Browsing by Author "Gassensmith, Jeremiah J."
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Item A Deterministic Model for Non-monotone Relationship Between Translation of Upstream and Downstream Open Reading Frames(2021-12-01T06:00:00.000Z) Milogorodskii, Aleksandr; Rachinskiy, Dmitry; Ruderman, Michael; Gassensmith, Jeremiah J.; Makarenkov, Oleg; Arnold, Maxim; Krawcewicz, WieslawThe TASEP modeling was shown to offer a parsimonious explanation for the experimentally confirmed ability of a single upstream Open Reading Frames (uORFs) to upregulate a downstream translation during the integrated stress response. As revealed by the numerical simulations, the model predicts that reducing the density of scanning ribosomes upstream of certain uORFs increases the flow of ribosomes downstream. To gain a better insight into the mechanism which ensures the counter intuitive non-monotone relation between the upstream and the downstream flows we propose a phenomenological deterministic model to approximate the modified TASEP model of the translation process. We establish the existence of a stationary solution featuring the decreasing density along the uORF for the deterministic model. Further, we find an explicit non-monotone relation between the upstream ribosome density and the downstream flow for the stationary solution in the limit of increasing uORF length and increasingly leaky initiation. The stationary distribution of the modified TASEP model, the stationary solution of the deterministic model and the explicit limit are compared numerically.Item A New Biomaterial for Vaccination: an Aqueous ZIF-8 Crystal Growth to Preserve Antigens and Enhance the Immune Activation(2020-12-01T06:00:00.000Z) Luzuriaga, Michael A; Gassensmith, Jeremiah J.; Winkler, Duane D.; Smaldone, Ronald A.; D'Arcy, Sheena; Ahn, Jung-MoBiomaterials encompass a broad range of applications for medical treatment and can be on the macroscale, such as heart valves or hip implants, to the microscale and nanoscale, such as stitches, dental fillings or particles for drug delivery. For the last thirty years, biomaterials with sustained release properties have been investigated as a method to improve the delivery of vaccines. Vaccines are considered one of the most significant inventions in the human history, as it has prevented hundreds of millions of deaths since its invention and is the main cause for extending the human life expectancy. However, a major issue with current methods of vaccination is their low stability at room temperature and the requirement for multiple injections to produce an immune response strong enough to develop long-term memory. For these reasons, the “cold chain” infrastructure keeps them refrigerated from manufacturer to clinic, ensuring the epitopes in the proteinaceous vaccines do not unfold. Any failures in this process can lead to the loss of billions of dollars and, even with this system, the proteinaceous material will denature over time. On top of that, the necessity of multiple injections to provide immunity it is clear how heavily we rely on the cold chain. To solve these issues, biomaterials with sustained release properties has been employed to “cage” proteins within a stabilizing polymer network that prevents conformational changes and enables storage at room temperature. Herein, methods to encapsulate proteinaceous material under aqueous conditions within zeolitic imidazole framework-8 (ZIF-8), a well-studied metal organic framework, is investigated for it’s ability to activate the immune system in mice and protect vaccines from denaturation.Item Biocompatible Tuning of Zeolitic Imidazolate Framework-8 and Encapsulation of Vaccine Model for Controlled Release(2021-05-01T05:00:00.000Z) Reyes, Cesar Alexis; Gassensmith, Jeremiah J.; D'Arcy, Sheena; Smaldone, Ronald A.Increased development of proteinaceous therapeutics and other biomolecules has signaled the demand for robust drug delivery technologies. Metal-organic frameworks have shown to meet the durability requirements but controlled delivery of their therapeutic-cargo and their long-term release profiles remain under-investigated. In this thesis, zeolitic imidazolate framework-8 (ZIF8) is explored as a vaccine delivery vehicle in combination with the highly-investigated polymer, poly(lactic-co-glycolic acid) (PLGA). Previous studies have shown their separate potential as immunogenic vaccine carriers, but a combined system remains unexplored. Using FITC-tagged ovalbumin as a model vaccine therapy, this combined vaccine delivery system is characterized and investigated to determine its’ release profile. In a separate study, a biocompatible method of tuning ZIF-8 micropores to create a hierarchically porous material is developed via defect formation using bovine serum albumin. Here, the cystine residues of the protein are alkylated to control the pore size and ultimately the release time of the encapsulated cargo. Together, these two research explorations serve as fundamental steps towards facilitating the establishment of ZIF-8 as a viable vaccine carrier platform.Item Catalytic Imine C–H Alkylation Methods and Novel Three-component Paths to Allylic Amines(December 2023) Kvasovs, Nikita; Tran, Anh; Gevorgyan, Vladimir; Gassensmith, Jeremiah J.; Smaldone, Ronald A.; Romiti, FilippoThe thesis describes development of C–H alkylation of imine-type compounds and three- component assembly of allylic amines. First, a general and efficient method of C–H alkylation of formaldoximes was developed. The protocol unifies light-induced hybrid palladium radical chemistry with oxidative properties of palladium. As a result, this protocol allows for coupling of primary, secondary and tertiary alkyl bromides or iodides with variety of terminal oximes under very mild conditions. Second, the logic was further translated onto glyoxylate-derived hydrazones, achieving C–H alkylation of the latter. In addition to alkyl halides, the method can also engage redox-active esters. Mildness of the method, as well as mechanistic features enabled synthesis of otherwise hard-to-access E isomers, and to approach unusual heterocycles. Next, a protocol for homologative three-component synthesis of allylic amines was developed. Merging of the light-induced alkyl-Heck reaction with the classical Tsuji-Trost chemistry enabled rapid access to allylic amines of various structures. Method also features potential for the stereoselective transformations.Item Chemically Tuned Virus Like Particles: From Cancer Therapy to Targeted Delivery(December 2021) Shahrivarkevishahi, Arezoo; Gassensmith, Jeremiah J.; Slinker, Jason D.; Zheng, Jie; Ahn, Jung-Mo; Dodani, SheelIn recent years, nanoparticle-based therapeutics have been increasingly applied in broad range of clinical applications from diagnosis to treatment of many diseases such as cancer, diabetes and neurodegenerative disorders. A wide range of synthetic and naturally occurring materials such as polymers, metal oxides, silicate, liposome, and carbon nanotubes have been developed to overcome some of the key barriers in free therapeutics including intracellular trafficking, cell/tissue targeting, poor biodistribution, and low efficiency. However, despite all achievements in creating these nanomaterials with different chemical and physical properties such as size, shape and surface properties, developing a nanoparticle to surmount these limitations all in one is a big challenge. Virus like particle (VLP) as protein-based nanomaterials that closely mimic the highly symmetrical and polyvalent conformation of viruses while lack the viral genomes have emerged as a solution for these limitations. Their unique features such as high biocompatibility, biodegradability, monodisperisty, intrinsic immunogenicity, and safety combined with interior and exterior modification capability offer new tool to scientists for careful design and engineering of multi-component therapeutic agent with intended biological behavior and pharmacological profiles. Herein, various chemistry strategies are introduced in combination with biology and immunology to turn virus like particle to a favorable engineered biomaterial for several functions such as cancer therapy and intracellular delivery. We showed how by modifying surface of VLP Qβ with NIR organic molecule we can make a highly efficient and stable photothermal agent that can cause thermal ablation of tumor while simultaneously activating the immune response. We found this immunophotothermal agent, suppress primary tumor, control metastasis, and prolong survival time in mice bearing breast cancer. We also addressed one of the biggest challenges in biologic delivery which is direct delivery of therapeutic cargo into cell cytoplasm. Using organic chemistry we designed a cytosolic targeting linker that when attached to surface of VLP Qβ, helps to escape endosomes and be released into cytoplasm, Moreover, this proteinaceous material is shown to have a great potential in combination with other materials such as metal organic framework to construct a multimodal cancer therapeutic agent enabling delivering mulit therapeutic agents such as immunotherapeutic drugs while taking advantage of all unique features of virus like particles. These works clearly show the significant potential of VLP in design and modification of new therapeutic platform.Item Design and Synthesis of Polymer Nanocomposites for Additive Manufacturing(May 2023) Perera, Sachini Dilinika 1992-; Penev, Kaloyan; Smaldone, Ronald A; Ferraris, John P.; Biewer, Michael C.; Gassensmith, Jeremiah J.Additive manufacturing or 3D printing is a process where the materials are deposited in a layer- by-layer fashion according to a pre-designed computer aided file to fabricate required geometries. There are a wide range of materials from thermoplastics, polymeric resins, metals, alloys, nanocomposites, to hydrogels that have been used in 3D printing, as well as several different types of processes are available for 3D printing. Fused filament fabrication, ink jet printing, stereolithography (SLA) and digital light projection (DLP) can be recognized as the most popular and affordable techniques. This manufacturing technique is very promising as a user friendly, customizable setup without the need to manufacture through expensive molding processes or producing waste from subtractive manufacturing methods such as milling. Even though it possesses all the advantages, poor interlayer adhesion, limited mechanical properties, low resolution and rough surface finish is limiting its applications at large scale. Vat photopolymerization 3D printing techniques provide better resolution as high as 10 μm for the printed parts from SLA and DLP compared to other 3D printing techniques. Here, a photo resin that contains photocurable monomers and oligomers, crosslinkers are polymerized in a print vat using UV irradiation in the presence of a photoinitiator. The photo printed structures show fine resolution and smooth surface finish, yet the mechanical properties of the printed parts are inadequate for end use applications. To address this limitation different approaches were taken and studied, thus the overall goal of this research was to enhance properties of 3D printed objects and to develop methodologies to improve photo printing processes that would ultimately improve intrinsic properties of the photo printed materials. Chapter 1 of the dissertation provides a literature review about 3D printing techniques, materials, and the limitations of additive manufacturing. This chapter further discusses the approaches taken to overcome the limitations by introducing ways to improve the mechanical properties. Chapter 2 describes our work in enhancing mechanical properties through a nanofiller derived from Kevlar and how we successfully 3D printed photoresin formulations using stereolithography without compromising printability. We discuss a methodology that can be used to incorporate unprocessable fibrous fillers in a resin formulation. Chapter 3 provides insights about how supramolecular interactions provide 3D printable materials with noncovalent cross-linking and stimuli-responsive properties to improve their processability and functionality. We evaluated urea formulations with aliphatic and aromatic sidechains and showed physical evidence for the presence of hydrogen bonding using variable temperature Fourier transform infrared (VT-ATR-FTIR) spectroscopy and van’t Hoff analysis. The self- healing efficiency of these formulations was characterized by measuring the recovery of their tensile mechanical properties. Chapter 4 describes our approach to process Metal Organic-Frameworks in vat photopolymerization. A new method is introduced to construct complex structures with fine features by integrating high loading weight percentages of MOF crystals to a photocurable acrylate formulation. Through free radical polymerization in a DLP setup, MOF loaded nanocomposites were 3D printed, and its catalytic performances were studied.Item Developing a Drug Delivery and Imaging System on a Virus-Like Particle Platform(2018-08) Chen, Zhuo; Gassensmith, Jeremiah J.Nanoparticle based therapeutics have been proved to improve therapeutic efficacy and reduce the off-target toxicity. However, poor monodispersity and long term bioaccumulation toxicity have been the obstacles for the biomedical applications. Viruses-like particles (VLPs) have emerged as promising natural nanoparticles, which are monodisperse, non-infectious and biodegradable. Each VLP is usually composed of hundreds of identical subunits, leading to a highly ordered quaternary structure and repetitive particle surface. These unique characteristics allow VLP to be chemically functionalized precisely and periodically. The proteinaceous viral capsids are a robust platform, and solvent exposed amino acids such as lysine, cysteine and tyrosine can be orthogonally modified with variety of bioconjugation techniques. Bacteriophage Qβ is one of the well-studied VLPs, which is 28 nm in diameter and composed of 180 identical coat proteins. In my study, Qβ was used as a robust platform for conjugation-induced fluorescent labelling for the application of in vitro cell tracking and developing a photocaged carrier for stimuli-responsive drug release.Item Development of a High-throughput Screening Method to Identify Chloride-sensitive Variants of the Fluorescent Protein mPapaya(2021-08-01T05:00:00.000Z) Liyanaarachchi, Sureshee Mekala; Dodani, Sheel; Meloni, Gabriele; Torabifard, Hedieh; Gassensmith, Jeremiah J.Engineering genetically encoded chloride-sensitive fluorescent biosensors to understand the behavior of chloride in biological systems is a promising area of research because these biosensors have the ability to target specific locations and the ability to express within specific cells in transgenic organisms while selectively enabling them to determine the behavior of chloride within a cell, both qualitatively and quantitatively. The GFP-derived fluorescent proteins are used in this engineering process as their unique structure enables them to act as anion recognition domain, mainly through the hydrophobic interactions and the formation of hydrogen bonds while the autocatalytic chromophore contributes to generating an optical output as a transducer. Despite the unique properties, these fluorescent proteins exist with certain limitations and therefore need to be modified through mutagenesis to get the desirable features to enable them to function as biosensors. To accomplish this process, detailed structural analysis of the protein structure including chloride binding coordination sphere and chromophore environment is required as the structural analysis will help to identify the sites for introducing mutations. After identifying the potential sites which would foster the desirable features, site-saturation mutagenesis will be used to generate the library. As the mutagenesis library contains a vast number of platforms of mutated variants, including the variants in which our desired feature (chloride-sensitivity) is enhanced/deprived or intact variants with no mutations, there should be a validated method for library preparation, screening and then identifying the chloride-sensitive variants from the library in high-throughput format. This thesis will explain about the development of a workflow to screen chloride-responsive variants in a high-throughput format while specifying every step, from selecting a vector, designing primers, library preparation, screening, and sequencing, as all these steps have a major impact on the protein engineering. We will use mPapaya, as the testing protein and describe a detailed structural analysis to provide a broad picture on selecting positions for engineering the chloride binding pocket in it. The process described in this thesis involves in the identification of an appropriate mutation site through sequence alignment with avYFP-H148Q and the identified site (H202/ p-position) is mutated using site-saturation mutagenesis where the chloride-sensitive variants can be identified based on confidence interval graphs. Future considerations will be to use this method to identify the mutated variants in other testing fluorescent proteins in our lab.Item Development of Transformations of Pyridotriazoles and Photoinduced Palladium Hydride-enabled Reactions(December 2023) Zhang, Ziyan; Gevorgyan, Vladimir; Lou, Xinchou; Smaldone, Ronald A.; Gassensmith, Jeremiah J.; Romiti, FilippoThe thesis describes the development of denitrogenative transformations of pyridotriazoles and photoinduced palladium hydride-enabled reactions. A general and efficient method for arylation, X–H insertions, and cyclopropanation reactions of pyridotriazoles was developed. This protocol proceeded via pyridyl carbene intermediates, under mild, transition metal-free, and light-induced conditions. Furthermore, a Co-catalyzed transannulation reaction of pyridotriazoles with isothiocyanates and xanthate esters was developed. This method features the conversion of pyridotriazoles into two N-fused heterocycles, such as imino-thiazolopyridines and oxo- thiazolopyridine derivatives, via one-step Co(II)-catalyzed transannulation reaction proceeding though a radical mechanism. Next, three protocols enabled by visible light and palladium hydride were developed. A variety of substrates, such as diazo compounds, N-tosylhydrazones, strained molecules, acrylic acids, acrylic amides, and Baylis-Hillman adducts were found to produce hybrid palladium alkyl radical intermediates under photoinduced and palladium hydride-catalyzed conditions. These alkyl radical species subsequently participated in diverse transformations, including Heck reaction, hydroalkenylation and cascade annulation reactions.Item Engineering and Biological Applications of Turn-on Fluorescent Protein-based Sensors for Chloride(August 2021) Tutol, Jasmine Nojadera; Dodani, Sheel C.; Choudhary, Pankaj; Meloni, Gabriele; Gassensmith, Jeremiah J.; Sibert IV, John W.Chloride is the most abundant anion in our body and is essential for all forms of life. The transport of chloride is linked to cellular functions including cell volume, pH regulation, cell division, muscle contraction, and neuroexcitation. However, dysregulation of cellular chloride transport has been implicated in human diseases such as cystic fibrosis, pancreatitis, and epilepsy suggesting that chloride could be a signal of cellular status. Moreover, we lack a clear molecular-level picture of what chloride is doing. In this thesis, I will discuss the various approaches researchers have used to study chloride in biological systems. Chapter 1 will review the different types of fluorescent proteins that have been used to develop sensors for chloride. To expand on the current state of the art, we have revealed the first examples of standalone turn-on fluorescent protein-based sensors for chloride using the naturally occurring yellow fluorescent protein from the jellyfish Phialidium sp., which is ratiometric and undergoes an excited state proton transfer in the presence of chloride (Chapter 2), and the engineered mNeonGreen protein from the cephalochordate Branchiostoma lanceloatum (Chapter 3). Given that the mNeonGreen sensor operates best at pH 4.5, Chapter 4 will describe how we designed and carried out double site-saturation mutagenesis of noncoordinating residues in the mNeonGreen chloride binding pocket. This protein engineering effort not only improved the chloride sensing properties of mNeonGreen at physiological pH but also generated sensors with the largest turn-on fluorescence responses to chloride thus far (ChlorON). Fluorescence imaging experiments in mammalian cells expressing a ChlorON sensor demonstrate how the advantage of using turn-on fluorescent sensors to provide spatial and temporal resolution for mapping chloride dynamics. Lastly, Chapter 5 will highlight an alternative de novo strategy to convert the membrane-bound, proton-pumping rhodopsin from the cyanobacterium Gloeobacter violaceus (GR) into a non-pumping, red-shifted, and turn-on fluorescent sensor for chloride that can be used to image chloride in bacteria. In this study, we identified how a single point mutation of a key residue in the proton transport pathway can create a new chloride binding site in GR while also altering the protein function and spectroscopic properties to sense chloride. Taken together, this body of work lays the foundation of building protein-based hosts for chloride recognition and illustrates how we can use and adapt naturally occurring proteins to expand the turn-on fluorescent imaging toolkit for chloride that can enable the discovery of new roles for chloride in biology.Item Fabrication of Core-Shell Bio-Nanocomposites via Biomimetic Mineralization of Metal Organic Frameworks(2018-08) Li, Shaobo; 0000-0002-4178-0897 (Li, S); Gassensmith, Jeremiah J.Biomineralization is one of the nature-owned masteries that the organisms have exploited for millions of years to produce organic-inorganic hybrid materials with highly customized compositions, microstructures, morphologies and functionalities. An emerging field called biomimetic mineralization arose from the idea of employing proteinaceous templates to fabricate novel biochemical composites that cannot be produced in nature. Among various biomimetic mineralization strategies, the core-shell fashioned fabrications are of great interest, due to mineralized shells serving as exoskeleton to protect inlaid biological specimens from external stresses. In addition, biomimetically mineralized shells are also functional materials that can be used for sensing, catalysis and drug delivery. Metal organic frameworks (MOFs) are a family of porous coordination complexes that possess a high surface area and well-defined porosity. The broad variety of composition, synthetic methods, and physicochemical properties make MOFs to be versatile functional materials. This dissertation summarizes exploration of fabricating core-shell fashioned biology@MOF bio-nanocomposites using the tobacco mosaic virus (TMV) and Escherichia coli (E. coli) as model biological templates. Zeolitic imidazolate framework-8 (ZIF-8), a widely studied MOF member that is featured in robust chemical and thermal stability, was chosen to compose the biomimetically mineralized shells. This dissertation will demonstrate i) success fabrication of TMV@ZIF-8 and E. coli@ZIF-8 core-shell bio-nanocomposites; ii) mechanistic understanding in regard to the impacts of synthetic conditions on morphology, crystallinity and stability of resultant products; iii) impact of synthetic conditions on cell viability.Item Growth of Zif-8 on Molecularly Ordered 2-Methylimidazole/Single-Walled Carbon Nanotubes to Form Highly Porous, Electrically Conductive Composites(Royal Soc Chemistry, 2018-10-25) Ellis, James E.; Zeng, Zidao; Hwang, Sean I.; Li, Shaobo; Luo, Tian-Yi; Burkert, Seth C.; White, David L.; Rosi, Nathaniel L.; Gassensmith, Jeremiah J.; Star, Alexander; 0000-0001-6400-8106 (Gassensmith, JJ); Li, Shaobo; Gassensmith, Jeremiah J.The combination of porosity and electrical conductivity in a single nanomaterial is important for a variety of applications. In this work, we demonstrate the growth of ZIF-8 on the surface of single-walled carbon nanotubes (SWCNTs). The growth mechanism was investigated and a molecularly ordered imidazole solvation layer was found to disperse SWCNTs and promote crystal growth on the sidewalls. The resultant ZIF-8/SWCNT composite demonstrates high microporosity and electrical conductivity. The ZIF-8/SWCNT composite displayed semiconducting electrical behavior and an increase in sensor sensitivity toward ethanol vapors versus pristine SWCNTs.Item Mild Methods for Alkyl Radical Generation and Their Translation to Radiochemistry for Molecular Imaging Probe Development(2022-12-01T06:00:00.000Z) Rivas, Monica; Gevorgyan, Vladimir; Rodrigues, Fabiano; Smaldone, Ronald A.; Gassensmith, Jeremiah J.; Romiti, FilippoThis thesis describes the development of mild methodologies toward alkyl radical formation and their application to positron emission tomography (PET) imaging probe development. The first transition metal- and light-free auxiliary enabled remote functionalization of unactivated aliphatic alcohols was developed. This protocol enabled the selective activation of inert tertiary, secondary, and even primary CH bonds of primary, secondary, and tertiary alcohols toward β-, - and - diazenes. Subsequent hydrogenation afforded the corresponding aminoalcohols in good to excellent yields. Aerobic oxidation of secondary diazenes yielded sterically bulky 1,3- hydroxyketones in good yields. Next, the alkyl Heck-type reaction of activated and unactivated tertiary alkyl halides was developed, building on previous work on the photoinduced palladium- catalyzed exogenous photosensitizer- and oxidant-free alkyl Heck-type reaction of primary and secondary alkyl halides with vinyl arenes and heteroarenes. The method featured a broad functional group tolerance toward valuable synthons bearing quaternary centers at the allylic position in good to excellent yields. When phenyl vinyl ether was used as a coupling partner, the reaction proceeded by radical-polar crossover (RPC) pathway to afford double addition mixed acetal products. This protocol was integrated with rapid, Markovnikov selective iodofluorination of alkenes in the first one-pot formal alkenylfluorination of alkenes as a modular prosthetic group (PG) toolkit for PET imaging probe development. A new class of aliphatic prosthetic groups was synthesized en route to valuable organofluorine compounds. The methodology was translated to radiochemistry in generally good radiochemical yields, and an automated protocol for PG- synthesis was developed. The RPC mechanism was expanded to afford -fluoroethers, in the first photoinduced Pd(0/I/II)-catalyzed direct alkyl(radio)fluorination of electron rich alkenes.Item Novel Monomer Designs for 2D Polymeric Materials With Directed Supramolecular Interactions(December 2022) Boardman, Samuel R; Smaldone, Ronald; Stefan, Mihaela C.; Nielsen, Steven O.; Gassensmith, Jeremiah J.Covalent organic frameworks (COFs) are a class of polymers that have garnered significant attention in recent years for their unique physical properties. Consisting exclusively of lightweight elements (C, N, O, B, H, etc.), COFs possess highly crystalline, low-density, and permanently porous architectures. Their synthesis is carried out under thermodynamic control, where through solvothermal approaches directionally oriented dynamic covalent bonds form between monomeric units. Diversity in the successful incorporation of boron ester, imine, azine, and hydrazone linkages in tandem with stabilization forces such as dipolar and π-π stacking interactions has demonstrated the crystallization problem can be overcome to confer COFs with highly crystalline structures. Reticular methodologies afford COFs with atomically precise molecular assembly, providing the ability to predefine pore size, geometry, and dimensionality. This fascinating feature of COF design suggests great advancements in long-range order, surface area, pore size, and sorption capabilities are possible. Thus, the goal of this research details the efforts made to leverage the ‘customizable’ utility of COF design, both in regards to the monomers employed in their fabrication and with respect to the stabilization of supramolecular complexes, for the purpose of improving materials properties of 2D-COFs and broadening the practical scope of COF materials in the future. In the first chapter, COF design is discussed through the lens of scientific literature. Progress in the underlying mechanisms of formation, monomer design, and COF application as materials in gas storage are discussed. In the second chapter, two novel monomers with intended application in 2D COFs are synthesized and discussed. The first monomer, 2,4,6-trihydroxy-1,3,5-tris(p-formylphenyl)benzene, possesses out-of-plane hydroxyl groups that may participate in interlayer hydrogen bonding to improve the stability between 2D COF sheets. The second monomer, N,N',N''-(benzene-1,3,5-triyl)tris(1,1-diphenylmethanimine), is a stable benzophenone imine protected analog of 1,3,5-triaminobenzene and is suitable for use in the synthesise of a 2D imine-linked COF. In the third chapter, preliminary reactions using the novel monomer discussed in Chapter 2 are detailed and the resultant polymers are characterized.Item Plasmonic Nanoparticles Enabled Rapid and Ultrasensitive Infectious Disease Diagnostics(December 2022) Liu, Yaning 1991-; Huynh, Dung T.; Qin, Zhenpeng; Kahn, Jeffrey; Gassensmith, Jeremiah J.; Dai, Xianming; Lu, HongbingIn vitro diagnosis of respiratory infectious diseases is of paramount importance as evidenced by the current COVID-19 pandemic. Standard diagnostic methods, such as polymerase chain reaction (PCR) and enzyme-linked immunoassay (ELISA), provide specific and sensitive detection yet cause delayed sample-to-answer time. In contrast, rapid methods such as lateral flow assays (LFAs), have compromised sensitivity and specificity. To address these issues, herein, we have developed new plasmonic nanoparticle-based techniques for rapid and ultrasensitive diagnostics of infectious diseases, including respiratory syncytial virus (RSV) and severe acute respiratory syndrome (SARS)-associated coronavirus 2 (SARS-CoV-2). First, we studied gold nanourchins for colorimetric detection of RSV with a one-step sample-to- answer homogeneous immunoassay. We found that nanourchins have improved plasmonic coupling and virus targeting properties. We further integrated this rapid detection method onto a smartphone-based spectrometer and realized a sensitive diagnosis of the intact virus at room temperature within 30 minutes. Second, we developed plasmonic sensing of loop-mediated isothermal amplification (termed as Plasmonic LAMP). We engineered gold and silver (Au-Ag) alloy nanoshells with strong extinction in the visible wavelengths for SARS-CoV-2 detection. It also provides an additional sequence identification enabled by the plasmonic recognition of the LAMP products, thus improving detection specificity and sensitivity over the conventional LAMP. Third, we utilize the unique photothermal effects of plasmonic gold nanoparticles to substantially lower the detection limit of conventional plasmonic coupling assay by innovative DIgitAl plasMONic nanobubble Detection (DIAMOND). Taking RSV as a model target, we endeavor to build a pump-probe two laser system to generate and detect plasmonic nanobubbles (PNB) synchronically. Upon digital counting of PNB signals, we achieved rapid and ultrasensitive diagnostics of intact viruses at the single molecular level, representing viral infections in the early phase. Last, we built a simplified digital photoacoustic (dPA) detection module based on the DIAMOND platform. The plasmonic nanoparticles generate acoustic waves at much lower pulse energy with a single pulse laser stimulation than the vapor nanobubble phenomenon. Also, the dPA detection approach only requires a low-cost ultrasound transducer in the setup, which significantly reduces the complexity of the device and is practical for both laboratory and POC testing. We integrated dPA detection technique in a benchtop device and realize RSV detection at high performance (i.e., a single copy equivalent detection) and high specificity. Collectively, our work provides new capabilities for rapid, sensitive, and specific detection of infectious and other diseases.Item Regulating Stimulus Responses of Functional Materials(2019-08) Lee, Hamilton; 0000-0003-2176-925X (Lee, H); Gassensmith, Jeremiah J.Functional materials are materials that are responsive to external stimuli. They appear in natural and synthetic forms and are crucial to the operation of many physical, chemical, and biological systems. An improved understanding and ability to regulate the stimulus responses of functional materials would unleash many insights on the development of novel functional materials and the optimization and application of existing ones. Materials responding to changes in pH and redox conditions are of particular interest to clinical applications owing to the ubiquity with which pH and redox regulation is found in biological systems. We report the development of a pH-responsive virus-like particle (VLP) to better understand the mechanisms behind cellular uptake. The uptake of this pH-responsive VLP by certain cell lines is inhibited by the presence of terminal carboxylic acid moieties attached onto the surface of the VLP. Exposure of the VLP to acidic conditions causes hydrolysis of a linker between the carboxylic acid moieties and the VLP, releasing the carboxylic acid moieties and allowing uptake of the VLP by cells. We also report the development of aminoxyl-based ORCAs for MRI that are able to resist reduction by ascorbate. These ORCAs rely on a macrocycle to encage the aminoxyl radical, shielding the radical from reduction by ascorbate while still allowing the H2O exchange necessary for contrast enhancement. The optimization of these aminoxyl-based ORCAs may someday lead to ORCA designs that can replace gadolinium and other metal-based contrast agents that currently find mainstream clinical usage.Item Supramolecular and Biomacromolecular Enhancement of Metal-Free Magnetic Resonance Imaging Contrast Agents(Royal Society of Chemistry) Lee, Hamilton; Shahrivarkevishah, Arezoo; Lumata, Jenica L.; Luzuriaga, Michael A.; Hagge, Laurel M.; Benjamin, Candace E.; Brohlin, Olivia R.; Parish, Christopher R.; Firouzi, Hamid R.; Nielsen, Stephen O.; Lumata, Lloyd L.; Gassensmith, Jeremiah J.; 0000-0003-2176-925X (Hamilton, L); 0000-0002-5420-1954 (Shahrivarkevishah, A); 0000-0002-4650-3768 (Lumata, JL)); 0000-0001-6128-8800 (Luzuriaga, MA); 0000-0003-1064-6991 (Hagge, LM); 0000-0002-9211-718X (Benjamin, CE); 0000-0003-3226-6711 (Brohlin, OR); 0000-0003-4537-5992 (Firouzi, HR); 0000-0003-3390-3313 (Nielsen, SO); 0000-0002-3647-3753 (Lumata, LL); 0000-0001-6400-8106 (Gassensmith, JJ); Lee, Hamilton; Shahrivarkevishah, Arezoo; Lumata, Jenica L.; Luzuriaga, Michael A.; Hagge, Laurel M.; Benjamin, Candace E.; Brohlin, Olivia R.; Parish, Christopher R.; Firouzi, Hamid R.; Nielsen, Stephen O.; Lumata, Lloyd L.; Gassensmith, Jeremiah J.Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further overcame the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.Item The Transition Metal Catalyzed Polymerization of Heteroallenes: Cholesteric Gels and Cyclopolymerizations(2020-12-01T06:00:00.000Z) Namal Arachchilage, Chamni Udeshika Jayarathna; Novak, Bruce M.; Zhang, Chuanwei; Ferraris, John P.; Sibert IV, John W.; Gassensmith, Jeremiah J.From microscopic to macroscopic scale, the helix is a distinctive structure which can be found in nature. Synthetically prepared helices impart a wide variety of applications such as chiral separations, chiral sensing materials, optical materials, liquid crystalline materials, drug application and asymmetric catalysis to mention but a few. Improved materials for these applications by development of novel helical frameworks can unlock doors to a wide expansion in the real world. Chapter 1 briefly discusses the synthetic helical polymers, more specifically polycarbodiimides and polyisocyanates with their monomer synthesis, polymerization methods developed over the years and their controlled chiral polymer backbone formations along with their applications. Chapter 2 explains how the chirality in an achiral polymer can be induced using copolymerization. In other words, how single-handed chiral segments of static helical polymers of carbodiimides can be used to convert a racemic mixture of dynamic helical polymers of polyisocyanates into adapting a chiral polymer backbone. Copolymers of isocyanates and carbodiimides using CpTiCl2OCH2CF3 as the catalyst have been synthesized. Effective random copolymerization can be observed in the resulting polymers which has improved the chirality and handedness of the otherwise achiral poly(hexylisocyanate). Chapter 3 focuses on inducing chirality of the achiral racemic mixture of polyisocyanates using light cross-linking to lock in their helical reversals along the polymer backbone. The liquid crystalline mesophases of these crosslinked polymers have been improved from nematic mesophases to cholesteric mesophases. Chapter 4 discusses the synthesis and characterization of unique polymers of 1,2-dicarbodiimides that possess a cyclic structure throughout the polymer backbone by tethering the two functional groups close enough to promote their intramolecular reaction during the polymerization. New cyclo-polycarbodiimide polymers have been synthesized and display significant differences when compared to their acyclic counterparts.Item Utilization of TMV-TEMPO as an in Vivo MRI Sensor of ROS Production in Liver Inflammation and as a DNP Agent(December 2023) Lumata, Jenica Laporca 1994-; Gassensmith, Jeremiah J.; Sanchez, Erica L.; Lumata, Lloyd; Meloni, Gabriele; Smaldone, Ronald A.; Zheng, JieMagnetic resonance imaging (MRI) is a powerful tool that can noninvasively generate exquisite images of soft tissues in living subjects for medical diagnostics. This PhD dissertation details the i) utility of a nitroxyl-modified tobacco mosaic virus (TMV) as an in vivo MRI contrast agent for detection of reactive oxygen species (ROS) in murine model of liver inflammation and ii) its feasibility as an agent in MRI signal-enhancing dynamic nuclear polarization (DNP) technology. Chapter 1 entails discussion of the basics of magnetic resonance and hyperpolarization. Chapter 2 involves discussion of the identification and physical characterization of spontaneous mutation of TMV in a laboratory environment. Chapter 3 shows the successful utility of reduced TMV- TEMPO as an in vivo T2 -weighted MRI sensor of ROS in lipopolysaccharide (LPS)-induced liver inflammation in Balb/c mice. Chapter 4 details the 13 C DNP testing of TMV-TEMPO as a polarizing agent and the use of gadolinium coordination polymer GduDEP as a 13 C DNP signal enhancer. Finally, the overall conclusion and outlook of these projects are detailed in Chapter 5. Overall, this PhD dissertation ties up the experimental details and discussions of a new in vivo MRI biosensor for ROS at least in the pre-clinical level, along with some details on improving the 13 C DNP signals.Item Virus-Like Particle Qβ : a Scaffold for Imaging and Plasmonic Gold Nanoparticles(2020-08) Benjamin, Candace E; 0000-0002-9211-718X (Benjamin, CE); Gassensmith, Jeremiah J.Nanoparticles, upon their introduction, seemed to be the key to solving many issues surrounding human illness, however, poor control of nanoparticle size distribution, long-term bioaccumulation, and toxicity are obstacles that continue to plague nanoparticle use in biomedical applications. To this end, nanoscopic virus-like particles (VLPs) have been continually developed to become a powerful tool in the fields of virology, drug delivery, and imaging applications. To date, more than 100 VLPs have been constructed to yield monodisperse capsids that mimic the native virus but lack the ability to self-replicate and are therefore considered noninfectious. These macromolecules possess interior cargo space and facile modification of solvent-exposed amino acid residues using chemical conjugation. Unlike semi-and fully synthetic nanoparticles such as dendrimers, liposomes, and solid nanoparticles, VLPs offer more precise cargo loading, ease of production, biocompatibility and innate immunogenicity – making an attractive system for further development. This work investigates the use of spherical VLP Qβ as a scaffold and drug carrier and delves into its ability to perform as a biomedically-relevant entity.