Molecular Biology Commons^™

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 …

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 …

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 …

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 …