2018 MU dissertations - Freely available online
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Item Structural/functional insights derived from studies of human and zebrafish CFTR orthologs(University of Missouri--Columbia, 2018) Zhang, Jingyao; Hwang, Tzyh-ChangCystic fibrosis transmembrane conductance regulator (CFTR) chloride channels play a critical role in regulating the trans-epithelial movement of water and electrolyte in exocrine tissues and the malfunctions of CFTR result in cystic fibrosis, the most prevalent lethal autosomal recessive hereditary disease among the Caucasian populations. Despite decades of biochemical and biophysical studies of CFTR, our understanding of CFTR’s structure and gating mechanism remains limited. My dissertation research started with a focus on the structural and functional contribution of the fifth transmembrane segment (TM5) of human CFTR in forming the chloride permeation pathway and continued into tackling the role of the electrostatic profile in the pore for anion flux. These studies have led to the following conclusions: First, TM5 indeed contributes to forming the chloride permeation pathway but TM7 does not line the pore. Unlike the well-studied TM1, TM6, and TM12, the six identified pore-lining residues (A299, R303, N306, S307, F310 and F311) in TM5 only line the internal vestibule of the pore, not the narrow region nor the outer vestibule. Second, changing the side-chain properties of several pore-lining residues along TM5 resulted in channels with two distinct subconductance levels, small conductance O1 state and large conductance O2 state, respectively. Intriguingly, the preferred gating transition C→O1→O2→C over C→O2→O1→C (namely the O1O2 phenotype) as reported previously for R352 mutations suggests the existence of an irreversible gating process attributed to the input of the free energy from ATP hydrolysis. Then the timely solution of the cryo-EM structures of human and zebrafish CFTR offers a great opportunity to interweave the structural and functional data for a comprehensive understanding of CFTR. Taking a full advantage of this opportunity, I launched a thorough investigation of zebrafish CFTR to fill the blank of functional data for this ortholog. Although current cryo-EM data show minimal structural differences between human and zebrafish CFTR, my electrophysiological characterization of zebrafish CFTR revealed unexpected functional differences, which subsequently offer novel mechanistic insights regarding the mechanism of CFTR gating by ATP binding and hydrolysis.Item The effects of metal contamination on larval white sturgeon in the upper Columbia River(University of Missouri--Columbia, 2018) Puglis, Holly Jane; Little, Edward E.Sturgeon populations are imperiled worldwide. Declines are attributed to many factors including habitat degradation and alteration, overexploitation, and contamination. In North America, white sturgeon (Acipenser transmontanus) populations in the upper Columbia River (UCR) face many of the same factors. White sturgeon in this system have been declining for decades due to a lack of recruitment to the population, despite evidence of spawning and early larval survival in the wild. Early life stage white sturgeon are among the most sensitive of aquatic species to copper. The UCR has been contaminated with metal laden slag and liquid effluents from smelter and mining activities, resulting in UCR sediment metal concentrations, including copper, being elevated. The goal of my dissertation was to understand the potential role of metal contamination in the decline of white sturgeon in the UCR. This effort included toxicity tests with copper contaminated water as well as sediments collected from the URC, with a focus on the behavioral responses of exposed fish as a consequence of sublethal exposures. In chapter 1, I exposed early life stage white sturgeon to sublethal concentrations of copper, one of the contaminants of concern in the UCR, to characterize and quantify the effects of copper on swimming and feeding behavior. I found that changes in larval sturgeon swimming behavior were apparent up to seven days earlier than mortality and that copper exposure reduced food consumption in juvenile sturgeon. Critical swimming performance, however, was not affected by copper exposure. While not directly lethal, these alterations in behavior would impair a sturgeon's ability to locate, capture, and consume prey, thus impacting survival. In chapter 2, I evaluated the toxicity of metal contaminated sediments from the UCR to larval white sturgeon. Sediment was collected from six sites in the transboundary reach of the UCR. All six sites had elevated metal concentrations above equilibrium partitioning sediment benchmarks, which would suggest the metals could pose a risk to benthic invertebrates. The overlying water metal concentrations were also above water quality criteria levels in three of the six sites. I found sediment at one site reduced survival and affected swimming behavior of larval sturgeon.In chapter 3, I provide a synthesis evaluation of white sturgeon life history with what has been reported about metal contamination in the UCR, including documented concentrations from the river, and what we know about how metals affect larval sturgeon behavior and survival. Of particular concern are reported field observations of large numbers of sturgeon larvae with empty guts at a critical point in their life cycle. By putting all these pieces together, I found that metal contamination in the UCR could be reducing the prey base of larval sturgeon, altering swimming behavior to increase likelihood of starvation or predation, and evidence of an additional exposure route when larval sturgeon ingest sediment. Thus, the most important findings from my dissertation include 1) low, environmentally relevant concentrations of copper alter swimming behavior of larval sturgeon indicative of sublethal injury, 2) sediments collected from the UCR can affect larval sturgeon survival and swimming behavior, and 3) metal concentrations measured at some sites in the UCR are above effect concentrations from laboratory studies. These findings suggest that metal contamination could be a factor in the decline of the upper Columbia River white sturgeon, and should be considered in recovery efforts for the population.Item A multisite case study of early alert implementation to adoption at the University of Missouri System(University of Missouri--Columbia, 2018) Balser, Christina Jean; Morris, Casandra HarperThis multisite case study of academic early alert warning system implementation explored the relationship between leadership, users (faculty and staff), technology, and the organization when implementing a campus wide early alert program facilitated by new technology at a university system in the Midwest. Early alert technology allows faculty and staff to raise academic concerns and outreach to students during the semester. The findings suggested that technology, people, task and structure were core components impacting implementation and adoption of tools. The findings also suggested that clear expectations from leadership, alignment of tasks within job duties, clearly defined processes, resources to support training, and data outcomes are essential to early alert adoption.Item Fabrication and optimization of three-dimensional metamaterials for terahertz energy-harvesting(University of Missouri--Columbia, 2018) Yang, Shendu; Pinhero, PatrickMetamaterials typically consist of metallic and dielectric repeating structures. Electrodeposition of copper is the preferred approach to fabricating the metallic part of the metamaterials of interest in this study. The highly-variant topography requires chemical additives, like chloride ions, 3-mercapto-1-propanesulfonic acid (MPSA), polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) to enhance bottom-up superfilling while maintaining terrace flatness. This study focuses on both experimental and computational investigations of the degradation potential of the additives and their adsorption mechanism in a highly-acidic copper electrolyte in order to optimally parameterize the copper electrodeposition process. Results show Cl-MPSAPEG-PVP additives perform well, but substitution of PVP with Janus Green B provides better terrace leveling. Additionally, NMR data show a quick and complete conversion of MPSA to bis(3-sulfopropyl) disulfide (SPS) in the acidic copper bath. Finally, FEM simulations further show that the accelerator species may initially accumulate and be transported vertically until overplating, whereby they are transported laterally. Our study also show that the electromagnetic performance of metamaterial is dependent on SU-8 related parameters, including pillar height, bottom side length and spacing between pillars. The metamaterial geometry is successfully optimized and it can localize the surface plasmon with the peak frequency of 1.2 THz to the nano-antenna and the highest terahertz absorption is 30%.Item Investigation of methods and factors influencing mouse oocyte quality, in vitro fertilization and embryo development(University of Missouri--Columbia, 2018) Wuri, Liga; Agca, YukselMice are one of the most commonly used rodent species as a model for biomedical research to better understand molecular, genetic, and cellular causes of human disease and disorders. The production of good quality oocytes is one of the important determinant factors for successful assisted reproductive technologies (ARTs) clinical outcome as well as reproductive biology research. Mouse oocyte quality, morphology and functions are influenced by a variety of the factors such as euthanasia methods of female donors, superovulation regimes and cryopreservation. The objectives of these studies were mainly to investigate the methods and factors that are influencing oocyte quality, in-vitro fertilization (IVF) and embryonic development. First, how different euthanasia methods including cervical dislocation (CD), high flow rate CO[2] (H CO[2]) and low flow CO2 (L CO[2]) would affect the quality and integrity of the metaphase II (MII) oocytes have been investigated. Cumulus oocyte complexes (COCs) were collected from female donors that were euthanized by three different methods and then the oocytes' subcellular structures including microtubules, F-actin, cortical granules (CGs) and mitochondria integrities were detected by specific fluorescence dyes. The results showed that L CO[2] caused significant increase in the incidence of premature cortical granule exocytosis (PCGE) which might be responsible for significantly reducing the in-vitro fertilization (IVF) and embryonic development rate compared to CD and H CO[2]. Secondly, how the superovulation methods would affect the resulting oocyte morphology, quality, IVF competence and embryonic development was investigated. The anti-inhibin serum (AIS) superovulation method produced a significantly higher number of oocytes compared to the pregnant mare serum gonadotrophin (PMSG). Overall, both methods yielded oocytes with similar sizes and comparable subcellular structures including microtubules, F-actin, cortical granules and mitochondria. However, superovulation with AIS produced significantly thinner zona pellucide than PMSG and the perivitelline space of the oocytes generated from AIS were significantly larger than PMSG. There were no differences in terms of two-cell embryo development, or morula and blastocyst formation rates between AIS and PMSG when the oocytes from two methods were in-vitro fertilized with fresh sperm. Morula and blastocyst development rates were significantly higher for AIS compared to PMSG when oocytes were fertilized with frozen-thawed sperm. Thirdly, clutches of mouse cumulus oocytes complexes (COCs) were cryopreserved by the cryoloop vitrification method after PMSG superovulation. The cryo-survival rate and integrity and distribution of subcellular structures including the meiotic spindles, F-actin, cortical granules and mitochondria were examined and compared with fresh MII oocytes. The vitrified-warmed oocytes maintained their subcellular structures to a high degree and resulted in acceptable IVF and embryonic development. In conclusion, for optimal research and clinical outcome, considerations should be given regard to euthanasia methods of oocyte donor mice and type of superovulation regimes. Despite of its high oocyte yield, superovulation of mice with AIS provides comparable quality oocytes to the PMSG method. Cryopreservation of the clutches of mouse COCs via cryo-loop vitrification should be considered for genome banking of genetically modified mice and biomedical research.