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Publication High-pressure Discovery of Novel Transition Metal Carbides(2024-09) Marshall, PaulTransition metal carbides are used in a large variety of industrial applications thanks to their high-resilience properties. Most of the industrially applicable transition metal car- bides belong to the early group transition metals, as the more electron-rich systems are difficult to isolate due to their poor stability. High-pressure techniques are proving to be a robust path for synthesizing metastable compounds, often exhibiting exotic crystal struc- tures. Herein, we report the high-pressure bulk synthesis of metastable Co3C (cementite- type), Cr3C (cementite-type), the substoichiometric CrCx (NaCl-type), and single crystals of the anti-NiAs-typeMnCx. The cobalt–carbon system was explored under high pressures using traditional diamond anvil cell techniques after first-principles calculations showed the cementite phase of cobalt carbide has a favorable formation enthalpy with pressure. The cementite phase of chromium carbide existed in a crowded phase space with a number of known stable compounds. In order to achieve the precision needed to target Cr3C in a diamond anvil cell, a novel preparation technique was established where chromium and carbon were co-deposited us- ing magnetron sputtering. Finally, single crystals of a novel phase of manganese carbide, MnCx with the anti-NiAs-structure type, were recovered to ambient conditions following high-pressure synthesis in a large volume press. This work shows that high-pressure techniques continue to offer a robust route for the dis- covery of novel materials. Several metastable phases of first-row transition metal carbides have been synthesized, with the successful isolation of single crystals of MnCx at ambient conditions. These results demonstrate the promising future of using high-pressure techniques for the synthesis of novel materials.Publication On the Non-Stoichiometry and Phase Stability of Transition Metal Carbides under High Pressures(2024-09) Thiel, ScottTransition metal carbides are industrially important materials that find use in a wide range of physically demanding applications such as thermal coatings and deep-earth drills. The carbides of the group 4, 5, and 6 transition metals tend to form very stable carbides while transition metals with more d-shell electrons tend to be unstable or metastable at best. High pressure is used to change the chemistry to unlock new structures of carbides that are not typically stable under standard conditions. Crystal structure prediction is used to theoretically investigate these carbide systems, and is shown to be quite reliable for supporting experimental efforts and predicting stable phases for syntheses at moderate temperatures in the Cr--C and Co--C systems. However, experiments that access higher temperatures have yielded metastable NaCl-type CrC and anti-NiAs-type MnC---results that were unexpected from the typical approach to predictions of metastable phases. These carbides are interstitial carbides which are known to exhibit significant substoichiometry at the carbon site, a feature that can significantly impact both bulk properties and stability. Likewise, it is also a feature that is often overlooked in crystal structure prediction because it enormously complicates the search space. The stabilizing effect of substoichiometry on these phases is explained through enthalpic and entropic contributions. Substoichiometry is identified as a potential blind spot in crystal structure prediction and potential improvements are outlined as possible directions for future work.Publication Polymorphism in Silver Delafossites under Extreme Pressures(2024-09) Manganaro, NicholasDelafossites are a class of metal oxides with the chemical formula ABO2 where A is a monovalent cation, and B is a trivalent cation. These materials have been notable for their high modularity, potential as transparent conducting oxides (TCOs), and potential as quantum spin liquids. Much work has been done to tune their electrical and optical properties. Copper delafossites have had excellent work examining how their structures evolve with pressure. Previous studies of copper delafossites have found polymorphs stable exclusively at pressure. Chapter 1 will introduce delafossites and why they interest the broader materials science community. It will also discuss their various applications, introduce the diamond anvil cell, and special considerations for studying materials under extreme pressure. Chapter 2 contains all of the experimental techniques, data, and data analysis of the first study of silver ferrite delafossite, AgFeO2, at pressure. This chapter is presented as it appears in Inorganic Chemistry. The text and images are unaltered from their published version but are formatted to be consistent with the rest of the thesis. This work includes Raman spectroscopy, infrared spectroscopy, phonon calculations, Xray diffraction, and nuclear resonant forward scattering experiments. Chapter 2 also reports a novel synthesis of AgFeO2 that yielded single crystals of a size never made before, allowing for high-quality data collection. These data allowed us to identify two new high-pressure polymorphs of AgFeO2 that are stable above ∼6GPa and ∼14GPa. We have assigned the first high-pressure structure as a monoclinic C2/c structure. Both the spin state and valence state of AgFeO2 are stable at the Fe3+ site up to 15.3(5)GPa. Chapter 3 contains experimental work on silver aluminum delafossite, AgAlO2. This work includes Raman spectroscopy, infrared spectroscopy, and X-ray diffraction data. This chapter provides a road map on the steps to improve the synthesis of AgAlO2 and collect high-quality data. A pressure-induced phase transition is reported above ∼19GPa. Chapter 4 provides a review of delafossites studied under pressure. The field’s limitations will also be reviewed. This chapter investigates new insights into the effect of the A site cation on pressure-induced phase transitions. Given the novel work presented in Chapter 2 and Chapter 3, we are in a unique position to compare the behavior of silver delafossites under pressure and copper delafossites under pressure. Additionally, the trends in silver delafossite phase transitions under pressure are explored. Chapter 5 summarizes the work presented in this dissertation and reevaluates the phase space of delafossites. Improvements in high-pressure single-crystal X-ray diffraction are highlighted. Future work and applications are also discussed, such as the synthesis of gold delafossites and potential computational studies to characterize high-pressure polymorphs.Publication Exploring the Chemistry of Shockwaves using Femtosecond X-ray Diffraction(2024-09) Pereira, Kimberly A.A rapidly growing area in the field of materials science is the study of how materials respond under extreme compression. High pressure forces atoms into higher densities, significantly reducing interatomic distances and increasing orbital overlap. These conditions often lead to the formation of new and exciting structures that are not stable under ambient pressures, thereby leading to exotic new physical and chemical properties. New materials with unique properties like ultrahardness, magnetism, or superconductivity are necessary to advance technology such as wind harvesting, high-speed transportation, advanced machining and drilling, and electricity distribution. For known materials, studying how the bulk properties respond to high pressure in the laboratory gives tremendous insight into how they will function under extreme conditions that arise in demanding applications. For example, understanding how steels and other alloys respond to elevated pressures and temperatures—and in particular under ultrafast conditions—is of special interest to those tasked with x safeguarding our nation’s stockpile of nuclear arms. In this dissertation work, I have used shockwaves to recreate ultrafast rates of pressure and temperature changes in the laboratory. I present two systems of interest studied at the Matter in Extreme Conditions instrument using the Linac Coherent Light Source at SLAC National Accelerator Laboratory. In both projects, my experimental goal was to study how the chemical bonding and crystallographic structure of the material changes upon compression to extreme conditions. I used in-situ X-ray diffraction to determine the precise crystallographic structures formed on a nanosecond timescale under shock conditions, and then mapped these phases and compare them to those observed with static measurements to quantify kinetic effects arising in the ultrafast regime. I also used the velocity interferometry system for any reflector (VISAR) as a method to characterize the particle velocity experienced by the sample and thereby back-calculate the peak stress experienced by the sample. My research on nickel has uncovered some surprising results. Nickel is a constituent of our earth’s core, occurring alongside iron at a ratio of 5-15%. While nickel has been well-characterized at low pressures, very little data exist at high pressures and temperatures. Through my analysis of the X-ray diffraction data, I have determined that nickel maintains the face-centered cubic structure up to ∼500GPa along the principal Hugoniot, or nearly twice the pressure at the center of our Earth. This is the first in situ X-ray diffraction data on shock-compressed nickel up to ∼500GPa and is particularly relevant to computational and experimental researchers studying nickel and other dense metals. The pressures I reached are some of the highest ever reported for shocked nickel, and my identification of a solid compressed phase up to 332GPa is significantly higher than expected by the majority of melt lines that have been proposed for nickel in the literature. The second project I discuss combines a novel sample preparation method with the first in situ X-ray diffraction study under shock compression on the manganese(II) oxide system. The samples were prepared in-house through a rolled slurry method that I designed. To address some of the limitations currently hindering study in this field, this method involves mixing a powder, such as manganese(II) oxide, with an epoxy matrix to produce a well-mixed slurry. This slurry can then be rolled into smooth sheets of sample with precisely controlled thicknesses. My goal was to reduce localized height and texture variances in the sample and to circumvent the need for additional glue layers whose thicknesses are often inconsistent and difficult to measure. These glue layers been shown to impact the quality of experimental data by making it difficult to precisely model shock transition times with hydrocode simulations. The ability to embed powders into an epoxy polymer matrix would allow us to test many materials that are not compatible with standard methods. Applying this method to samples of manganese(II) oxide, I have observed the formation of the B2 phase at ∼150 GPa. While the B2 phase is well-documented in other systems such as magnesium oxide, iron oxide, and nickel oxide, it has not been previously observed in manganese(II) oxide. My investigation combines VISAR data and in situ X-ray in order to map out the phase diagram of manganese(II) oxide under dynamic compression conditions.Publication Designing Polymer-based Nanomaterials for Enhanced Intracellular Therapeutic Delivery(2024-09) Goswami, RitabritaPharmaceutical technology has advanced significantly with the advent of polymer-based nanocarriers, transforming drug delivery by enabling controlled release, improved bioavailability, and targeted delivery to specific tissues or cells. These systems, engineered at the molecular level, facilitate the encapsulation and precise targeting of a wide range of therapeutics, including drugs, proteins, and nucleic acids. This has led to more effective treatments for complex diseases. However, translating these polymeric vehicles into clinical applications remains challenging due to the complex biological environments they must navigate. Overcoming extracellular barriers like the extracellular matrix and intracellular obstacles such as lysosomal degradation requires sophisticated polymer designs and advanced strategies to ensure both stability and effective cargo delivery. My dissertation explores these challenges through the engineering of guanidinium-functionalized poly(oxanorbornene)imide (PONI-Guan) polymers for the efficient delivery of therapeutics into the cytosol. I developed PONI-Guan-based nanoparticles with varying structural configurations to optimize their hydrophobicity and surface characteristics, enabling targeted delivery for anti-inflammatory and anti-cancer therapies. Additionally, I expanded the PONI-Guan polymer library to improve siRNA delivery efficiency and explored a gelatin-based nanoemulsion system for macrophage polarization. These studies collectively demonstrate the potential of polymeric nanomaterials for therapeutic delivery, offering promising solutions for overcoming the challenges associated with drug delivery in complex biological systems.Publication Controlling Reactivity and Triggerability Using Intramolecular and Ad Hoc Electrostatic Interactions(2024-09) Das, RitamElectrostatic interactions play a vital role in natural design principles. An extraordinary amount of impact of such interactions also can be observed in evolutionary biology. The profound impact of these interactive forces underscores their pivotal role, yet there remains ample scope to develop our grasp of the bases that regulate and manipulate these interactions. Enriched intellectual capacity regarding electrostatic interactions will enable their more sophisticated and effective application across diverse platforms, unlocking new avenues and innovations. This dissertation reviews charge-based interactions at the molecular level and presents three perspectives on their fundamental and practical applications. The dissertation focuses on three key aspects i) studying intramolecular electrostatic interactions in tertiary amine-based zwitterionic structures, showing how these interactions affect reaction kinetics and nanostructure formation ii) developing hybrid lipid-polymer nanoparticles for RNA delivery using a ‘de-cationizable’ non-viral design, highlighting the separate roles of encapsulation and intracellular trafficking in optimizing delivery vectors. It also creates a unique opportunity to impose targeted delivery capabilities for selective treatment of excruciating diseases like triple-negative breast cancer, using a two-factor authentication approach for enhanced selectivity and efficacy. iii) Additionally, the dissertation explores improving electroporation in T-cells with anionic polymers attached to targeting antibodies, aimed at enhancing targeted cell therapies. Finally, this thesis summarizes these findings and discusses prospects, emphasizing the multifaceted roles of electrostatic interactions in biomedical research.Publication Dynamically-Driven Allosteric and Specific Inhibition of Sika Virus Protease(2024-09) Cruz, KristalleDue to the lack of effective vaccines and antivirals, flaviviruses continue to pose a significant threat to public health. The Dengue virus is now a leading cause of hospitalization among children in developing countries while Zika virus outbreak resulted in severe congenital abnormalities. Most drug development research for flaviviruses targets the viral protease due to its crucial role in virus maturation. This dissertation presents the discovery of an allosteric inhibitor, MH1, which selectively binds to the Zika virus protease (ZVP). Our combined biochemistry and structural biology approach demonstrated that the dynamic C-terminal region of the NS2B of ZVP dictates the selectivity and efficacy of MH1. Our data suggested that MH1 disrupts the interaction between the C-terminal residue of NS2B and NS3pro, resulting in protease inhibition while selectivity stems from the differences in the dynamic properties of the NS2B of ZVP and Dengue virus protease (DVP). We believe that we are the first to report that the dynamics of the NS2B can influence the selectivity of an allosteric inhibitor. This discovery opens a new avenue that may be exploited to overcome selectivity issues that some allosteric inhibitors encounter. It is always of interest to develop a broad-spectrum antiviral to address future outbreaks and resistance mutations that commonly occurs in viruses.Publication Engineered Polymer-Based Nanomaterials for the Treatment of Biofilm-Associated Infections(2024-09) Nabawy, AhmedBiofilm-associated infections present a clinical challenge, with biofilms protecting resident bacteria from host immune response and therapeutic agents. Severe biofilm infections annually afflict 300 million people worldwide, with treatment costing $25B in the US alone.Clinical treatment of refractory chronic wound infections combines surgical removal of infected tissues with long-term antibiotic therapy. Debridement is an invasive process and use of antibiotics selects for drug resistance, further increasing therapeutic challenges with chronic wound infections. Polymeric nanomaterials provide a promising opportunity to effectively address bacterial and biofilm infections.Polymers can be engineered to combat biofilm infections by tuning their morphological and physicochemical properties, including size, shape, and surface chemistry. In this dissertation, I demonstrate polymer-based strategies for the treatment of biofilm-associated infections, with a focus on wound biofilms. In the initial studies, we leveraged poly(oxanorborneneimide)-based biodegradable polymeric nanoemulsion to deliver plant-derived essential oil, including carvacrol, that can penetrate and eliminate bacterial biofilms. Next, we build upon this nanoemulsion platform and encapsulate two hydrophobic antimicrobial agents (eugenol and triclosan) into this nanoemulsion for synergistic treatment of wound biofilms. Notably, this combination nanoemulsion mitigates resistance development of antimicrobial triclosan and clears 99% of bacterial load in severe wound biofilm infections in mice. In a related system, I developed an antimicrobial nanoemulsion composed of all nature-derived materials. This nanoemulsion uses gelatin as a scaffold and carvacrol (from oregano oil) as the active antimicrobial phytochemical. Crosslinking of the gelatin scaffold using riboflavin (vitamin B2) led to a formation of a stable nanoemulsion with excellent antifungal activities against C. albicans biofilms. In a following study, I have leveraged this all-natural gelatin nanoemulsion to encapsulate transition metal catalysts (TMCs) for bioorthogonal catalysis in biofilms. This emulsion nanocatalyst can efficiently penetrate biofilms and eradicate mature bacterial biofilms through bioorthogonal activation of a pro-antibiotic, providing a highly biocompatible platform for antimicrobial therapeutics. The last parts of this dissertation focus on developing polymers with inherently antimicrobial activity for topical applications in wound biofilms. Our strategies include 1) integration of antimicrobial poly(oxanorborneneimide)-based polymer into hydrogel materials, 2) development of cationic conjugated polymers for simultaneous biofilm imaging and therapy. In summary, Polymer nanotherapeutics offer a promising alternative to antibiotics, alleviating challenges faced in the post-antibiotic era.Publication Reactions of Metal and Metal Oxide Cluster Cations with Small Organic Molecules(2024-09) Phasuk, ApakornThe interactions between metal and metal oxide cations and small organic molecules are important in solvation and catalysis. Here, we explore three different gas-phase reactions: C‒H bond activation in ethane by AlxOy+, acetone C=O bond activation by Al+, and solvation of metal cations and dications by acetone. The entrance channel complexes, reaction intermediates and products of these reactions were characterized using photodissociation vibrational spectroscopy coupled with DFT calculations. The results reveal information about the structures of, and the covalent bonds in, the complexes and how the nature of the metal, ion charge, and the number of ligands influence the perturbation of bonds in the ligand. For the C-H activation of ethane by AlxOy+, oxygen-rich species and open-shell cluster ions have smaller barriers, except for Al3O4+. Only entrance channel complexes are observed for oxygen-deficient clusters (Al2O+ and Al3O2+) due to large C-H activation barriers. For Al3O4+(C2H6)3, both the entrance channel complex and C‒H activation intermediate are observed. For oxygen-rich Al4O7+, ethane is favored to bind far from the reactive superoxide group, reducing the reactivity. The observed red shift of the C‒H symmetric stretch in ethane is ~200 cm-1, indicating significant weakening of the proximal C‒H bonds. In the investigation of Al+(acetone)n complexes, computations predict the thermodynamic favorability of the pinacol coupling reaction for n≥3. However, our experimental results reveal the characteristic peak of the pinacolate C-O stretch solely at n=5. Furthermore, the red shift of the C=O stretch and the concurrent blue shift of the CC antisymmetric stretch are also observed. Consistent with anticipated trends, the magnitudes of these shifts decrease with increasing cluster size. When acetone solvates M+/2+ ions, a red shift of the C=O stretch and blue shift of the antisymmetric CC stretch are observed, corresponding to a weakening of the C=O bond and strengthening of the C-C bonds. These shifts decrease as the size of clusters increases, and increase with the charge on the metal ion. A strong correlation of the calculated red shift in the C=O stretch in M+/2+(Ace) with the ionization energy of M+ and M2+ was also discovered.Publication Applying Nanopore Tweezers in Enzyme Analytical Chemistry(2024-09) Shorkey, SpencerCellular activities depend on the continual synthesis, modification, and breakdown of molecules. In this self-sustaining network of chemical activity we refer to as life, proteins organize and catalyze almost every reaction process. Proteolysis is a general process whereby one protein, known as a protease, cuts a peptide bond in another protein. Proteases have been well characterized over the years, however, there remain some proteases where biochemical activities are relatively unknown or may benefit from more detailed studies. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. The focus of this collective work has been the development of nanopore methods useful for 1) analyzing the composition of ubiquitin polymers and their regulation by deubiquitinase proteolytic enzymes, and 2) analyzing the conformation dynamics of the West-Nile virus NS2B/NS3 protease. In the process, we also explored strategies for optimization of nanopore-analyte interactions. These nanopore methods developed here have the advantages of being real-time and label-free approaches, which may be applied using high-throughput nanopore arrays to studies of protein and enzyme conformations, activities, and inhibition.Publication Advanced DNA Probes for Imaging and Modulating Cell Membrane Dynamic Interactions(2024-09) Ali, Ahsan AusafThe cell membrane is a critical structure which serves as a natural barrier between cells and their external environment. It plays a vital role in enabling cells to communicate with each other and transduce signals vital for healthy cellular function. To carry out such functions, the various components of the cell membranes dynamically interact with one another. Most of these interactions are very transient and therefore difficult to study and visualize. In this dissertation, we aim to use DNA as a tool to stabilize and visualize the dynamic and transient interactions which take place on the cell membrane to understand more about them as well as the cell membrane’s structure and biophysical properties. Initially, we introduce the current methods which exist in visualizing transient and dynamic cell membrane interactions, followed by the current advances in DNA based probes which have been used to study the cell membrane biophysics. This will be followed by the development of a ‘DNA Zipper’ probe to visualize and quantify dynamic cell membrane interactions and their role in T-cell signaling. The ‘DNA Zipper’ is further expanded upon and improved into various pairs of probes which are comprehensively used to visualize cell membrane heterogeneity and investigate the presence of both ordered and disordered regions of live cell membranes. Lastly, we expand the ‘DNA Zipper’ based system to membrane proteins by optimizing workflows for the targeted labelling of membrane proteins and use the probes to image ligand induced membrane protein dimerization on live cells with potential applications in drug screening and cell membrane modulation and regulation. Therefore, this dissertation will serve to shed light on the excellent properties of DNA as a tool to visualize and stabilize dynamic interactions on live cell membranes and to regulate these interactions for biological manipulation and function, thereby expanding our understanding of the membrane.Publication CHEMICALLY DOPED CONJUGATED POLYMERS: ELUCIDATING FACTORS THAT IMPACT CHARGE TRANSPORT(2024-05) Lu Diaz, MichaelOrganic semiconductors offer numerous advantages over inorganic semiconductors, including cost-effective fabrication, a versatility of applications, and an environmentally sustainable alternative. Among these materials, conjugated polymers suggest promising results. Conjugated polymers rely on dopants to produce charge carriers, but dopants also create an unanticipated tradeoff between trapped and free carriers, changes to the electronic structure and packing of the polymer, and instability of the material properties over time. In this dissertation, I explored different methods to study the effects of dopants in a variety of systems, including pristine polymers and blends of polymers with other polymers, small molecules, and nanoparticles. We primarily studied thermoelectric properties such as Seebeck coefficient and electrical conductivity since they depend on the doping level and are sensitive to the chemical structure, electronic structure, and morphology of the material. Our work contributed to unrevealing the role of dielectric permittivity, types of morphologies, and other aspects of conjugated polymers in charge transport. We pave the way towards a holistic material design that favors dopant-generated free carriers but also eliminates many detrimental dopant-induced effects.Publication LIPID-DNA CONJUGATES FOR EFFICIENT AND TARGETED CELL MEMBRANE MODIFICATION AND INTERCELLULAR TENSILE FORCE VISUALIZATION(2024-05) Tian, QianAs a physical barrier of cells, the cell membrane plays a vital role in cell communications and signaling. Engineering cell membranes have attracted a great amount of attention in the field of biosensing, tissue engineering, and cell therapy, etc. Recently, synthetics DNAs have attracted considerable attention to remodel and functionalize live cell membranes. In particular, a type of amphiphilic lipid-DNA conjugate has been rationally designed and synthesized for this purpose. These conjugates have enabled a rapid, straightforward, and efficient cell membrane modification due to the hydrophobic-hydrophobic interaction between lipid moiety and lipid bilayer. Taking advantage of the highly precise and programmable self-assembly of DNAs, lipid-DNA conjugates have been used for membrane bioanalysis, therapeutics, building artificial membrane structures, and regulating cell–surface and cell–cell interactions. In this thesis, I would mainly focus on how we have applied lipid-DNA conjugates for selective modification of cell membranes and for the investigation of intercellular mechanotransduction. First, we described the development of a simple, fast, and highly efficient system to engineer bacterial membranes with designer DNA molecules by using lipid-DNA conjugates. We have constructed a small library of synthetic lipid-DNA conjugates and characterized their membrane insertion properties on various Gram-negative and Gram-positive bacteria. Simply after incubation, these lipid-DNA conjugates can be rapidly and efficiently inserted onto target bacterial membranes. Based on the membrane selectivity of these conjugates, we have further demonstrated their applications in differentiating bacterial strains and potentially in pathogen detection. These lipid-DNA conjugates are promising tools to facilitate the possibly broad usage of DNA nanotechnology for bacterial membrane analysis, functionalization, and therapy. Secondly, we applied these lipid-DNA conjugates for the development of molecular tension probes to visualize tensile forces between cells. Mechanical forces are important stimuli in signaling pathways and regulate cell proliferation, adhesion, and differentiation, etc. We designed several ratiometric DNA tension probes to study the roles of intercellular tensile force during Notch1 activation. Our data indicated that the mechanical force Notch1 receptor exerts on its different ligands were quite different. In addition, our data indicated that the force could be generated when ligand endocytosis was not available, indicating that the force could be induced both by Notch ligands and receptors. Finally, we reported the first-time usage of DNA molecular tension probes in visualizing and detecting mechanical forces within 3D spheroids and embryoid bodies (EBs). By varying the concentrations of these DNA probes and their incubation time, we have first ix characterized the kinetics and efficiency of probe penetration and loading onto tumor spheroids and stem cell EBs of different sizes. After optimization, we have further imaged and measured E-cadherin-mediated forces in these 3D spheroids and EBs for the first time. Our results indicated that these DNA-based molecular tension probes can be used to study the spatiotemporal distributions of target mechanotransduction processes. Besides, these powerful imaging tools may be potentially applied to fill the gap between ongoing research of biomechanics in 2D systems and that in real 3D cell complexes. In sum, we described the applications of lipid-DNA conjugates on efficient and targeted membrane modification and intercellular tensile force visualization in this dissertation. The high efficiency and selectivity of lipid-DNA conjugates on bacterial membrane may allow a broad applications of DNA nanotechnology on prokaryotic cells, such as cell surface engineering, manipulation of cell-cell communications, and drug delivery, etc. Besides, the selectivity of lipid-DNA conjugates on bacterial membranes could provide some insights on achieving targeted modification on cell membranes, which is a current challenge in the field. On the other hand, the studies of intercellular mechanical forces in 2D and 3D cell models indicated the versatility of molecular DNA tension probes, which might be feasible to investigate mechanotransductions in real tissue with further optimization. The investigation of intercellular force during Notch activation may shed some light on revealing the regulation mechanisms or patterns of Notch signaling pathways. In addition, the direct visualization and flexibility of molecular DNA tension probes would allow the imaging of a great amount of mechano-sensitive ligand-receptor interaction in molecular level.Publication Multiplexed Mass Spectrometric Methods to Detect, Quantify, and Image Polymer-Based Drug Delivery Vehicles in Biological Samples(2024-05) Agrohia, Dheeraj KrishanPolymeric nanocarriers represent versatile platforms for drug delivery, exhibiting significant promise in facilitating the cytosolic delivery of cell membrane-impermeable protein therapeutics. Understanding the impact of their diverse structural designs on biological accumulation, both in terms of bulk distribution and site-specific localization, is essential for designing potent delivery vehicles. However, the traditional approach of analyzing individual designs in cells and animal models, followed by side-by-side comparisons, encounters challenge due to inherent biological variability associated with cells and animals. To address this issue, an innovative analytical method has been developed, aiming to simultaneously and quantitatively monitor both bulk and site-specific biodistributions of polymeric nanocarriers. While mass spectrometry techniques are increasingly being applied to monitor various kinds of nanocarriers, their quantitative application for PNCs has been largely unexplored. This dissertation introduces metal- coded mass tags (MMTs) as a solution. When combined with inductively coupled plasma mass spectrometry, MMTs enable the quantitative monitoring (both bulk and site-specific) of the fate of various PNC designs concurrently. Firstly, the development of MMTs involved a thorough assessment to accurately screen the cellular uptake of multiple polymeric carriers simultaneously. Subsequent evaluation focused on accurately screening the cellular uptake of protein delivered by various PNCs. After successfully establishing the method at the cellular level, the final step included simultaneously and quantitatively assessing the site-specific fate of both the polymeric carrier and its cargo protein in tissues. This was achieved by combining MMTs with laser ablation inductively coupled plasma mass spectrometry imaging. Overall, this dissertation presents a robust method for the side- by-side quantitative comparison of multiple PNC designs, significantly reducing biological variability. This innovative approach not only reduces the costs, time, and effort but also minimizes the need for multiple animals, facilitating the development of potent delivery systems more efficiently and ethically.Publication DESIGN AND FABRICATION OF ANTIBODY-NANOGEL CONJUGATES FOR TARGETED DELIVERY OF THERAPEUTICS(2024-05) Huynh, Uyen Gia ThucThe utilization of antibody–drug conjugates (ADCs) represents a promising avenue for achieving precise drug delivery, thereby facilitating the targeted eradication of cancer cells while minimizing the adverse effects on healthy tissues. This approach seeks to ameliorate the side effects typically associated with conventional chemotherapy. Striking examples of ADCs include Kadcyla and Trodelvy, which have exhibited remarkable efficacy in the treatment of HER2-positive and triple-negative breast cancer (TNBC), respectively. Nonetheless, the complete realization of the clinical potential of ADCs faces substantial challenges. The prevailing ADC format exhibits a low drug-to-antibody ratio (DAR), necessitating the deployment of highly potent yet inherently toxic drugs, thereby increasing the risk of off-target toxicity. Additionally, stringent criteria regarding the stability and degradability of the antibody–drug linker introduce intricate design considerations and impose constraints on the selection of drugs compatible with ADC components. To address these challenges, we developed a versatile drug delivery platform known as antibody-nanogel conjugates (ANCs). This nanocarrier platform features (i) an easily functionalized surface for antibody decoration; (ii) simple preparation protocols; (iii) high drug loading capacity for a broad range of drugs thereby, significantly increases the DAR; (iv) low vehicle toxicity; and (v) triggerable on-demand release of cargo at targeted sites. The efficacy of our antibody-nanogel conjugate (ANC) systems was demonstrated through the fabrication of two ANCs containing identical cytotoxic payloads and targeting antibodies, analogous to Kadcyla and Trodelvy. in vitro assessments reveal a substantial increase in efficacy with the ANC system compared to the ADCs with several-folds improvement. Moreover, we have also presented a dual chemo-immunomodulator approach employing the ANC system. This strategy involves the conjugation of an anti-PD-L1 antibody to a nanogel encapsulating a chemotherapeutic agent. The aPDL1 antibody serves as an inhibitor for the PD-1/PD-L1 interaction, thereby activating the immune system's response against cancer cells. Concurrently, the chemotherapeutic drug exerts its effect by impeding cancer cell proliferation and promoting apoptosis in a synergistic fashion.Publication Polymeric Systems for Active Targeted Therapeutics Delivery(2024-05) Kanjilal, PintuThe nanomedicine field has advanced significantly and current progress in nanotechnologies has enabled translational research across therapeutic landscape. Recent trends in clinical studies and FDA approvals have demonstrated the potential of using biologics as therapeutic modalities. Ongoing fundamental research across therapeutic modalities have set foundations to treat multiple diseases, including the ones that were hard-to-cure and assures the promising future of nanomedicine. However, the therapeutic expansion from small molecule drug to biologics comes with their unique delivery challenges. It requires strategic designing of delivery platforms to overcome biological barriers associated with each modality and demands fundamental understanding of formulation factors to predetermine the fate of delivered therapeutics. Each formulation requires stability in blood circulation, ability to reach specific organs or tissue types, have enough tissue penetrations and importantly, and have cytosolic accessibility. These different aspects of delivery challenges can be addressed by tuning formulation properties, and understanding these structural and formulation factors are essential in improving therapeutic efficacy. In this dissertation, we have developed polymeric systems focusing on two different aspects of drug delivery viz, targeted tissue accumulation and cellular internalization. An antibody guided polymeric system is established to address the challenges with current antibody drug conjugates (chapter 2). Then we understood the effect of antibody polymer conjugation process towards cell receptor binding activity by varying polymer molecular weights and number of polymers per antibody (chapter 3). We also designed disulfide-based polymeric nanogel to fundamentally understand the effect of disulfide bonds in endosomal escape (chapter 4). Finally, we designed stimuli-responsive self-immolative protein-PEGylation strategy with terminal disulfide functionality to leverage thiol-mediated internalization of biologics.Publication VIBRATIONAL SPECTROSCOPIC STUDIES OF GAS-PHASE EARLY TRANSITION METAL AND METAL CLUSTER CATIONS WITH METHANE(2024-05) Kozubal, Justine RThe study of interactions between metal ions and methane is key to understanding the C-H activation reactions involved in the generation of liquid fuels from methane. Gas phase studies serve as a model to understand these metal-ligand interactions. The interaction of the metal with methane weakens the C-H bonds and produces a substantial reduction in the C-H stretching frequencies which can be determined by measuring the vibrational spectra. This work investigates the interaction of early transition metals and metal cluster ions with methane to learn about the geometry and bonding of the reactants, intermediates, and products using photofragment spectroscopy and density functional theory. Chapters 1 and 2 discuss the motivations and techniques. Chapter 3 discusses the vibrational spectra of complexes of Ti+ and V+ with methane. The M+(CH4)1-2 complexes have different methane orientations while the M+(CH4)3-4 (M=Ti,V) are similar. Comparison of complexes for the two metals shows that methanes orient to minimize repulsion with singly- or doubly-occupied orbitals. Chapter 4 discusses the intermediates and reaction products of sequential reactions of Zr+ with CH4. Spectra are measured for Zr+(CH4)1-4 and for four dehydrogenation products. The spectra of [ZrCH4]+ and [ZrC2H8]+ are a combination of entrance and exit channel complexes, and possibly intermediates [H-Zr-CH3]+(CH4)0-1. The dehydrogenation products are observed when Zr+ sequentially reacts with three or four methanes. The products formed come from loss of H2 from n=3-4, ZrC3H +10 and ZrC4H + 14 , and loss of H and H2+H from n=4, ZrC4H +13 and ZrC4H + 15 . All of the products have methyl groups: Zr(CH3)m+(CH4)n, except for ZrC4H + 15 , which has an agostic carbene: HZrCH + 2 (CH4)3. Chapter 5 discusses the spectroscopy of vanadium cluster ions with methane. Vibrational spectra are measured for V2+(CH4)1-4, V3+(CH4)1-3, and Vx+(CH4) (x=4-8). The larger red shifts of x=5-8 suggest they are more reactive with methane than x=2-3. Chapter 6 summarizes the findings and suggests possible extensions of the experiments. The metal-methane studies can be extended to study the interaction of ethane with Ti+, Zr+, and Nb+, which can dehydrogenate ethane at room temperature. The Vx+(CH4)n cluster studies can be extended to Nbx+ as metal clusters can be more reactive than atoms.Publication IMPROVED COVALENT LABELING-MASS SPECTROMETRY WITH DIETHYLPYROCARBONATE FOR STUDYING MEMBRANE PROTEINS AND NUCLEIC ACID BINDING(2024-05) Kirsch, Zachary JohnUnderstanding protein higher order structure (HOS) is important because it is directly related to a protein’s function. These functions are wide-ranging, and misfolding of proteins can have significant adverse effects. Consequently, biophysical and biochemical approaches have been developed to study protein HOS in numerous different contexts. Covalent labeling (CL) coupled with mass spectrometry (MS) has become a powerful tool for probing protein HOS information in recent years, owing to its throughput, sensitivity, and sample efficiency which set it apart from traditional biophysical methods. This dissertation focuses primarily on the use of diethylpyrocarbonate (DEPC) as a CL-MS reagent. DEPC reacts with the side chains of multiple amino acids, allowing for coverage of 30% of the average protein’s sequence. Owing to its ease of use, DEPC has been used extensively by our group to study protein structure and interactions. DEPC readily reacts in solution to produce a single reaction product which is easily identified by tandem MS. The goal of this dissertation is to further the development and application of DEPC CL-MS tools to study protein structure and interactions. x The first part of this dissertation addresses the current experimental constraints placed on DEPC CL-MS studies. Through modeling the kinetics of the labeling reaction, bottom-up site mapping, molecular dynamics simulations, and ligand binding experiments, we show that proteins can accommodate more than one label at a time, allowing for improvement to the obtainable information from DEPC CL-MS experiments. Next, we apply DEPC to new systems: membrane proteins in live cells and nucleic acid binding to proteins. We show that DEPC can be applied in live cells in order to obtain information about membrane protein interactions and that DEPC can be used to probe protein-nucleic acid binding interactions. Lastly, we investigate the αα,ββ-unsaturated carbonyl (ABUC) scaffold as a CL-MS reagent specifically for ligand-directed labeling approaches. Similarly to DEPC, the ABUC reacts readily in solution and produces a single reaction product, but has a wider range of reactivity. This reagent provides better amino acid coverage compared to traditional ligand-directed approaches, expanding the current tools that are available.Publication Intermolecular Electron Transfer Reactivity and Dynamics of Cytochrome c – Nanoparticle Adducts(2009-09) Carver, Adrienne M.Interprotein electron transfer (ET) is crucial for natural energy conversion and a fundamental reaction in the pursuit of understanding the broader problem of proteinprotein interactions and reactivity. Simplifying the complicated nature of these natural systems has driven development of biomimetic approaches. Functionalized gold nanoparticles offer simplified, tunable surfaces that can serve as a proxy to study the reactivity and dynamics of proteins. Amino-acid functionalized gold nanoparticles (Au-TX) served as a complementary partner to cytochrome c (Cyt c) and catalyzed its ET reactivity without altering the native structure. Redox mediator and EPR experiments confirmed that the redox potential and coordination environment of the heme were unaltered. Varying the functionality of Au-TX under limiting redox reagent concentrations resulted in distinct ET reactivity. These conditions reflected the collision of a small redox reagent with the Cyt c/Au-TX adduct, introducing the possibility of Cyt c/Au-TX dynamics to modulate ET. Under high ionic strength conditions, the rate enhancement ranged from 0.0870 " 1011 for Cyt c/Au-TAsp to 1.95 " 1011 M-1 s-1 for Cyt c/Au-TPhe. Au-TAsp binds to a larger surface of the front face of Cyt c than Au-TPhe, likely reducing heme access and resulting in attenuated ET reactivity.Site-directed spin-labeling characterized the dynamic interactions and motion of Cyt c with Au-TX. Several mutants of Cyt c were utilized to extract information about the different dynamics of the Cyt c/Au-TPhe and Cyt c/Au-TAsp systems. Cyt c appeared to have a highly dynamic binding interaction with the surface of Au-TPhe while binding to Au-TAsp resulted in a more rigid interface, particularly at the heme crevice. The dynamic interaction of Cyt c/Au-TX at the heme crevice could promote a gated ET mechanism between Cyt c and its redox partner. Thus, the reduced reactivity of Cyt c/Au-TAsp is likely a result of both slower global dynamics and more rigid binding near the heme crevice.Publication Development of Mass Spectrometry-Based Methods for Quantitation and Characterization of Protein Drugs: Transferrin as a Model Drug Delivery Vehicle(2013-09) Wang, ShunhaiIn the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.