Theses
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Browsing Theses by Discipline "Biochemistry"
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- 1.9A Crystal Structure of the Rap1a GTPase Bound to its Natural Ligand, GTP(2007-01-21) Miller, Christopher Michael; Carla Mattos, Committee Chair; Clay Clark, Committee Member; Robert Rose, Committee MemberRap1a is a small GTPase in the Ras superfamily whose most well known function is to antagonize the Ras. Rap1a and Ras share common effectors which allow Rap1a to either unproductively bind Ras' effectors forming an inactive complex or sequester Ras' effectors away from the plasma membrane where Ras is inserted by C-terminal post-translational modifications. To date, a 2.2Å crystal structure of Rap1a bound to the non-hydrolyzable GTP analogue, GMPPNP, and one of its effectors, Raf-1, has been solved. This thesis presents the 1.9Å monomeric form of Rap1a bound to its natural ligand, GTP. Comparisons made between the previously published Rap—Raf structure, Rap2a, H-Ras, and RalA shed some light on the functions for conserved areas of Rap1a. The presence of a unique salt bridge at the Rap⁄Raf interface, a new conformation of threonine 61, a possible link for switch the II residue phenylalanine 64 with GAP-induced GTPase activity, and a suggested role for α helix 4 contribute to the Rap1a story.
- A Diversified Approach to Geminivirus Research.(2022-01-07) Spalango, Vendela; Jose Ascencio-Ibanez, Chair; Michael Goshe, Member; Peter Thompson, Member
- A Study of Hyaluronidases and UDP-Glucose Dehydrogenase as Contributing Factors to Prostate Cancer Progression.(2021-08-09) Brownfield, Andrew; Melanie Simpson, Chair; Joseph Barycki, Member; Arion Midgett, Member
- Accuracy and Sensitivity Comparison of ddPCR and qPCR for mtDNA.(2022-10-28) Moore, David Lewis; Melanie Simpson, Co-Chair; Kelly Meiklejohn, Co-Chair; Joshua Strable, Member
- Action spectra for UV- light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome.(2002-08-28) Zhirnov, Oksana Vladimirovna; Paul L. Wolenzien, Committee ChairUV- light induced crosslinking has been used to study RNA structures since the 1970s. The idea behind the method is that if there is an interaction between two nucleotides in the RNA tertiary structure, either as a part of the same strand or as a part of intermolecular interaction, they sometime will form covalent bond or "crosslink" with each other upon exposure to the UV light. Importantly, these interactions seem to be specific, limited in number and the crosslinking efficiencies are sufficient to allow their use in monitoring conformational changes of macromolecules. The occurrence of crosslinks within macromolecules depends on a combination of factors, including the inherent photoreactivity of the nucleotides that are joined, the correct distance and geometrical arrangement of the nucleotides, the molecular dynamics at the site of the crosslink and the wavelength of UV light that is used for irradiation. In this work, action spectra for eight out of fifteen 16S rRNA crosslinks were determined. For six of the crosslinks the action spectra correspond to the absorption spectra of at least one of the participating nucleotides. For two of them: C967 x C1400 and C1402 x C1501 the absorption maximum differs from the spectra of cytidine in aqua solution. The spectra for the C1402 x C1501 crosslink may be shifted because C1402 is posttranscriptionally modified to m4Cm. This difference can also result from the influence of molecular environment at the absorption of nucleotides, participating in those crosslinks formation.
- Analysis of Temporal and Tissue Specific Humulus lupulus Isoforms by Long-Read Sequencing.(2020-04-01) Liebelt, Donna J; Colleen Doherty, Chair; Flora Meilleur, Member; Michael Goshe, Member
- Antioxidant Defense: Effects of an NRF2 ARE Polymorphism on the Activation of Phase II Enzymes(2006-11-02) Marzec, Jacqueline; Dr. William Miller, Committee Co-Chair; Dr. Bob Rose, Committee Co-Chair; Dr. Hosni Hassan, Committee Member; Dr. Steven Kleeberger, Committee MemberThe purpose of the research put forth in this thesis has been to critically evaluate the functional effects of a single nucleotide polymorphism in the antioxidant response element (ARE) of NRF2. NRF2 is a transcription factor that regulates numerous antioxidants in response to oxidative stress, and we believe mutation within the ARE may impact both transcription and translation of downstream antioxidants. Initial studies focus on quantitative differences in NRF2 expression between ARE clones with and without mutation using transient transfection assays as well as diminished NRF2:ARE binding by gel shift analysis. Effects of decreased function are further analyzed using lymphoblast cell lines with naturally occurring NRF2 ARE mutations (wildtype, ARE heterozygote and ARE variant) exposed to oxidative stress. Differences in mRNA expression of NRF2 and downstream antioxidants are assessed by reverse transcriptase PCR and quantitative real-time PCR, and protein level changes are analyzed by western blot. These studies show that NRF2 function is decreased with mutation to the ARE and NRF2:ARE binding is diminished in the presence of variant alleles. Furthermore, transcription of NRF2 and several downstream antioxidants (NQO1, HO-1 and GPx2) is significantly upregulated in heterozygotes with one copy of the variant allele during serum starvation and following exposure to several oxidative stress agents [hyperoxia (95% oxygen) and lipopolysaccharide]. However, increases in mRNA transcripts do not directly correlate with protein levels. Nonetheless, we did observe differential protein expression between NRF2 genotypes for NRF2, the accessory protein small Maf, as well as ARE-bearing downstream antioxidants (NQO1, HO-1, GPx2 and GSR). The consequences of these functional differences are likely to impact the cellular response to oxidative stress.
- Caspase-8 and FLIP Form a Dimer After Urea-Induced Unfolding.(2014-03-26) Rogers, Bryan M; Allan Clark, Chair; Robert Rose, Member; Earl Maxwell, Member
- Characterization Of OleTJE-SOR Fusion Enzymes For Use In Biofuel Production.(2021-01-07) Garcia, Enrique; Amy Grunden, Co-Chair; Robert Rose, Co-Chair; Michael Goshe, Member; Katie Maloney, Technical Consultant
- Characterization of the Redox Transition of the XRCC1 N-Terminal Domain.(2015-03-23) Smith, Cassandra Elizabeth; James Knopp, Chair; Michael Goshe, Member; Paul Wollenzien, Member; Geoff Mueller, External
- Characterizing the Interactions Between the Cytoplasmic Domains of Arabidopsis BRI1 and BAK1.(2018-05-08) Robinson, Jeffrey Lee; Michael Goshe, Chair; Guozhou Xu, Member; Robert Rose, Member
- A comparative analysis of Bacillus subtilis and Bacillus anthracis AbrB using Circular Dichroism and NMR Spectroscopy(2009-04-20) Perry, Nicole Alana; William Miller, Committee Member; John Cavanagh, Committee Chair; Dennis Brown, Committee MemberThe transition state regulators are DNA-binding proteins found in many bacterial species and are involved in the regulation of processes related to the organism’s defense. Each transition state regulator binds multiple DNA targets with affinity in the nanomolar range, in spite of not recognizing a consensus sequence. Structural studies of the N-terminal DNA-binding domain have revealed a mechanism by which the proteins bind DNA, however, to date no NMR structure of a full-length transition state regulator exists. AbrB is the most well-characterized transition state regulator, and homologs are found in both Bacillus subtilis and Bacillus anthracis. The sequences of AbrB from B. subtilis and B. anthracis are overall 85% identical, and understanding the sequence differences from a structural standpoint is key to understanding the function of each protein. The far-UV circular dichroism spectra of each were deconvoluted using a number of programs, and the estimates of secondary structure content compared to the predictions by PSIPRED. Multiple NMR experiments were performed in order to assign the backbone chemical shifts of AbrB from both B. subtilis and B. anthracis. Chemical shift analysis has recently proven to be a powerful tool to investigate protein structure, providing information on backbone dihedral angles, aromatic ring orientation and other parameters. A comparative analysis of the homologous proteins was undertaken by determining the change in chemical shifts (Άδ) of conserved residues. The Chemical Shift Index (CSI) was used to determine the secondary structure of AbrB based on chemical shift information. Results show that the N-terminal DNA-binding domain adopts the same structure as observed in the NMR structure of the isolated AbrB DNA-binding domain. The appearance of the TROSYs indicates that neither AbrBBA nor AbrBBS adopts a largely random coil structure, and likely contains the C-terminal α-helix predicted by PSIPRED.
- D-type Cyclins, Epidermal Patterning and Their Roles in Geminivirus and Cell Cycle Interface(2017-06-28) Isaksson, Jonatan Dan; Jose Ascencio-Ibanez, Chair; Colleen Doherty, Member; Michael Goshe, Member
- Defining the Mannitol Dehydrogenase (MTD) Interactome during Unconventional Protein Secretion.(2016-03-04) Ho, Tricia Christine; John Williamson, Co-Chair; Michael Goshe, Co-Chair; Paul Wollenzien, Member; James Knopp, Member
- Deletions of Sindbis Virus E2 Endodomain Affect Virus Assembly and Identify the Virus Membrane as a Determinant of Host Range.(2012-10-30) Ribeiro, Mariana Tavares; Dennis Brown, Chair; Raquel Hernandez, Member; Frank Scholle, Member
- Di-metal Enzymes Responsible for Para-aminobenzoic Acid Biosynthesis.(2022-03-22) Mosley, Dontae Allen; Thomas Makris, Chair; Robert Rose, Member; Paul Swartz, Member
- Effect of Therapeutic Florfenicol Treatment of Steers on the Prevalence and Antimicrobial Resistance of Campylobacter and Impact of Antimicrobial Resistance Profiles on Survival of Campylobacter in Feces.(2020-08-16) Campos, Ashley; Sophia Kathariou, Co-Chair; Robert Rose, Co-Chair; Michael Goshe, Member; Derek Foster, Member; Driss Elhanafi, Minor; Jonathan Olson, Member
- Elucidating the Secretome of Lung Spheroid Cells using LC/MS/MS.(2018-03-22) Paudel, Dipti; Michael Goshe, Chair; Ke Cheng, Member; Robert Rose, Member; Colleen Doherty, Member
- Elucidation of the BRI1/BAK1 Heterodimer Using Chemical Crosslinking and Multidimensional Liquid Chromatography-Tandem Mass Spectrometry.(2014-08-01) Argo, Andrew Scott; Michael Goshe, Co-Chair; Steven Clouse, Co-Chair; Linda Hanley, Member; Benjamin Bobay, Member
- Evaluation of Microbial Defluorination and Biosequestration of PFAS Precursors by Pseudomonas putida.(2025-08-13) Mccullough, Reina Chardrice; Bob Rose, Co-Chair; Michael Hyman, Co-Chair; Arion Kennedy, Member
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