Matching Items (21)
Description
Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is to make a variety of mutant CV-Ns to specifically bind other glycan targets. Such a set of lectins may be used as screening reagents to identify biomarkers and other glycan motifs of interest. As proof of concept, a T7 phage display library was constructed using P51G-m4-CVN genes mutated at positions 41, 44, 52, 53, 56, 74, and 76 in binding Domain B. Five CV-N mutants were selected from the library and expressed in BL21(DE3) E. coli. Two of the mutants, SSDGLQQ-P51Gm4-CVN and AAGRLSK-P51Gm4-CVN, were sufficiently stable for characterization and were examined by CD, Tm, ELISA, and glycan array. Both proteins have CD minima at approximately 213 nm, indicating largely β-sheet structure, and have Tm values greater than 40°C. ELISA against gp120 and RNase B demonstrate both proteins' ability to bind high mannose glycans. To more specifically determine the binding specificity of each protein, AAGRLSK-P51Gm4-CVN, SSDGLQQ-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN were sent to the Consortium for Functional Glycomics (CFG) for glycan array analysis. AAGRLSK-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN, have identical specificities for high mannose glycans containing terminal Manα1-2Manα. SSDGLQQ-P51Gm4-CVN binds to terminal GlcNAcα1-4Gal motifs and a subgroup of high mannose glycans bound by P51G-m4-CVN. SSDGLQQ-wt-CVN was produced to restore anti-HIV activity and has a high nanomolar EC50 value compared to wt-CVN's low nanomolar activity. Overall, these experiments show that CV-N Domain B can be mutated and retain specificity identical to wt-CVN or acquire new glycan specificities. This first generation information can be used to produce glycan-specific lectins for a variety of applications.
ContributorsRuben, Melissa (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2013
Description
ABSTRACT Peptide microarrays may prove to be a powerful tool for proteomics research and clinical diagnosis applications. Fodor et al. and Maurer et al. have shown proof-of-concept methods of light- and electrochemically-directed peptide microarray fabrication on glass and semiconductor microchips respectively. In this work, peptide microarray fabrication based on the abovementioned techniques were optimized. In addition, MALDI mass spectrometry based peptide synthesis characterization on semiconductor microchips was developed and novel applications of a CombiMatrix (CBMX) platform for electrochemically controlled synthesis were explored. We have investigated performance of 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) derivatives as photo-labile protecting group. Specifically, influence of substituents on 4 and 5 positions of phenyl ring of NPPOC group on the rate of photolysis and the yield of the amine was investigated. The results indicated that substituents capable of forming a π-network with the nitro group enhanced the rate of photolysis and yield. Once such properly substituted NPPOC groups were used, the rate of photolysis/yield depended on the nature of protected amino group indicating that a different chemical step during the photo-cleavage process became the rate limiting step. We also focused on electrochemically-directed parallel synthesis of high-density peptide microarrays using the CBMX technology referred to above which uses electrochemically generated acids to perform patterned chemistry. Several issues related to peptide synthesis on the CBMX platform were studied and optimized, with emphasis placed on the reactions of electro-generated acids during the deprotection step of peptide synthesis. We have developed a MALDI mass spectrometry based method to determine the chemical composition of microarray synthesis, directly on the feature. This method utilizes non-diffusional chemical cleavage from the surface, thereby making the chemical characterization of high-density microarray features simple, accurate, and amenable to high-throughput. CBMX Corp. has developed a microarray reader which is based on electro-chemical detection of redox chemical species. Several parameters of the instrument were studied and optimized and novel redox applications of peptide microarrays on CBMX platform were also investigated using the instrument. These include (i) a search of metal binding catalytic peptides to reduce overpotential associated with water oxidation reaction and (ii) an immobilization of peptide microarrays using electro-polymerized polypyrrole.
ContributorsKumar, Pallav (Author) / Woodbury, Neal (Thesis advisor) / Allen, James (Committee member) / Johnston, Stephen (Committee member) / Arizona State University (Publisher)
Created2013
Description
ABSTRACT
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
ContributorsMorales Betanzos, Carlos (Author) / LaBaer, Joshua (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Glycosaminoglycans (GAGs) are a class of complex biomolecules comprised of linear, sulfated polysaccharides whose presence on cell surfaces and in the extracellular matrix involve them in many physiological phenomena as well as in interactions with pathogenic microbes. Decorin binding protein A (DBPA), a Borrelia surface lipoprotein involved in the infectivity of Lyme disease, is responsible for binding GAGs found on decorin, a small proteoglycan present in the extracellular matrix. Different DBPA strains have notable sequence heterogeneity that results in varying levels of GAG-binding affinity. In this dissertation, the structures and GAG-binding mechanisms for three strains of DBPA (B31 and N40 DBPAs from B. burgdorferi and PBr DBPA from B. garinii) are studied to determine why each strain has a different affinity for GAGs. These three strains have similar topologies consisting of five α-helices held together by a hydrophobic core as well as two long flexible segments: a linker between helices one and two and a C-terminal tail. This structural arrangement facilitates the formation of a basic pocket below the flexible linker which is the primary GAG-binding epitope. However, this GAG-binding site can be occluded by the flexible linker, which makes the linker a negative regulator of GAG-binding. ITC and NMR titrations provide KD values that show PBr DBPA binds GAGs with higher affinity than B31 and N40 DBPAs, while N40 binds with the lowest affinity of the three. Work in this thesis demonstrates that much of the discrepancies seen in GAG affinities of the three DBPAs can be explained by the amino acid composition and conformation of the linker. Mutagenesis studies show that B31 DBPA overcomes the pocket obstruction with the BXBB motif in its linker while PBr DBPA has a retracted linker that exposes the basic pocket as well as a secondary GAG-binding site. N40 DBPA, however, does not have any evolutionary modifications to its structure to enhance GAG binding which explains its lower affinity for GAGs. GMSA and ELISA assays, along with NMR PRE experiments, confirm that structural changes in the linker do affect GAG-binding and, as a result, the linker is responsible for regulating GAG affinity.
ContributorsMorgan, Ashli M (Author) / Wang, Xu (Thesis advisor) / Allen, James (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015
Understanding the Current State of Vaccination Coverage in Arizona Schools: A Correlational Analysis
Description
The purpose of this study was to investigate what correlations exist between the immunization and personal belief exemption (PBE) rates and selected characteristics of Arizona schools. The demographic information of a school's student body, the percentage of student who are on free or reduced lunch, the presence of a Title I program at the school, the median household income of the zip code the school resides in, and the presence of a school nurse were all compared with immunization and PBE rates. Using data provided by the Arizona Department of Health Services (AZDHS), the National Center for Education Statistics (NCES), and the United States Census Bureau, these factors were investigated for kindergarten and 6th grade students. It was found that a higher percentage of white students in a student body was correlated with an higher rate of PBE and a lower immunization rate for measles, mumps, and rubella (MMR), polio, and hepatitis B. A higher percentage of Hispanic students in a student body was correlated with a lower rate of PBE and a higher immunization rate for measles, mumps, rubella (MMR), polio, and hepatitis B. There was little to no correlation between the percentage of students on free or reduced lunch and immunization or PBE rates. A higher median household income was correlated with a higher rate of PBE in public and private schools. Additionally, the immunization rates at schools with a nurse were significantly higher and the rate of PBE was significantly lower than at schools without a nurse. Finally, schools with a Title I program had mean immunization rates that were significantly higher and a mean PBE rate that was significantly lower than schools that did not have a Title I program.
ContributorsSellers, Abigail Leigh (Author) / Hendrickson, Kirstin (Thesis director) / Lefler, Scott (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Sandra Day O'Connor College of Law (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
With the new independence of adulthood, college students are a group susceptible to adopting unsupported, if not harmful, health practices. A survey of Arizona State University undergraduate students (N=200) was conducted to evaluate supplement use, trust in information sources, and beliefs about supplement regulation. Of those who reported using supplements, college students most frequently received information from friends and family. STEM majors in fields unrelated to health who were taking a supplement were found to be less likely to receive information about the supplement from a medical practitioner than those in health fields or those in non-STEM majors (-26.9%, p=0.018). STEM majors in health-related fields were 15.0% more likely to treat colds and/or cold symptoms with research-supported methods identified from reliable sources, while non-health STEM and non-STEM majors were more likely to take unsupported cold treatments (p=0.010). Surveyed students, regardless of major, also stated they would trust a medical practitioner for supplement advice above other sources (88.0%), and the majority expressed a belief that dietary supplements are approved/regulated by the government (59.8%).
ContributorsPerez, Jacob Tanner (Author) / Hendrickson, Kirstin (Thesis director) / Lefler, Scott (Committee member) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The Heliobacterial Reaction Center (HbRC) is the simplest Type I Reaction Center (RC) known today. However, upon illumination it has been found to produce menaquinol, and this has led to experiments investigating the function of this reduction scheme. The goal of the experiment was to investigate the mechanisms of menaquinol production through the use of Photosystem II (PSII) herbicides that are known to inhibit the QB quinone site in Type II RCs. Seven herbicides were chosen, and out of all of them terbuthylazine showed the greatest effect on the RC in isolated membranes when Transient Absorption Spectroscopy was used. In addition, terbuthylazine decreased menaquinone reduction to menaquinol by ~72%, slightly more than the reported effect of teburtryn (68%)1. In addition, terbuthylazine significantly impacted growth of whole cells under high light more than terbutryn.
ContributorsOdeh, Ahmad Osameh (Author) / Redding, Kevin (Thesis director) / Woodbury, Neal (Committee member) / Allen, James (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Each family approaches a cancer diagnosis differently. While some families pursue traditional treatments to the fullest extent, others attempt to refuse chemotherapy, often in favor of alternative medicines. When the patient is a minor, his or her parents have the authority to make medical decisions on their behalf, and this authority is constitutionally protected and socially upheld. However, when the decision to forgo chemotherapy does not comply with minimum standard of care and puts the minor's life in danger, legal action can and has been taken to force the minor to undergo chemotherapy. Legal precedent and biomedical ethics principles guide the decision-making process of the physicians and judges involved, although there is no official framework by which to prioritize these principles. Neglect and abuse procedures, as well as capacity determinations, mature minor doctrines, and religious convictions, add complexity to each forced chemotherapy case. These complexities were explored through the context of four case studies: Cassandra Callendar, who was not granted mature minor status and was forced into treatment by the Connecticut Supreme court; Starchild Abraham Cherrix, who was allowed to pursue the alternative Hoxsey therapy with the consent of his parents and the local court; Dennis Lindberg, a 14-year-old Jehovah's Witness who was permitted to refuse blood transfusions under the Mature Minor Doctrine; and Daniel Hauser, a developmentally delayed teen who was forced to undergo therapy against his parents' religious convictions. In the analysis and comprehensive comparison of these cases, it was concluded that an attempt to establish a protocol by which to determine the ethics of forcing chemotherapy, while well-intended, would ultimately be ineffective and extremely complex. Thus, each forced chemotherapy case must be evaluated on an individual basis.
ContributorsNelson, Sarah Gabrielle (Author) / Hendrickson, Kirstin (Thesis director) / Lynch, John (Committee member) / Jaramillo, Andres (Committee member) / School of Molecular Sciences (Contributor) / Sanford School of Social and Family Dynamics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases.
Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo.
Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity.
Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo.
Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity.
ContributorsSchmierer, Margaret (Author) / Hecht, Sidney M. (Thesis advisor) / Allen, James (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016