Master of Science, The Ohio State University, 2021, Microbiology

The cell envelope of gram-negative bacteria is characterized by two membranes and an aqueous compartment that create a barrier between the cell and its outside environment. The asymmetric outer membrane is essential to protecting the cell against small, hydrophobic antibiotics. This function is accomplished by lipopolysaccharide (LPS), an amphipathic molecule whose biogenesis occurs in the cytoplasm. Because of its amphipathic nature, LPS must be actively transported across the inner membrane and periplasm to its destination at the outer membrane. This transit is accomplished by the Lipopolysaccharide transport (Lpt) system of proteins, which features an ABC transporter at the inner membrane, a continuous periplasmic bridge, and an outer membrane translocon. The ABC transporter is composed of LptB2FG and couples an ATP hydrolysis cycle to extraction of LPS from the inner membrane via the interaction of coupling helices from LptFG cytoplasmic loop 1 and the groove region of LptB. LptFG cytoplasmic loop 2 is also implicated in coupling these functions and in ATP-binding through an essential arginine residue, according to cryo-EM structures of a recent study. This work demonstrates that the cytoplasmic loop 2 arginine residues are not essential for LPS transport. We also demonstrate that the cytoplasmic loop 2 arginine residues are still important for LPS transport and ATP-binding as demonstrated by combinations of variants to these residues with other well-characterized defective lptB alleles. The resulting synthetic phenotypes of these combinations suggest that the cytoplasmic loop 2 of LptF and LptG, and more specifically its conserved arginine residue, may play a role in re-opening the LptFG cavity in the ABC transporter cycle and still be involved in ATP binding by LptB.

Committee: Natividad Ruiz Ph.D. (Advisor); Jane Jackman Ph.D. (Committee Member); Kurt Fredrick Ph.D. (Committee Member) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2020, Microbiology

The dual-membrane architecture of the Gram-negative bacterial cell envelope provides a robust protective barrier between the cell and its environment. These membranes segment the cell up into two aqueous compartments, the cytoplasm and the periplasm. The inner membrane encapsulates the cytoplasm and is an symmetrical phospholipid bilayer. Beyond the inner membrane lies the periplasm, which is surrounded by the outer membrane and contains the peptidoglycan cell wall. Unlike the inner membrane, the outer membrane is a highly asymmetrical bilayer with an inner leaflet made of phospholipids and an outer leaflet composed of the glycolipid, lipopolysaccharide (LPS). It is this layer of LPS that makes Gram-negative bacteria uniquely resistant to hydrophobic antibiotics. In fact, LPS is essential for the survival of numerous Gram-negative bacteria. LPS is synthesized at the inner membrane and must then be trafficked to the outer membrane. Transport of LPS is mediated by the LPS transport (Lpt) machine, LptB2FGCADE. These proteins form a trans-envelope complex that facilitates LPS extraction from the inner membrane, transport across the periplasm, and insertion into the outer leaflet of the outer membrane. The inner membrane complex, LptB2FGC, is an ATP-binding cassette (ABC) transporter responsible for powering LPS transport. The cytoplasmic nucleotide-binding domains (NBDs) LptB2 bind and hydrolyze ATP, and through protein-protein interactions control the movement of the transmembrane domains, LptFG, which extract LPS from the inner membrane. Specifically, there is a groove region in LptB that accommodates the coupling helices of LptFG to link the movement of these proteins. The proper coordination of the ATP-hydrolysis cycle of LptB2 with LPS extraction by LptFG is essential for successful LPS transport. The process of coupling the ATP-hydrolysis cycle to LPS transport is both complex and poorly understood. This dissertation encompasses work that illuminates several aspec (open full item for complete abstract)

Committee: Natividad Ruiz PhD (Advisor); Irina Artsimovitch PhD (Committee Member); Michael Ibba PhD (Committee Member); Ross Dalbey PhD (Committee Member) Subjects: Microbiology

MS, University of Cincinnati, 2017, Engineering and Applied Science: Chemical Engineering

The role of cell membrane permeability in the biodegradation of organic compounds has been investigated in the present research study. A mechanistic model has been developed that explains the overall biodegradation process as the sum total of three phases: (1) molecular diffusion of the chemical from the bulk liquid phase, through the liquid-phase boundary layer, to the surface of the cell membrane; (2) diffusion of the chemical through the cell membrane; and (3) biotransformation of the chemical through a sequential, enzyme-based kinetic reactions. The biodegradation kinetics data of an exhaustive list of over 500 chemicals has been presented. For the first time, the cell membrane permeability rate and the bulk transport rate contribution towards the overall biodegradation rate has been shown in this study. The rate limiting step in the overall biodegradation process is determined using non-linear regression and correlation analysis. The overall biodegradation rate of randomly selected 50 chemicals was then predicted using the correlation findings. The present study concludes that the diffusion of the chemical through the cell membrane is the rate determining step in the overall biodegradation process. This sheds new light into the field of biodegradation and how researchers could use it to potentially enhance the biodegradation rates by altering the cell membrane permeability and also predict rates to take careful decisions towards the manufacturing of new chemicals.

Committee: Rakesh Govind Ph.D. (Committee Chair); Anastasios Angelopoulos Ph.D. (Committee Member); Stephen Thiel Ph.D. (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2023, Chemistry

Proteins have immense therapeutic potential, including enzyme replacement, targeted protein degradation, protein-protein interaction (PPI) inhibition, and genome editing. However, selective permeability of the cell membrane poses a major obstacle for intracellular delivery of such large hydrophilic macromolecules. Extensive research has led to significant advances in the field of drug delivery, yet ~85% of human proteome is currently deemed undruggable. In this study, we engineered a family of membrane translocating domains (MTDs) by replacing surface loops of fibronectin type III (FN3) scaffold with cell-penetrating peptide (CPP) motifs. One of the domains, MTD4 displayed exceptional cytosolic entry efficiency in mammalian cells in various assays. Protein cargos with diverse size, sequence and physiochemical properties were genetically fused to MTD4 resulting in their intracellular delivery with unprecedented efficacy. Moreover, fused protein constructs broadly biodistributed in mice post systemic administration, a highly desirable property for biomedical applications. We also showed that MTD4 is capable of translocating across the plant cell membrane. Harpin protein (HrpZ) was genetically fused at the C-terminus of MTD4, the construct successfully enhanced growth and pathogen resistance in tomato plants. The MTD platform should enable the development of cell-permeable proteins as a new class of therapeutic agents and agricultural products.

Committee: Dehua Pei (Advisor); Ross Dalbey (Committee Member); Jane Jackman (Committee Member) Subjects: Chemistry

Doctor of Philosophy, Case Western Reserve University, 2023, Applied Mathematics

Two of the many functions supporting life, pH regulations and gas exchange, appear to be related and many studies have been conducted on the gas exchange across cell membrane. After decades where the passage of gas through a cell membrane was modeled by Fick's law, recently there has been a body of work aiming to test the hypothesis that gas can enter a cell also utilizing gas channels present on the membrane, namely aquaporins (AQPs) and Rhesus (Rh) proteins. In the case of CO2 solutions, the gas exchange is also facilitated by the presence of carbonic anhydrase (CA), an enzyme that accelerates the reaction rates of association and dissociation of H2CO3 in water, increasing the gradient between the intracellular and the external solution. Many studies used Xenopus laevis as a biological model. A standard experiment consists of placing an oocyte of X. laevis inside an aqueous solution of CO2, and measuring the pH changes on the cell membrane by means of a micro-electrode. The hypothesis that pH change are greatly influenced by the presence of AQPs, Rh and CA on the membrane surface can be tested by injecting the oocyte with RNA encoding the different proteins of interest, then comparing the response of the wild type and modified membrane to exposure to a high-concentration CO2 aqueous solution. Several mathematical models have been proposed in the literature to describe this experiment, however simpler models do not fully match the data, only providing qualitative validation, while the more detailed ones require such a large computational effort to make them unsuited to solve the inverse problem of estimating the properties of the cell membrane, in particular its permeability. Estimating the permeability from pH measurements is a powerful tool to analyse the effects of AQPs, Rh proteins and CA on the gas transport, and could be used to confirm or reject the hypothesis of gas exchanges through preferential channels. In this thesis, a com (open full item for complete abstract)

Committee: Daniela Calvetti (Committee Chair); Wanda Strychalski (Committee Member); Bryan Schmidt (Committee Member); Erkki Somersalo (Advisor) Subjects: Applied Mathematics

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, The Ohio State University, 2010, Chemistry

The challenges to reveal the molecular organization and interactions at the biological and atmospheric relevant interfaces were confronted in this dissertation by using vibrational sum frequency generation (VSFG) spectroscopy. In particular, the interfaces of biological membrane represented by model phospholipid monolayers, and the aqueous organosulfur species (dimethyl suloxide, DMSO and methanesulfonic acid, MSA) are studied. A condensing effect is observed for the model phospholipid (dipalmitoylphosphatidylcholine, DPPC) monolayer on concentrated DMSO subphases. When the DMSO molecules interact with the phospholipid membranes, DMSO molecules squeeze aside the phospholipids, which cause them to form tightly packed domain structures as well as causing the membrane to expand. In addition, the miscibility of DMSO with water and its powerful solvation of many substances make the formed pores a transportation corridor across the membrane, which as a result accounts for the enhanced permeability of membranes upon exposure to DMSO. Similar effects were found through “in-situ” Brewster angle microscopy (BAM) on dipalmitoylphosphatidyl ethanolamine (DPPE), glycerol (DPPG), and serine (DPPS) phospholipids, indicating that the condensing effect is not dependant upon the phospholipid headgroup structure. Novel structural features of water confined in phospholipid monolayers are revealed. At the air/D2O/monolayer interface, the dangling OD stretching mode showed a marked frequency red-shift as well as spectral structure upon increasing the monolayer surface coverage. Furthermore, the dangling OD was found to exist even when a D2O surface was fully covered by the lipid molecules. This phenomenon was observed in monolayers formed with DPPC and with palmitic acid. The frequency red-shift of the dangling OD is interpreted to be due to the perturbation imposed by the lipid hydrophobic tail groups. In addition, phase sensitive vibrational sum frequency generation is employed to i (open full item for complete abstract)

Committee: Heather Allen (Advisor); Dennis Bong (Committee Member); Sherwin Singer (Committee Member); James Waldman (Committee Member) Subjects: Chemistry

Master of Science, The Ohio State University, 2007, Chemistry

The ability of Rh2(μ-O2CCH3)4 (1), cis-[Rh2(μ-O2CCH3)2(CH3CN)6]2+ (2), cis-[Rh2(µ-O2CCH3)2(dppz)(η1-O2CCH3)(CH3OH)]+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine) (3), and cis-[Rh2(µ-O2CCH3)2(bpy)(dppz)]2+ (bpy = 2,2'-bipyridine) (4) to transverse a membrane bilayer was determined through quenching of DNA-bound SYBR Green I (SG) encapsulated within the interior of a vesicle. For comparison, quenching studies were performed with the known DNA binders ethidium bromide (EtBr), methyl viologen (MV2+), Hoechst 33258 and 2-methyl-antrhacene (2-Me-An). Emission quenching experiments were performed with DNA-bound SG (SG-DNA) free solution to determine the ability of the known DNA-binding compounds and the dirhodium series to affect the luminescence of the dye. The known minor groove binder Hoechst 3328 produced the most quenching of SG-DNA, likely due to displacement of the dye from the duplex. Within the dirhodium series, the degree of quenching correlates to the DNA binding constant of each complex. Complex 3, with the largest kb, produced the greatest quenching of SG-DNA. Each of the complexes in the dirhodium series can quench SG-DNA emission through various mechanisms, including displacement, energy transfer, and charge transfer. In each case, close proximity to the SG-DNA emissive species is a requirement. Therefore, a correlation between the association of each complex in the series with SG-DNA and its quenching ability is not unexpected. When SG-DNA was encapsulated in POPC vesicles, compounds added to the outside of the vesicles can only quench the SG-DNA emission if they are able to penetrate the bilayer membrane. The vesicles with encapsulated DNA-bound SG, SG-DNA@POPC, were incubated for 4 hours and the emission was monitored at 520 nm. Quenching was retained for the known DNA-binders, EtBr and Hoechst 33258. In the dirhodium series, complexes 1, 2, and 4 maintained at least some of their quenching ability, showing that these complexes are able to transverse the l (open full item for complete abstract)

Committee: Claudia Turro (Advisor) Subjects: Chemistry, Inorganic

Doctor of Philosophy, Case Western Reserve University, 1991, Macromolecular Science

This work is a study of the structure and properties of solutions of hydroxypropyl cellulose (HPC) and its films crosslinked under various conditions. Temperature, concentration and shear flow field were found to be important parameters in the formation of HPC mesomorphic structures. Photoinitiated crosslinking of HPC solutions in both the isotropic and liquid crystalline states preserved their structures permanently. The crosslinked HPC films have much better thermal properties than the uncrosslinked ones. Higher tensile modulus and strength were obtained from films crosslinked in the liquid crystalline state compared to those crosslinked in the isotropic state or to uncrosslinked films. A shear-crosslinking device was used to prepare crosslinked films from different concentration solutions and under various shear conditions. X-ray diffraction measurements reveal that these HPC films show a nematic liquid crystal structure when the solution concentration is above the critical concentration. The molecular chain direction in the nematic LC films is parallel to both the surface of the film and the shear flow direction. The degree of orientation has been quantitatively expressed using Herman's orientation function. Band texture formation and loss, and degree of orientation were interpreted in terms of the competition between orientation and relaxation processes of the rigid-rod polymers. This further clarifies the conflicting reports in the literature regarding the origin of the band texture and its relationship to the orientation. Permselectivity of HPC membranes crosslinked in the isotropic or liquid crystalline states was characterized by using small organic penetrants and long chain macropenetrants. The diffusivities decrease as the interaction between penetrant molecules and membranes increases, and with the increase of the effective size of the penetrants. The molecular weight cut-off of the membranes corresponds to a radius of gyration between 14 A and 22 A. Phe (open full item for complete abstract)

Committee: Morton Litt (Advisor) Subjects: Chemistry, Polymer