Biochemistry, Biophysics, and Structural Biology Commons^™

Novel Post-Translational Modification And Function Of Fus: The Relevance To Amyotrophic Lateral Sclerosis, Alexandra Arenas

Theses and Dissertations--Toxicology and Cancer Biology

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in Sarcoma (FUS) is a ubiquitously expressed RNA binding protein implicated in familial ALS and frontotemporal dementia (FTD). FUS is ubiquitously expressed in cells and has a variety of functions in the nucleus and cytoplasm. FUS mutations in the nuclear localization sequence (NLS) causes mislocalization of FUS in the cytoplasm, where it can undergo liquid-liquid phase separation and become stress granules or protein inclusions. Although FUS inclusion bodies can be found in …

The Effects Of Membrane Physicochemical Properties On Huntingtin Membrane Association And Downstream Aggregation, Maryssa Beasley

Graduate Theses, Dissertations, and Problem Reports (ETD)

Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by an expanded glutamine repeat region (polyQ) within the huntingtin protein (htt). As a result of the expanded polyQ domain, htt associates into a variety of toxic aggregate species. The polyQ domain of htt is flanked at the N-terminal end by 17 amino acids (Nt17) that adopt an amphipathic α-helical structure in the presence of binding partners such as lipid membranes. In addition to comprising a lipid binding domain, the Nt17 amphipathic α -helix has been directly implicated in htt aggregation initiation via self-association with other Nt17 α -helices. Due to …

A Eukaryotic Signature Based Method For Identification Of Saliva, Feces, And Urine, Alyssa Daniels

Master of Science in Forensic Science Directed Research Projects

The conception of the Human Microbiome Project advanced the understanding of microbial communities in the human body and previous research has established that unique microbial signatures can help distinguish each body fluid. While these signatures have been developed for the prokaryotic microbiome, the next step is the examination of the eukaryotic microbiome. Eukaryotic signatures could provide a greater specificity and statistical weight when discerning between body fluids. These microbial markers can be implemented to develop a confirmatory assay for body fluid identification that works in tandem with other DNA based methods in the forensic workflow. Using a VCU approved IRB …

Engineering Ph-Dependent Antibody Interactions Through Linked Protonation Events In Interdomain Interfaces, Hyeyoung Eom

Graduate Research Theses & Dissertations

Engineered antibodies are frequently used in life science applications, such as therapeutics and clinical diagnostics. These customized antibodies typically possess enhanced binding affinity and/or specificity, yet these interactions are only one-way binding events that cannot be easily controlled without extreme changes in conditions, such as pH, denaturant, and/or temperature that likely irreversible damage the antibody and antigen. Here, we explore the design of proton-linked binding events in the context of both antibody/hapten (i.e., a low molecular weight ligand target) and antibody/antigen complexes. Specifically, this work looks to overcome the challenges of engineering pH-dependent small ligand/protein interactions, as well as develop …

Consciousness As A Factor In Evolution, Kenneth A. Augustyn

Michigan Tech Publications, Part 1

What I call the mind began as a non-conscious robotic biochemical process control system in the very earliest forms of life. As life evolved, problems in control became more difficult and exceeded the computational capabilities of the organisms. Nature discovered a means of transcending computable physical processes resulting in non-computational subjective mental capabilities that, while still not conscious, had a degree of genuine autonomy from the physical world. These autonomous subjective wants and goals now affected the course of (but not the mechanism of) evolution. The integrated amalgam of robotic and transrobotic unconscious capabilities eventually gave rise to consciousness, which …

Evaluation Of Salmonella Enteritidis And Escherichia Coli O157:H7 Volatile Organic Compound Analysis For Food Safety Monitoring: A Preliminary Study, Jessica E. Lemley

Graduate Theses, Dissertations, and Problem Reports (ETD)

Food borne illness has the potential to impact every point of production from producers to consumers. Food product recalls are common due to the presence of food borne illness causing bacteria. Detection of volatile organic compounds (VOC) have the potential to reduce the number of illnesses and recalls through application of additional monitoring of food products like those most commonly recalled. The objective of this research was to 1) explore the use of closed-loop headspace analysis coupled with gas chromatography-mass spectrometry (GC-MS) on food borne illness causing bacteria and 2) examine the VOC profile of Escherichia coli serotype O157:H7 in …

Characterization Of The Biochemical Properties Of Nudt8, A Novel Coa-Degrading Enzyme That Localizes To The Mitochondria, Evan W. Kerr

Graduate Theses, Dissertations, and Problem Reports (ETD)

Coenzyme A (CoA) is a vital cofactor that is required for a variety of metabolic reactions including the TCA cycle and the synthesis and oxidation of fatty acids, amino acids and ketone bodies. The importance of CoA is underscored by its tight regulation, as prolonged elevations or inability to synthesize adequate amounts of this cofactor lead to severe metabolic dysfunction. Regulation of CoA biosynthesis has been extensively characterized, however less is known about regulation of CoA and its thioesters via degradation. Presently, two CoA-degrading enzymes, Nudt7 and Nudt19 have been identified as regulators of the peroxisomal pool of (acyl-)CoA in …

Protein/Peptide Characterization Using Mass Spectrometry And Molecular Dynamics Simulations, Ahmad Kiani Karanji

Graduate Theses, Dissertations, and Problem Reports (ETD)

Mass spectrometry (MS) based-techniques and molecular dynamics (MD) simulations have been used to characterize protein/peptide structure as well as their interactions with lipid vesicles and detergents. Chapter 1 introduces an introduction to the concepts and tools that were used in this work. In Chapter 2, the dominant gas-phase conformer of [M+3H]3+ ions of the model peptide Acetyl-PSSSSKSSSSKSSSSKSSSSK are examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. This section furthers the development of a protein structural prediction tool by providing information about gas-phase ion conformers of two model peptides having different solution conformational …

The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd

Graduate Theses, Dissertations, and Problem Reports (ETD)

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry …

Role Of Ciliary Proteins Adp Ribosylation Factor Like Gtpase 13b (Arl13b) And Bardet-Biedl Syndrome-8 (Bbs8) In Photoreceptor Outer Segment Morphogenesis, Maintenance, And Viability, Tanya L. Dilan

Graduate Theses, Dissertations, and Problem Reports (ETD)

Photoreceptor neurons are modified primary cilia with an extended ciliary compartment known as the outer segment (OS). The mechanisms behind the elaboration of photoreceptor cilia, OS morphogenesis, and maintenance remain poorly understood. In this work, we focused on dissecting the role of two ciliary proteins, the small GTPase ADP-ribosylation factor-like GTPase 13B (ARL13B) and Bardet-Biedl Syndrome-8 (BBS8) in the context of photoreceptor biology. Both BBS8 and ARL13B are linked to defects in ciliogenesis (cilia development) and Retinitis Pigmentosa (vision loss). ARL13B is implicated in regulating ciliary length, and BBS8 is part of the Bardet-Biedl Syndrome complex (BBSome); the BBSome is …

Investigation Of Glutamate Carboxypeptidase Ii Roles In The Folate Cycle Dependent Reproduction And Development Of Caenorhabditis Elegans, Jessica M. Derham

Masters Theses

Glutamate Carboxypeptidase II (GCPII) is a transmembrane, zinc metallopeptidase that is expressed in a wide range of organisms, including roundworms, mice, and humans. In humans, GCPII is primarily expressed in the prostate, kidneys, small intestine, and central nervous system. Within the small intestine, the expected function of GCPII is to aid in the absorption of dietary folate from the intestinal lumen. GCPII cleaves excess glutamates from folate to yield monoglutamated folate which is then readily transported into the enterocyte. Folate can then be used through the one carbon metabolic cycle for the synthesis of nucleotides, conversion of homocysteine to methionine, …

Reprograming Neuronal Cells By Overexpression Of Fibroblast-Specific Transcription Factors, Abdulmohsen Alanazi

Masters Theses

In mammals, a complex system of regulatory signals distinguishes tissues, structures and functions. Combinations of transcription factors and co-factors regulate activation and repression of genes that result in cellular differentiation. Whole genome arrays allow the monitoring of genomic expression in specific tissues. Fibroblast microarray studies have shown candidate genes that may be involved in fibroblast identification, including genes that express transcription factors Prrx1, Snai2 and Twist1. A previous study showed that the Prrx1 and Snai2 could reactivate a fibroblast phenotype in hybrid cells that had lost fibroblast identity. Furthermore, overexpression of these factors in liver-derived cells strongly repressed liver gene …

Identifying Metabolites As Markers Of Fatigue In Athletes, Christopher D. George

Honors Theses

Fatigue in athletes caused by inadequate rest and other stressors can be severely detrimental to their health, and yet there is no reliable way to measure and track fatigue. Many classical measures of fatigue like body mass and resting heart rate are not reliable ways to track the physical fatigue of an athlete because they can change with many factors. In addition, it is not known how academic stress on top of physical stress affects fatigue. Metabolomics is a relatively new area of study and tracking metabolites offers the possibility to produce biomarkers to assess fatigue. This study collected saliva …

A Biophysical Investigation Of Stability, Ligand Binding, And Iron State Of Cyp102a1, Catherine A. Denning-Jannace

Theses and Dissertations--Chemistry

Cytochrome P450s (CYPs) are cysteine ligated Fe-heme monooxygenases that are found in all domains of life. In mammals, they have a role in xenobiotic metabolism and steroid synthesis, making them a fundamental requirement for survival. In addition, their ability to perform a variety of chemical reactions on an array of substrates makes CYPs highly sought for biotechnical applications such as wastewater remediation, production of potential drug candidates, and creation of drug metabolites. By mutating specific amino acids, these enzymes can be engineered to change their substrate binding profiles and achieve stereo- and regio-specific chemistry. While these mutations are essential to …

New Single Molecule In Vivo And Ex Vivo Methods To Understand The Physiological Effects Of Nicotine Use, Xu Fu

Theses and Dissertations--Chemistry

The consumption of tobacco products leads to several health risks, including respiratory diseases, cardiovascular diseases, and many different cancers. Nicotine, the primary addictive compound in tobacco, acts on the cholinergic system through nicotinic acetylcholine receptors (nAChRs), which is believed to be an essential component of addiction. However, the lack of a complete understanding of the neurobiological mechanisms of nicotine abuse is one of the primary barriers to finding potent therapeutics for smoking cessation. Single-molecule imaging is an ideal tool to study molecular mechanisms of protein and physiological changes both in vitro and in vivo. In this dissertation, our research …

Deconvolution Tools For Extracting Insight From Challenging Two-Flavin Systems, Dallas Michael Bell

Theses and Dissertations--Chemistry

Flavoproteins have long been explored for their ubiquity among a number of metabolic and energetic reactions. The flavin cofactor has the inherent benefit of distinct spectral changes associated with redox transitions; however, the double-edged sword is incurred as these distinct signatures overlap and take up much of the UV-vis spectral range. Therefore, it is crucial to create a method to demarcate the expressed redox transitions for studying these systems. The first portion of these studies discusses the creation of a program that deduces spectra for redox transitions in a single-flavin containing model protein: flavodoxin. The latter portions discuss the application …

Single-Domain Vhh Antibodies As Protein Stabilization Modules, Nourhan Mohamed Disouky Mostafa

Graduate Research Theses & Dissertations

VHH domains are the antigen-binding units found in heavy-chain-only antibodies from the Camelidae family (e.g., camels and llamas). They are considered a unique minimalist antibody scaffold due to the lack of a variable light chain domain. Despite having half the antigen-binding interface of conventional antibodies, these VHH antibodies bind their target antigens with affinities comparable to conventional antibodies. This project explored how flexible linkers connecting an anti-GFP VHH (αGFP VHH) domain to its antigen (EGFP) enhanced antigen stability and influenced oligomeric assembly. It was hypothesized that linking the anti-GFP VHH antibody to its target antigen (EGFP) will increase the local …

A Bioinformatic Approach To Exploring Land Plant Evolution, Lauren Margaret Orton

Graduate Research Theses & Dissertations

Today, researchers are inundated with an overwhelming amount of data due to advances in DNA sequencing technologies. This has benefited the fields of both bioinformatics and phylogenomics substantially. With the ability to increase sampling at both the taxonomic and molecular levels, we are now able to produce robust phylogenomic reconstructions and better discern relationships between taxa.

The goals of this dissertation are in two distinct parts: 1) to advance the understanding of land plant terrestrialization and evolution through examination of the closest relative to the land plant lineage, a species of green algae; and 2) to explore evolution among a …

Seasonal Variation In Mitochondrial Bioenergetics Of The Bluegill Sunfish, Lepomis Macrochirus, From A Shallow Midwest River, Derick Isaac Lamptey

Masters Theses

As average global temperature increase, the frequency and magnitude of extreme temperatures in shallow aquatic ecosystems are more ubiquitous. In order to understand how these changing thermal regimes affect aquatic ectotherms, it is essential to develop studies evaluating the response of ectotherms to seasonal fluctuating thermal regimes. Previous studies on fluctuating temperature regimes have reported an increased physiological stress leading to morphological, behavioral and biochemical adaptations. From the latter, the adaptive capacity and seasonal performance associated with optimal function of the oxidative phosphorylation system (OXPHOS) are key for species persistence. However, studies on this matter are scarce. This study explores …

Flavodoxin, The Hydrogen Atom Of Flavoproteins: A 19f Nmr Study Of Dynamics And Conformational Changes Utilizing Flavodoxin From Rhodopseudomonas Palustris, Taylor Varner

Theses and Dissertations--Chemistry

Flavodoxin is a small, highly stable protein that contain a single FMN cofactor used to transfer single electrons at low potentials. The organism Rhodopseudomonas palustris contains a long-chain flavodoxin. Long-chain flavodoxins are characterized by a 20 amino acid loop that is proposed to allow interactions with partner proteins. We plan to utilize this protein as a model to build our repertoire with protein fluorination and fluorine NMR in flavoproteins. This tool kit will then be applied to the study of a partner protein of interest that is capable of performing electron bifurcation. We have incorporated m-fluoro tyrosine into flavodoxin and …

Hijacking The Fusion Complex Of Human Parainfluenza Virus As An Antiviral Strategy, T. C. Marcink, E. Yariv, K. Rybkina, V. Más, F. T. Bovier, A. Des Georges, A. L. Greninger, C. A. Alabi, M. Porotto, N. Ben-Tal, A. Moscona

Advanced Science Research Center

The receptor binding protein of parainfluenza virus, hemagglutinin- neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein …

Correction: Visible-Light Photooxidation In Water By 1o2-Generating Supramolecular Hydrogels, Sankarsan Biswas, Mohit Kumar, Andrew Levine, Ian Jimenez, Rein V. Ulijn, Adam Braunschweig

Advanced Science Research Center

Correction for ‘Visible-light photooxidation in water by ¹O₂-generating supramolecular hydrogels’ by Sankarsan Biswas et al., Chem. Sci., 2020, 11, 4239–4245, DOI: 10.1039/C9SC06481H.

Polymer Brush Hypersurface Photolithography, Carlos Carbonell, Daniel J. Valles, Alexa M. Wong, Andrea S. Carlini, Mollie A. Touve, Joanna Korpanty, Nathan C. Gianneschi, Adam B. Braunschweig

Advanced Science Research Center

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. Here we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are …

Extraction Of Protein From Four Different Seaweeds Using Three Different Physical Pre-Treatment Strategies, Jack O'Connor, Steve Meaney, Gwilym A. Williams, Maria Hayes

Articles

Seaweeds are a rich source of protein and can contain up to 47% on the dry weight basis. It ischallenging to extract proteins from the raw biomass of seaweed due to resilient cell-wall complexes.Four species of macroalgae were used in this study-two brown,Fucus vesiculosusandAlaria esculenta,and two red,Palmaria palmataandChondrus crispus. Three treatments were applied individually tothe macroalgal species: (I) high-pressure processing (HPP); (II) laboratory autoclave processing and(III) a classical sonication and salting out method. The protein, ash and lipid contents of the resultingextracts were estimated. Yields of protein recovered ranged from 3.2% forFucus vesiculosuspre-treatedwith high pressure processing to 28.9% protein recovered …

Synthesis And Optimisation Of P3 Substituted Vinyl Sulfone-Based Inhibitors As Anti-Trypanosomal Agents, William Doherty, Nikoletta Adler, Andrew Knox, Thomas J. Butler, Paul Evans

Articles

A series of lysine-based vinyl sulfone peptidomimetics were synthesised and evaluated for anti-trypanosomal activity against bloodstream forms of T. brucei. This focused set of compounds, varying in the P3 position, were accessed in a divergent manner from a common intermediate (ammonium salt 8). Several P3 analogues exhibited sub-micromolar EC50 values, with thiourea 14, urea 15 and amide 21 representing the most potent anti-trypanosomal derivatives of the series. In order to establish an in vitro selectivity index the most active anti-trypanosomal compounds were also assessed for their impact on cell viability and cytotoxity effects in mammalian cells. Encouragingly, all compounds only …

A Novel Pool Of Microparticle Cholesterol Is Elevatedin Rheumatoid Arthritis But Not In Systemic Lupus Erythematosus Patients, Shuai Shuai Hu, Brenton L. Cavanagh, Robert Harrington, Muddassar Ahmad, Grainne Kearns, Steve Meaney, Claire Wynne

Articles

Microparticles are sub-micron, membrane-bound particles released from virtually allcells and which are present in the circulation. In several autoimmune disorders their amountand composition in the circulation is altered. Microparticle surface protein expression has beenexplored as a differentiating tool in autoimmune disorders where the clinical pictures can overlap.Here, we examine the utility of a novel lipid-based marker—microparticle cholesterol, present in allmicroparticles regardless of cellular origin—to distinguish between rheumatoid arthritis (RA) andsystemic lupus erythematosus (SLE). We first isolated a series of microparticle containing lipoproteindeficient fractions from patient and control plasma. There were no significant differences in thesize, structure or protein content of …

A Proteomic Analysis Of Corydoras Sterbai Secretions And Tissues, Erik Powell Wictor

University of the Pacific Theses and Dissertations

Defensive mechanisms vary widely in the animal kingdom ranging from physical defenses like spines to chemical defenses such as toxins. Toxins in these secretions and tissues can fluctuate from enzymes to lipids to uncharacterized chemicals. Next generation -omics technology and mass spectrometry are extremely important in analyzing these samples because of their ability to distinguish minute amounts of toxic substance within a complicated sample. The goal of this experiment was to look at secretions and tissues from Corydoras sterbai. All samples in this study were proteolyzed using a mixture of Trypsin and Lys-C, fractionated, and run through nanoLC-MS/MS analysis using …

Proteomic Analysis Of Fetal Rat Neural Stem Cells After Treatment With Hericium Erinaceus, Bright Adam Test

University of the Pacific Theses and Dissertations

The fungus, Hericium erinaceus, has outstanding chemical properties, displaying health benefits in digestive, hepatic, and nervous tissues. Its ease of accessibility and use makes it one of the most common substances used for treatment in Eastern medicine. More and more recent research is confirming the incredible health benefits of this fungus, especially the impact that is seen on nervous tissue growth and recovery post-treatment. Such neurite outgrowth and myelin sheath regeneration could illustrate the beginning of the cure to lifelong neurodegenerative diseases such as Multiple Sclerosis. In this first-of-its-kind study, we cultured and differentiated fetal rat neural stem cells while …

Tools To Evaluate Nanodiamond-Mediated Delivery Of Tiopronin For Cataract Prevention, Justin Beltz

Doctoral Dissertations

”There is a growing demand for non-surgical means of cataract treatment. This dissertation presents three bodies of work that reflect the early-stage development of eye drop formulations aimed at delaying cataract progression. These formulations consist of the antioxidant 2-mercaptopropionylglycine (MPG) loaded onto nanodiamond particles.

Cataractogenesis is linked to oxidative damage to lens proteins. To investigate the potential of MPG for protection against oxidative damage, A549 cells were incubated in 0.6 mM tert-butylhydroperoxide (tBHP). Cells exposed to tBHP without MPG exhibited elevated levels of reactive oxygen species, which led to the depletion of the vital antioxidant glutathione and, ultimately, apoptosis. Co-administration …

Factors Influencing Huntingtin Aggregation At Surfaces: Implications For Huntington’S Disease, Sharon E. Groover

Graduate Theses, Dissertations, and Problem Reports (ETD)

Huntington’s Disease (HD) is a genetic, neurodegenerative disease characterized by an abnormal polyglutamine (polyQ) expansion in the first exon of the huntingtin protein (htt). The polyQ domain facilitates aggregation and initiates the formation of a diverse collection of aggregate species, including fibrils, oligomers and annular aggregates. The first 17 amino acids of htt (Nt17) directly flank the polyQ domain and is a key factor in htt’s association to membranous structures. In addition to Nt17 being an amphipathic αhelix, it also promotes aggregation through self-association and contains numerous posttranslational modifications (PTMs) that can modulate toxicity and subcellular localization. For in depth …