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 85
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
Background: Evidence about the purported hypoglycemic and hypolipidemic effects of nopales (prickly pear cactus pads) is limited. Objective: To evaluate the efficacy of nopales for improving cardiometabolic risk factors and oxidative stress, compared to control, in adults with hypercholesterolemia. Design: In a randomized crossover trial, participants were assigned to a 2-wk intervention with 2 cups/day of nopales or cucumbers (control), with a 2 to 3-wk washout period. The study included 16 adults (5 male; 46±14 y; BMI = 31.4±5.7 kg/m2) with moderate hypercholesterolemia (low density lipoprotein cholesterol [LDL-c] = 137±21 mg/dL), but otherwise healthy. Main outcomes measured included: dietary intake (energy, macronutrients and micronutrients), cardiometabolic risk markers (total cholesterol, LDL-c, high density lipoprotein cholesterol [HDL-c], triglycerides, cholesterol distribution in LDL and HDL subfractions, glucose, insulin, homeostasis model assessment, and C-reactive protein), and oxidative stress markers (vitamin C, total antioxidant capacity, oxidized LDL, and LDL susceptibility to oxidation). Effects of treatment, time, or interactions were assessed using repeated measures ANOVA. Results: There was no significant treatment-by-time effect for any dietary composition data, lipid profile, cardiometabolic outcomes, or oxidative stress markers. A significant time effect was observed for energy, which was decreased in both treatments (cucumber, -8.3%; nopales, -10.1%; pTime=0.026) mostly due to lower mono and polyunsaturated fatty acids intake (pTime=0.023 and pTime=0.003, respectively). Both treatments significantly increased triglyceride concentrations (cucumber, 14.8%; nopales, 15.2%; pTime=0.020). Despite the lack of significant treatment-by-time effects, great individual response variability was observed for all outcomes. After the cucumber and nopales phases, a decrease in LDL-c was observed in 44% and 63% of the participants respectively. On average LDL-c was decreased by 2.0 mg/dL (-1.4%) after the cucumber phase and 3.9 mg/dL (-2.9%) after the nopales phase (pTime=0.176). Pro-atherogenic changes in HDL subfractions were observed in both interventions over time, by decreasing the proportion of HDL-c in large HDL (cucumber, -5.1%; nopales, -5.9%; pTime=0.021) and increasing the proportion in small HDL (cucumber, 4.1%; nopales, 7.9%; pTime=0.002). Conclusions: These data do not support the purported benefits of nopales at doses of 2 cups/day for 2-wk on markers of lipoprotein profile, cardiometabolic risk, and oxidative stress in hypercholesterolemic adults.
ContributorsPereira Pignotti, Giselle Adriana (Author) / Vega-Lopez, Sonia (Thesis advisor) / Gaesser, Glenn (Committee member) / Keller, Colleen (Committee member) / Shaibi, Gabriel (Committee member) / Sweazea, Karen (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
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
Effect of a Wii Fit® intervention on balance, muscular fitness, and bone health in middle-aged women
Description
Sustaining a fall can be hazardous for those with low bone mass. Interventions exist to reduce fall-risk, but may not retain long-term interest. "Exergaming" has become popular in older adults as a therapy, but no research has been done on its preventative ability in non-clinical populations. The purpose was to determine the impact of 12-weeks of interactive play with the Wii Fit® on balance, muscular fitness, and bone health in peri- menopausal women. METHODS: 24 peri-menopausal-women were randomized into study groups. Balance was assessed using the Berg/FICSIT-4 and a force plate. Muscular strength was measured using the isokinetic dynamometer at 60°/180°/240°/sec and endurance was assessed using 50 repetitions at 240°/sec. Bone health was tracked using dual-energy x-ray absorptiometry (DXA) for the hip/lumbar spine and qualitative ultrasound (QUS) of the heel. Serum osteocalcin was assessed by enzyme immunoassay. Physical activity was quantified using the Women's Health Initiative Physical Activity Questionnaire and dietary patterns were measured using the Nurses' Health Food Frequency Questionnaire. All measures were repeated at weeks 6 and 12, except for the DXA, which was completed pre-post. RESULTS: There were no significant differences in diet and PA between groups. Wii Fit® training did not improve scores on the Berg/FICSIT-4, but improved center of pressure on the force plate for Tandem Step, Eyes Closed (p-values: 0.001-0.051). There were no significant improvements for muscular fitness at any of the angular velocities. DXA BMD of the left femoral neck improved in the intervention group (+1.15%) and decreased in the control (-1.13%), but no other sites had significant changes. Osteocalcin indicated no differences in bone turnover between groups at baseline, but the intervention group showed increased bone turnover between weeks 6 and 12. CONCLUSIONS: Findings indicate that WiiFit® training may improve balance by preserving center of pressure. QUS, DXA and osteocalcin data confirm that those in the intervention group were experiencing more bone turnover and bone formation than the control group. In summary, twelve weeks of strength /balance training with the Wii Fit® shows promise as a preventative intervention to reduce fall and fracture risk in non-clinical middle aged women who are at risk.
ContributorsWherry, Sarah Jo (Author) / Swan, Pamela D (Thesis advisor) / Adams, Marc (Committee member) / Der Ananian, Cheryl (Committee member) / Sweazea, Karen (Committee member) / Vaughan, Linda (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
Description
Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 ⁰C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic γ-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability.
ContributorsFlory, Justin David (Author) / Fromme, Petra (Thesis advisor) / Yan, Hao (Committee member) / Buttry, Daniel (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014