Biochemistry, Biophysics, and Structural Biology Commons^™

Development Of A Hek293 Cell Line To Show Inhibition Of Tau Aggregation, Justin Ray Shady

Electronic Theses and Dissertations

Intracellular deposition of aggregated tau is the hallmark of several different tauopathies, the most widespread of these being Alzheimer's disease. Tau is a highly soluble, intrinsically disordered, microtubule associated protein. Tau's native function is to stabilize microtubule formation in the axons of neurons. Post translational modification such as hyperphosphorylation as well as several familial mutations allow tau to nucleate and form fibrils. These fibrils can recruit healthy monomers onto their ends in a fashion described as template-assisted growth. Tau has 6 isoforms that vary by the inclusion or exclusion of two N-terminal repeats and the inclusion or exclusion of the …

Translation Of Partially Decayed Messenger Rnas In Yeast, Ana Luisa Franklin

Electronic Theses and Dissertations

Flaviviruses are positive-strand single-stranded RNA viruses that are known to form pseudo-knot RNA structures that halt the progression of 5’→3’ exonuclease Xrn1. We show that these viral Xrn1-resistant structures (xrRNAs) can be used to protect specific homologously-expressed messenger RNAs from 5’→3’ degradation. We investigated the effects of addition of xrRNAs, artificially-installed into the intergenic region of bicistronic mRNA reporters, in the observed levels of protein expression in yeast. The reporters also contain an internal ribosome entry site from the cricket paralysis virus (CrPV IRES) to allow for cap-independent translation of the decay-protected gene, LacZ, encoding the enzyme β-galactosidase. Through …

Brønsted Acid Catalyzed Carbon-Carbon Bond Forming Reactions, Jiangyue Miao

Electronic Theses and Dissertations

The focus of this research project lies in the development of new methodology in the field of Brønsted acid catalysis enabling rapid synthesis of medicinally relevant compounds. It is foreseen that small molecule sulfonic acids evaluated in this research project will unveil new asymmetric carbon carbon bond forming reactions between substrates hitherto unexplored with Brønsted acid catalysis. It has been established that strong Brønsted acids, such as phosphoric acids, are capable of mediating highly selective transformations operating through unique mechanistic manifolds.

Specific focus for the sulfonic acid catalysts was geared towards asymmetric coupling reactions with synthetically useful precursors such as …

Relationship Between Tdp-43 Toxicity And Aggregation In Saccharomyces Cerevisiae, Martin Anthony Aguilar

Electronic Theses and Dissertations

Protein aggregation and inclusion body formation are hallmarks of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis (ALS). These neurodegenerative diseases share a common pathology in that all include accumulation of insoluble protein aggregates in the brain. TAR-DNA-binding protein (TDP-43) is the major component found in the pathological inclusions of two of these diseases, ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). This thesis focuses upon the biophysical basis for TDP-43 aggregation in S. cerevisiae. Current in vitro evidence indicates that TDP-43 is a natively dimeric protein and that binding to RNA inhibits aggregation. Corresponding …

Identifying The Relevance Of C-Reactive Protein Conformers And The Advancement Of A Membrane Curvature Binding Assay, Carrie Leine Moon

Electronic Theses and Dissertations

Lipid membranes play a vital role in cell signaling processes. Membrane shape and lipid content affect interactions between cellular membranes and proteins. This research focuses on characterizing those interactions and their impact by using various biochemical and biophysical assays. These assays were applied to C-reactive protein (CRP), an immune system protein that interacts with lipid membranes and has at least two forms with different properties. Native, pentameric CRP (pCRP) is found in blood serum and is commonly used as a marker for inflammation. The modified form of CRP (mCRP) binds to the protein C1q, which activates the complement immune response. …

Lipid-Coated Gold Nanoparticle As A Biosensor For Lipid-Protein Interactions, Yuheng Cai

Electronic Theses and Dissertations

Gold nanoparticles have been intensively studied for their unique optical features. Due to surface plasmon resonance phenomenon, gold nanoparticles can respond to the refractive index change of the environment near the particles. This phenomenon gives gold nanoparticles the potential to become biosensors that detect the biological interactions at or near the particles. In this work, gold nanospheres were coated with phosphatidylcholine as the substrate for lipoxygenase. It is demonstrated, in our work, that lipid coated gold nanospheres can be used to detect the activity of lipoxygenase and provide more information of this reaction than common assays, like conjugated diene assay …

Species Preference Of Viral Deubiquitinating Proteases Toward Isg15 Through Structural And Enzymatic Characterization, Michelle Kay Deaton

Electronic Theses and Dissertations

Proteases from the Ovarian Tumor domain (OTU) superfamily of deubiquitinating enzymes (DUBs) are expressed by a range of RNA viruses. Viral OTUs (vOTUs) are found in nairoviruses such as Crimean Congo Hemorrhagic Fever virus (CCHFV), Nairobi sheep disease virus (NSDV), the Erve virus (ERVEV), and the Dugbe virus (DUGV), as well as in the areterivirus Porcine Reproductive and Respiratory Syndrome virus (PRRSV), among others. vOTUs, which interfere with host innate immune response through editing of host Ub and Ub-like molecules such as interferon stimulated gene 15 (ISG15), have been identified as a potential virulence factor through their role in evading …

Analyzing The Interactions Between Xenopus Tropicalis Mc2r, Mc5r, And The Mraps: Modeling The Regulation Of Frog Interrenal Cells, Yesenia Garcia

Electronic Theses and Dissertations

The role of Melanocortin 2 Receptor (MC2R) in adrenal/interrenal glucocorticoid secretion has been well documented in many organisms. Studies in mammals have shown that in the adrenal gland two melanocortin receptors and two melanocortin receptor accessory proteins are expressed: MC2R, MC5R, MRAP, and MRAP2. The MRAPs have an opposite effect on the cell surface expression of MC2R and MC5R. In mammals, MRAP aids MC2R but inhibits MC5R cell surface expression. This thesis aims to explore the functional relationship between MC2R, MC5R, MRAP, and MRAP2 in Xenopus tropicalis to determine if the MRAPs have a similar effect on amphibian MC2R and …

Mitochondrial Involvement In Neuronal Cell Death, Josie J. Gray

Electronic Theses and Dissertations

Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. In this thesis, I demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from …

Biophysical Characterization Of The Votu Proteases From The Cchf And Dugbe Nairoviruses, Glenn C. Capodagli

Electronic Theses and Dissertations

My research focuses on understanding the substrate specificity of the viral homolog of the ovarian tumor domain protease superfamily (vOTU) from nairoviruses, and the structural reasons for their specificities. The vOTU from the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) has been implicated in the down-regulation of the innate immune response through its ability to cleave post-translational modifications via Ubiquitin (Ub) as well as the Ub-like interferon-stimulated gene 15 (ISG15). vOTU homologs have been found in numerous viruses across several families. Moreover, the effects of these viruses range in severity from mild flu-like symptoms to mortality depending on the species of the …

Development Of A Biosensor For Investigating Membrane Curvature Sorting, Joshua C. Black

Electronic Theses and Dissertations

The physical structure of cellular membranes plays a critical role in lipid and protein sorting. A novel biosensor was developed to probe the influence of curvature on sorting. This biosensor mimics large, two-dimensional membranes in dynamic equilibrium, achieves high spatial resolution between curvature and molecules of interest, and has high sensitivity, enough for single particle detection. The biosensor consists of continuous supported lipid bilayer formed over nanoparticles (40 to 200 nm diameter) deposited on a glass substrate. The nanoparticles determine the extent of curvature. This biosensor is the first to observe large-scale 2-dimensional diffusion of biomolecules on a supported lipid …

Regulation Of Actin Dynamics During Drosophila Germband Extension, Ashley M. Motlong

Electronic Theses and Dissertations

Convergent extension is a process that occurs in the development of a wide variety of organisms, including gastrulation in the Drosophila embryo to begin to lay out the adult body plan. In fly embryos, this is known as germband extension and is mainly driven by cell intercalation or neighbor exchange by planar polarized cell-cell interface contraction to shorten the tissue along the dorsal-ventral axis. In this thesis, I show that interface contraction consists of phases of fast interface shortening and intervals of stable interface size. My data also suggests that regulation of F-actin aggregates at these shrinking interfaces is important …

X-Band Rapid-Scan Epr, Deborah Gale Mitchell

Electronic Theses and Dissertations

The advantages of rapid-scan EPR relative to CW and pulse techniques for samples with long longitudinal relaxation time T₁ (N_s⁰ defects in diamond, N@C₆₀, and amorphous hydrogenated silicon), heterogeneous samples (crystalline 1:1 α,γ-bisdiphenylene-β-phenylallyl (BDPA):benzene), lossy samples (aqueous nitroxyl radicals), and transient radicals (5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)-superoxide adduct) were studied.

For samples with long relaxation times, CW (continuous wave) EPR is challenging due to power saturation and distortions from passage effects. In rapid-scan EPR, the field is swept through resonance in a time that is short relative to T₂. In rapid-scan EPR, the magnetic field is …

Mitochondrial Dna Analysis By Denaturing High-Performance Liquid Chromatography For The Characterization And Separation Of Mixtures In Forensic Samples, Richard Kristinsson

Electronic Theses and Dissertations

A mixture of different mtDNA molecules in a single sample is a significant obstacle to the successful use of standard methods of mtDNA analysis (i.e., dideoxy dye-terminator sequencing). Forensic analysts often encounter either naturally occurring mixtures (e.g., heteroplasmy) or situational mixtures typically arising from a combination of body fluids from separate individuals. The ability to accurately resolve and interpret these types of samples in a timely and cost efficient manner would substantially increase the power of mtDNA analysis and potentially provide valuable investigative information by allowing its use in cases where the current approach is limited or fails. Therefore, this …

Altered Calcium Handling In Cerebellar Purkinje Neurons With The Malignant Hyperthermia Mutation, Ryr1-Y522s/+, George C. Talbott

Electronic Theses and Dissertations

To investigate the etiology of malignant hyperthermia and central core disease, mouse models have recently been generated and characterized (Chelu et al., 2006). These RyRY522S/+ knock-in mutant mice provide an excellent tool to investigate calcium dysregulation, its pathological consequences, and potential therapeutic approaches. Skeletal muscle harboring this mutation exhibits calcium leak from internal stores and an increased sensitivity to activation by caffeine, voltage, and temperature (Durham et al., 2008). Although alterations in RyR1 channel function and resultant changes in cellular function have been characterized in skeletal muscle, the effects of MH mutations in RyR1 on central nervous system function have …

Structure-Function Analysis Of Endoproteolytic Cleavage: Site-Directed Mutagenesis Studies Of The Α-Msh Cleavage Site In Silurana Tropicalis Pomc, Quinn Kun Chen

Electronic Theses and Dissertations

The maturation process of many prohormone molecules typically requires endoproteolytic cleavage C-terminal of dibasic residues, such as the K¹⁴¹R¹⁴²↓ site N-terminal of the α-melanocyte stimulating hormone (α-MSH) sequence in Silurana tropicalis proopiomelanocortin (POMC). In order to determine the absolute requirement of basic amino acid residues in the cleavage process, site-directed mutagenesis was employed to substitute alanine for the wild-type residues in the frog POMC open reading frame. Specifically, the following underlined residues were individually targeted for alanine substitution: R ¹³⁷ Q ¹³⁸ E ¹³⁹ N ¹⁴⁰ K ¹⁴¹ R ¹⁴²↓. The mouse pancreatic αTC1.9 cell …

Role Of Pro-Apoptotic Bcl2-Homology-3 Domain (Bh3)-Only Proteins In The Mutant Sod1 Mouse Model Of Als, Anna George Andrianakos

Electronic Theses and Dissertations

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease involving rapid degeneration of motor neurons in the spinal cord and retraction of their axonal projections to the neuromuscular junctions. Several known mutations linked to some familial cases of ALS have been linked to mutations in Cu/Zn superoxide dismutase (SOD1), resulting in mitochondrial oxidative stress and intrinsic apoptosis. Transgenic mice expressing a G93A mutant of SOD1 provide an in vivo model to investigate motor neuron death during disease progression. The principal regulators of intrinsic apoptosis are the Bcl-2 family proteins. While some members of this family are pro-survival, the Bcl-2 homology-3 …

The Evolution Of Opioid/Orphanin Receptors In Chordates, Jazalle Deshaun Mcclendon

Electronic Theses and Dissertations

The interest in the opioid/orphanin gene family stems from functional similarities that these peptides have to the narcotic opium. Opiates have been extensively studied because of their analgesic properties; however, the reason that these plant products can affect the human central nervous system was a mystery until the discovery of opiate-like peptides. The endogenous opioid peptides are well understood today because they have been fully cloned and characterized in several different organisms including lower chordates. On the other hand, the opioid/orphanin receptors have not been fully cloned or characterized in lower chordates; therefore, to better understand the past and present …

Soil Amino Acids At Upper Tree Line, Montane And Lower Tree Line, Kelly C. Owens

Electronic Theses and Dissertations

The classic view of the nitrogen cycle in soils is for plants to take up inorganic N in solution for N nutrition. More recent studies reveal plants can take up low molecular weight dissolved organic N such as amino acids directly from the soil. In ecosystems where the rate of microbial mineralization is limited, plants may take up 10 to 200 percent more amino acid N than mineral N. It is not known if plants take up amino acids in all ecosystems, however recent research shows that plants generally take up amino acids when they are present in high quantities …

Polymer Supported Optical Biosensors, Ignacio Joseph Garcia

Electronic Theses and Dissertations

The use of various polymer supports adhering phospholipid multibilayers to an internal reflection element have been investigated. The polymer supports studied range from polystyrene (PS), and 100 nm wide PS nanospheres to triethylaminated poly(vinyl benzyl chloride) (PVBC). The PS nanospheres showed the most promise as an adhesion layer since they appear to be the most robust with repeated washings. They also appear to stabilize their adjacent phospholipid multibilayer by increasing the lipid melting temperature. The triethylaminated DVBC also provided an increase of lipid melting temperature, but not quite to the same degree as the PS nanospheres. The cooperativity of such …