Molecular 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. …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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, …

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 …

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 …

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 …

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 …

Dense Core Vesicle Heterogeneity In Anterior Pituitary Cells, Kelly Sinak

Electronic Theses and Dissertations

Peptides, which are packaged in dense core vesicles, are an integral part of the function of the endocrine and neurological systems. The dense core vesicles function as an efficient form of peptide storage prior to regulated exocytosis. Two different dense core specific transmembrane proteins traffic different when comparted to retained prolactin cores, offering evidence of heterogeneity of vesicles within a single cell. By comparing synaptotagmin 1 and 7 distribution in male rat and lactating female lactotrophs, a distinct pattern emerges. Cells that retain prolactin cores after exocytosis correspond with those that contain synaptotagmin 1. This finding is a reversal for …

Akap150 Dynamics In Anterior Pituitary Cells, Kristen E. Dew

Electronic Theses and Dissertations

Cellular communication occurs as a result of changes in signaling pathways. A well-studied signaling pathway is through G protein coupled receptors (GPCRs). In gonadotropes, GPCR stimulation by GnRH leads to the activation of protein kinase A (PKA). Activated PKA can phosphorylate ion channels, potentially causing an influx of calcium, depolarization and secretion of hormones. A scaffolding protein known as AKAP150 anchors PKA near L-type calcium channels. In addition, AKAP150 anchors phosphatases, which provides temporal control during signaling events. It was recently shown that AKAP150 is mobile in neuronal dendrites, providing regulation to where the signaling cascade occurs in the cell. …

Regulation Of Synaptogenesis By The Mirna Pathway And Fmr/P Bodies, Jacqueline Rochelle Furlong

Electronic Theses and Dissertations

Post-transcriptional regulation of mRNA is facilitated by different mechanisms, such as microRNA (miRNA) induced gene silencing or fragile X mental retardation protein (FMRP) mediated repression either independent of or acting through cytoplasmic RNA Processing bodies (P bodies). DPTP99A, Lar, and Wg have known functions during synaptogenesis and may be targets of miR-8. Here, we provide evidence that miR-8 regulates DPTP99A in vitro. Non-endogenous miR-8 expressed using an UAS driver regulates Lar. Endogenous miR-8 may regulate DPTP99A in vivo. Here we show that FMRP is capable of colocalizing with the P body components: DCP1, HPat, and Me31B, but not …

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 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 …

Acute Synaptic Activity Causes Differential Mirna Expression In The Drosophila Melanogaster Larval Central Nervous System, Robert Ian Sand

Electronic Theses and Dissertations

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Alpha-Msh Regulated Cell Signaling In Pancreatic Alpha Cells, Liang Liang

Electronic Theses and Dissertations

Peripheral injection of α-MSH promotes glucagon secretion in POMC knockout mice, suggesting the role of α-MSH in regulation of pancreatic α cells. However, the underlying mechanisms remain unknown. This study investigated the stimulatory effect of α-MSH on mice pancreatic α cell line αTC1-6 cells in cell signaling pathway as well as glucagon secretion. In normal level of glucose, application of α-MSH stimulated Ltype Ca2+ current induced Ca2+ induced Ca2+ release (CICR) and membrane hyperpolarization. Increase of [cAMP]c was also observed when α-MSH was applied with IBMX. Acute hypoglycemia-induced CICR via N- and L-type Ca2+ channels was overridden by typical α-MSH …

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 …

Amino Acid Residues Implicated In The Interaction Of Melanocortin Ligands And Their Receptors: A Study Of Mc2r Selectivity, Kristopher D. Veo

Electronic Theses and Dissertations

Melanocortin receptor ligand selectivity has been a question not easily answered. The inability to functionally express melanocortin 2 receptor (MC2R) has inhibited the study of why MC2R is only stimulated by ACTH, a melanocortin hormone. With the recent discovery of the MC2R accessory protein (MRAP), creating a heterologous system is now feasible. Using a general cell line like CHO-K1 cells, which do not express endogenous MCRs, we were able to create a heterologous expression system and test the selectivity of MC2R using analog variants of ACTH(1-24). Our results indicate an amino acid requirement in the C-terminal portion of ACTH(1-24) for …