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Item Characterization of a novel Escherichia coli exopolysaccharide and its biosynthesis by NfrB(2024) Fernando, Sashika Hansini Lakmali; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biofilms are made from an association of bacterial cells and extracellular products dominated by a plethora of exopolysaccharides. Accumulating evidence have demonstrated that the bacterial second messenger cyclic-di-guanosine monophosphate (c-di-GMP) promotes the synthesis of these exopolysaccharides through direct allosteric activation of glycosyltransferase enzymes. The Escherichia coli inner membrane protein NfrB, which together with the outer membrane protein NfrA acts as a receptor system for phage N4, contains a N-terminal glycosyltransferase domain and C-terminal c-di-GMP binding domain. Recent research revealed that NfrB is a novel, c-di- GMP controlled glycosyltransferase that is proposed to synthesize a N-acetylmannosamine containing polysaccharide product, though the exact structure and function of this remains unknown. Nfr polysaccharide production impedes bacterial motility, which suggests a possible role of the Nfr proteins in bacterial biofilm formation. Here, we carry out in-vivo synthesis of novelNfr polysaccharide followed by its structural characterization. Preliminary data from MALDI- TOF mass spectrometry and Solid State 13C NMR spectroscopy indicated that the Nfr polysaccharide is mainly a homo polymer of poly-?-(1®4)-N-acetylmannosamine, bound to an aglycone. In addition, we report efforts to develop of a Nfr polysaccharide binding and detection tool, through the mutation of YbcH, a putative Nfr polysaccharide hydrolase enzyme. These studies advance the understanding of Nfr polysaccharide biosynthesis and could offer potential new targets for the development of antibiofilm and antibacterial therapies.Item ESOTAXIS: IDENTIFYING THE FACTORS THAT INFLUENCE NANOTOPOGRAPHIC GUIDANCE OF THE DYNAMICS AND ORGANIZATION OF THE ACTIN CYTOSKELETON AND OTHER MOLECULES INVOLVED IN DIRECTED CELL MIGRATION(2024) Hourwitz, Matt; Fourkas, John T.; Losert, Wolfgang; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Directed migration is a crucial capability of cells in developmental and immunological processes. Defects in cell migration can lead to negative health outcomes. Cell motion depends on the organization and dynamics of internal components, especially the actin cytoskeleton, and the extracellular environment. Microscale and nanoscale topographical cues, with at least one dimension that is much smaller than most cells, can bias cell motion over long distances, due to the guidance of the organization and dynamics of the cytoskeleton and other molecules and assemblies within the cell. In this work, I describe a technique to reproduce patterned nanotopographic substrates for use in the study of esotaxis, the guided organization and dynamics of the actin cytoskeleton and other cellular components in response to nanotopographic cues. The guidance of actin drives directed cell motion along a pattern with dimensions much smaller than the cell. The dimensions of the nanotopography determine the extent to which cellular components are guided. Differences in the physical properties of the plasma membrane and the actin cytoskeleton among cell lines will influence the extent of guidance by nanotopography. Asymmetric patterns can accentuate the distinctions in esotactic responses among cell lines and drive contact guidance in different directions. The cytoskeletal response to nanotopography is a local phenomenon. A cell in contact with multiple nanotopographic cues simultaneously will show distinct organization of actin in the different regions of the cell. The importance of local actin dynamics requires an analysis method, optical flow, that can identify and track the distinct cytoskeletal motions in different parts of the cell. The formation of adhesions attached to the extracellular matrix is a characteristic of the migratory behavior of many types of cells and these adhesions are credited with allowing the cell to sense and interact with the underlying substrate. Actin can sense nanotopographic cues without the widespread availability of adhesive ligands. Although adhesion to the substrate strongly increases the extent of cell spreading and migration on nanoridges, epithelial cells can align with and migrate along nanotopography even with a dearth of adhesive cues. Therefore, actin is a supreme sensor of nanotopography that can drive directed cell migration.Item Optimized simulations of fermionic systems on a quantum computer(2024) Wang, Qingfeng; Monroe, Christopher; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quantum computing holds promise for simulating microscopic phenomena, offering profound implications across disciplines such as chemistry, condensed matter physics, and high-energy physics, particularly in the accurate simulation of fermions. However, practical implementation requires the optimization of quantum programs to mitigate quantum noise and decoherence effects. Given the constraints of near-term quantum computers, the Variational Quantum Eigensolver (VQE) emerges as a key approach for estimating molecular ground state energies, crucial for determining chemical properties. This work aims to present advancements in optimizing VQE simulations to minimize quantum computational resources. Specifically, this work explores various optimization strategies, including the utilization of second-order perturbation correction to recover additional energy beyond VQE estimates and select critical ansatz terms. Additionally, circuit optimization techniques are investigated, focusing on achieving shorter equivalent ansatz circuits, particularly for physically-inspired VQE ansatz, through methods such as generalized fermion-to-qubit transformations and Pauli string orderings. Furthermore, this work demonstrates the advantage of a better initial state on a trapped-ion quantum computer.Item Using Electric Fields to Modulate Polymeric Materials: Electro-adhesion, Electro-gelation and Electro-carving(2023) XU, WENHAO; Raghavan, Srinivasa R.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation concerns the effects of electric fields on aqueous polyelectrolytes (solutions and gels), including those of polysaccharides and proteins. Electrical effects on such polymeric systems have not been studied in detail thus far. In this work, we apply electric fields as stimuli to trigger responses in these materials. We have discovered three novel responses: electro-adhesion of a gel to a solid electrode; electro-gelation of a polymer solution, which allows gels to be made in 3D, and localized electro-disruption of gels, which allows gels to be carved or sculpted. In our first study, we show that it is possible to adhere a soft ionic conductor (like a polymeric hydrogel) to a hard, electronically conductive electrode using a low DC voltage without any adhesive. When 5 to 10 V DC is applied between a pair of electrodes (e.g., graphite, copper, etc.) spanning a cylindrical hydrogel (e.g., acrylamide, gelatin, etc.), in 3 to 15 min, the gel strongly adheres to either or both electrodes. The ultimate adhesion strength can exceed 150 kPa and is only limited by the strength of the soft material. This hard-soft electro-adhesion applies to not only lab-synthesized hydrogels but also animal or plant tissues, such as beef, pork, apples, bananas, etc. We show that this adhesion results from electrochemical reactions that form chemical bonds between the polymers in the gel backbone and the electrode surface. Hard-soft electro-adhesion can be used to assemble hybrid materials with hard and soft compartments, which could be useful in robotics, energy storage, underwater adhesion etc. Next, we demonstrate how an electric field can be used to gel a polymer solution with spatial control thereby, we can ‘print’ gels in 3D. When a solution of alginate (an anionic biopolymer) is subjected to a DC electric field (~ 10 V) using a platinum (Pt) needle as the anode, a gel is formed right around the anode within seconds. By using a mobile anode, gel “voxels” can be formed sequentially and these merge into 3D structures. Similar electro-gelation can also be done with the cationic biopolymer chitosan, but at the cathode instead of the anode. The mechanism for gelation with both alginate and chitosan involves the polymer chains losing their charge next to the electrode. A loss of charge leads to insolubility, and insoluble domains act as crosslinks and connect the chains into networks. We have built a prototype for a 3D-printer that can translate a 3D design into a robust biopolymer gel formed by electro-gelation. Lastly, we show that an electric field applied by an electrode can be used like a knife to carve or sculpt hydrogels into 3D shapes. When we apply a DC electric field across certain gels, the gel shrinks near the anode, while water is expelled out of the gel near the cathode. Ultimately the gel shrinks by more than 50% of its original size. Such shrinkage is observed with a range of anionic gels, including both physical gels of biopolymers like agar and alginate as well as covalent gels such as sodium acrylate. If the ionic strength of the gel is high, the shrinkage does not occur. The origin of this effect lies in a combination of electroosmosis as well as pH changes near the electrodes. Finally, we show that with a focused electric field, the shrinkage can be limited to a specific location in a gel, thereby allowing us to electro-carve gels in 3D.Item Spatiotemporal proteomic approaches for investigating patterning during embryonic development(2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.Item How Non-Hermitian Superfluids are Special? Theory and Experiments(2024) Tao, Junheng; Spielman, Ian Bairstow; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ultracold atoms emerge as a promising advanced platform for researching the principles of quantum mechanics. Its development of scientific understanding and technology enriches the toolbox for quantum simulations and quantum computations. In this dissertation work, we describe the methods we applied to build our new high-resolution 87Rb Bose-Einstein condensate (BEC) machine integrated with versatile quantum control and measurement tools. Then we describe the applications of these tools to the research of novel superfluidity and non-Hermitian physics. Superfluids and normal fluids were often studied in the context of Landau’s two-fluid model, where the normal fluid stemmed from thermally excited atoms in a superfluid background. But can there be normal fluids in the ground state of a pure BEC, at near zero temperature? Our work addressed the understanding of this scenario, and then measured the anisotropic superfluid density in a density-modulated BEC, where the result matched the prediction of the Leggett formula proposed for supersolids. We further considered and measured this BEC in rotation and found a non-classical moment of inertia that sometimes turns negative. We distinguished the roles of superfluid and normal fluid flows, and linked some features to the dipolar and spin-orbit coupled supersolids. As a second direction, we describe our capability to create non-Hermiticity with Raman lasers, digital-micromirror device (DMD), and microwave, and present our work in engineering the real space non-Hermitian skin effect with a spin-orbit coupled BEC. By use of a spin-dependent dissipative channel, we realized an imaginary gauge potential which led to nonreciprocal transport in the flat box trap. We studied the system dynamics by quenching the dissipation, and further prepared stationary edge states. We link our discoveries to a non-Hermitian topological class characterized by a quantized winding number. Finally, we discuss the exciting promises of using these tools to study many-body physics open quantum systems.Item Magnetic and Toroidal Symmetry of Lithium Transition Metal Orthophosphates(2024) Gnewuch, Stephanie Kardia; Rodriguez, Efrain; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)LiCoPO4 is the foremost candidate material for a novel type of ferroic ordering calledferrotoroidicity. In this work, the synthesis of polycrystalline sample of LiCoPO4 is discussed, along with the structural analog LiMnPO4. Their magnetic susceptibility and magnetic structure were determined and analyzed and found to be consistent with previous reports on single crystal materials. This work also provides a thorough introduction to ferrotoroidicity, a history of its theoretical development, and a summary of the most studied candidate materials. The work then presents a detailed methodology for determining the toroidal structure which would result for the magnetic structure in candidate ferrotoroidal materials. The model provides a method for determining how many toroidal moments would be present, where they would be located within the unit cell, and along which crystallographic direction they would be oriented. Detailed examples for determining the magnetic structure are provided for LiCoPO4 and analogous structures with the olivine structure type, as well as several structures with the pyroxene structure type. The results demonstrate a method for understanding ferrotoroidal arrangements, anti-ferrotoroidal arrangements and non-toroidal structures.Item UNRAVELING THE ROLE OF LASSA VIRUS TRANSMEMBRANE DOMAIN IN VIRAL FUSION MECHANISM(2024) Keating, Patrick Marcellus; Lee, Jinwoo; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa and recently isolated cases worldwide, no current therapeutics or vaccines pose LASV as a significant global public health threat. One of the key steps in LASV infection is the delivery of its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Investigating how the TM participates in membrane fusion will provide new insights into the LASV fusion mechanism and uncover a new therapeutic target sight to combat the dangerous infection. Here, we describe our protocols for expressing and purifying the isolated TM and our GP2 constructs which we use to probe the relationship between the structure of the TM and its influence on the function of GP2.We express TM as a fusion protein with a Hisx9 tag and a TrpLE tag using E. coli bacterial cells. We purify the TM using Nickel affinity chromatography and enzymatic cleavage to remove the tags. Since the TM is prone to aggregation, we must use a strong denaturant, trichloroacetic acid (TCA), to remove the TM from the resin. The isolated TM is then buffer exchanged to a detergent solution for structural studies. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, our structural studies revealed a pH-dependent structural change resulting in an N-terminal extension of the alpha helix in the postfusion state. To test the importance of this structural change, we used the GP2 construct to perform a modified lipid mixing fusion assay. Our results from the fusion assay and a combined mutational study revealed that this structural change is important for the fusion efficiency of GP2. Loss of this extension resulted in lower fusion activity. To further understand these structural changes and to probe the TM’s environmental interactions, we turned to fluorine NMR. This method gives us a unique and highly sensitive probe to monitor changes in the structure and membrane environment. We describe our incorporation protocol of fluorine into the TM and our method for incorporating the TM into a lipid bilayer system. We describe preliminary results showing sensitive changes in the structure of the TM and the implications this method has to enhance our understanding of the LASV membrane fusion mechanism.Item Nature-Inspired Polymeric Materials: Unveiling Unique Responsive Properties(2023) Rath, Medha; Woehl, Taylor J.; Raghavan, Srinivasa R.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In nature, biological systems are able to respond autonomously to environmental cues. Drawing inspiration from nature, scientists have been creating materials that change their appearance, shape, or properties (e.g., optical or mechanical) in response to various stimuli. This work is our contribution to the field - we have designed a range of nature-inspired polymeric materials that reconfigure their properties in response to either physical cues (e.g., temperature) or chemicals in the external medium. In our initial study, our point of inspiration is the natural pearl, which displays a bright sheen (called ‘pearlescence’) due to light reflection from plate-like particles. We show, for the first time, that pearlescence can be reversibly induced in soft capsules that contain no plate-like particles. Our millimeter-sized capsules have an outer shell (~ 500 µm thick) of N-isopropylacrylamide (NIPA) gel, which shrinks above its lower critical solution temperature (LCST) of ~ 32°C. When a transparent capsule is heated above this LCST, it turns pearlescent, and the transparent state is recovered upon cooling. Specular reflectance measurements confirm that the pearlescence of the capsules is comparable to that of natural pearls. We attribute the pearlescence to light reflection from nanoscale domains in the shrunken NIPA shell above the LCST. Next, we draw inspiration from the skin of chameleons - the brilliant colors of the skin are due to ordered arrays (photonic crystals) of particles within the skin cells. To mimic this structure, we first create ‘photonic capsules’ with silica nanoparticles (NPs) in their liquid cores. When the capsules are placed in a polymer solution, the shell is impermeable to the polymer chains but is permeable to water. The resulting osmotic gradient induces the silica NPs to form close-packed arrays, i.e., photonic crystals, which deposit on the inner wall of the capsule. The capsules thereby show brilliant colors (iridescence), with the exact color depending on the NP size. We then further use these capsules as building blocks and fuse them together to form a free-standing sheet. The sheet is thus analogous to a tissue, with the capsules analogous to the constituent cells. We are thereby able to create a sheet of colored capsules, resembling the chameleon skin. Lastly, we take a step towards creating an ‘artificial muscle’. The muscles in our body are nature’s ideal machines as they can expand and contract at will. To mimic this ability, materials that change their size autonomously are of interest. With this goal in mind, we start with an anionic hydrogel with microscale pores - the gel expands by 300% when placed in water. When a carbodiimide is added to the water, it converts the carboxylates on the gel strands to anhydrides, and the loss of charge makes the gel shrink by 50%. The anhydrides are metastable, however, and hydrolyze over time - thereby, the charge on the chains is restored and the gel expands back to its initial size. A cycle of gel expansion and contraction is completed in ~ 40 min, which is ~ 10x faster than any previous soft autonomous material. The rapid response moves our gels closer to the timescales required for use in practical actuators or soft robots.Item Design and Assembly of Block Copolymer-Modified Nanoparticles into Supracolloidal, Molecular Mimics(2023) Webb, Kyle; Fourkas, John T; Nie, Zhihong; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Large strides have been achieved in nanoparticle self-assembly, using various strategies to achieve ordered, supracolloidal structures, ranging from dimers to chains and vesicles to 3-D lattices. However, these methods, while expanding the scope and accessibility of design, face inherent limitations in targeting complex structures with high yields, particularly when using isotropic building blocks (e.g. gold nanoparticles and polystyrene nanoparticles). Additionally, research studying the reversibility of nanoparticle assemblies is mostly limited to small-ligand-modified particles rather than polymer-modified nanoparticles. Polymers are particularly advantageous as they provide a higher degree of functionality to the nanoparticle surface and allow for increased control in directing particle interactions. This control is necessary to continue furthering the advancement of gold nanoparticles in plasmonics, sensors, and catalysts. Here, we introduce two strategies to assemble gold nanoparticles into supracolloidal nanostructures. Gold nanoparticles are modified with complementary, functionalized-block-copolymers that drive the assembly of the nanoparticles. The first strategy uses a diblock copolymer composed of a hydrophilic outer block and an acid or base-functionalized inner block. Upon mixing, particles are assembled due to the acid–base neutralization between the complementary block copolymers. The resultant supracolloids consist of nanoparticles precisely arranged in space, which mimic the geometries of small molecules. The particle interactions are fine tuned by varying the size and feeding ratio of the nanoparticles, along with the length and composition of the block copolymers. Careful tuning of these parameters yields nanostructures with different valences that were produced in high yield. Additionally, the implementation of a long outer, hydrophilic polymer block provided the assembled nanostructures stability when transferred from THF to water. Colloidal stability in an aqueous medium could allow for expanded use of these nanostructures in cellular uptake studies and biomedical applications. The second strategy uses a diblock copolymer composed of a hydrophilic outer block and an inner block containing either complementary host or guest moieties. Particularly, we take advantage of the well-established interactions between β-cyclodextrin and adamantane as the host and guest molecules. Upon the slow addition of water, particles assemble due to the host–guest interactions between the complementary block copolymers, as the hydrophobic adamantane moieties are driven within the β-cyclodextrin macrocycles. Fine tuning of the nanoparticle sizes and feeding ratios and the block copolymer lengths and compositions results in high yields of targeted supracolloids that also mimic the geometries of molecules. Interestingly, the size difference between the host and guest-modified particles led to different types of nanostructures. In addition, due to the reversibility of the host–guest interactions, we demonstrate the ability of our system to reorder in response to competitive host moieties. Upon addition of free β-cyclodextrin, the host–guest interactions are disrupted, resulting in disassembly of the nanostructures, which we could reassemble upon removal of the free cyclodextrin. Finally, due to the strength of the nanoparticle interactions, we also tested the selectivity of the nanoparticle interactions by assembling the host building block with different guest building blocks. We showed that when assembled with competing guest building blocks, the β-cyclodextrin building blocks preferentially interact the adamantane building blocks due to the stronger particle interactions. This reversibility and selectivity make our system a potential candidate for use in biosensors.Item Crystallographic Studies of Intercalated Transition Metal Chalcogenides(2023) Balisetty, Lahari; Rodriguez, Efrain; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Crystallography, in simplest terms, is the study of crystals, their atomic arrangement,symmetry, and morphology. This knowledge is applied across disciplines ranging from determining the structure of geological minerals to biologically important compounds such as deoxyribonucleic acid(DNA) to high-temperature superconductors and quasi-crystals. The work presented in this dissertation involves structure and property studies of iron chalcogenide heterostructures prepared via intercalation. The tetragonal iron chalcogenides, FeCh (Ch = S, Se) studied in this work possess a layer structure made by linking edge-shared tetrahedra with chalcogen atoms at the corners and an iron atom at the center of each tetrahedron. The weak van der Waals forces holding these layers together make introducing intercalants into the interlayer space feasible. Iron chalcogenide heterostructures were prepared by bottom-up solvothermal growth technique and primarily focused on two kinds of intercalants: layered double hydroxides (LDHs) and metal-amine complexes. Both the intercalants are positively charged and can interact with the chalcogenide layer through electrostatic interactions. Along with electrostatics, the intercalants are strong hydrogen bond donors (O-H, N-H, respectively), and can participate in H-bond formation with chalcogenide anions of the host layers. In this work, we provide structural descriptions for two kinds of intercalated iron heterostructures. First is a twisted layer structure of ethylenediamine intercalated FeS. Layered van der Waals materials are susceptible to disorder and can form various stacking polymorphs through translations and twisted structures due to rotations between layers. We study the influence of intercalation of ethylenediamine molecules on such layer-to-layer twist stacking behavior in iron sulfide. The second type is LDH-iron chalcogenide heterolayered materials. They belong to the family of naturally occurring mineral, tochilinite. A new structure model for the heterolayered material is built using prior knowledge of individual components. The structural insights into this material were applied to synthesize new compositions of tochilinite. A synergistic relationship between the host layers and intercalants can lead to superior properties than constituent components or new emergent properties that are not present in the individual bulk compounds, making these compounds interesting.Item Analyzing Dynamical Processes with Local Molecular Field Theory(2023) Zhao, Renjie; Weeks, John D; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Local molecular field (LMF) theory provides a framework for describing the collective response of a system to long-range interactions in nonuniform liquids. Based on this theory, different roles played by the short and long-range components of the intermolecular interactions can be disentangled in determining relevant structural and thermodynamic properties in equilibrium. Furthermore, in dynamical processes, nonlocal long-range interactions are often associated with long relaxation times, and can contribute significantly to the stability of the system in different phases. In this thesis, LMF theory is utilized to quantify and analyze the dynamical effects arising from long-range Coulomb interactions in aqueous solutions, while elucidating how they are connected to strong local forces and fluctuations. The first half of the work concerns ionic and dipolar solvation dynamics, which plays an essential role in many solution phase chemical reactions. The physical models of Gaussian-smoothed charge and dipole distributions are conceptualized from LMF theory to investigate the molecular origins of linear and nonlinear effects in solvation dynamics. The long-range component of the solute-solvent electrostatic interaction is shown to underlie the linear response behavior of the system, while the short-range interactions introduce additional nonlinear effects. The LMF-based solvation models further demonstrate their functionality in probing the intrinsic dielectric dispersion of solvent water. The second half of the work is focused on the nucleation processes in the aqueous environment. Simulating crystal nucleation from solutions requires efficient treatments for intermolecular interactions to drive the transitions on time scales affordable to molecular dynamics simulations. For this purpose, a LMF-based molecular model is employed to capture the renormalized long-range interactions, and well-tempered metadynamics is adopted to enhance the fluctuations arising from short-range interactions. By comparing to a short-range reference model, the necessity of long-range interactions in explaining metastability is revealed. Temporal fluctuations and direct evidence for the two-step nucleation mechanism are observed through the analysis using a deep learning-based approach. The results about these two types of dynamical processes contribute to a deeper understanding of the roles of short and long-ranges interactions in the aqueous systems.Item UNVEILING THE SELF-ASSEMBLY OF POLYMER-GRAFTED NANOPARTICLES IN SELECTIVE SOLVENTS(2023) Lamar, Chelsey; Nie, Zhihong; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The self-assembly of inorganic nanoparticles (NPs) has garnered considerable attention due to the potential for fabricating functional structures with unique collective properties. In recent years, polymers have emerged as valuable candidates in assisting the organization of NPs into complex architectures with multiple capabilities. Researchers have shown that polymer-grafted nanoparticles (PGNPs) facilitate the use of advanced nanostructures with tailored properties in biomedical applications. Although, continued exploration of the rational design and tailoring of PGNP assemblies is needed to expand our understanding before we can fully realize the potential of these structures in desired applications. My dissertation aims to investigate the fundamental aspects and elucidate the underlying mechanisms in the self-assembly of PGNPs for modern biomedical applications. A facile and versatile solution-based strategy was utilized to explore the individual self-assembly of PGNPs with anisotropic NPs and the co-assembly of binary PGNPs with distinct sizes. We focused on designing, characterizing, and exploring the optical properties of hierarchical assembly structures produced from inorganic NPs tethered with amphiphilic block copolymers (BCPs). Individual PNGPs with anisotropic NPs and binary mixtures of small and large PGNPs produce vesicle structures with well-defined packing arrangements. My work shows how key parameters, including polymer chain length, nanoparticle size, and concentration, influence the self-assembly behavior and the formation of vesicles in each system. Through a combination of experimental observations and theoretical considerations, I highlight the significance of polymer shell shape in dictating the self-assembly behavior of individual anisotropic PGNPs. Moreover, I demonstrate that elevated temperatures impacted the stability and optical responses of the vesicle structures. In co-assembly studies, my work describes the macroscopic segregation of PGNPs with different sizes in the vesicular membrane, which is attributed to the conformation entropy gain of the grafted copolymer ligands. This research will provide valuable insights into the self-assembly behavior and fundamental design of PGNP structures relevant to biomedical applications.Item Photoredox-Active Tertiary N-Oxyammonium Reagents For Selective sp3 C-H Oxidative Functionalization(2023) Hitt, Michael James; Vedernikov, Andrei N; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The C(sp3)-H bond functionalization under photoredox catalysis has been a topic of active research over two last decades. Photoredox catalysis involves the use of light to access catalysts’ excited states allowing for facile single electron transfer (SET) with a hydrogen atom transfer (HAT) agent precursor. One prominent HAT agent precursor is quinuclidine (Q), of which the active radical is the electrophilic radical cation (Q●+) able to abstract a hydrogen atom from a C(sp3)-H bond, so allowing for the bond functionalization. Q has been used by researchers since 2015 in the reductive photoredox catalytic C(sp3)-H functionalization (Chapter 1). In this work we focus primarily on the design of novel N-acyloxyquinuclidinium reagents with the goal of the development of new reaction protocols for the selective oxidative C(sp3)-H bond functionalization that utilize our new reagents under photoredox catalysis. In Chapter 2 we present our new catalytic system that allows for the selective oxidative C(sp3)-H trifluoroacetoxylation of donors of 1o, 2o and 3o benzylic C-H bonds using N-trifluoroacetoxyquinuclidinium trifluoroacetate that can be conveniently generated in situ by mixing quinuclidine N-oxide and trifluoroacetic anhydride in DCM solutions. Under photoredox catalyst under blue LED light, this reagent allows for the unique high-yielding (up to >99%) selective oxidative trifluoroacetoxylation of various (functionalized) alkylarenes used as limiting reactants (22 examples overall, including a pharmaceutical-derived substrate). The proposed reaction mechanism involves Q●+ as a highly selective HAT agent and benzylic carbocations resulting from oxidative radical polar crossover of transient benzylic radicals. In Chapter 3 we introduce a series of isolable N-aroyloxyquinuclidinium tetrafluoroborates (Q-Bz) that allow for the preparation of N-alkylimides in a first of its kind Ritter-Mumm type three-component oxidative imidation of donors of benzylic and cycloalkane C(sp3)-H bonds. In this reaction carbonitriles serve as the source of an imide nitrogen atom and a solvent, whereas the third reaction component, Q-Bz, acts as the oxidant, the source of HAT agents and one of two acyl groups of the imide products. All three reaction components can be varied. 33 different N-alkylimides were prepared using (substituted) alkylarenes as limiting reagents, with product yields up to 94%, and cycloalkanes taken in 3-fold excess with respect to the oxidant. The proposed reaction mechanism involves either Q●+ or aroyloxy radicals as HAT agents, depending on the identity of the aroyl group. Chapter 4 discusses the first example of a Balz-Schiemann – type C(sp3)-H fluorination of alkylarenes and cycloalkanes using N-aroyloxyquinuclidinium tetrafluoroborates (Q-Bz), with tetrafluoroborate anion as the source of the fluorine atom of the resulting alkyl fluorides. The proposed reaction mechanism involves either Q●+ or aroyloxy radicals as HAT agents. Chapter 5 discusses the use of N-trifloxypyridinium salts for Minisci type cross dehydrogenative coupling (CDC) of alkane C-H bonds and pyridine C(sp2)-H bonds. Some limitations and possible future development of this chemistry are presented. Finally, Chapter 6 gives a summary of the results of this work and suggests future directions for the advancement of oxidative C(sp3)-H functionalization chemistry using various N-oxyammonium salts as HAT agent precursors, oxidants, and co-reagents.Item New method for kinetic isotope effect measurements(2023) Kljaic, Teodora; Poulin, Myles B; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precision. In this thesis I described the development of a novel competitive KIE measurement method using MALDI-TOF-MS and the investigation of the transition state of glycosyltransferase enzyme BshA from B. subtilis. We developed a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). This approach enabled quantitative measurements of both relative isotope abundance of an analyte and fractional conversion F in single measurements without the need for purification prior to analysis. The application of this MALDI-TOF MS approach has demonstrated the precision of KIE measurements comparable to those obtained using competitive radioisotope labelling, and NMR based approaches while requiring smaller amounts of stable isotope labelled substrates. Using two chemoenzymatic approaches, we then synthesized 5 substrates for the application of our method to investigate the transition state of BshA: UDP-GlcNAc (3.1), [1''-13C]UDP-GlcNAc (3.2), [2''-13C]UDP-GlcNAc (3.3), [13C6]UDP-GlcNAc (3.4) and [2''-2H]UDP-GlcNAc (3.5). Finally, we have begun to work on the synthesis of [1''-18O]UDP-GlcNAc and describe an approach to prepare this substrate that is currently underway in the lab. Application of the quantitative whole molecule MALDI-TOF MS approach enabled us to determine multiple competitive KIEs for the enzymatic reaction catalyzed by BshA. While previous studies suggested a front-face SNi (DNAN) TS for the conjugation of UDP-GlcNAc and L-malate, our KIE results show that a stepwise mechanism resulting in the formation of a discrete, though likely short lived, oxocarbenium ion intermediate is more likely. Our method be applied to study other glycosyltransferases whose mechanisms still remain to be elucidated and to design TS based inhibitors for enzymes involved in different bacterial infections. Future work on automation of this method would simplify the KIE measurement process and increase reproducibility making the measurement of KIEs for TS analysis a more experimentally accessible technique for the broader enzymology research community.Item DISCOVERYING THE ROLES OF SOLUBLE DHH-DHHA1 TYPE PHOSPHODIESTERASES IN RNA DEGRADATION AND CYCLIC DINUCLEOTIDE SIGNALING(2023) Myers, Tanner Matthew; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis and degradation of RNA is a fundamental and essential process for all life forms. It is imperative that cells utilize multiple mechanisms to modulate the lifetimes of RNA molecules to ultimately control protein synthesis, to maintain homeostasis or adapt to environmental challenges. One mechanism to directly alter the stability or half-life of an RNA is through the direct enzymatic activity of ribonucleases (RNases). Bacterial organisms encode for many different RNases that possess distinct functions in RNA metabolism. It is through the actions of multiple cellular RNases that a long RNA polymer corresponding to an mRNA, rRNA, tRNA, or sRNA can be fully degraded back into nucleotide monophosphate precursors. The nucleoside monophosphate precursors are recognized by kinases that ultimately recycle these molecules for use in the synthesis of other long RNA polymers. The processing of RNA polymers by endo- and exoribonucleases generates nucleoside monophosphates as well as short RNA oligonucleotides ranging from 2-6 nucleotides in length. Previously, a subset of enzymes broadly referred to as “nanoRNases” were found to process these short RNA fragments. Oligoribonuclease (Orn), NanoRNase A (NrnA), NanoRNase B (NrnB), and NanoRNase C (NrnC) were previously ascribed the function of indiscriminately processing nanoRNA substrates. However, recent analyses of the evolutionarily related DnaQ-fold containing proteins Orn and NrnC have provided compelling evidence that some nanoRNase protein families possess distinct dinucleotide substrate length preferences (Kim et al., 2019; Lormand et al., 2021). To determine whether all nanoRNases are diribonucleotide-specific enzymes, we utilized a combination of in vitro and in vivo assays to conclusively elucidate the substrate specificity and intracellular roles of the DHH-DHHA1 family proteins NrnA and NrnB. Through an in vitro biochemical survey of many NrnA and NrnB protein homologs, including from organisms of varying degrees of relatedness, we have determined that there are many functional dissimilarities contained within the DHH-DHHA1 protein family. Furthermore, we have conducted a rigorous investigation into the biological and biochemical functions of NrnA and NrnB in the Gram-positive model organism Bacillus subtilis. These analyses have shown that B. subtilis NrnA and NrnB are not redundant in biochemical activities or intracellular functions, as previously believed. In fact, we have found that B. subtilis NrnA is a 5’-3’ exoribonuclease that degrades short RNAs 2-4 nucleotides in length during vegetative growth, while B. subtilis NrnB is specifically expressed within the developing forespore and functions as a 3’-5’ exoribonuclease that processes short RNA in addition to longer RNA substrates (>40-mers). Our collective data provide a strong basis for the subdivision of the DHH-DHHA1 protein family on the basis on their diverse substrate preferences and intracellular functions.Item CHEMOENZYMATIC SYNTHESIS OF FUCOSYLATED OLIGOSACCHARIDES AND ANTIBODY GLYCOFORMS FOR ELUCIDATING BIOLOGICAL FUNCTIONS(2023) Lunde, Grace Henry; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fucosylation is critical for molecular recognition events in the immune system, microbial interactions, and cancer metastasis. Terminal fucosylation (α1,2, α1,3, and α1,4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis) and is expressed on many N- and O-glycans and most human milk oligosaccharides (HMOs). Core fucosylation (α1,6) exists primarily on the sixth position of the core GlcNAc of N-glycans and plays a profound role in modulating the biological functions of IgG antibodies. Due to their diverse biological properties, structurally well-defined fucosylated oligosaccharides and glycoconjugates are highly demanded for detailed structure-function relationship studies and translational applications. Three main approaches exist to meet this end: metabolic engineering, traditional chemical synthesis, and chemoenzymatic synthesis. Metabolic engineering has unmatched scalability but can suffer from limitations in many factors, including the availability of various sugar nucleotides, genetic instability, and inherent microheterogeneity due to the non-template-driven multiple-step assembly of glycans. Traditional chemical synthesis is ideal for producing a diverse array of pure targets due to its flexibility but may require prohibitively tedious multistep protocols. Alternatively, chemoenzymatic synthesis harnesses the precision of traditional chemical synthesis and marries it to the usually regio- and stereo-selective enzymatic transformations. Glycosyltransferases (GTs), the natural enzymes for synthesizing glycosidic bonds, are most widely applied in chemoenzymatic routes yet some glycosidases, which naturally hydrolyze glycosidic bonds, can be used in transglycosylation mode for chemoenzymatic synthesis. Generally, glycosidases can be easier to express, have a more relaxed acceptor substrate specificity, and utilize simpler synthetic donor substrates than GTs. However, glycosidases possess an inherent propensity toward product hydrolysis. This has led to the design of mutant glycosidases, known as glycosynthases and glycoligases, which have diminished hydrolysis and enhanced transglycosylation activity. A glycosynthase is a catalytic nucleophile mutant that inverts the anomeric configuration of the donor substrate (α→β or β→α) in glycosylation and a glycoligase is a mutant at the general acid/base residue that retains the anomeric configuration (α→α or β→β) in its catalytic glycosylation. Both require an activated glycosyl donor substrate with a suitable leaving group at the anomeric position. Readily synthesized glycosyl fluorides are frequently used. Given their superior stability in aqueous conditions, α-glycosyl fluorides are preferred over β-glycosyl fluorides. Therefore, the glycoligase approach is favored in the synthesis of α-glycosidic bonds, like the α-fucosides found in mammalian systems. Our group has successfully applied the glycoligase strategy for robust synthesis of core fucosylated (α1,6) N-glycans and glycoproteins. For my first project, I aimed to expand our fucoligase toolbox and designed an α1,3/4-fucoligase (AfcB E746A) for the synthesis of α1,3 and α1,4-fucosylated oligosaccharides. AfcB E746A very efficiently catalyzed the synthesis of the Lewis X (LeX) and A (LeA) trisaccharides and two HMOs: 3-fucosyllactose (3FL) and lacto-N-fucopentaose (LNFP) III with only slight excess (1.5 eq.) of the αFucF donor substrate. I concluded that AfcB E746A prefers to synthesize α1,3- over α1,4-fucosidic bonds, demonstrates a unique specificity for acceptors with a reducing end GlcNAc over glucose, and seems to require a free terminal Gal in its acceptor substrate. In my second project, I aimed to characterize the enzyme activity of two of our lab’s fucoligases, AfcB E746A and BfFucH E277G, in the synthesis of novel difucosylated tetrasaccharides. I concluded that AfcB E746A requires a free terminal galactose in the acceptor substrate given that AfcB E746A cannot efficiently utilize BfFucH E277G’s monofucosylated products. Alternatively, BfFucH E277G utilizes AfcB E746A’s monofucosylated products. BfFucH E277G synthesized several difucosylated tetrasaccharides, which were characterized by mass spectroscopy and one- and two-dimensional NMR analysis. I concluded that BfFucH E277G catalyzes the synthesis of an α1,3-fucosidic bond on the terminal galactose of 3FL and LeA to yield two novel HMO/Lewis antigen-like structures: 3’3-lactodifucotetraose (LDFT) and 3’-fucosyl-Lewis A (3’-FLeA), respectively. This study further elucidates BfFucH E277G’s unique acceptor substrate driven regioselectivity. In my first two projects, I meticulously characterized the transfucosylation activity of AfcB E746A and BfFucH E277G to provide insight into how these fucoligases may be integrated into synthetic schemes. Enzyme-catalyzed synthesis is an indispensable synthetic strategy given its reliability in substrate specificity and stereo- and regioselectivity. Pivoting from the study of fucoligases, in my third project I prepared highly pure and structurally well-defined IgG antibody glycoforms by chemoenzymatic remodeling with enzymes discovered and designed by our group. The objective of this work was to demonstrate our lab’s expertise in the chemoenzymatic remodeling of the IgG Fc N297 glycan. Furthermore, these antibodies will be applied in future experiments to more confidently characterize the effect of antibody core (α1,6) fucosylation on their binding affinity for Fcγ receptors (FcγRs) and demonstrate how the allosteric effect of immune complex formation contributes to this phenomenon. Core fucosylation of the N297 glycan reduces antibody-dependent cellular cytotoxicity (ADCC) by decreasing the antibody’s affinity for the FcγIIIa receptor. In fact, by removing core fucose, binding can be enhanced up to 50-fold, leading to improved ADCC and enhanced therapeutic efficacy. However, the current data is considerably disparate with enhancements ranging from 3- to 53-fold. These discrepancies may be attributed to glycoform heterogeneity and contamination with afucosylated glycoforms. Additionally, some studies have reported that, in the presence of core fucosylation, sialylation negatively impacts ADCC. This is accompanied by only a modest decrease in FcγRIIIa binding affinity which cannot solely account for the large difference in ADCC. This phenomenon may be explained by conformational allosteric cooperativity where a conformational change in Fab, upon antigen binding, is transmitted to the Fc to alter its affinity for the FcγRs. We hypothesize this phenomenon explains the discrepancy between FcγRIIIa binding affinity and the degree of ADCC activated by the sialylated and core fucosylated IgG antibodies. These detailed studies on the IgG-FcγRIIIa interaction are important for basic research and translational science. Exceedingly pure glycoforms are required for these experiments. My final project demonstrates the Wang group’s exceptional position in the field of basic antibody research and the indispensable nature of our work for the improved therapeutic application of monoclonal antibodies.Item RELAXATION TIME FLUCTUATIONS IN TRANSMONS WITH DIFFERENT SUPERCONDUCTING GAPS(2023) Li, Kungang; Lobb, Christopher; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this thesis, I discuss the fabrication and measurement of Al/AlOx/Al transmons that have electrodes with different superconducting gaps. With gap-engineering, the tunneling of single quasiparticle from the low-gap side to the high-gap side can be suppressed, hence increasing the relaxation time T1. The best gap-engineered device showed T1 exceeding 300 μs. Large T1 fluctuations in my devices were also observed. I proposed a mechanism for exploring the T1 fluctuation data and discuss the possible underlying cause of the T1 fluctuations. I first discuss the theory of the loss in gap-engineered transmons, with a focus on the loss from non-equilibrium quasiparticles. The model yields the quasiparticle-induced loss in transmons and its dependence on temperature. I also discuss how multiple Andreev reflection (MAR) effects might alter these conclusions, leading to a further reduction in T1. I then describe the design, fabrication and basic characterization of the transmon chip SKD102, which features two transmons – one with thin-film electrodes of pure Al and another that had one electrode made from oxygen-doped Al. I next examined T1 vs temperature and how the T1 fluctuations depended on temperature. I compare my results to a simple model and find reasonable agreement in transmons on chip SKD102, KL103 and KL109, which had different electrode and layer configurations. Finally, I analyze T1 fluctuations in different devices and as a function of temperature and propose a model to explain this behavior. Over the different devices, the T1 fluctuation magnitude roughly scaled as T13/2. With increasing temperature, T1 decreases due to a higher density of thermally generated quasiparticles. In contrast, for an individual device measured from 20mK to 250 mK, the fluctuation magnitude appears to be proportional to T1. I present a model of quasiparticle dissipation channels that reproduces both of these observed scaling relationships.Item Versatile Strategies for Multifunctional Polyolefins(2023) Fischbach, Danyon Miles; Sita, Lawrence R; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polyolefins have quickly become one of the world’s most utilized products since their discovery in the 1950s. With 350 million tons produced each year, it is clear that the use of polyolefins is not subsiding in the near future. Instead, it is imperative to develop novel materials that are more efficient than their current counterparts. As the function of a plastic is derived from its properties, creating polyolefins with designable and targetable attributes is a major priority. The Sita group has played a huge role in the development of ‘precision’ polyolefins. The techniques employed allow for the scalable synthesis of a plethora of polyolefins. To do this, input variables such as the monomer, tacticity, molar mass, and molar mass distribution are controlled in an organized manner to affect output properties such as crystallinity, elasticity, and tensile strength. The ability to create diverse plastics is necessary for the functions asked of them, however, the missing element in almost all polyolefin synthesis is chemical functionality. The inert nature of polyolefins leads to limited reactivity, therefore, reducing possible chemical reactions, such as recycling. The goal of this work is to increase the scope of functional polyolefins so that new materials with improved properties can be produced. The first step in adding functionality is choosing the proper functional group. A drawback to many polyolefin functionalities currently under study is that they have a very limited scope. Functional groups are designed and used individually, requiring different compounds for each target functionality. To overcome this obstacle, aryl functional groups were targeted in this report. Phenyl functionalities are known for undergoing a range of chemical transformations leading to a wide variety of possible materials. Described in this report, aryl-functionalized polyolefins were synthesized using three different techniques. Each method has been shown to later undergo post-synthetic transformations to yield new functional groups that can either be used as contact points for macromolecular building blocks or as chromophores for optical observation. The single use or combination of these techniques has led to polyolefin-based materials that may in fact lower the barrier for the next-generation of functional polyolefins.Item Structural and Biophysical Explorations of Protein Degradation Tags(2023) Bonn, Steven Michael; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin is a 76-amino acid, well-folded and highly stable protein that is highly conserved across all eukaryotes. It is a post-translational modifier of other proteins through itsattachment via an isopeptide bond to a substrate lysine sidechain. Multiple ubiquitin units can be stacked to form a polyubiquitin chain, with chain topologies and cellular outcomes varying based on which of ubiquitin’s lysine residues they are attached to. The most common and well-studied outcome of polyubiquitin attachment is degradation by the proteasome, a massive barrel-shaped protease complex responsible for general protein quality control as well as cell cycle progression. Proteasomes have been discovered in archaea and bacteria, and are controlled by the small archaeal modifier protein (SAMP) and the disordered prokaryotic ubiquitin-like protein (Pup), respectively. Recently, a second bacterial proteasome operon was discovered with a new putative signaling protein, ubiquitin bacterial (UBact). Here, the first investigation of the UBact proteasomal operon is presented. Using nuclear magnetic resonance (NMR) spectroscopy and a variety of biophysical techniques, UBact is demonstrated to be disordered in solution and interact with its putative proteasomal receptor. This sets the groundwork for further studies of the UBact system. Additionally, NMR is used to explore the activity and directionality of various deubiquitinase enzymes responsible for breaking down polyubiquitin chains, and for exploring small molecule binding to ubiquitin chains themselves. This likewise provides a groundwork for further studies of the ubiquitin system, whose dysregulation is responsible for many diseases and is an area of intense therapeutic development.