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The environmental remediation of water and air sources is a challenging field, due to widespread contamination, pollutant variety, and efficiency of existing treatment options. Remediation of certain pollutants, such as per- and polyfluorinated chemicals (PFAS), polychlorinated biphenyls (PCBs), volatile organic compounds (VOCs), and viral aerosols, has shown promise via adsorptive, degradative, or filtration-based processes, each with their respective advantages and limitations. Membrane technology is one option that has exhibited considerable pollutant removal from environmental sources via reverse osmosis (RO) filtration, but is limited by low permeabilities and lack of pollutant detoxification. With the support of NIEHS and NSF, the functionalization of microfiltration (MF) membrane systems with high water/air permeabilities was investigated to increase remediation efficiency, decrease operation cost, and create a flexible platform that could adapt to different pollutant environments. Depending on the intended pollutant, membranes were functionalized with stimuli-responsive polymers, biological components, and catalytic nanoparticles to incorporate adsorptive and/or degradative properties. For enhancing the remediation of perfluorooctanoic acid (PFOA), adsorptive capabilities were incorporated in flat-sheet polyvinylidene fluoride (PVDF) membranes via functionalization with poly-N-isopropylacrylamide (PNIPAm), a temperature-responsive polymer that can exhibit both hydrophilic and hydrophobic properties below and above its lower critical solution temperature (LCST) of 32°C, respectively. Over 5 cycles of convective flow, PNIPAm-functionalized PVDF membranes showed stable PFOA adsorption of ~1800 μg/m2 of membrane above the LCST (hydrophobic expression) with 50-60% desorption of PFOA below the LCST (hydrophilic expression), displaying the temperature swing adsorption/desorption capabilities of the membrane/polymer matrix. Membrane systems with bimetallic nanoparticle catalysts, which are limited by low mass transfer between the pollutant and catalyst active site, benefited from the incorporation of stimuli-responsive polymer hydrogels as well. The addition of PNIPAm into a Fe/Pd-functionalized membrane matrix allowed for increased interaction between the pollutant, such as PCB-1, and catalyst site above the LCST, resulting in first-order kSA values of 0.28 and 1.36 L/m2/g at 25 ºC and 45 ºC, respectively. This functionalization was additionally applied to highly-scalable hollow fiber membranes, which yielded a 30% and 420% increase in kSA above 32 °C for the dye methyl orange and the VOC trichloroethylene, respectively. As a cost-effective option for increasing degradation rates, precise heating of the catalytic membrane domain was also studied via the palladium nanoparticle’s plasmonic properties. This work extended to viral aerosols by utilizing enzyme-functionalized membranes for the denaturation of SARS-CoV-2 spike glycoproteins. PVDF membranes provided a protection factor of 540 ± 380 for coronavirus-sized particle, above the Occupational Safety and Health Administration’s standard of 10 for N95 masks. SARS-CoV-2 spike glycoprotein on the surface of virus-sized particles was denatured in 30 s by subtilisin enzyme-functionalized membranes with 0.02-0.2% water content on the membrane surface. For the environmental remediation of both water and air, stimuli-responsive functionalized membranes exhibit a promising field for high-efficiency capture and detoxification of harmful pollutants.

Hydrophobic deep eutectic solvents (DESs) have emerged as excellent extractants. A major challenge is the lack of an efficient tool to discover DES candidates. Currently, the search relies heavily on the researchers’ intuition or a trial-and-error process, which leads to a low success rate or bypassing of promising candidates. DES performance depends on the heterogeneous hydrogen bond environment formed by multiple hydrogen bond donors and acceptors. Understanding this heterogeneous hydrogen bond environment can help develop principles for designing high performance DESs for extraction and other separation applications. This work investigates the structure and dynamics of hydrogen bonds in hydrophobic DESs using molecular dynamics (MD) simulations. The results show the diversity of hydrogen bonds in the eight DESs and their impact on diffusivity and molecular association. The dominating hydrogen bonds determine whether the DESs are governed by intra- or inter-component associations. The DES-aqueous liquid–liquid interface also plays a vital role in the extraction ability of DESs. One question is how the DES compositions affect the structural features of the interface. This work also investigates the density profile, dipole moment, and hydrogen bonds of hydrophobic DES-aqueous interfaces using MD simulations. The results show the variations of dipole moment and hydrogen bond structure and dynamics at the interfaces. These variations could influence the extraction ability of DES through adjusting the partition and kinetics of organic substrates in the DES-aqueous biphasic systems. Recognizing the central role that hydrogen bonds play in the DES formation, this work aims to decipher the hydrogen bond features for DESs and develop machine learning models to predict the potential of a system to be DES based on the hydrogen bond-based descriptors. Based on our MD simulation results, we developed 30 machine learning models using ten algorithms and three types of hydrogen bond-based descriptors. The model performance is first benchmarked using their average and minimal ROC-AUC values. We also analyze the importance of individual features in the models and the results are consistent with the simulation-based statistical analysis. Finally, we validate the prediction ability of the models using the experimental results of 34 systems. Our work iterates the importance of hydrogen bond in DES formation and shows the potential of machine learning in discovering new DESs. Large protein language models (PLMs) have presented excellent potential to reshape protein research. The trained PLMs encode the amino acid sequence of a protein to a mathematical embedding that can be used for protein design or property prediction. It is recognized that protein 3D structure plays an important role in protein properties and functions. However, most PLMs are trained only on sequence data and lack protein 3D structure information. The lack of such crucial 3D structure information hampers the prediction capacity of PLMs in various applications. We utilize contrastive learning to develop a 3D structure-aware protein language model (S-PLM). The model encodes the sequence and 3D structure of proteins separately and deploys a multi-view contrastive loss function to enable the information exchange between the sequence and 3D structure embeddings. Our analysis shows that contrastive learning effectively incorporates 3D structure information into sequence-based embeddings.

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall formation requires the coordination of many metabolic pathways. Additionally, complex traits such as lignin are highly polygenic. While there are several methods to analyze lignin structure, 2D HSQC NMR is a powerful analytical tool that elucidates many structural traits of lignin simultaneously. This work examined the lignin structure of 409 unique Populus trichocarpa genotypes via HSQC NMR. Twelve lignin phenotypes were subsequently utilized for a genome-wide association study (GWAS) – a powerful approach for identifying loci contributing to natural phenotypic diversity. This deep phenotyping enabled GWAS to identify 756 candidate genes associated with at least one lignin phenotype. Many of these candidate genes have not been previously reported to be associated with lignin or cell wall biosynthesis. These results provide a valuable resource for gaining insights into the molecular mechanisms of lignin biosynthesis and offer new targets for future genetic improvement in poplar.

The lignocellulosic biorefinery is a visionary concept that endeavors to provide an alternative to fossil-based refineries by producing biobased fungible fuels and specialty chemicals almost exclusively derived currently from petroleum refineries. This vision of the lignocellulosic biorefinery can only be realized if all fractions of lignocellulosic biomass are efficiently deconstructed and valorized to generate a diverse portfolio of products to sustain it against market vicissitudes. Of the three main structural constituents of lignocellulosic biomass (i.e., cellulose, hemicellulose, and lignin), lignin is underutilized despite being the most abundant renewable source of aromatic platform chemicals, representing a growing 250 billion dollar market. One pathway for lignin valorization includes its efficient fractionation followed by controlled deconstruction. Through the thermochemical deconstruction route, the focus of this dissertation project, greater understanding of the thermal deconstruction or depolymerization reactions and their associated kinetics is necessary to control competing reaction pathways to improve selectivity toward desirable products and increase yields. In this project, we seek to deepen our mechanistic understanding of the prevalent reaction pathways during the thermal deconstruction of lignin by computationally and experimentally investigating model lignin oligomers with important linkages found in lignin. Insight of the thermal deconstruction pathways of oligomeric lignin fragments with diverse linkages is a key missing piece of the puzzle required to develop fuller and generalizable lignin thermal deconstruction mechanisms. We will employ density functional theory (DFT), a computational quantum chemistry investigative tool, to assess the reactivity of a combination of important lignin linkages in the model oligomers. We will then experimentally pyrolyze commercially available model compounds with closely related to the model compounds used in the DFT investigation using a pulse heated pyrolysis reactor (PHPR) system developed for this project. The PHPR system overcomes transport limitations found in commercial pyroprobe systems used in milligram-scale biomass pyrolysis studies. We will use this system to identify major products and intermediates and validate the results obtained from the computational calculations. The computational and experimental findings will be used to identifying general reactivity trends between the major interunit linkages of lignin and propose and validate reaction mechanisms and kinetics for each model oligomer.

Although the exact cause of Alzheimer’s disease (AD) is still unknown, it is widely considered that the accumulation of amyloid plaques composed of the amyloid-β (Aβ) peptide in the brain is linked to neurodegeneration. Co-localization of viral DNA with Aβ plaques, the association of brain infection and AD, and research indicating the protective effect of Aβ against bacteria and fungi in mice and human cells have led to the hypothesis that Aβ expression and deposition may be central to its function as an antimicrobial peptide (AMP). In my thesis research, we seek to elucidate how Aβ functions as an AMP by investigating its toxicity toward bacteria, particularly the role aggregation plays in its antimicrobial activity. A computational study was also undertaken to acquire a better understanding of Aβ aggregates' interactions with neuronal and bacterial-mimicking lipid membranes. Later, a molecular dynamics (MD) simulation was performed to examine the effect of methionine (Met35) oxidation on the destabilization of Aβ aggregates, which can potentially lead to its clearance. This research also focused on evaluating and modulating conjugated polyelectrolytes' antibacterial efficacy in solution, as a disinfectant spray, and in hydrogel platforms. In summary, this study revealed the antimicrobial effect of conjugated polyelectrolytes and Aβ aggregates.

Over the past decade, use of electronic cigarettes (e-cigarettes) has increased in the younger generations of the United States. With the broad range of flavors and devices distributed on the market, American youth are prime marketing targets for the e-cigarette industry. To create a more regulated market, research of this thesis has been conducted on newer generations of e-cigarette “MOD” devices to examine how e-cigarette battery power output and coil temperature, concentrations of propylene glycol and vegetable glycerin, added flavorings (strawberry, mango, and menthol), and the presence of nicotine affect generation of aerosol particles and aldehydes in aerosols emitted by later versions of e-cigarettes. A 50 mL syringe and Tedlar bags were used to standardize and collect the vapor produced by the e-cigarette. Gas chromatography-mass spectrometry (GC-MS) was utilized to analyze the amounts and concentrations of aldehydes in the collected e-cigarette aerosols. Various tests were run using different e-liquid flavors, nicotine concentrations, and power-temperature settings. Tests were conducted on two separate heating coils with resistances of 1.4 Ω and 0.6 Ω. The results indicate that an increase in power and a decrease in resistance of the heating coil generated more aldehydes. Given the wide variety of e-cigarette device structures, flavor types, and nicotine concentrations on the market, it is likely that e-cigarettes produce broad ranges of toxic aldehydes, like formaldehyde, acrolein, glyoxal, and methylglyoxal, that react with proteins linked to respiratory diseases such as cardiovascular disease, chronic obstructive pulmonary disease (COPD), and early onset cancers.

Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts. Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures. I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells. Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes. I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer.

The use of lignin in the fabrication of soft composites has become an emerging area of research in polymer science and polymer chemistry. These lignin-based materials present numerous benefits, notably, a reduction in the use of petroleum-based precursor, improved structural benefits to otherwise soft host polymers, as well as the inherent antimicrobial and antioxidant properties of lignin, making it suitable for biomaterials. Herein, we present two chemical reaction pathways of incorporating lignin that was fractionated and cleaned using the Aqueous Lignin Purification with Hot Agents (ALPHA) process into poly(vinyl alcohol) (PVA) hydrogel composites for aqueous-based separations. By leveraging the ALPHA process, we can obtain lignins of prescribed molecular weights (MWs) with narrow dispersity (Ð) and low ash content – i.e., low concentrations of sodium and potassium. In one reaction pathway, lignin was first functionalized with vinyl-containing acrylate groups that enabled free radical chemical crosslinking of lignin chains. Notably, both the lignin MW and chemical functionality had an impact on the permeability of methylene blue (MB), where the breakthrough time of the MB across the membrane was significantly longer for lignin with higher hydroxyl content. Further, the permeability of MB was seen to decrease by over two orders of magnitude with the introduction of just 20 wt % lignin. In addition, the importance of leveraging lignin of narrow Ð in the development of structure–processing–property relationships for these materials was underscored by the consistent, repeatable permeation experiments obtained for these membranes versus their counterparts made with unfractionated lignins. In the second reaction pathway, both the lignin and PVA chains were chemically crosslinked via a condensation reaction using glutaraldehyde (GA). In general, increases in the GA content and lignin MW resulted in improved mechanical properties, including increases in ultimate tensile strength, storage modulus, and Young’s modulus, which was attributed to a tightening of the hydrated network structure and supported by decreases in equilibrium water uptake and molecular weight between crosslinks. Enhanced mechanical properties were also observed in hydrogel composites containing ALPHA-fractionated lignin as compared to unfractionated, stock lignin, underscoring the impact of the ALPHA process on the resulting properties.

Cell therapy is a technology that relies on replacing diseased or dysfunctional cells with healthy functioning ones. One of the cells used for such advanced therapies are stem cells, owing to their ability to differentiate into specific cells required for repairing damaged or defective tissues or cells. The majority of cell-based products are intended to transiently persist in the patient, secreting factors which then allow the patient’s body to heal; in these products, the cells are subsequently eliminated from the body. Furthermore, unique manufacturing platforms, in addition to novel commercialization strategies, will be required to create a successful, sustainable cell therapy industry. Currently in cell manufacturing companies, need to produce adherent cells, that bind to a solid surface such as tissue culture- treated plastic. In some cases, the growth surface needs to be treated or coated with a matrix (such as natural proteins) to facilitate cell adhesion. Cells can be grown on large flat surfaces or on spherical microcarriers for suspension?based culture in bioreactors. However, those proteins leading to batch-to-batch variability and concerns regarding contamination and affects the cell therapeutic potency. This thesis aims to develop techniques based on the merger of novel materials approaches to manipulate cell microenvironments in culture. For controlling cell-cell interaction and cell-soluble factor interactions, layer-by-layer deposition of ionic biopolymers were developed (Heparin as a negative charge polymer, collagen and Poly-L-Lysin are used as positive charge polymers). In addition, techniques were developed to control the viability of cells under hypoxic conditions within collagen hydrogels by controlling the three-dimensional properties of hydrogels and oxygen delivery (Perfluorocarbon-based oxygen carriers). In addition, to control cell-soluble factor interactions, Metal-Organic-Frameworks (MOFs) nanocarriers application was discussed as a carrier for delivering interferon-gamma (IFN-γ) to cells.

Dissipative particle dynamics (DPD), a coarse grain simulation method, was applied to the membrane formation process of non-solvent induced phase separation (NIPS) to gain further insight on the mechanism of certain variables and how they affect the final morphology. NIPS involves two solutions, an organic polymer dissolved in an organic solvent colloquially called the dope and an aqueous coagulation bath, brought into contact with one another. The solvents then mix, causing the polymer to fall out of solution as an asymmetric membrane with a dense surface layer and a more open subsurface layer in response to the decreasing solubility. Polyethersulfone (PES), a common industrial choice we have previously studied, was utilized as the polymer with N-methyl-2-pyrrolidone (NMP) as the organic solvent and water as the coagulation bath. In this study, our previous model construction was altered in several ways. Firstly, the simulation area was enlarged, allowing for a better sampling of subsurface behavior. Secondly, polymer chain length was increased to bring it more in line with the high molecular weight of industrially common polymers, with our experimental systems ranging from 100 to 200 monomers. Lastly, polyvinylpyrrolidone (PVP) was introduced as an additive to the polymer solution in concentrations varying from 1 to 10% by volume of the dope. PVP is a common polymer additive utilized in industry to produce larger pores, a result that was successfully replicated. We also investigated the effects of adding solvent to the coagulation bath as well as the effects of adding water to the polymer solution.

Volumetric muscle loss (VML) remains one of the few skeletal muscle injuries without a reliable and repeatable treatment. In large volume muscle injuries, muscle fibers as well as the surrounding connective tissue are damaged, preventing therapeutic muscle stem cells, called myogenic progenitor cells (MPCs), from reaching the injury site and initiating repair. There is a clinical need to rapidly fabricate in vitro muscle tissue constructs that mimic the native tissue organization, with aligned myotubes, for insertion and integration at the patient’s injury site. In this dissertation, we utilize the MPC’s natural propensity to close gaps across an injury site to guide alignment on collagen I, an extracellular matrix (ECM) protein substrate. Through the binding affinity of biotin and streptavidin molecules, we pre-position MPCs in temporary straight-line patterns separated with small gaps, mimicking small-scale muscle injuries. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps. Additionally, we have developed a novel technique to wash the substrate once it is seeded with cells using centrifugation to maximize the cells adhered in the desired patterned structures, while minimizing the number of cells adhered in the gaps between them. The patterning system we have designed allows for long-range myotube ordering, confirmed through the expression of myosin heavy chain (MyHC). The next part of this dissertation expands our work on cell migration and adhesion to study the properties of 3 pairs of epithelial cancer cell lines (breast, endometrium, tongue) with varying metastatic potential. According to the epithelial-to-mesenchymal transition, cancer cells lose their cell-cell and cell-matrix adhesion junctions as they become more metastatic, contributing to increased detachment from the primary tumor and a migratory response into nearby tissue and vasculature. Current in vitro strategies for characterizing a cell’s metastatic potential heavily favor quantifying the presence of cell adhesion biomarkers; however, little attention is placed on quantitative analysis of metastatic function across well-known cancer cell lines. To place cell lines more accurately within the spectrum of metastasis, we deploy two functional metrics of cancer aggression (wound closure migration velocity and cell detachment from a culture surface) on each cell line. We demonstrate that there is not a relationship between cell migration velocity and metastatic potential. Additionally, we test cell adhesive function through a shear flow assay to confirm that cell lines with higher metastatic potential had greater detachment from a culture surface compared to low metastatic potential cell lines in homogenous populations. Normalizing the data to the highest value within each metric reveals that cell lines with low metastatic potential rank higher wound closure migration velocity while cell lines with high metastatic potential rank higher in detachment from a culture surface. Finally, this dissertation concludes with a study initiated during the COVID-19 pandemic to help local Kentucky distillers transition between hand sanitizer and spirits production. Hand sanitizer introduces the bitter tasting compound denatonium benzoate to production equipment, requiring appropriate cleaning solutions for removal to prevent changes in spirit flavors. Materials compatibility with high proof ethanol dominant in hand sanitizer plays a large role in the effectiveness of the tested cleaning solutions. Metals and rigid polymers do not retain denatonium benzoate due to being compatible with high proof ethanol, so they can be cleaned easily using any of the cleaning solutions. However, elastomers permeate denatonium benzoate into and out of the material due to their poor compatibility with high proof ethanol. Ethanol proves to be a useful cleaner, but can cause irreversible damage to these materials over time.

Optical 3D printing is a branch of Additive manufacturing (AM) that utilizes low-waste production leading to efficient and rapid prototyping. Currently, this method of AM is associated with mainly petroleum-based resins. Bio-resin polymerization is an emerging research area for finding replacements for petroleum-based resins, but curing the resin with laser energy is not normally a practical use case. Current optical 3D printing uses lasers to cure liquid resins to form solid 3D objects in a layer-by-layer process which is time consuming, defect prone, and a leading limiting factor for high volume production; so, finding a way to incorporate bio-resin into the 3D printing industry while eliminating the necessity of layers in a 3D printed object is a long-term research goal for this study. One way of removing the layer lines is through polymerizing the resin below the surface, suspended by non-cured resin for structure and support. This idea has been dubbed Sub-Surface Laser Additive Manufacturing (SSLAM), and this study is the first stepping stone to the end goal; optimizing the optical polymerization of a bio-resin through the addition of energy absorption enhancers. In this study, a tung oil based bio-resin is used to study how visible wavelength lasers can cure the resin only in localized areas, what parameters are necessary to consider for process optimization, and are there any additives that can be used to increase the curing rate of the resin. Using a bio-resin and visible wavelength lasers combats the inherent problems associated with petroleum-based resins and the safety risks of using UV light sources. This study involved three main concept areas: the tung oil bio-resin and what initiators to use, three lasers of differing wavelengths and power levels, and two different types of additives to increase energy absorbance into the resin. A thermal initiator of Di-tert-butyl Peroxide (DTB) with a 5% (wt.) concentration was used as the main initiator in this study. It was concluded that combining this bio-resin with complimentary colorant additives, local polymerization is possible. Ceramic additives and other resin constituents like polymerization catalysts should also be researched to further enhance this polymerization process.

Traditional chemical processes are made inefficient by the generation of side products and reactions that fail to reach completion. Bioprocesses, on the other hand, lose product potential due to the necessity of growing the organism required to produce the desired compounds. The field of metabolic engineering often seeks to alter this balance between product formation and cell growth to generate more product from the same quantity of feed (reagent). In addition to balance, such organisms may also be engineered to produce the desired products from nontraditional substrates, such as waste compounds. In resource-poor environments, the ability to produce a wanted compound from a waste material is exceptionally valuable. One such application of this value is in human spaceflight; all needed material must be brought with the astronauts from the beginning of their trip. For short missions, this is possible, but it becomes difficult or impossible on longer missions due to substantial mass requirements. NASA’s solution to this is to produce needed supplies in situ and one method they have chosen to utilize is 3D printing. Printers have already been used to fabricate tools and components out of plastic aboard the International Space Station (ISS), however the plastic must be sent from Earth. The bioproduction of plastic in space provides one solution to this requirement by manufacturing what is needed in orbit. Polyhydroxyalkanoates (PHAs) are a class of natural, biodegradable, fully-biosynthetic polyesters. They possess varying thermal and mechanical properties on par with many petroleum-derived plastics and these properties can be manipulated by altering the monomer composition of the final PHA. To that end, the work in this dissertation demonstrates multiple means by which the bioproduction of PHA can be carried out more efficiently, with limited resources, through metabolic engineering of the host organism. In this case, Y. lipolytica was engineered to heterologously produce PHA via exploitation of its peroxisome. Peroxisome morphology, expression systems, β-oxidation, and the greater upstream carbon metabolism of Y. lipolytica were modified. The results of these efforts serve to improve the understanding of peroxisome-associated metabolic engineering in Y. lipolytica. Application of this knowledge resulted in a doubling of PHA accumulation efficiency on the non-ideal substrate oleic acid. Finally, this work demonstrates the ability of terrestrial engineered Y. lipolytica to effectively carry out heterologous production of PHA in the hostile environment of space and identified areas of future strain health concern as targets for mitigation via synthetic biology efforts.

Industrial Chinese hamster ovary (CHO) production of therapeutics requires cell lines with high secretory capacity to avoid an accumulation of improperly folded proteins, or endoplasmic reticulum (ER) stress. This presents a challenging engineering bottleneck. The unfolded protein response (UPR) is initiated to overcome ER stress and reestablish homeostasis. In this dissertation, the impacts of ER stress and the UPR on protein production in mammalian cells are detailed, and both selection- and rational-based strategies for enhancing the ER stress response in productive CHO cell lines are reviewed. This project aims to expand on recent research efforts for engineering ER stress-related responses in CHO cell lines to augment higher productivity in biomanufacturing. This work demonstrates recombinant protein overexpression and higher specific productivity results in unavoidable ER stress in fed-batch culture of two distinct CHO cell lines, one producing immunoglobulin G (IgG1) and one producing erythropoietin (EPO-Fc). High throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) were used to conduct differential gene expression analyses and study the effect of protein production over time versus a non-producing host cell line during fed-batch culture. These analyses showed enrichment and transient upregulation of mRNAs involved in ER stress, the UPR, and oxidative protein folding and processing. Analysis of temporal differential expression profiles indicated dependency on ATF6 pathway activation. The RNASeq analysis led to the identification of two engineering targets, ATF6β and WFS1, known regulators of the UPR. The IgG1-producing CHO cell line was used to analyze the effect of stable knockdown of either ATF6β or WFS1 on CHO cell growth and IgG1 production. This dissertation presents the first study to analyze the effect of WFS1 knockdown in producing CHO cells. Knockdown of ATF6β was found to improve specific productivity; however knockdown of WFS1 resulted in no improvement. Knockdown of ATF6β improved the UPR specifically during ER stress, but knockdown of WFS1 had negative impacts on UPR activation and product mRNA expressions. This dissertation closes with conclusions and future work in engineering the CHO cell ER stress response. It is anticipated this work will lead to advancements in CHO cell engineering for enhanced protein production of therapeutics.

GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of β-galactosidase (βgal) and subsequent accumulation of GM1 ganglioside in lysosomes. The rare nature of this disorder presents challenges with prognosis and treatment. Enzyme replacement therapy (ERT) can effectively treat systemic deficiencies, but the intravenously administered enzyme cannot cross the blood-brain barrier (BBB) to treat the central nervous system. A delivery system to encapsulate and deliver the enzyme can facilitate enzyme transport across the BBB with suitable surface modifications. One of the pathological aspects of GM1-affected cells is the upregulation of the lysosomal enzyme hexosaminidase A (HexA). In this dissertation, I evaluate a biohybrid poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymersome system as an enzyme-responsive fluorescence probe to monitor the disease progression during the treatment. The polymersomes are synthesized using the solvent injection method and self-assembled in a size range of < 200 nm. The effect of synthesis parameters such as infusion rate and needle gauge on PEG-PLA polymersome properties was studied and shown to be negligible. I also designed a hyaluronic acid-b-polylactic acid (HA-PLA) polymersome system as a therapeutic tool that can achieve an enzyme-responsive and controlled delivery of βgal in response to upregulated HexA in diseased cells. HexA can degrade the hyaluronic acid in polymersomes and induce a higher drug release. HA-PLA polymersomes were synthesized with an average diameter of 138.0 ± 17.6 nm and ζ-potential of -18.0 ± 3.8 mV. βgal was encapsulated in polymersomes with an efficiency of 77.7 ± 3.4 %. In the presence of model enzyme hyaluronidase, HA-PLA polymersomes demonstrate an enzyme-specific degradation and a two-fold higher release of encapsulant than without cognate enzyme. Another pathological aspect of GM1 gangliosidosis, among other lysosomal storage disorders, is impaired autophagy, i.e., a reduced fusion of autophagosomes and lysosomes to degrade cellular waste. Reduced autophagy in a cellular model of GM1 Gangliosidosis (GM1SV3) compared to healthy cells was demonstrated using immunofluorescence. Upon treating the GM1SV3 cells with βgal-loaded polymersomes, autophagy was enhanced in a controlled manner. Most notably, the fusion of lysosomes and autophagosomes in GM1SV3 cells returned to normal levels comparable to healthy cells after 24 hours of polymersome treatment as opposed to the 6 hours of treatment with the free enzyme. The studies in my dissertation demonstrate that HA-PLA polymersomes can potentially deliver βgal in response to disease pathology and provide a promising delivery system to treat GM1 Gangliosidosis.

As the supply of phosphorus decreases, nutrient recovery in municipal wastewater treatment has become an important area of research. Recovery methods for phosphorus-containing fertilizers, such as struvite, and other products are currently being developed and assessed for their efficiency and economic feasibility. Potential environmental impacts from nutrient recovery are also being assessed as design for the environment is important, especially with regards to the eutrophying potential of phosphorus in phosphorus-limited freshwater aquatic systems. Life cycle assessment (LCA) is a tool that can be used to estimate potential environmental impacts of a product or service focusing on its entire life cycle. This research applies LCA to determine potential environmental benefits, as well as unforeseen burdens, that could be realized from implementation of nutrient recovery at the treatment facility scale and national scale. First, this dissertation provides an LCA of a novel electrochemical struvite precipitation method using a sacrificial Mg anode at the facility scale, the first analysis to do so (Chapter 2). Second, spatially explicit eutrophication potential from wastewater treatment discharge in the U.S. including potential changes from nutrient recovery are modeled (Chapter 3). Additionally, the work presents a new model in which eutrophication potential is disaggregated to determine whether phosphorus loading is local or resulting from upstream phosphorus emissions. In addition to expanding the body of knowledge in the realm of phosphorus recovery, these two studies also employ LCA methods that are growing areas of research: prospective and spatially explicit LCA. As emerging nutrient recovery technologies evolve, employing these developing fields of LCA can help guide decisionmakers in developing strategies to not only conserve phosphorus through nutrient recovery, but also to effectively mitigate eutrophication potential where point-sources of phosphorus are significant.

When a peripheral nerve injury (PNI) occurs, the gold standard for tissue regeneration is the use of autografts. However, due to the secondary effects produced by multiple surgeries involved in the removal and implantation of autografts for very small lesions, it is possible to replace them with the use of Nerve Guide Conduits (NGCs). However, NGCs are limited to short lesions (less than 1 cm). This limitation is caused by the absence of compounds in the extracellular matrix (ECM) that autografts can provide. Since much of the regenerative process takes place on the NGC surface, our work aims to modify its surface using heparin and collagen coatings, two natural polymers present in the ECM. Heparin/collagen bilayers (HEP/COL) will increase biocompatibility, cell adhesion and migration, and allow the incorporation of neurotrophic factors such as Nerve Growth Factor (NGF). This research evaluates the effects on human Schwann cells (hSCs) when cultured on a commercial collagen NGC under four conditions: without coatings and with six heparin/collagen bilayers (HEP/COL)6 with and without NGF supplemented to the culture medium (10 ng/ml). We have exhaustively characterized (HEP/COL)6 coatings. The chemical structures of the polymers, the morphology, and the surface charge of the coatings could be determined. Real-time analysis of hSCs behavior showed three main phases: adhesion, proliferation, and adaptation. The adhesion phase was increased in the conditions with (HEP/COL)6, and the viability studies showed an increase of up to 250% compared to cells grown on polystyrene without coatings. The cell morphology has never been altered by (HEP/COL)6, as well as the morphology of the coated NGC. Cell migration was favored, and (HEP/COL)6 also increased the expression of some proteins such as BDNF, which promotes the duration of the myelination process, or TNF alpha, which is a mediator of inflammatory and immune functions. Concerning the surface modification of a commercial NGC, these studies showed an increase of more than 200% in cell viability when compared to uncoated NGCs. We also determined the presence of heparin and evidenced the cell adhesion on (HEP/COL)6 coated NGCs. In conclusion, (HEP/COL)6 seems to be a promising strategy to improve the performance of NGCs.

Soft poly-ethylene-glycol (PEG)-based soft nanoparticles (NPs) including cylindrical (CNPs) micelles, spherical (SNPs) micelles, and lipid bilayer vesicles (LNPs) are thought to be treated as foreign objects by mammalian phagocytes. If this hypothesis is true, NPs should trigger a proinflammatory, autophagic phenotype that is similar to the one seen when macrophages phagocytose pathogens or when macrophage surface expressed proteins bind pathogen surface factors such as lipopolysaccharide (LPS). Here, we show that macrophage responses to the above NPs are almost completely unique from those triggered by group A streptococcus (GAS) pathogens (JRS4 cells) and LPS. Instead, macrophages treat these soft NPs more like high-density lipoprotein (HDL) and low-density lipoprotein (LDL). RNA sequencing of macrophage transcripts after hHDL, hLDL, JRS4 cell, LPS, and LNP incubation showed three diverse response clusters triggered by JRS4 cells (1), LPS (2), and hHDL-hLDL-LNP-PBS (3). Of these reagents, LNPs triggered the fewest post-incubation transcript changes from macrophages to which PBS was added (control). LNPs did not increased the transcription of factors associated with foreign object identification including Fc and complement receptors. LNPs did not increase transcripts whose translated proteins are involved in phagocytosis, autophagy, lysosome biogenesis, or inflammation. LNPs did not increase Tfeb transcripts, which is a master regulator of lysosome biogenesis and a necessary component of autophagy. To further determine the effects of these NPs on cells, we performed fluorescence microscopy and flow cytometry experiments. CNPs, SNPs, and LNPs lowered macrophage autophagosome levels that were raised by the canonical challenges: starvation, rapamycin, and LPS. CNPs, SNPs, and LNPs lowered reaction oxygen species (ROS) levels, did not increase lysosome acidification, and the reduced the secretion of proinflammatory cytokines compared to basal conditions and LPS addition to macrophage cultures. CNPs and SNPs triggered low lysosome acidification and LNPs did not increase Tfeb levels, the master regulator of lysosome biogenesis and a necessary component of autophagy. Thus, the terminology that macrophages "clear" NPs, which has been used over many decades, is most likely misleading. Our findings challenge the hypothesis that the main uptake mechanism of soft PEG-NPs by M1-polarized murine macrophages in vitro is phagocytosis.

With the ongoing pandemic, there have been increasing concerns recently regarding major public health issues such as abuse of organophosphorus compounds, pathogenic bacterial infections, and biosecurity in agricultural production. Biosensors have long been considered a kernel technology for next-generation diagnostic solutions to improve food safety and public health. Significant amounts of effort have been devoted to inventing novel sensing mechanisms, modifying their designs, improving their performance, and extending their application scopes. However, the reliability and selectivity of most biosensors still have much to be desired, which holds back the development and commercialization of biosensors, especially for on-site and point-of-care (POC) usages. Herein, we introduce an innovative two-phase sensing strategy based on tunable AC electrokinetics and capacitive sensing. By dividing the detection process into a sensitivity-priority step and a selectivity-priority step, the specificity and sensitivity of a biosensor can be significantly improved. A capacitive POC aptasensor is fabricated for the implementation of the 2-phase detection and a quasi-single-cell level detection of limit together with an excellent selectivity is achieved simultaneously. The sensor is capable of handling real-world clinic samples without sophisticated pretreatment. Just after a simple one-step dilution, the developed sensor can detect bacteria no less than 2~3 bacteria/10 µL in raw milk samples within 100 s. Alongside whole bacteria detection, the biosensor can also detect endotoxin, the lipopolysaccharide, in bovine serum samples, with a limit of detection of 10 pg/mL. The biosensor is low-cost and easy to use. This work not only demonstrates a biosensor with significant advantages in sensitivity, selectivity and assay time but also opens up a new horizon for further research of all affinity-based biosensors.

Precise detection and monitoring of nuclear fuel cycle, enrichment, and weapon development activities are critical for supporting warfighter preparation in chemical, biological, radiological, nuclear, and explosives (CBRNE) operations, clandestine activities, and nuclear compliance. A biological sensing system could serve as an alternative to traditional detection methods by using organic material naturally present in the environment to discreetly detect residual trace nuclear material. Microorganisms provide an optimal platform for an alternative sensing system; however, their response to low levels of ionizing radiation is poorly characterized. Combining the power of next-generation sequencing and transcriptomic analysis, this dissertation takes an approach to obtain a comprehensive picture of microbial response to low-dose ionizing radiation and, through characterization analysis, elucidate transcriptional signatures unique to radiation for future RNA-based sensing applications. In this work, we aim to characterize the response of model and environmental microorganisms to in situ low-dose ionizing radiation. This dissertation is the first to describe the transcriptional response of stationary phase microorganisms, Escherichia coli, Pseudomonas putida, Saccharomyces cerevisiae, and Rhodosporidium toruloides, exposed to in situ radionuclide sources at an absorbed dose rate of 9 mGy d-1, where the biological response of microorganisms is poorly characterized. Furthermore, internal radionuclides representative of the nuclear fuel cycle, plutonium-239 (239Pu), tritium (3H), and iron-55 (55Fe), were selected to represent three major radiation types: alpha, beta, and gamma respectively. Initially, two bacteria, E. coli and P. putida, were selected to characterize the microbial response to low-dose radiation. Cultures were subjected to internal radiation exposures for a 15-day period before preparing samples for next-generation sequencing. RNA-Seq was used to process sequencing data, and differential expression analysis was performed to identify significant changes from non-irradiated controls. Global transcriptional analysis found exposure to 239Pu initiated a moderate change in gene expression following one-day, but a negligible response after 15-days in both E. coli and P. putida. The beta emitter, 3H, had the opposite response with a minimal change after one-day of exposure in E. coli and P. putida, but the most substantial change of the radionuclides studied after 15-days. Exposure to 55Fe resulted in similar changes in gene expression in the bacteria after both one and 15-days. Differential expression analysis revealed key similarities and differences in the gene expression of E. coli with the three radionuclide sources, 239Pu, 3H, and 55Fe, and two times, one and 15-days, studied. The transcriptional response was broad with genes encoding biosynthesis pathways of nuclear envelope components, amino acids, and siderophores, transport systems such as ABC transporters and Type II secretion proteins, and initiation of stress response and regulatory systems of temperature stress, the RpoS regulon, and oxidative stress. Differential expression analysis of the P. putida data identified significant changes in gene expression to membrane components, central metabolic processes, and motility by multiple radiation types. In addition, genes uniquely changed by a single radiation type were also observed. The next step was to expand the comprehensive picture to a simple eukaryotic system. Again, a model yeast, S. cerevisiae, and an environmental organism, R. toruloides, were selected. Utilizing a similar analysis, the yeast systems did not produce as large of a response as their bacterial counterparts. We hypothesize the robust and adaptive nature of R. toruloides prevented any significant changes in gene expression with exposure to the internal radionuclides at this dose rate. Similarly, S. cerevisiae had a smaller response to 239Pu and 55Fe with significant gene expression changes in Ty1 transposons and metal ion processes. This dissertation demonstrates an initial characterization of the effects of low levels of ionizing radiation on microbial species. Genes unique to a single radiation type and those commonly differentially expressed by multiple radiation types provide components for future engineering of discriminatory and general RNA-based detection of nuclear material.

Nanoparticles have been of interest to the pharmaceutical industry since the 1980s. The first FDA approved nanoparticle-based therapies included liposomal anesthesia agents. Since then, the amount of FDA-approved nanoparticle therapies remains low. This is because nanoparticle-patient interactions can be very complex and are not well understood. Complicating factors also include increasing obesity rates among the patient population and many small animal pre-clinical trials are completed with healthy, lean animals. The biochemical differences between lean and obese patients prevents early studies from accurately predicting nanoparticle clinical behaviors. Many nanoparticles fail in trails. In this thesis, I aimed to uncover how nanoparticles interact with cells in an obesity-like environment. I focused mainly on nanoparticles made from PEO-PBD di-block polymers (synthesized by Jimmy Mays’s group in chemistry). The di-block polymers form into long, flexible cylinders (“filomicelles” or more generally, cylindrical nanoparticles (CNPs)). CNPs have been previously shown to have favorable circulation times compared to spherical nanoparticles, but I wanted to know how CNP enter cells. Surprisingly, this mechanism is not well-known for any nanoparticle. I focused on the potential interaction between CNPs and the major HDL receptor SR-BI. This was done with in vitro experiments involving the small molecule inhibition and binding site competition of SR-BI. In vivo experiments were also conducted with SR-BI deficient mice and co-injection of CNPs and HDL. I then wanted to study the effects obesity would have on the pharmokinetics profile and biodistribution of CNPs and liposomal nanoparticles (LNPs). LNPs are the most used NPs in the clinic, so they provide a good control to evaluate the possibility of using CNPs in the clinic. I used a combination of controlled diet and leptin deficiency in mice to conduct this study. Interestingly, obesity caused CNPs to localize to the liver faster than lean controls. LNPs had the opposite effect. Further experimentation suggested this was due to different destination cell types within the liver for CNP vs LNP. In the third study, I developed a nucleic acid therapy that can trigger the lipolysis (breakdown) of neutral lipids in mammalian cells using a split-intein technology that I learned as an undergraduate.

The world’s energy demands are projected to increase by nearly 50% by the year 2040, and consumption of carbon-based fuels continues to release greenhouse gases such as carbon dioxide and methane into the atmosphere. This has been causally linked with climate change and increased extreme weather events, which has been further linked to adverse health outcomes and negative effects on biodiversity, food security, and increased disease transmission. Clearly, there is a need for a sustainable, carbon-free, and cost-effective method of energy production to meet growing energy production demands. The sun irradiates Earth’s surface annually with ~80,000 terawatts (TW), making solar energy conversion (photovoltaics) a probable part of any plan to meet our growing needs. The biological process of photosynthesis is responsible for the annual generation of ~ 130 TW of energy through solar energy conversion to drive essentially all primary production on Earth. Photosynthesis does so with widely available elements and with a quantum efficiency approaching unity, both of which are challenges to current commercially available photovoltaic devices. The growing field of applied photosynthesis hopes to utilize the biological process of photosynthesis to help meet these growing energy demands through the development of bio-hybrid electronic and photovoltaic devices. The study of the bio-inorganic interface in these devices remains one of the largest areas for improving the outputs and efficiencies of these devices while simultaneously allowing for more detailed insight into fundamental biological photosynthetic processes.

The intracellular environment is crowded with macromolecules that can occupy a significant fraction of the cellular volume. This can give rise to attractive depletion interactions that impact the conformations and interactions of biopolymers, as well as their interactions with confining surfaces. We used computer simulations to study the effects of crowding on biologically-inspired models of polymers. We showed that crowding can lead to attractive interactions between two flexible ring polymers, and we further characterized the adsorption of both flexible and semiflexible polymers onto confining surfaces. These results indicate that crowding-induced depletion interactions could play a role in the spatial organization of biopolymers in cells, and they also suggest that macromolecular crowding could be used to alter the spatial organization of cell-free synthetic systems. A major limitation of cell-free expression systems, which are widely used to study gene expression, is the lack of means to achieve spatial control of gene expression components. With a coarse-grained model of DNA plasmids and crowders, we showed that plasmids were uniformly distributed at low levels of crowding but, due to depletion interactions, became strongly adsorbed to confining surfaces at high levels of crowding. These results were experimentally validated by our collaborators using DNA and crowders in cell-sized vesicles. We used kinetic Monte Carlo simulations to study the effect of crowding and confinement on gene expression dynamics and noise, giving insight into experiments. Our work provides insights into the role of crowding and confinement on the spatial organization and dynamics of gene expression in cellular and cell-free systems.

In this work, the synthesis of protein-functionalized nonporous silica nanoparticles and their application to capture antibodies was demonstrated. Silica nanoparticles were chosen as they offered particle and pore size tunability, low toxicity, thermal stability, ease of synthesis and ease of surface functionalization. A biotin-streptavidin-biotin bridge was constructed on the surface of the particles and used to attach biotinylated Protein A/G (PAG), which has high affinity for antibodies. The biotin-streptavidin-biotin bridge is hypothesized to orient the PAG in a favorable manner for efficient antibody capture. Nonporous silica nanoparticles (approximately 270 nm in diameter) were synthesized via the Stöber method. Following amine functionalization, biotin was covalently attached to the SNPs. Attachment of streptavidin, followed by biotinylated PAG was achieved using the strong biotin-streptavidin interaction. 42.8 mg streptavidin was loaded onto per gram particles, which was estimated to correspond to monolayer surface coverage. Due to their similar size, 1:1 binding between streptavidin and PAG was assumed. The uptake of IgG by PAG attached particles was measured by solution depletion. The equilibrium isotherm was fit to the Langmuir adsorption model by performing nonlinear regression. Maximum uptake capacity, qmax was found to be 110 mg antibody/g PAG particles, and the equilibrium association constant KA was found to be 6.05×107 L/mol. The maximum uptake capacity of IgG is consistent with 1:1 binding with Protein A/G on the SNP surface. Experiments of antibody binding in the presence of bovine serum albumin, which served as a model nonspecific protein, showed insignificant reduction of antibody binding. The biotin-streptavidin-biotin bridge is a versatile approach to attach and orient biomolecules at the surface of SNPs. This technique can be extended to biomolecule capture for a range of affinity pairs, building on the prevalence of biotin and streptavidin functionalization strategies of proteins and nucleic acids. Advantages of nanoparticle-enhanced biomolecule detection and immunoassays can be further explored based on the versatility and specificity of affinity capture using SNPs.

Soluble enzymes have the unique ability to assist the conversion of complex substrates in fewer steps compared to conventional chemical synthesis; however, when immobilized to a surface, the insoluble enzyme's activity and stability can suffer in its new environment. To improve upon future immobilized enzyme efficacy, a fundamental understanding of the interactions across the enzyme, linker, and surface during covalent, site-specific immobilization that can be detrimental to its activity is required. More specifically, using a well-characterized and model enzyme, T4 lysozyme (T4L), 28 different cysteine attachment points' effects on site-specifically immobilized activity are examined. Different combinations of heterobifunctional amine-to-sulfhydryl crosslinkers and hydrophobic and hydrophilic aminated polymer surfaces are used to immobilize T4L site-specifically via amine-to-sulfhydryl conjugation. The cysteine mutations availability and accessibility result in heuristics that minimize nonspecific adsorption while maximizing site-specific conjugation. Based on the findings, an improved method for site-specifically immobilizing biotinylated T4L on streptavidin-coated polymer beads is developed. Innate structural characteristics such as cysteine accessibility, amino acid consensus, and enzyme surface concentration enhance immobilized enzyme’s specific catalytic efficiency regardless of the attachment site and orientation.

In lieu of chemical and physical methods, biologically guided synthesis is increasingly used as a cost-effective medium for the fabrication of nanoparticles (NP). Recently, a palladium metal binding sequence Pd4 (TSNAVHPTLRHL) has been demonstrated to be instrumental in the production of palladium (Pd) nanoparticles. Although, by eliminating the additional cost of purification of the protein, the crude lysate of E. coli containing Pd specific protein has been proven to be a viable cost-effective means for the synthesis of Pd NP, studies have not be done to ascertain the comparative catalytic activity of nanoparticles synthesized from both clarified lysate and pure protein sample. This thesis aims to provide a comparative catalytic analysis of the turnover frequency of NP derived from pure Pd43-GFPuv and turnover ratio derived from NP synthesized by Mosleh et al. An E. coli expression system is employed for production of three copies of palladium binding sequence fused with green fluorescence protein [(Pd4)3-GFP] as a tag. This protein was purified with a Fast Protein Liquid Chromatography (FPLC) apparatus coupled with Immobilized Metal Affinity Column (IMAC). Transmission electron microscopy (TEM) was utilized to characterize the synthesized NPs from clarified lysate and pure protein sample. The catalytic property of fabricated NP from the purified protein was analyzed by a model Suzuki-Miyaura coupling reactions. It was observed that the purified metal binding protein had are three times higher turnover frequency compared to the crude lysate previously published.

Growing population demand and challenges brought on by climate change have spurred the need for more resilient fruit and vegetable supply chains. One agricultural technology of significant interest is the use of greenhouses for food production. Greenhouses create a stable and adaptable environment for crops such as tomatoes to grow year-round. Fresh tomatoes are the second most consumed vegetable per capita in U.S. diets, currently averaging 20.7 pounds. The growing consumption of fresh tomatoes has been the result of increasing cultural diversity in the United States. To meet the growing demand, Venlo-type greenhouses have been frequently used by growers. It provides an economical solution to produce multiple crops in various climate environments while withstanding severe weather conditions. While there have been many studies and advancements in using greenhouse technology to grow tomatoes in Europe, production has yet to be analyzed in the U.S. This study seeks to fill the gap of greenhouse tomato production by simulating growing scenarios using the openly accessible Modelica Greenhouse Library in 10 select locations across the mainland USA. Two growing scenarios were explored, a base case without CO2 enrichment and a CO2+ case with a continuous flow of CO2 being externally supplied to the system. All simulations had yields above 3-8 kg/m2/yr, which is expected of field-grown tomatoes. However, a few locations were below the expected range of 50-80 kg/m2/yr yield for greenhouse-grown tomatoes. CO2 enrichment in most cases resulted in increased fresh weight yield, reduced the use of resources which improved Product Water Use (PWU), Electrical and Thermal Energy Efficiency. Factors influencing the model such as Temperature, Supplemental Lighting, and CO2 enrichment were discussed.