Biochemistry, Biophysics, and Structural Biology Commons^™

Examining The Roles Of The Conserved Mrna Deadenylase Complex On Drosophila Neuronal Structures, Megan Mccaughey, Scott Barbee

DU Undergraduate Research Journal Archive

The most common cause of inherited mental deficiency and monogenetic cause of autism is Fragile X Syndrome (FXS). There is little known about the origins of this disease which will be investigated in the present study. The series of experiments conducted examined the potential role of mRNA deadenylation proteins as contributing factors to the pathogenesis of FXS using Drosophila melanogaster as a model organism. One of the main complexes involved in deadenylation is the CNOT complex, which is comprised of many proteins, including POP2, TWIN, and NOT3. Each protein plays a unique role within the CNOT complex. This goal of …

Faculty Spotlight—Dr. Phil Danielson, Hannah Stanley, William Moody

DU Undergraduate Research Journal Archive

Interview with Dr. Phil Danielson

Analyzing The Effect On Ligand Sensitivity For Xenopus Tropicalis Mc1r, Mc3r, Mc4r, And Mc5r As A Result Of Coexpression With Gallus Gallus Mrap1 In Chinese Hamster Ovary Cells, Avery Niemann, Robert Dores

DU Undergraduate Research Journal Archive

Some melanocortin receptors and the accessory protein MRAP1 have been found to interact in novel ways when co-expressed in the same cells following stimulation with either ACTH (1-24) or αMSH. These interactions have been seen for mammalian, bird, and some bony fish melanocortin receptors. Such analysis has not been done yet on the amphibian melanocortin receptors, MC1R, MC3R, MC4R, and MC5R. To this end, this study was done on the effects regarding ligand sensitivity for ACTH (1-24) and αMSH, when the MC1R, MC3R, MC4R, and MC5R paralogs of the amphibian, Xenopus tropicalis were expressed in Chinese Hamster Ovary cells either …

Faculty Spotlight—Dr. Robert Dores, Anit Tyagi, William Moody

DU Undergraduate Research Journal Archive

Robert Dores Interview

Edited by Will Moody

Examining Artifacts Of The Watershed Segmentation, Emily Jo Armitage

Electronic Theses and Dissertations

The watershed segmentation is an algorithm used to systematically track cell intercalary behaviors during germ band extension of the Drosophila embryo. Neighboring cells share a contracting vertical interface, called a T1, which continues contracting to a single point, a T2, and extending in the horizontal direction to create what is called a T3 interface (Fig. 1). Additionally, higher order vertices called rosettes occur when five or more cells meet at a common vertex. Simulated T2 events demonstrate that cell angle and not noise level in the image contributes to the incorrect detection of artifactual T1s in more acute angled cells …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Cellular And Developmental Insights Into The Early Evolution Of Muscle, Jeffrey J. Colgren

Electronic Theses and Dissertations

Whereas a great deal has been learned about the molecular underpinnings of morphological evolution in animals, much less is known about the origin of novel cell and tissue types. During the time in which the earliest animal lineages were diversifying, fundamental cell and tissue types, such as muscles, arose. Sponges are one of two animal lineages that lack muscles, yet they undergo coordinated full body contractions. Whereas the signaling processes have been studied, the physical mechanisms of contraction are completely uncharacterized. The main purpose of this work is to understand the primary contractile tissue of the sponge Ephydatia muelleri, …

Characterization Of The Dimerization And Salt Dependent Aggregation Of The Als-Linked Protein Tdp-43, Elizabeth Ehrhardt

Electronic Theses and Dissertations

Trans-active response (TAR) DNA-binding protein 43 (TDP-43) is essential for RNA processing but can also form toxic cytoplasmic inclusions in neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA-binding has been shown to have the potential to decrease or inhibit the aggregation of TDP-43, indicating that supplying RNA therapeutics may be a solution to treat these neurodegenerative disorders. However, the mechanism of aggregation, transitioning from TDP-43’s native dimeric state to small oligomers to toxic aggregates, is still relatively unknown. This needs to be established before determining how to target and disrupt this aggregation. Using GFP-TDP-43, …

Rab39 And Klp98a Are Required For Furrow Formation During Early Drosophila Embryogenesis, Megan R. Millage

Electronic Theses and Dissertations

The formation of a plasma membrane furrow is an essential process during development. Furrow formation is necessary for successful cell division and cytokinesis in addition to the ability to create multicellular tissues. Here, I will explore the role of the Golgi-associated Rab protein Rab39 in furrow formation during early Drosophila embryogenesis. Rab39 is one of eight Rab proteins that has been shown to localize to discrete puncta by live imaging in early Drosophila embryos, but its function and pathway have not been well characterized. In this thesis will I show that Rab39 forms dynamic, tubular structures that colocalize with trans-Golgi …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Quantification Of Dynamic Epithelial Sheet Architecture In Botryllus Schlosseri Using 2-D & 3-D Image Analysis, Roopa Madhu

Electronic Theses and Dissertations

Epithelial tubules form critical structures in various body tissues; how- ever, since they are difficult to access experimentally, their architecture and dynamics are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel and uniquely accessible model system: the extracorporeal vasculature of Botryllus schlosseri (sea squirt). In Botryllus, massive retraction of blood vessels can be triggered without loss of barrier function, through (i) disrupting collagen crosslinking in the basement membrane using β-aminopropionitrile (BAPN); or (ii) disrupting the integrin pathway through inhibition of focal adhesion kinase (FAK). We performed stereographic projections of 3-dimensional …

Cellular And Organismal Ramifications Of De Novo Purine Synthesis Dysregulation, Randall Craig Mazzarino

Electronic Theses and Dissertations

Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, …

Amelioration Of Alzheimer's Disease Pathology In 12-Month-Old Happ(Sweind) Transgenic Mice After Treatment With A Cysteine Rich Whey Supplement, Immunocal®, Srivalli Puttagunta

Electronic Theses and Dissertations

Deficits in Reelin expression and signaling play a pathogenic role in Alzheimer’s disease (AD). Thus, strategies aimed at correcting Reelin deficits may provide a novel therapeutic approach to treating AD. The cysteine-rich, whey protein supplement, Immunocal®, has recently been shown to rescue Reelin expression in a mouse model of Schizophrenia. Given that Reelin-expressing neurons of the entorhinal cortex region are a highly vulnerable population of cells that are lost early in AD, we examined the effects of Immunocal® in the hippocampal-entorhinal cortex formation in a mouse model of AD. Glutathione levels and Reelin expression in the hippocampal-entorhinal cortex formation (entorhinal …

Targeted-Ion Mass Spectrometry For The Identification Of Forensically Relevant Biological Fluids And Samples From Sexual Assault Evidence, Heather Erin Mckiernan

Electronic Theses and Dissertations

Forensic practitioners have long sought efficient and reliable means for identifying those samples that are best suited for successful genetic profiling. Traditional serological screening methodologies rely upon enzyme activity and antibody-based serological tests. These tests can be consumptive, laborious and costly while reliance on antibody-based serological testing can be prone to error. Positive results resulting from non-target biological fluids, the potential for cross- reactivity and non-specific binding events yield merely presumptive results. This has led forensic biologists to omit serological testing, at least in the case of sexual assault kit samples, in favor of Y-Screen assays. While these Y-Screen approaches …

Qualitative And Quantitative Fitness Consequences Of Advanced Maternal Age, Claudia Jean Hallagan

Electronic Theses and Dissertations

Parental age can affect offspring fitness across taxa and through various mechanisms. However, the effect(s) of advanced maternal age on offspring, particularly in insects, has not been comprehensively reviewed making it difficult to draw conclusions about the effects of advanced maternal age on offspring in insects. In my first chapter, I reviewed maternal age literature and found overall negative effects of advanced maternal age on offspring fitness. However, results vary depending on which fitness measures were used, the life stages at which offspring were measured, and the experimental design of the study. In my second chapter, I conducted an experiment …

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 …

Mechanistic Insight Into Tau Fibril Cross-Seeding Barriers: Structural Order And Disorder, Hilary Ann Weismiller

Electronic Theses and Dissertations

Filamentous deposition of microtubule-associated protein tau is a hallmark for a number of neurodegenerative diseases collectively termed tauopathies. While tau fibrils are directly linked to the etiology and pathogenesis of these diseases, fibril morphology and their phenotypic presentation can be quite disparate. Alternative splicing of tau results in two main isoform groups, four-repeat (4R) tau and three-repeat (3R), identified by their number of microtubule binding repeats. Some diseases show deposition of both 4R and 3R isoforms, while others show preferential deposition of only one type. The conformational templated growth scheme and trans-synaptic spreading of fibrils is influenced by fibril conformation. …

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 …

Cooperative Regulation Of Translational Repression By Fmrp And The Mirna Pathway, Navneeta Kaul

Electronic Theses and Dissertations

Fragile X syndrome (FXS) is the most common inherited monogenic cause of intellectual disability. FXS patients exhibit social and language deficits, hyperactivity, seizures, growth abnormalities, macroorchidism, anxiety, and epilepsy. FXS is caused by the transcriptional silencing of the fragile X mental retardation gene 1 (Fmr1), resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP is a selective mRNA binding protein that plays a role in translation repression. Studies suggest that FMRP utilizes the miRNA pathway to repress translation of its target mRNAs through an unknown mechanism. The aim of my thesis is to investigate …

Development Of Low Frequency Electron Paramagnetic Resonance Methods And Instrumentation For Biological Applications, Laura A. Buchanan

Electronic Theses and Dissertations

EPR is a powerful biophysical tool that can be used to measure tumor physiology. With the addition of magnetic field gradients, the spectral properties of paramagnetic species can be mapped. To facilitate EPR imaging, methods and instrumentation at frequencies between 250 MHz and 1 GHz were developed.

At low spin concentrations, the rapid scan background signal is often many times larger than the EPR signal of interest. To help remove the background contribution, a data acquisition procedure that takes advantage of a cross-loop resonator and bipolar power supplies was developed at 250 MHz. In this procedure, two scans are collected. …

Learning From Disorder And Noise In Physical Biology, Taylor Emil Firman

Electronic Theses and Dissertations

Stochasticity, disorder, and noise play crucial roles in the functioning of many biological systems over many different length scales. On the molecular scale, most proteins are envisioned as pristinely folded structures, but intrinsically disordered proteins (IDPs) have no such folded state and still serve distinct purposes within the cell. At the scale of gene regulation, realistic in vivo conditions produce stochastic fluctuations in gene expression that can lead to advantageous bet-hedging strategies, but can be difficult to characterize using a deterministic framework. Even at the organismal scale, germband extension (GBE) in Drosophila melanogaster embryos systematically elongates the epithelial tissue using …

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 …

Tau Aggregation, Conformational Selection, And Inhibition, Michael R. Holden

Electronic Theses and Dissertations

Tau fibrils are a pathological hallmark of over 20 neurodegenerative disorders, including Alzheimer's disease. There currently is no cure for these diseases and treatments are limited. Once Tau fibrils form in the brain, they propagate down neuronal networks, and this spreading is linked to disease progression. Studying the behavior and structure of Tau monomer and Tau aggregates therefore may give insight into methods by which the spread of Tau fibrils can be inhibited. The structures of the Tau fibrils from different diseases are thought to vary, partially giving rise to the different disease phenotypes. Tau natively binds to microtubules by …

Improving The Detection Limit Of Tau Aggregates For Use With Biological Samples, Emily Rickman Hager

Electronic Theses and Dissertations

The protein Tau is found in neurofibrillary tangles in Alzheimer's disease and over 20 other neurodegenerative diseases. An assay has been developed to detect minute amounts of fibrils from human brain tissue. This assay subjects brain tissue extract and recombinant Tau to several rounds of sonication and incubation. Incubation allows recombinant Tau to add itself to the ends of the existing fibrils in brain tissue extract. Sonication breaks the existing fibrils in the brain tissue extract offering more ends for Tau to add onto. Cycles of sonication and incubation have been shown to allow for amplification of Tau fibrils from …

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 …

Superresolved Three-Dimensional Analysis Of The Spatial Arrangement Of The Human Immunodeficiency Virus Type-1 (Hiv-1) Envelope Glycoprotein At Sites Of Viral Assembly, Carmen Anne Buttler

Electronic Theses and Dissertations

Human Immunodeficiency Virus type 1 (HIV-1) replicates by forcing infected host cells to produce new virus particles, which assemble form protein components on the inner leaflet of the host cell's plasma membrane. This involves incorporation of the essential viral envelope glycoprotein (Env) into a structural lattice of viral Gag proteins. The mechanism of Env recruitment and incorporation is not well understood. To better define this process, we seek to describe the timing of Env-Gag encounters during particle assembly by measuring angular positions of Env proteins about the surfaces of budding particles. Using three-dimensional superresolution microscopy, we show that Env distributions …

The Investigation Of Lactoferrin Nitration: Quantification, Function, And Inhibition, Amani Yahya Alhalwani

Electronic Theses and Dissertations

Lactoferrin (LF) is an iron-binding glycoprotein of molecular mass ca. 80 kDa that is predominantly found in mammalian body fluids. Lactoferrin is a multifunctional protein that has a wide range of properties such as antibacterial, antiviral, anti-inflammatory, and anti-allergic functions. Tyrosine residues in the protein play a part in many lactoferrin functions. Protein tyrosine nitration modification represents an oxidative and nitrosative stress process which can be caused by the exposure of proteins to oxidants from air pollution or disease. Understanding the way nitrated lactoferrin interacts with the biochemical environment of the body is thus important to the broader goal of …

Evaluating The Interaction Between The Human Melanocortin-2 Receptor And The Accessory Protein, Mrap1: Chimeric Receptor And Alanine Substitution Studies On Transmembrane Domain 4, Extracellular Loop 2, And Transmembrane Domain 5, Perry Victoria Davis

Electronic Theses and Dissertations

The melanocortin-2 receptor (MC2R) is the most complex due to its trafficking and ligand selectivity requirements for proper activation. The MC2R requires the melanocortin receptor accessory protein-1 (MRAP1) for proper trafficking and activation of the receptor by the melanocortin hormone, ACTH. MRAP1 is a single transmembrane-spanning domain protein that creates a homodimer with another MRAP1 protein. Furthermore, MRAP2 creates a heterodimer with the MC2R. Previous studies have shown that the MRAP1 protein contains an activation motif required for activation of MC2R and this activation motif located on the extracellular space side of the plasma membrane of the cell. The objective …

Validation Of Minimally-Invasive Sample Collection Methods For Measurement Of Telomere Length, Stephanie A. Stout, Jue Lin, Natalie Hernandez, Elysia Poggi Davis, Elizabeth Blackburn, Judith E. Carroll, Laura M. Glynn

Psychology: Faculty Scholarship

Objective: The discovery of telomere length (TL) as a biomarker of cellular aging and correlate of age-related disease has generated a new field of research in the biology of healthy aging. Although the most common method of sample collection for TL is venous blood draw, less-invasive DNA collection methods are becoming more widely used. However, how TL relates across tissues derived from these sample collection methods is poorly understood. The current study is the first to characterize the associations in TL across three sample collection methods: venous whole blood, finger prick dried blood spot and saliva.

Methods: TL …

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. …