Biochemistry, Biophysics, and Structural Biology Commons^™

Small Gtpase Regulated Intracellular Protein Trafficking In Endothelium, Caitlin Francis

Electronic Theses and Dissertations

Intracellular protein trafficking is the movement of membrane-bound organelles to and from requisite locations within the cell. Small GTPases are a critical component to the spatiotemporal accuracy of intracellular trafficking pathways as they determine the specificity and direction of organelle transport. There exists over 150 small GTPases categorized into 5 sub-families and are employed across all cell types. Despite their universal expression and relevance to cellular function, small GTPases remain incompletely understood across tissue types. In various instances, the trafficking pathway of a particular Rab in one cell type may belong to a completely disparate pathway in another cell type. …

Uncovering Zn2+ And Ca2+ Homeostasis And Crosstalk In Primary Hippocampal Neurons, Chen Zhang

Electronic Theses and Dissertations

Zn²⁺ has been suggested to act as an intracellular signaling molecule due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular Zn²⁺ concentration is tightly maintained at steady levels under natural physiological conditions. Dynamic changes in intracellular Zn²⁺ concentration have only been detected in cells exposed to pathologic stimuli or upon receptor ligand binding. In the first project of my dissertation, for the first time, we revealed that developing neurons fire spontaneous and synchronous Zn²⁺ spikes at a specific time window (from 14 …

Quantification Of Nuclear Dynamics During Epithelial Remodeling, Noah De Leeuw

Electronic Theses and Dissertations

The morphogenesis of developing tissues is contingent on an extensive array of rearrangements in cellular shape, position and identity at large and small scales. One commonly used process to reshape tissues is the cell intercalation-driven elongation of a tissue in a common axis, in which rows of epithelial cells undergo oriented intercalation in a directional fashion. In most models of intercalation, cells are treated as homogeneous objects directed in their shape changes by cortical forces localized along cell-cell interfaces or tricellular junctions. However, less attention has been paid to how inhomogeneities in mechanical resistance of their own internal structures affects …

Mechanistic Insights Into Mftr-Dependent Regulation Of The Redox Cofactor Mycofactocin, Aigera Mendauletova

Electronic Theses and Dissertations

Organic redox cofactors are essential for life. While classic flavins and nicotinamides are widely distributed across all domains of life, nature has also evolved niche cofactors in subsets of life domains. For example, in Actinobacteria, coenzyme F420 is commonly used in place of flavin mononucleotide in enzymes associated with carbon fixation and oxidation of secondary alcohols. The importance of niche cofactors has long been recognized however, detailed understanding about their biosynthesis and physiological uses has been lagging. One class of niche cofactors is derived from ribosomally synthesized and posttranslationally modified peptides (RiPPs). To achieve their mature form, the genetically …

Developing Red Fluorescent Zn2+ Sensors And Characterizing Zn2+ Homeostasis In Mucolipidosis Type Iv Disease, Anna M. Dischler

Electronic Theses and Dissertations

Zinc (Zn²⁺) is a divalent, redox-inert metal that plays a vital role in many cellular processes either by acting as a catalytic cofactor or a labile signaling molecule in the cytoplasm. Cytosolic Zn²⁺ levels are tightly regulated to stay within a narrow window of concentrations, but fluctuations in Zn²⁺ signals have been detected in a variety of cells. We developed a genetically encoded, single red fluorescent protein (RFP) based Zn²⁺ sensor, RZnP1, that can be used alongside green-wavelength sensors for the simultaneous detection of different signaling molecules within the same cells or among different subcellular …

Post-Translational Modification Of Proteins Via Ambient Air Pollutants And Endogenous Reactive Species, Rachel Lauren Davey

Electronic Theses and Dissertations

Proteins can react with reactive oxygen species (ROS) and reactive nitrogen species (RNS) to form post-translational modifications (PTMs), which can affect protein structure and function. The formation of 3-nitrotyrosine (NTyr) and dityrosine (DiTyr) upon reaction of proteins with ROS/RNS are two common PTMs studied due to their stability and irreversibility, as well as their ability to enhance the allergenicity of pollen allergens upon formation. Many common techniques used to study the formation of these PTMs can reliably detect the PTMs but can only provide semi-quantitative information due to many assumptions and limitations. In Chapter 2 we present an analysis of …

Identifying The Role Of Phospholipase D1 And Phosphatidic Acid In Exocytosis, Broderick L. Bills

Electronic Theses and Dissertations

Exocytosis is an essential process for intercellular communication in eukaryotic cells. This process involves significant changes in membrane curvature, and lipids and curvature-sensing proteins can assist these processes. One protein in particular, phospholipase D1 (PLD1), and its product, the lipid phosphatidic acid (PA), are involved in multiple exocytic processes. However, PLD1 and PA’s role in this process has remained unclear. In this work, PLD1 and the production of PA were visualized during exocytosis, and PA localization to regions of membrane curvature was established. Together, these results support the hypothesis that PLD1 production of PA stabilizes negative curvature during membrane fusion. …

Du Undergraduate Showcase: Research, Scholarship, And Creative Works: Abstracts, Emma Aggeler, Elena Arroway, Daisy T. Booker, Justin Bravo, Kyle Bucholtz, Megan Burnham, Nicole Choi, Spencer Cockerell, Rosie Contino, Jackson Garske, Kaitlyn Glover, Caroline Hamilton, Haley Hartmann, Madalyne Heiken, Colin Holter, Leah Huzjak, Alyssa Jeng, Cole Jernigan, Chad Kashiwa, Adelaide Kerenick, Emily King, Abigail Langeberg, Maddie Leake, Meredith Lemons, Alec Mackay, Greer Mckinley, Ori Miller, Guy Milliman, Katherine Miromonti, Audrey Mitchell, Lauren Moak, Megan Morrell, Gelella Nebiyu, Zdenek Otruba, Toni V. Panzera, Kassidy Patarino, Sneha Patil, Alexandra Penney, Kevin Persky, Caitlin Pham, Gabriela Recinos, Mary Ringgenberg, Chase Routt, Olivia Schneider, Roman Shrestha, Arlo Simmerman, Alec Smith, Tessa Smith, Nhi-Lac Thai, Kyle Thurmann, Casey Tindall, Amelia Trembath, Maria Trubetskaya, Zachary Vangelisti, Peter Vo, Abby Walker, David Winter, Grayden Wolfe, Leah York

DU Undergraduate Research Journal Archive

Abstracts from the DU Undergraduate Showcase.

Foldamers Reveal And Validate Therapeutic Targets Associated With Toxic Α-Synuclein Self-Assembly, Jemil Ahmed, Tessa C. Fitch, Courtney M. Donnelly, Johnson A. Joseph, Tyler D. Ball, Mikaela M. Bassil, Ahyun Son, Chen Zhang, Aurélie Ledreux, Scott Horowitz, Yan Qin, Daniel Paredes, Sunil Kumar

Chemistry and Biochemistry: Faculty Scholarship

Parkinson’s disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (αS) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of αS aggregation. However, the bottleneck towards achieving this goal is the identification of αS domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified αS sequences-based targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays …

The Role Of Ehd2 In Endothelial Cells, Rachael Judson

Electronic Theses and Dissertations

The role of EHD2 in terms of Cav1 interaction had not been previously characterized and was the main goal of this research. Cav1 has been shown to lead to the ubiquitination of Rac1. Because of this, the role of EHD2 in Rac1 regulation was investigated. An increase in long-term migration in endothelial cells was observed and suggested that EHD2 impacts the RhoA pathway instead of the Rac1 pathway. This lead to the hypothesis that EHD2 controls a part of the RhoA pathway. This hypothesis is supported by the loss of filamentous actin and an increase in serrated junctions in cells …

Characterization Of Cyclopropyl Synthases Involved In The Maturation Of Ribosomally Synthesized And Posttranslationally Modified Peptides, Yi Lien

Electronic Theses and Dissertations

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products with significant human health implications. RiPPs are synthesized from a genetically encoded precursor peptide that undergoes significant modifications by maturing enzymes, or maturases. Recently, radical-S-adenosylmethionine (rSAM) enzymes have emerged as an important family of RiPP maturases. rSAM enzymes have been shown to install ether, thioether, and carbon-carbon bonds on the precursor peptide. These modifications usually define the backbone structure of the mature RiPP. This thesis describes the characterization of a novel RiPP modification catalyzed by the radical S-adenosylmethionine enzyme TigE. TigE belongs to the TIG biosynthetic …

Inhibition Of De Novo And The Prion-Like Spread Of Amyloidogenesis Using In Vitro And In Vivo Disease Models, Johnson Anazoba Joseph

Electronic Theses and Dissertations

The aberrant fibrous, extracellular, and intracellular proteinaceous deposits in cells, organs and tissues are referred to as amyloids. These deposits are dominated by β-sheet structures that have been implicated in several neurodegenerative diseases and cancer. In this work, the types of amyloidosis studied include Parkinson’s disease (PD) using UA196 and NL5901 strains of Caenorhabditis elegans (C. elegans), Alzheimer’s disease (AD) using GMC101 strain of C. elegans, and cancer-associated mutant p53 aggregation in MIA PaCa-2 mutant cells. Several molecules including SK-129, NS132, NS163, bexarotene, a polyphenol (-)-epi-gallocatechine gallate (EGCG), ADH40, RD148, and RD242 were screened in vitro and in …

Lipophilic Probes For Cellular Ethylene Detection, Morgan R. Brown

Electronic Theses and Dissertations

The structure of ethylene is simple, yet its biological effects are significant. When considering its role in biology it is almost exclusively regarded as a plant hormone. Research on ethylene from plants was progressed by several advancements in analytical instrumentation, from its discovery to elucidation of its signaling pathway. There is currently limited understanding of ethylene’s role in mammals, but evidence suggests that it may be a biomarker for oxidative stress! Additional tools and technology are crucial to study this surprising and important signaling role in mammals. Our group has developed molecular ethylene probes as a strategy to detect ethylene …

The Pathophysiological Mechanisms Of Alzheimer's Disease; Investigating Therapeutic Interventions For Disease Onset, Alexandra A. Sandberg

Electronic Theses and Dissertations

Alzheimer’s Disease is a multifarious disease that progressively affects more people as both the proportion of older adults in the population and life expectancy increase in both the United States and worldwide. This devastating disease is a result of rampant neuronal loss in the memory centers of the brain that robs the independence of those who are diagnosed and places a heavy burden on those who care for them. Traditionally speaking, research has focused on the hallmark pathology of amyloid plaques, targeting them to try and prevent disease onset. However, countless failures in clinical trials aimed at this said pathology …

Antioxidant Biomarkers And Nutraceutical Therapeutics In Neurodegeneration And Neurotrauma, Lilia A. Koza

Electronic Theses and Dissertations

Mild traumatic brain injury (mTBI), yielding a Glascow Coma Scale of 13-15, is the most commonly occurring severity of TBI. Pathology from mTBI consists of blood brain barrier disruption, neuroinflammation, oxidative stress, excitotoxicity, mitochondrial dysfunction, protein aggregation, axonal degeneration, and resulting neuronal death. These processes deplete the body’s endogenous antioxidant system. We report a retrospective analysis of antioxidant blood biomarkers in patients with a history of mTBI from a local sports medicine clinic, Resilience Code. We found persistent sex-specific antioxidant depletions in mTBI patients associated with worsened symptomology.

Certain populations, such as athletes, are at high risk for repetitive mTBI …

Organellar Zn2+ Homeostasis And The Role Of Trpml Channels In Neuronal Lysosome Physiology And Axonal Transport, Taylor Franklin Minckley

Electronic Theses and Dissertations

Zinc (Zn²⁺) is crucial for proper cellular function, and as such it is important to measure and track Zn²⁺ dynamics in living cells. Fluorescent sensors have been used to estimate Zn²⁺ content of subcellular compartments, but little is known about endolysosomal Zn2+ homeostasis. Similarly, although numerous sensors have been reported, it is unclear whether and how Zn²⁺ can be released from intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn²⁺. My dissertation started with comparing and characterizing different Zn²⁺ sensors including the …

Investigating Spatiotemporal Kinetics, Dynamics, And Mechanism Of Exosome Release, Anarkali Mahmood

Electronic Theses and Dissertations

Exosomes are small lipid-based vesicles that can carry biomolecules from one cell to another. While exosomes are crucial to maintain homeostasis in healthy cells, they are exploited by unhealthy cells to aid disease progression. Exosomes likely facilitate disease progression via the transfer of disease-causing biomolecules from unhealthy to healthy cells. Exosomes are generated in Multivesicular endosomes (MVEs) and are then secreted into the extracellular space to travel to other cells. Despite being a crucial step, very little is known about exosomes release mechanism and dynamics. To further our understanding of exosomes, specifically their secretion, my work has focused on investigating …

Phosphatidylserine Externalization On Non-Apoptotic Cells And Lipid Scramblases In C. Elegans, Julia Frondoni

Electronic Theses and Dissertations

Cell corpse clearance is key for animals to avoid inflammation and autoimmune disease. To better understand the “eat me” signal of phosphatidylserine (PS) exposure for non-apoptotic corpse clearance, we attempted to disrupt the externalization of PS on C. elegans polar bodies. The second polar body undergoes a non-apoptotic form of cell death, loses membrane integrity, externalizes PS and is cleared by embryonic cells during early development. During apoptotic and necrotic cell death, PS asymmetry is disrupted by lipid scramblases, which translocate phospholipids between the leaflets of the lipid bilayer. We found neither apoptotic nor necrotic scramblases are required for PS …

Vps54 Regulates Lifespan And Locomotor Behavior In Adult Drosophila Melanogaster, Emily C. Wilkinson, Emily L. Starke, Scott A. Barbee

Biological Sciences: Faculty Scholarship

Vps54 is an integral subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). A destabilizing missense mutation in Vps54 causes the age-progressive motor neuron (MN) degeneration, muscle weakness, and muscle atrophy observed in the wobbler mouse, an established animal model for human MN disease. It is currently unclear how the disruption of Vps54, and thereby the GARP complex, leads to MN and muscle phenotypes. To develop a new tool to address this question, we have created an analogous model in Drosophila by generating novel loss-of-function alleles of the …

Modeling Disorder In Proteins Yields Insights Into The Evolution Of Stability And Function, Jonathan Huihui

Electronic Theses and Dissertations

The central dogma of molecular biology dictates that a DNA sequence codes for an RNA sequence, which in turn codes for a sequence of amino acids that comprises a protein. Proteins are responsible with performing myriad functions within living organisms and most proteins require a folded structure in order to perform their function. The protein's structure is the direct link from sequence to function. This is known as the sequence - structure - function paradigm. However, this does not mean that the unfolded state is unimportant. In order to properly model the stability of the folded state, one needs to …

Notch Regulates Vascular Collagen Iv Basement Membrane Through Modulation Of Lysyl Hydroxylase 3 Trafficking, Stephen J.B. Gross

Electronic Theses and Dissertations

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have …

Development Of Endoplasmic Reticulum Targeted Probes And Red Fluorescent Probes For Detecting Zinc, Drew Maslar

Electronic Theses and Dissertations

Zinc (Zn²⁺) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn²⁺ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn²⁺, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn²⁺ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design …

Validation Of A Deployable Proteomic Assay For The Serological Screening Of Sexual Assault Samples, Catherine O'Sullivan Brown

Electronic Theses and Dissertations

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing …

Nucleic Acids Promote Oligomerization Of Immunoglobulin G, Alexa Gomez

Electronic Theses and Dissertations

Nucleic acids have been found to prevent aggregation as chaperones, as well as act as co-factors and promote aggregation of amyloidogenic proteins leading to various diseases. Immunoglobulin G, IgG, are prone to aggregate as therapeutic proteins, and light chains of IgG can form amyloid fibrils, causing a disease known as light chain amyloidosis. Here we discuss the effect nucleic acids have on full-length immunoglobulin G aggregation. Our results show G-quadruplex DNA, and bulk DNA lead to oligomerization of full-length IgG, and induce increases in secondary structure. Tryptophan fluorescence indicates structural changes are occurring in the presence of DNA. Additionally, IgG …

Characterization Of The Whale Shark (Rhincodon Typus) Melanocortin-2 Receptor, Brianne Hoglin

Electronic Theses and Dissertations

Among bony vertebrates, the melanocortin-2 receptor ortholog is unique among the family of five melanocortin receptors on the basis that it is dependent on its accessory protein, MRAP1, for trafficking and activation, and is selective for activation by ACTH alone. Previous studies on the MC2R orthologs of select cartilaginous fish, the elephant shark (Callorhinchus milii) and the red stingray (Dasyatis akajei), revealed divergent traits in a less obligatory relationship on MRAP1 and its ability to be activated by ACTH or the MSH-sized peptides. However, observed traits were not consistent between these two cartilaginous fish species, posing …

A Biophysical Investigation Of Hiv-1 Envelope Cytoplasmic-Tail Mediated Retention In The Gag Lattice, Nicholas Scott Groves

Electronic Theses and Dissertations

The infectivity of Human Immunodeficiency Virus is critically dependent on successful incorporation of the trimeric viral envelope glycoprotein, Envelope (Env), into Gag lattice assembly sites on the host-cell plasma membrane during virus biogenesis. The mechanistic nature of interaction between Gag, the structural protein, and Env, the viral spike, are dependent on the Gag matrix (MA) and Env cytoplasmic tail (CT) domains. Env laterally diffuses on the host cell plasma membrane until encountering a budding Gag lattice, where it is retained until particle scission and release. The mechanism of Env entry and retention in the lattice remains elusive and enigmatic. Here …

Optimized Microbial Recombinant Production Of Hiv-1 Anti-Envelope Antibody Fragments With Applications To Single Particle Tracking Of Virus Assembly, Merissa Michelle Bruns

Electronic Theses and Dissertations

In my findings, I have established a set series of protocols to recombinantly produce, purify and apply various fluorescent probes in vitro for the fluorescent labeling and study of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein during HIV viral assembly. There remains insufficient knowledge about the molecular dynamics and interactions of HIV-1 Env protein with its counterpart, Gag, on the inner host cell surface during assembly of a mature virus particle. There also remains an insufficient amount of data for the understanding and clarification of the mechanism of action of a known host cell HIV-1 restriction factor, …

Rab35 Centered Membrane Trafficking Pathway Directs Apical Constriction During Drosophila Gastrulation, Hui Miao

Electronic Theses and Dissertations

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating high-tension contractile forces at the cell cortex before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be cellular processes to extract unidirectional changes that drive processive transformations in cell shape. During Drosophila melanogastergastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a …

Fxs-Causing Point Mutations In Fmrp Disrupt Neuronal Granule Formation And Function, Emily L. Starke

Electronic Theses and Dissertations

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by the disruption of Fragile X Mental Retardation Protein (FMRP) function in neurons, affecting nearly 1 in 7,500 individuals. Although FXS typically occurs from a complete loss of FMRP expression due to a CGG trinucleotide expansion within the 5’UTR of the FMR1 gene, single nucleotide polymorphisms (SNPs) within the KH domains of FMRP have been shown to severely disrupt FMRP function. FMRP is an RNA-binding translation repressor that interacts with ~4% of the neuronal transcriptome. Many target mRNAs encode for proteins important for regulating synaptic processes and modulate synaptic plasticity. It …

Broadening The Capability Of Kinetics Analysis In Biomechanics, Nicholas Nelson

Electronic Theses and Dissertations

Two studies are discussed in this manuscript each preceded by a literature review of the topic. The first review and study explore agility movements and the effect that alternative upper designs in shoes might have on ground reaction force measures of performance. The second review and study evaluate methods of predicting ground reaction forces without the use of a force platform. A method of using effective forces and ways of improving its accuracy are evaluated in depth.