Theses and Dissertations
This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students.
Displaying 1 - 10 of 92
Description
The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration.
ContributorsMaini, Rumit (Author) / Hecht, Sidney M. (Thesis advisor) / Gould, Ian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
This research examines the current challenges of using Lamb wave interrogation methods to localize fatigue crack damage in a complex metallic structural component subjected to unknown temperatures. The goal of this work is to improve damage localization results for a structural component interrogated at an unknown temperature, by developing a probabilistic and reference-free framework for estimating Lamb wave velocities and the damage location. The methodology for damage localization at unknown temperatures includes the following key elements: i) a model that can describe the change in Lamb wave velocities with temperature; ii) the extension of an advanced time-frequency based signal processing technique for enhanced time-of-flight feature extraction from a dispersive signal; iii) the development of a Bayesian damage localization framework incorporating data association and sensor fusion. The technique requires no additional transducers to be installed on a structure, and allows for the estimation of both the temperature and the wave velocity in the component. Additionally, the framework of the algorithm allows it to function completely in an unsupervised manner by probabilistically accounting for all measurement origin uncertainty. The novel algorithm was experimentally validated using an aluminum lug joint with a growing fatigue crack. The lug joint was interrogated using piezoelectric transducers at multiple fatigue crack lengths, and at temperatures between 20°C and 80°C. The results showed that the algorithm could accurately predict the temperature and wave speed of the lug joint. The localization results for the fatigue damage were found to correlate well with the true locations at long crack lengths, but loss of accuracy was observed in localizing small cracks due to time-of-flight measurement errors. To validate the algorithm across a wider range of temperatures the electromechanically coupled LISA/SIM model was used to simulate the effects of temperatures. The numerical results showed that this approach would be capable of experimentally estimating the temperature and velocity in the lug joint for temperatures from -60°C to 150°C. The velocity estimation algorithm was found to significantly increase the accuracy of localization at temperatures above 120°C when error due to incorrect velocity selection begins to outweigh the error due to time-of-flight measurements.
ContributorsHensberry, Kevin (Author) / Chattopadhyay, Aditi (Thesis advisor) / Liu, Yongming (Committee member) / Papandreou-Suppappola, Antonia (Committee member) / Arizona State University (Publisher)
Created2013
Description
Single molecule DNA Sequencing technology has been a hot research topic in the recent decades because it holds the promise to sequence a human genome in a fast and affordable way, which will eventually make personalized medicine possible. Single molecule differentiation and DNA translocation control are the two main challenges in all single molecule DNA sequencing methods. In this thesis, I will first introduce DNA sequencing technology development and its application, and then explain the performance and limitation of prior art in detail. Following that, I will show a single molecule DNA base differentiation result obtained in recognition tunneling experiments. Furthermore, I will explain the assembly of a nanofluidic platform for single strand DNA translocation, which holds the promised to be integrated into a single molecule DNA sequencing instrument for DNA translocation control. Taken together, my dissertation research demonstrated the potential of using recognition tunneling techniques to serve as a general readout system for single molecule DNA sequencing application.
ContributorsLiu, Hao (Author) / Lindsay, Stuart M (Committee member) / Yan, Hao (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2013
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
Aluminum alloys and their composites are attractive materials for applications requiring high strength-to-weight ratios and reasonable cost. Many of these applications, such as those in the aerospace industry, undergo fatigue loading. An understanding of the microstructural damage that occurs in these materials is critical in assessing their fatigue resistance. Two distinct experimental studies were performed to further the understanding of fatigue damage mechanisms in aluminum alloys and their composites, specifically fracture and plasticity. Fatigue resistance of metal matrix composites (MMCs) depends on many aspects of composite microstructure. Fatigue crack growth behavior is particularly dependent on the reinforcement characteristics and matrix microstructure. The goal of this work was to obtain a fundamental understanding of fatigue crack growth behavior in SiC particle-reinforced 2080 Al alloy composites. In situ X-ray synchrotron tomography was performed on two samples at low (R=0.1) and at high (R=0.6) R-ratios. The resulting reconstructed images were used to obtain three-dimensional (3D) rendering of the particles and fatigue crack. Behaviors of the particles and crack, as well as their interaction, were analyzed and quantified. Four-dimensional (4D) visual representations were constructed to aid in the overall understanding of damage evolution. During fatigue crack growth in ductile materials, a plastic zone is created in the region surrounding the crack tip. Knowledge of the plastic zone is important for the understanding of fatigue crack formation as well as subsequent growth behavior. The goal of this work was to quantify the 3D size and shape of the plastic zone in 7075 Al alloys. X-ray synchrotron tomography and Laue microdiffraction were used to non-destructively characterize the volume surrounding a fatigue crack tip. The precise 3D crack profile was segmented from the reconstructed tomography data. Depth-resolved Laue patterns were obtained using differential-aperture X-ray structural microscopy (DAXM), from which peak-broadening characteristics were quantified. Plasticity, as determined by the broadening of diffracted peaks, was mapped in 3D. Two-dimensional (2D) maps of plasticity were directly compared to the corresponding tomography slices. A 3D representation of the plastic zone surrounding the fatigue crack was generated by superimposing the mapped plasticity on the 3D crack profile.
ContributorsHruby, Peter (Author) / Chawla, Nikhilesh (Thesis advisor) / Solanki, Kiran (Committee member) / Liu, Yongming (Committee member) / Arizona State University (Publisher)
Created2014
Description
This research focuses on the benefits of using nanocomposites in aerospace structural components to prevent or delay the onset of unique composite failure modes, such as delamination. Analytical, numerical, and experimental analyses were conducted to provide a comprehensive understanding of how carbon nanotubes (CNTs) can provide additional structural integrity when they are used in specific hot spots within a structure. A multiscale approach was implemented to determine the mechanical and thermal properties of the nanocomposites, which were used in detailed finite element models (FEMs) to analyze interlaminar failures in T and Hat section stringers. The delamination that first occurs between the tow filler and the bondline between the stringer and skin was of particular interest. Both locations are considered to be hot spots in such structural components, and failures tend to initiate from these areas. In this research, nanocomposite use was investigated as an alternative to traditional methods of suppressing delamination. The stringer was analyzed under different loading conditions and assuming different structural defects. Initial damage, defined as the first drop in the load displacement curve was considered to be a useful variable to compare the different behaviors in this study and was detected via the virtual crack closure technique (VCCT) implemented in the FE analysis.
Experiments were conducted to test T section skin/stringer specimens under pull-off loading, replicating those used in composite panels as stiffeners. Two types of designs were considered: one using pure epoxy to fill the tow region and another that used nanocomposite with 5 wt. % CNTs. The response variable in the tests was the initial damage. Detailed analyses were conducted using FEMs to correlate with the experimental data. The correlation between both the experiment and model was satisfactory. Finally, the effects of thermal cure and temperature variation on nanocomposite structure behavior were studied, and both variables were determined to influence the nanocomposite structure performance.
Experiments were conducted to test T section skin/stringer specimens under pull-off loading, replicating those used in composite panels as stiffeners. Two types of designs were considered: one using pure epoxy to fill the tow region and another that used nanocomposite with 5 wt. % CNTs. The response variable in the tests was the initial damage. Detailed analyses were conducted using FEMs to correlate with the experimental data. The correlation between both the experiment and model was satisfactory. Finally, the effects of thermal cure and temperature variation on nanocomposite structure behavior were studied, and both variables were determined to influence the nanocomposite structure performance.
ContributorsHasan, Zeaid (Author) / Chattopadhyay, Aditi (Thesis advisor) / Dai, Lenore (Committee member) / Jiang, Hanqing (Committee member) / Rajadas, John (Committee member) / Liu, Yongming (Committee member) / Arizona State University (Publisher)
Created2014
Description
Natural hydrogenases catalyze the reduction of protons to molecular hydrogen reversibly under mild conditions; these enzymes have an unusual active site architecture, in which a diiron site is connected to a cubane type [4Fe-4S] cluster. Due to the relevance of this reaction to energy production, and in particular to sustainable fuel production, there have been substantial amount of research focused on developing biomimetic organometallic models. However, most of these organometallic complexes cannot revisit the structural and functional fine-tuning provided by the protein matrix as seen in the natural enzyme. The goal of this thesis is to build a protein based functional mimic of [Fe-Fe] hydrogenases. I used a 'retrosynthetic' approach that separates out two functional aspects of the natural enzyme. First, I built an artificial electron transfer domain by engineering two [4Fe-4S] cluster binding sites into an existing protein, DSD, which is a de novo designed domain swapped dimer. The resulting protein, DSD-bis[4Fe-4S], contains two clusters at a distance of 36 Å . I then varied distance between two clusters using vertical translation along the axis of the coiled coil; the resulting protein demonstrates efficient electron transfer to/from redox sites. Second, I built simple, functional artificial hydrogenases by using an artificial amino acid comprising a 1,3 dithiol moiety to anchor a biomimetic [Fe-Fe] active site within the protein scaffold Correct incorporation of the cluster into a model helical peptide was verified by UV-Vis, FTIR, ESI-MS and CD spectroscopy. This synthetic strategy is extended to the de novo design of more complex protein architectures, four-helix bundles that host the di-iron cluster within the hydrophobic core. In a separate approach, I developed a generalizable strategy to introduce organometallic catalytic sites into a protein scaffold. I introduced a biomimetic organometallic complex for proton reduction by covalent conjugation to biotin. The streptavidin-bound complex is significantly more efficient in photocatalytic hydrogen production than the catalyst alone. With these artificial proteins, it will be possible to explore the effect of second sphere interactions on the activity of the diiron center, and to include in the design properties such as compatibility with conductive materials and electrodes.
ContributorsRoy, Anindya (Author) / Ghirlanda, Giovanna (Thesis advisor) / Yan, Hao (Committee member) / Gust, Devens (Committee member) / Arizona State University (Publisher)
Created2014