Matching Items (280)
Description
Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.
ContributorsBozeman, Trevor C (Author) / Hecht, Sidney M. (Thesis advisor) / Chaput, John (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
In this thesis, we present the study of several physical properties of relativistic mat- ters under extreme conditions. We start by deriving the rate of the nonleptonic weak processes and the bulk viscosity in several spin-one color superconducting phases of quark matter. We also calculate the bulk viscosity in the nonlinear and anharmonic regime in the normal phase of strange quark matter. We point out several qualitative effects due to the anharmonicity, although quantitatively they appear to be relatively small. In the corresponding study, we take into account the interplay between the non- leptonic and semileptonic weak processes. The results can be important in order to relate accessible observables of compact stars to their internal composition. We also use quantum field theoretical methods to study the transport properties in monolayer graphene in a strong magnetic field. The corresponding quasi-relativistic system re- veals an anomalous quantum Hall effect, whose features are directly connected with the spontaneous flavor symmetry breaking. We study the microscopic origin of Fara- day rotation and magneto-optical transmission in graphene and show that their main features are in agreement with the experimental data.
ContributorsWang, Xinyang, Ph.D (Author) / Shovkovy, Igor (Thesis advisor) / Belitsky, Andrei (Committee member) / Easson, Damien (Committee member) / Peng, Xihong (Committee member) / Vachaspati, Tanmay (Committee member) / Arizona State University (Publisher)
Created2013
Description
Numerical simulations are very helpful in understanding the physics of the formation of structure and galaxies. However, it is sometimes difficult to interpret model data with respect to observations, partly due to the difficulties and background noise inherent to observation. The goal, here, is to attempt to bridge this gap between simulation and observation by rendering the model output in image format which is then processed by tools commonly used in observational astronomy. Images are synthesized in various filters by folding the output of cosmological simulations of gasdynamics with star-formation and dark matter with the Bruzual- Charlot stellar population synthesis models. A variation of the Virgo-Gadget numerical simulation code is used with the hybrid gas and stellar formation models of Springel and Hernquist (2003). Outputs taken at various redshifts are stacked to create a synthetic view of the simulated star clusters. Source Extractor (SExtractor) is used to find groupings of stellar populations which are considered as galaxies or galaxy building blocks and photometry used to estimate the rest frame luminosities and distribution functions. With further refinements, this is expected to provide support for missions such as JWST, as well as to probe what additional physics are needed to model the data. The results show good agreement in many respects with observed properties of the galaxy luminosity function (LF) over a wide range of high redshifts. In particular, the slope (alpha) when fitted to the standard Schechter function shows excellent agreement both in value and evolution with redshift, when compared with observation. Discrepancies of other properties with observation are seen to be a result of limitations of the simulation and additional feedback mechanisms which are needed.
ContributorsMorgan, Robert (Author) / Windhorst, Rogier A (Thesis advisor) / Scannapieco, Evan (Committee member) / Rhoads, James (Committee member) / Gardner, Carl (Committee member) / Belitsky, Andrei (Committee member) / Arizona State University (Publisher)
Created2012
Description
Understanding the temperature structure of protoplanetary disks (PPDs) is paramount to modeling disk evolution and future planet formation. PPDs around T Tauri stars have two primary heating sources, protostellar irradiation, which depends on the flaring of the disk, and accretional heating as viscous coupling between annuli dissipate energy. I have written a "1.5-D" radiative transfer code to calculate disk temperatures assuming hydrostatic and radiative equilibrium. The model solves for the temperature at all locations simultaneously using Rybicki's method, converges rapidly at high optical depth, and retains full frequency dependence. The likely cause of accretional heating in PPDs is the magnetorotational instability (MRI), which acts where gas ionization is sufficiently high for gas to couple to the magnetic field. This will occur in surface layers of the disk, leaving the interior portions of the disk inactive ("dead zone"). I calculate temperatures in PPDs undergoing such "layered accretion." Since the accretional heating is concentrated far from the midplane, temperatures in the disk's interior are lower than in PPDs modeled with vertically uniform accretion. The method is used to study for the first time disks evolving via the magnetorotational instability, which operates primarily in surface layers. I find that temperatures in layered accretion disks do not significantly differ from those of "passive disks," where no accretional heating exists. Emergent spectra are insensitive to active layer thickness, making it difficult to observationally identify disks undergoing layered vs. uniform accretion. I also calculate the ionization chemistry in PPDs, using an ionization network including multiple charge states of dust grains. Combined with a criterion for the onset of the MRI, I calculate where the MRI can be initiated and the extent of dead zones in PPDs. After accounting for feedback between temperature and active layer thickness, I find the surface density of the actively accreting layers falls rapidly with distance from the protostar, leading to a net outward flow of mass from ~0.1 to 3 AU. The clearing out of the innermost zones is possibly consistent with the observed behavior of recently discovered "transition disks."
ContributorsLesniak, Michael V., III (Author) / Desch, Steven J. (Thesis advisor) / Scannapieco, Evan (Committee member) / Timmes, Francis (Committee member) / Starrfield, Sumner (Committee member) / Belitsky, Andrei (Committee member) / Arizona State University (Publisher)
Created2012
Description
This thesis deals with the first measurements done with a cold neutron beam at the Spallation Neutron Source at Oak Ridge National Laboratory. The experimental technique consisted of capturing polarized cold neutrons by nuclei to measure parity-violation in the angular distribution of the gamma rays following neutron capture. The measurements presented here for the nuclei Chlorine ( 35Cl) and Aluminum ( 27Al ) are part of a program with the ultimate goal of measuring the asymmetry in the angular distribution of gamma rays emitted in the capture of neutrons on protons, with a precision better than 10-8, in order to extract the weak hadronic coupling constant due to pion exchange interaction with isospin change equal with one ( hπ 1). Based on theoretical calculations asymmetry in the angular distribution of the gamma rays from neutron capture on protons has an estimated size of 5·10-8. This implies that the Al parity violation asymmetry and its uncertainty have to be known with a precision smaller than 4·10-8. The proton target is liquid Hydrogen (H2) contained in an Aluminum vessel. Results are presented for parity violation and parity-conserving asymmetries in Chlorine and Aluminum. The systematic and statistical uncertainties in the calculation of the parity-violating and parity-conserving asymmetries are discussed.
ContributorsBalascuta, Septimiu (Author) / Alarcon, Ricardo (Thesis advisor) / Belitsky, Andrei (Committee member) / Doak, Bruce (Committee member) / Comfort, Joseph (Committee member) / Schmidt, Kevin (Committee member) / Arizona State University (Publisher)
Created2012
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Bacteria have been shown to possess a large array of regulatory mechanisms to not just respond to a diverse array of environmental stresses, but to injurious artificial proteins as well. A previous investigation introduced DX, a man-made ATP sequestering protein into Escherichia coli (E. coli) which resulted in the formation of novel endoliposome structures and induced a viable but non-culturable state (VBNC) that was not easily reversed. It was hypothesized that the broadly conserved bacterial stringent response pathway may have been responsible for the observed phenotypic changes. With the goal of unveiling the molecular mechanism behind this novel response, changes in cellular morphology and physiology upon DX expression were assessed in a population of E. coli encoding a dysfunctional relA gene, one of the two genes controlling the induction of the stringent response. It was ultimately shown that RelA directly contributed to cellular filamentation, endoliposome structure formation, and the induction of a VBNC state. While the stringent response has been extensively shown to induce a VBNC state, to our knowledge, relA has not yet been shown to induce filamentation or coordinate the formation of endoliposome structures in bacteria. As the stringent response has been shown to be increasingly involved in antibiotic tolerance, this study provided an exciting opportunity to further characterize this adaptive response pathway to aid in the future development of novel therapeutics. In addition to this, this study continued to highlight that the DX protein may serve one of the first tools to allow for the direct selection of bacteria in a VBNC state by morphologically distinguishing non-culturable cells through cellular filamentation.
ContributorsFrost, Fredrick Charles (Author) / Chaput, John (Thesis director) / Wachter, Rebekka (Committee member) / Korch, Shaleen (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
Description
Identifying disease biomarkers may aid in the early detection of breast cancer and improve patient outcomes. Recent evidence suggests that tumors are immunogenic and therefore patients may launch an autoantibody response to tumor associated antigens. Single-chain variable fragments of autoantibodies derived from regional lymph node B cells of breast cancer patients were used to discover these tumor associated biomarkers on protein microarrays. Six candidate biomarkers were discovered from 22 heavy chain-only variable region antibody fragments screened. Validation tests are necessary to confirm the tumorgenicity of these antigens. However, the use of single-chain variable autoantibody fragments presents a novel platform for diagnostics and cancer therapeutics.
ContributorsSharman, M. Camila (Author) / Magee, Dewey (Mitch) (Thesis director) / Wallstrom, Garrick (Committee member) / Petritis, Brianne (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor) / Virginia G. Piper Center for Personalized Diagnostics (Contributor) / Biodesign Institute (Contributor)
Created2012-12
Description
Cell-free protein synthesis (CFPS) is becoming an increasingly popular method of in vitro protein expression for biotechnology applications. However, there is still no comprehensive resource that outlines the most effective lysate and template combinations for efficient eukaryotic CFPS. To address this issue, expression vectors were constructed and assayed in order to determine their activity within three commercial eukaryotic CFPS systems: Wheat Germ Extract (WGE), Rabbit Reticulocyte Lysate (RRL), and HeLa Cell Lysate (HCL). Previously in the Chaput lab, a luciferase reporter vector was expressed in each lysate system, testing different template variables impacting protein expression, including the 5' UTR sequence, presence of poly(A) tail, and DNA type. It was found that plasmid DNA templates generally yielded ~500-fold greater amount of protein than linear DNA templates and the inclusion of a poly(A) tail did not significantly increase protein expression in the plasmid systems. Additionally, the incorporation of a viral translation enhancing sequence into the 5' UTR increased translation in a lysate-specific manner. The HCL system had a strong preference for the EMCV sequence, WGE had a preference for the sequences from AMV and TMV, and RRL showed no specific preference. Overall, the HCL-EMCV system generated the greatest amount of protein per volume, producing 10-fold more protein than the second best template-lysate combination tested. Here, four human genes fused with a c-Myc tag were expressed in each lysate using the EMCV 5' UTR sequence in order to test the generality of the previous results. Protein synthesis was assayed using a luciferase construct with a c-Myc tag to recapitulate the previous luminometer data and western blotting of the human proteins. These analyses showed the same EMCV expression trends across all systems, with the HCL system synthesizing the greatest amount of each protein. In the future, when choosing commercial eukaryotic CFPS systems for gene expression, these template variables should be considered when performing cost analysis for cell-free protein production.
ContributorsHartsough, Emily Mae (Author) / Chaput, John (Thesis director) / Chandler, Douglas (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Arts, Media and Engineering (Contributor)
Created2015-05