutional analysis and LCA (the IA-LCA) is demonstrated using a case study of passenger transportation in the Phoenix, Arizona metropolitan area. A retrospective LCA is developed to estimate how roadway investment has enabled personal vehicle travel and its associated energy, environmental, and economic effects. Using regional travel forecasts, a prospective life cycle inventory is developed. Alternative trajectories are modeled to reveal future "savings" from reduced roadway construction and vehicle travel. An institutional analysis matches the LCA results with the specific institutions, players, and policies that should be targeted to enable transitions to these alternative futures. The results show that energy, economic, and environmental benefits from changes in passenger transportation systems are possible, but vary significantly depending on the timing of the interventions. Transition strategies aimed at the most optimistic benefits should include 1) significant land-use planning initiatives at the local and regional level to incentivize transit-oriented development infill and urban densification, 2) changes to state or federal gasoline taxes, 3) enacting a price on carbon, and 4) nearly doubling vehicle fuel efficiency together with greater market penetration of alternative fuel vehicles. This aggressive trajectory could decrease the 2050 energy consumption to 1995 levels, greenhouse gas emissions to 1995, particulate emissions to 2006, and smog-forming emissions to 1972. The potential benefits and costs are both private and public, and the results vary when transition strategies are applied in different spatial and temporal patterns.
ContributorsKimball, Mindy (Author) / Chester, Mikhail (Thesis advisor) / Allenby, Braden (Committee member) / Golub, Aaron (Committee member) / Arizona State University (Publisher)
Created2014
Description
Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the source of spider silk's unique mechanical properties by investigating the protein composition, molecular structure and dynamics. Chemical compositional heterogeneity of spider silk fiber is critical to understand as it provides important information for the interactions between MaSp1 and MaSp2. Here, the amino acid composition of dragline silk protein was precisely determined using a solution-state nuclear magnetic resonance (NMR) approach on hydrolyzed silk fibers. In a similar fashion, solution-state NMR was applied to probe the "13"C/"15"N incorporation in silk, which is essential to understand for designing particular solid-state NMR methods for silk structural characterization. Solid-state NMR was used to elucidate silk protein molecular dynamics and the supercontraction mechanism. A "2"H-"13"C heteronuclear correlation (HETCOR) solid-state NMR technique was developed to extract site-specific "2"H quadrupole patterns and spin-lattice relaxation rates for understanding backbone and side-chain dynamics. Using this technique, molecular dynamics were determined for a number of repetitive motifs in silk proteins - Ala residing nanocrystalline &beta-sheet; domains, 3"1"-helical regions, and, Gly-Pro-Gly-XX &beta-turn; motifs. The protein backbone and side-chain dynamics of silk fibers in both dry and wet states reveal the impact of water on motifs with different secondary structures. Spider venom is comprised of a diverse range of molecules including salts, small organics, acylpolyamines, peptides and proteins. Neurotoxins are an important family of peptides in spider venom and have been shown to target and modulate various ion channels. The neurotoxins are Cys-rich and share an inhibitor Cys knot (ICK) fold. Here, the molecular structure of one G. rosea tarantula neurotoxin, GsAF2, was determined by solution-state NMR. In addition, the interaction between neurotoxins and model lipid bilayers was probed with solid-state NMR and negative-staining (NS) transmission electron microscopy (TEM). It is shown that the neurotoxins influence lipid bilayer assembly and morphology with the formation of nanodiscs, worm-like micelles and small vesicles.
ContributorsShi, Xiangyan (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Levitus, Marcia (Committee member) / Marzke, Robert F (Committee member) / Arizona State University (Publisher)
Created2014
Description
Increasing computational demands in data centers require facilities to operate at higher ambient temperatures and at higher power densities. Conventionally, data centers are cooled with electrically-driven vapor-compressor equipment. This paper proposes an alternative data center cooling architecture that is heat-driven. The source is heat produced by the computer equipment. This dissertation details experiments investigating the quantity and quality of heat that can be captured from a liquid-cooled microprocessor on a computer server blade from a data center. The experiments involve four liquid-cooling setups and associated heat-extraction, including a radical approach using mineral oil. The trials examine the feasibility of using the thermal energy from a CPU to drive a cooling process. Uniquely, the investigation establishes an interesting and useful relationship simultaneously among CPU temperatures, power, and utilization levels. In response to the system data, this project explores the heat, temperature and power effects of adding insulation, varying water flow, CPU loading, and varying the cold plate-to-CPU clamping pressure. The idea is to provide an optimal and steady range of temperatures necessary for a chiller to operate. Results indicate an increasing relationship among CPU temperature, power and utilization. Since the dissipated heat can be captured and removed from the system for reuse elsewhere, the need for electricity-consuming computer fans is eliminated. Thermocouple readings of CPU temperatures as high as 93°C and a calculated CPU thermal energy up to 67Wth show a sufficiently high temperature and thermal energy to serve as the input temperature and heat medium input to an absorption chiller. This dissertation performs a detailed analysis of the exergy of a processor and determines the maximum amount of energy utilizable for work. Exergy as a source of realizable work is separated into its two contributing constituents: thermal exergy and informational exergy. The informational exergy is that usable form of work contained within the most fundamental unit of information output by a switching device within a CPU. Exergetic thermal, informational and efficiency values are calculated and plotted for our particular CPU, showing how the datasheet standards compare with experimental values. The dissertation concludes with a discussion of the work's significance.
ContributorsHaywood, Anna (Author) / Phelan, Patrick E (Thesis advisor) / Herrmann, Marcus (Committee member) / Gupta, Sandeep (Committee member) / Trimble, Steve (Committee member) / Myhajlenko, Stefan (Committee member) / Arizona State University (Publisher)
Created2014