ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Displaying 1 - 8 of 8
Filtering by
- All Subjects: Biochemistry
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
Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation.
ContributorsNakano, Asuka (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2014
Description
The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
ContributorsRoy, Basab (Author) / Hecht, Sidney M. (Thesis advisor) / Jones, Anne (Committee member) / Levitus, Marcia (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
Description
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM.
ContributorsBreen, Isabella (Author) / Wachter, Rebekka (Thesis advisor) / Chiu, Po-Lin (Thesis advisor) / Levitus, Marcia (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2020
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
For cold chain tracking systems, precision and versatility across varying time intervals and temperature ranges remain integral to effective application in clinical, commercial, and academic settings. Therefore, while electronic and chemistry/physics based cold chain tracking mechanisms currently exist, both have limitations that affect their application across various biospecimens and commercial products, providing the initiative to develop a time temperature visual indicator system that resolves challenges with current cold chain tracking approaches. As a result, a permanganate/oxalic acid time temperature visual indicator system for cold chain tracking has been proposed. At thawing temperatures, the designed permanganate/oxalic acid reaction system undergoes a pink to colorless transition as permanganate, Mn(VII), is reduced to auto-catalytic Mn(II), while oxalate is oxidized to CO2. Therefore, when properly stored and vitrified or frozen, the proposed visual indicator remains pink, whereas exposure to thawing conditions will result in an eventual, time temperature dependent, designed color transition that characterizes compromised biospecimen integrity. To design visual indicator systems for targeted times at specific temperatures, absorbance spectroscopy was utilized to monitor permanganate kinetic curves by absorbance at 525 nm. As a result, throughout the outlined research, the following aims were demonstrated: (i) Design and functionality of 1x (0.5 mM KMnO4) visual indicator systems across various time intervals at temperatures ranging from 25°C to -20°C, (ii) Design and functionality of high concentration, 5x, visual indicator systems across varying targeted time intervals at temperatures ranging from 25°C to 0°C, (iii) Pre-activation stability and long-term stability of the proposed visual indicator systems.
ContributorsLjungberg, Emil (Author) / Borges, Chad (Thesis advisor) / Levitus, Marcia (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2024