Doctor of Philosophy, The Ohio State University, 2019, Microbiology

The cell envelope of bacteria mediates their interaction with the outside world and determines what can enter the cell. Gram-negative bacteria have a cell envelope defined by two membranes: an inner membrane, which surrounds the cytoplasm, and an outer membrane, which together with the inner membrane delimits an additional cellular compartment termed the periplasm. The inner membrane of Gram-negative bacteria is primarily composed of a phospholipid bilayer. The outer membrane, in contrast, contains phospholipids in its inner leaflet, and the essential glycolipid lipopolysaccharide (LPS) in the outer leaflet. The presence of LPS in the outer leaflet renders the outer membrane relatively impermeable, and therefore grants the cell resistance to noxious compounds in the environment, such as antibiotics. LPS is synthesized in the cytoplasmic face of the inner membrane, though it can also undergo non-stoichiometric modifications in the periplasmic face, and must thereafter be transported across the rest of the cell envelope. Transversal of the inner membrane by LPS is mediated by the ATP-binding cassette transporter MsbA. LPS extraction from the inner membrane, and subsequent transport across the rest of the cell envelope, is mediated by the LPS transport, or Lpt, complex. The Lpt complex is composed of eight proteins: a dimer of LptB in the cytoplasm that binds and hydrolyzes ATP to drive LPS transport; two transmembrane domains, LptF and LptG, which form a cavity in the inner membrane that accepts LPS and extracts it; LptC, LptA, and LptD, which form a bridge across the periplasm to allow the hydrophobic portion of LPS to traverse the aqueous periplasm; and LptE, which in conjunction with LptD facilitates the transport of LPS across the outer membrane. Here, we describe work in which we dissect the molecular mechanisms by which the Lpt system's inner membrane complex, LptB2FGC, interacts with LPS. In chapter two, we describe the identification of a residue within LptG, (open full item for complete abstract)

Committee: Natividad Ruiz (Advisor); Irina Artsimovitch (Committee Member); Ross Dalbey (Committee Member); Patrice Hamel (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2018, Microbiology

Gram-negative bacteria coat their cell surface with the glycolipid, lipopolysaccharide (LPS), that provides stringent permeability characteristics. This layer of LPS comprises the outer leaflet of the outer membrane, and prevents entry of toxic compounds, like antibiotics. After it is synthesized at the inner membrane, LPS must be extracted from the inner membrane, transported across the periplasm, and translocated across the outer membrane to the outer leaflet. This process is accomplished by seven Lpt (LPS transport) proteins which have been best studied in Escherichia coli. An unusual ATP-binding cassette (ABC) transporter, LptB2FG, powers LPS transport. The LptB dimer utilizes conserved features of ABC-motor domains to bind and hydrolyze ATP causing conformational movements. These conformational changes are then transmitted to transmembrane partners LptFG to drive LPS extraction. The objective of this dissertation is to unravel how conformational movements of ATP hydrolysis are coupled to LPS extraction in LptB2FG. Interactions between subunits of ABC transporters utilize a conserved mechanism that consists of a groove in the ATPase domains that interacts with cytoplasmic coupling helices of the transmembrane domains. The tight interactions between these conserved structures allow movements to be coupled between the ATPase and transmembrane domains and is critical for their function. In chapter 2, we identify the coupling helices of LptFG and demonstrate they interact with a groove previously identified in LptB. Using structure-function and suppressor analyses, we demonstrate that a cluster of residues in the groove of LptB and conserved Glu residues in the coupling helices of LptFG are important for coupling ATP hydrolysis and LPS extraction. In chapter 3, while selecting for suppressors of a strain with antibiotics sensitivity conferred by a mutant lptB allele, we serendipitously identified a suppressor that is only resistant to novobiocin. The suppressor mu (open full item for complete abstract)

Committee: Natividad Ruiz Ph.D. (Advisor) Subjects: Microbiology

Doctor of Philosophy (PhD), Ohio University, 2017, Biological Sciences (Arts and Sciences)

Cancer is the second leading cause of death in the US. Despite the endeavors and achievements made in treating cancers during the past decades, resistance to classical chemotherapeutic agents and/or novel targeted drugs has been remaining to be a major problem in cancer therapies. Drug resistance, either existing before treatment (intrinsic) or generated after therapy (acquired), is responsible for most relapses of cancer, one of the major causes of death of the disease. Heterogeneity among patients and tumors, and the versatility of cancer to circumvent therapies make drug resistance more challenging to deal with. Better understanding the mechanisms of drug resistance is required to provide guidance to future cancer treatment and achieve better outcomes. Dysfunctional energetics and opportunistic uptake of extracellular molecules have been named as emerging hallmarks of cancer and cancer metabolism, respectively. Cancer cells are able to uptake extracellular ATP (eATP) via macropinocytosis to elevate intracellular ATP (iATP) levels, enhancing their survival in drug treatment. However, the involved drug resistance mechanisms are unknown. Here we investigated the roles of eATP as either an energy-providing or a phosphorylating molecule in general drug resistance mediated by ATP internalization and iATP elevation. We report that eATP increased iATP levels and promoted drug resistance to various tyrosine kinase inhibitors (TKIs) and chemo-drugs in human cancer cell lines of five cancer types. In A549 lung cancer cells, the resistance was downregulated by macropinocytosis inhibition or siRNA knockdown of PAK1, an essential macropinocytosis enzyme. The elevated iATP upregulated the efflux activity of ABC transporters as well as phosphorylation of PDGFRa and proteins in the PDGFR-mediated Akt-mTOR and Raf-MEK signaling pathways. Similar phosphorylation upregulations were found in A549 tumors. Meanwhile, the resistance cannot be attributed to the overall purinergic recepto (open full item for complete abstract)

Committee: Xiaozhuo Chen (Advisor); Shiyong Wu (Committee Member); Robert Colvin (Committee Member); Fabian Benencia (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2017, Translational Plant Sciences

Transgenic plants containing genes from Bacillus thuringiensis have been used as an alternative to chemical insecticides for insect pest control. The vegetative insecticidal proteins (Vip) secreted during the vegetative growth phase of bacteria are considered a second generation of insecticidal proteins since they do not share any structural or sequence homology with previously used crystal proteins (Cry) as well as having a wide insecticidal spectrum. One of the target pests for this protein is the fall armyworm (FAW) (Spodoptera frugiperda), the most important corn pest in South America. Previously it has been controlled by insecticides and corn expressing Cry proteins, but has rapidly evolved resistance to many control practices and remains a top concern for sustainable biotechnology control efforts. Thus, resistance characterization involving mode of action and genetics of resistance can help with Insect Resistance Management strategies, and improve the durability of control. In this dissertation, using selected FAW population resistant to Vip3Aa20 Bt protein (Vip-R1and Vip-R2) we generated comparative proteomic and transcriptomic data among resistant and susceptible colonies. In the chapter 2, we bring FAW biology/ecology and Brazilian agriculture landscape data to support the high adaptive potential of this pest to genetically modified corn expressing Bt Cry proteins in Brazil. Proteomics studies in the chapter 3 revealed that neither Vip-R1 nor Vip-R2 showed difference between resistant and susceptible colonies either for Vip3Aa20 activation through proteolysis assay nor protein binding to the receptor. Transcriptomic sequencing and RNA-seq analysis in the chapter 4 showed strong evidence of ABC transporter genes associated with resistance as well as genes related to G-protein signaling pathway as downregulated. These results will be discussed in context of providing best management practices for managing FAW resistance to Vip, and extending the durability o (open full item for complete abstract)

Committee: Andrew Michel Dr (Advisor) Subjects: Agriculture; Biochemistry; Entomology; Molecular Biology

PhD, University of Cincinnati, 2004, Medicine : Pathobiology and Molecular Medicine

Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity and mortality in the United States due to consumption of a high-fat “Western” diet. A major indication of these disease states is excess lipid deposition, particularly cholesterol, in the arterial walls. It has been well documented that high levels of circulating high-density lipoprotein (HDL) and its main protein component apolipoprotein AI (apoA-I) are associated with lowered risk of cardiovascular disease. The protective effects of HDL are thought to be mediated by a process called reverse cholesterol transport - in which HDL takes up excess cholesterol from peripheral tissues and transports it to the liver for excretion in the bile. It is now widely accepted that the interaction of apoA-I with the cell membrane protein ATP-binding cassette transporter AI (ABCAI) is critical for the formation of nascent HDL particles. Since sphingomyelin maintains a preferential interaction with cholesterol in membranes, the breakdown of sphingomyelin may regulate the availability of cellular cholesterol utilized by ABCAI. Furthermore, the catabolite of sphingomyelin, ceramide, is a potent signaling molecule and may play an important role in ABCAI regulation or function. The following study examines the potential contribution of sphingolipids in ABCAI-mediated cholesterol removal from the cell. It was discovered that treatment with C2-ceramide enhances cholesterol release to apoA-I. This effect appeared to becaused by an increase in cellular ABCAI content with enrichment at the cell surface. These findings may lead to new ways to increase cellular ABCAI and further promote cholesterol removal from regions of excess cholesterol such as the atherosclerotic lesion.

Committee: Dr. William Davidson (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Chemistry

In this research, we primarily focus on the structure and function of both the nucleotide binding domain, and the full length membrane-spanning transporter. First, the soluble nucleotide binding domain of Atm1 (Atm1-C), an ABC transporter in yeast mitochondria, that has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins, has been overexpressed in E. coli, purified, and characterized. The full length version of Atm1 from Saccharomyces cerevisiae has been cloned, over-expressed, purified from a yeast expression system, and characterized. A fluorescent assay of liposome-loaded reconstituted Atm1p suggested that Atm1p only allowed small molecules and/or metal complexes to cross the channel. Both pH gradient and fluorescent assays also indicated that ADP-bound Atm1p existed in an open state that is different from the closed state for ATP-bound Atm1p. The further discovery of an iron carrying peptide, hepcidin, provides the first step toward understanding iron trafficking in living cells. With eight, well-conserved cysteine residues in the sequence, hepcidin may not only be a signal peptide, but could potentially serve as an iron carrier. The iron binding properties have been determined by UV-vis spectroscopy, mass spectroscopy, and isothermal titration calorimetry (ITC). The iron binding affinity has been determined in the micromolar range. Studies by circular dichroism (CD) reveal varying degrees of secondary structure within an apparent dynamic tertiary fold. Taken together, hepcidin clearly binds iron, and the secondary structure change induced by iron binding may be required for the full function of the peptide in iron homeostasis and antimicrobial activity. In Part II, a novel fluorescent assay has been developed for monitoring the cleavage of a target RNA by cooper kanamycin in vivo. However, demonstration of the efficacy of such reagents in vitro is only a first step. To demonstrate in vivo cleavage chemistry we have designed a (open full item for complete abstract)

Committee: James Cowan (Advisor) Subjects: Chemistry, Biochemistry