Life Sciences 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 …

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 …

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 …

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 …

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 …

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, …

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 …

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 …

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, …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

A Hydrodynamic Method For Measuring Aqueous Nanoparticle Surface Interactions, Kellen John Sorauf

Electronic Theses and Dissertations

The objectives of this research dissertation were to develop and present novel analytical methods for the quantification of surface binding interactions between aqueous nanoparticles and water-soluble organic solutes. Quantification of nanoparticle surface interactions are presented in this work as association constants where the solutes have interacted with the surface of the nanoparticles. By understanding these nanoparticle-solute interactions, in part through association constants, the scientific community will better understand how organic drugs and nanomaterials interact in the environment, as well as to understand their eventual environmental fate. The biological community, pharmaceutical, and consumer product industries also have vested interests in nanoparticle-drug …

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 …

Assessing Crosstalk Between Calcium And Camp In Pancreatic Alpha Cells, Julianne Michelle Imseis

Electronic Theses and Dissertations

Blood glucose levels are regulated by the interactions between insulin and glucagon. Pancreatic alpha cells release glucagon in response to a drop in blood sugar. Pancreatic alpha cells appear to be regulated through multiple signaling pathways. One potential method of regulation occurs from α-MSH or epinephrine stimulated production of the intracellular messenger cyclic AMP. Exocytosis of dense core vesicles containing glucagon ultimately requires an increase in cytosolic calcium. This study focused on the link between cyclic AMP and calcium in pancreatic alpha cells which allows alpha cell regulation. Interactions between calcium and cyclic AMP in pancreatic alpha cells consists 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 …