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Advisor: Stephanie Bryant

All non-biological biomaterials trigger an foreign body response (FBR) that can lead to chronic inflammation and formation of a fibrous capsule around the material. This capsule creates a physical barrier that walls off the implant from the rest of the body, hinders communication with surrounding tissues, and can ultimately lead to implant failure. Innate immune cells called macrophages are the key drivers of the FBR, but their cell signaling response to different materials is not well understood. Macrophage polarization and fusion into foreign body giant cells (FBGCs) play a crucial role in the progression of the FBR, but we do not currently know what receptors drive their behavior. The purpose of this study was to elucidate the role of the EP2 receptor in macrophage polarization and fusion in order to determine its role in the FBR. In vitro models support previous research that the hormone PGE 2 suppresses macrophage fusion through the EP2 receptor on the surface of glass coverslips. However, we also show that PGE 2 is not able to suppress macrophage fusion on the surface of MGS, revealing that the role of PGE 2 and EP2 in macrophage fusion is material dependent. While Lipopolysaccharide (LPS) is able to induce classical activation of macrophages on MGS regardless of whether or not the EP2 receptor is present, removal of EP2 appears to delay the transition of macrophages into an alternative activation state after 8 hours. Furthermore, the molecule, evatanepag, is found to be an unsuitable EP2 agonist in peritoneal macrophages and thus eliminates its potential use on the surface of a therapeutic biomaterial aimed at exploiting the EP2 receptor. Ultimately, EP2 plays a complex role in macrophage polarization and fusion, and further exploring this role would allow us to better understand macrophage behavior so that we can ultimately modulate the FBR.

Advisor: Jerome Fox

Drug discovery and development is a lengthy and expensive process. It takes well over a decade to develop a single FDA-approved drug. Discovery of functional and safe compounds account for a significant portion of the time, money, and energy. Genetically encoded biosensors with the ability to detect inhibition of target proteins offer a tool for accelerating the discovery of pharmaceutically relevant natural products. These sensors use transcription of a reporter protein to determine inhibitors of the target protein. Use of a fluorescent reporter reduces the dependence on growth in comparison to survival-based systems and enables a closer inspection of toxicity, a common side effect of exogenously expressed proteins. Development of a biosensor for a difficult to inhibit on-target protein such as Protein Tyrosine Phosphatase 1B (PTP1B) allows for identification of inhibitory properties but not selectivity of the compounds. Complementing such a system with an off-target biosensor for a protein from a separate family such as 3-chymotrypsin-like protease (3CLpro) helps to distinguish selective inhibitors from general protein denaturants. The project seeks to develop genetically encoded detection systems that link inhibition of drug targets – PTP1B and 3CLpro – to a fluorescent output. Once established, these systems could serve as a platform to identify selective inhibitors of more closely related proteins.

Advisor: Edward Chuong

Transposable elements (TEs) are increasingly recognized as key drivers of species differentiation. However, as we seek to further characterize the role TEs play in both evolution and development, the dynamics of their movement and the potential consequences of these jumps require further study. To investigate the potential mobility of various bovine TEs including BovB, an ancient long interspersed nuclear element (LINE) transposon comprising nearly 30% of the cow genome, a combination of experimental and computational techniques were employed. Pilot experiments of a retrotransposition assay were undertaken in HeLa, MDBK, and HEK293T cell lines using an assay-specific plasmid (pMT525) with an active human LINE1 element. The plasmid drives expression of EGFP in response to successful retrotransposition, which was observed in human cells using the control human TE. The consensus sequence of BovB was cloned into the assay plasmid and is being

evaluated in vitro for activity, with promising results observed in HEK293T cells. Re-analysis of publicly available long-read, trio-binning whole genome sequencing data has uncovered novel TE polymorphisms in Bos taurus, some of which will be cloned into pMT525 to test for extant copy activity. The discovery of active BovBs, L1-BTs, and/or BovA2s would revolutionize our understanding of modern bovine evolution as these elements could play a significant role in genome regulation and development.

Advisor: Kristi Anseth

Intestinal organoids are important models of disease and development. Their highly irregular morphology is physiologically relevant but defies easy characterization. While existing tools for organoid morphology characterization are often inadequate, transformation approaches like the Euler Characteristic Transform (ECT) have shown promise. However, there's a need for more physically meaningful shape descriptors, leading to the development of a novel 'polar autocorrelation' metric. This work demonstrates the use of this metric to quantify the number of features of real and contrived datasets. This end goal was chosen as a nod to our group’s work creating organoids with a deterministic feature number. Several classification approaches using polar autocorrelation are demonstrated, with both analytical and whole-dataset methods giving high accuracy, especially in comparison to shape descriptors more typical to morphometry. A sizable contrived dataset with added noise was used to generate simulated bounds of a four-featured organoid, and experimental data of mature organoids are generally contained within these bounds. This metric provides an objective basis to quantify radially distributed features.

Advisor: Kristi Anseth

Viscoelasticity is an important characteristic of biomaterials, influencing many cellular responses such as morphology, proliferation, migration, and extracellular matrix (ECM) deposition. As a result, interest has grown in designing viscoelastic biomaterials as synthetic matrices for more physiologically relevant in vitro tissue models. While current synthetic ECMs cover a wide range of stiffnesses and rates of stress relaxation, fast-relaxing materials (half times on the order of seconds, resembling the native properties of brain or adipose tissue) are relatively under-explored, partly due to a limited number of chemical and physical crosslinking methods that can achieve such fast relaxation. In some applications, covalent adaptable networks utilizing boronate ester crosslinks have been developed to access these material properties. While these systems enable fast-relaxing synthetic ECMs, most modern viscoelastic biomaterials have two fundamental limitations: lack of homogeneity due to fast crosslink formation and difficulty tuning the degree of stress relaxation. In this work, we design and characterize a fast-relaxing dual-crosslinked PEG hydrogel that addresses both limitations and apply this system to cell and organoid culture. Uniquely, our system utilizes a combined competitor/crosslinker molecule which produces highly homogeneous photopolymerizable hydrogels, affording spatiotemporal control over network formation and facile manipulation of material (visco)elasticity by altering the ratio of elastic to viscoelastic crosslinks. Cells encapsulated in this material display remarkable cellular remodeling and contraction of the matrix when cell-adhesive peptides are incorporated into the hydrogel network. Finally, Murine intestinal organoids show crypt formation after encapsulation in this material, despite a lack of typical matrix-incorporated proteins.

Advisor: Wyatt Shields

This research investigates the use of functionalized microparticles for label-free biomolecule detection through changes in electrophoretic behavior and their underlying mechanics. The microparticles used are two-sided Janus particles comprised of a spherical dielectric material with a biofunctionalized gold cap over one hemisphere. When the microparticles are placed in an aqueous fluid and subjected to an AC electric field at certain frequencies, they propel in a direction opposite of the gold hemisphere by induced-charge electrophoresis (ICEP). The project builds upon our previous work demonstrating that capturing biomolecules with these induced-charge electrophoretic microsensors (ICEMs), suppresses particle speed. This previous work focused on use of ICEMs as biosensors. It confirmed that capturing a target biomolecule, streptavidin, with ICEMs leads to a decrease in their speed. This finding suggests a potential application for ICEMs in rapid and simple biomolecule detection without the need for complex labeling procedures. However, the underlying mechanisms of this ICEM behavior have not yet been elucidated. The work presented here explores the influence of surface modifications on ICEM behavior. The ultimate goal of the study is to compare experimentally measured ICEM motion with theoretical predictions of ICEM speeds based on experimentally measured particle characteristics measured using dielectrophoretic trapping. Here, we show that the observed trends in ICEM motion suppression were consistent with previous findings. We also show the preliminary analysis of dielectrophoretic (DEP)-trapped particles, which will be used to determine theoretical ICEP speed. Future work will focus on finalizing the DEP trapping experiments and comparing theoretical predictions with experimental data. Overall, this research strengthens the potential of ICEMs for label-free biomolecule detection and paves the way for further development and understanding of this technology.

Advisor: Wyatt Shields

Microscale robots, or microrobots, are a class of engineered particles capable of harvesting energy from their environment or external stimuli to change shape or propel. Given their ability to access hard-to-reach environments, there is significant interest in using these systems in medicine for sensing, medical imaging, or drug delivery. As there has been a large focus on proof-of-concept studies with using non-translatable materials that limits their use in applications such as delivering living cells, there is a need for studies that focus on optimizing design components for biomedical applications. As such, we have designed magnetic helical microparticles for a biocompatible cell therapy system that utilizes macrophage attachment to microrobots. Once attached, macrophages are transported to a target site via particle swimming; helices in particular enable transport through biological non-Newtonian fluids (e.g., blood, mucus) in order to direct a therapeutic cytokine response by carrier macrophages at hard-to-reach sites. We have established the design parameters of helical particles that promote optimal macrophage attachment and transport. By developing a comprehensive understanding of this cell therapy system, we aim to enable the microrobotic field to move towards clinical translation of medical microrobots. A tunable cell therapy system that can be applied to difficult to reach target regions has been demonstrated, ultimately reducing the limitations of adoptive cell transfers.

Advisor: Kristi Anseth

Current biofabrication methods to generate interior void geometries rely on the usage of sacrificial templating or targeted photodegradation using confocal microscopy. We present a multilayer hydrogel construct consisting of varying allyl sulfide crosslinks amenable to rapid orthogonal degradation and fabrication of interior geometries. Allyl sulfide crosslinkers employing both the 3-mercaptopropionate (alkyl) and 4-mercaptophenylacetamide (phenyl) functionality are first synthesized to form a hydrogel using the strain-promoted azide-alkyne cycloaddition (SPAAC). In-situ rheology is used for modulus characterization, and swollen degradation rheology is used to determine optimal degradation conditions with methyl 3-mercaptopropionate (3MMP) and 4-mercaptophenylacetic acid (4MPAA) thiols. Layered hydrogel constructs are then fabricated and sequentially patterned with inlet and outlet ports using 3MMP degradation solution and interior channels with 4MPAA degradation solution using lithographic irradiation. Intestinal stem cells are then seeded into the hydrogel constructs and are cultured to grow a confluent monolayer within the channels. This biofabrication method could support the culture of traditionally lumenized organoid models, promoting long-term viability, while also allowing for spatiotemporal alteration over the course of culture.

Advisor: Kayla Sprenger

Alzheimer's disease (AD) is a devastating and complex disease, with no singular genetic cause identified. Potential therapeutics fail to fully mitigate disease progression, let alone cure the disease. Several genetic risk factors have been associated with the development of AD, including mutations on a family of transmembrane microglial proteins called triggering receptor expressed on myeloid cells (TREM). The role of TREM2 in AD pathogenesis is highly studied, but there is little work regarding TREM1’s potential therapeutic impact. Previous studies identified that TREM1’s proinflammatory immunological function induces microglial phagocytosis of Aβ plaques, one of the hallmarks of AD, yet the mechanisms underlying TREM1 function in AD are unknown.

Understanding how both TREM1 and TREM2 conversely act in the presence of AD-based ligands may help clarify how they can be targeted for AD therapeutics. The application of molecular dynamics (MD) simulations allows for atomistic-level insights on the short-term behavior of protein-ligand interactions. Herein, we describe the use of the MD engine, GROMACS, to provide mechanistic insights on TREM1 and TREM2 behavior and structure due to the presence of AD-risk variants and AD-associated ligands. Comparisons of TREM1 and TREM2 simulations over the span of 1 microsecond, combined with alignment analyses of their amino acid sequences, revealed key differences in TREM1 and TREM2 behavior. Simulations of the homomeric form of TREM1 provided insight into the function of TREM1’s monomeric and homomeric forms, and how these structures impact ligand binding capabilities. Ultimately, uncovering these mechanisms will help elucidate how binding causes conformational changes and downstream signaling events that impact microglial function during disease pathology.

Advisor: Robert Davis

Droplet-based microfluidic systems are involved in numerous applications, including medical diagnostic devices, microreactors, lab-on-a-chip devices, and high-throughput cell screening. Imperative to designing these systems is an understanding of droplet dynamics, especially within straight rectangular microchannels and microchannel constrictions. Important parameters affecting droplet dynamics and microfluidic flow include the Reynolds number (Re), capillary number (Ca), viscosity ratio between drop/bulk phases (λ), channel geometry, and drop-to-channel size ratio. This investigation replicated microfluidic droplet behavior in an experimental flow cell, which has been scaled up macroscopically while preserving nondimensional parameter values and density-matching fluid phases. These experiments are compared to in-house numerical simulations, which employ a boundary-integral algorithm with a moving frame that reduces computational intensity for simulating viscous droplets in channels. Results indicate good qualitative and quantitative agreement between experiments and simulations. For straight channels, increasing Ca results in more deformable drops that move faster, while increasing λ results in more deformable drops but with slower motion. Increasing drop size interestingly has different effects on steady-state velocity depending on λ. High λ droplets go slower as drop size increases, while very low λ droplets speed up with increasing drop size. Increasing channel aspect ratios predictably result in slower-moving droplets due to a lower maximum velocity of the Boussinesq flow profile. We also found that increasing the drop size leads to more deformation. For constrictions, we found that decreasing the length of an abrupt constriction leads to different breakup patterns, with a longer constriction eliciting droplet breakup within the constriction and a shorter constriction causing drop breakup while exiting. We also found that increasing Ca and drop size increases break-up frequency, even causing large drops to have two breakup events in series. Our findings suggest that it is possible to accurately replicate microfluidic phenomena on a macroscopic scale, making it easier and more cost-effective to prototype microfluidic systems.

Advisor: Kayla Sprenger

Protein-polymer bioconjugates are a promising class of hybrid biomaterials formed from the covalent attachment of polymer molecules to a protein or peptide. However, presently there are few computational design tools available to predict their properties prior to in vitro experimentation. Computational approaches such as molecular dynamics (MD) simulations provide an excellent entry point for such studies, via the development of accurate molecular models for studying variations of the protein-polymer bioconjugate system.

Herein, we showcase the development of a streamlined computational framework to systematically model and analyze the molecular behavior of protein-polymer bioconjugates using MD. This methodological workflow utilizes a breadth of in silico tools such as Python, CGENFF, Gaussian, ChemDraw, VMD, and GROMACS. Specifically, we built and tested this framework on a model bioconjugate constructed from hen egg white lysozyme (1LYZ) and polyethylene glycol (PEG). Atomistic MD simulation of the bioconjugate in water revealed that the addition of polymer resulted in altered dynamics of the protein compared to a control simulation. The results of this research serve as a proof of concept for the expansion of this computational framework to higher complexity bioconjugate systems.

Advisor: Ankur Gupta

Supercapacitors are known for their high power density, but low energy density. Adding pore network geometries increases energy density by decreasing the mass of the device while increasing the surface area of the device where charge can be stored. The geometry of the pore network has varying affects on charging/ discharging time of these devices. These effects can be characterized by Nyquist plots of these pore networks. Generating these Nyquist plots requires a model that accurately describes how electrolytes flow into pore network geometries made in these electrodes. The project solves a pore charging model derived in (Henrique, Zuk, & Gupta, 2023) that describes the flow of electrolytes with electrochemical potential at any given position and time. The model is extended to pore networks by using current and electrochemical potential matching conditions at junctions: a location where pores connect. Some insights that can be gained from the model are charge transfer resistance in electrical double layer, charge transfer resistance in the pore bulk, characteristic charging time, and overall double layer capacitance. Some factors that are considered during analysis are:

1. Varying pore sizes at a given junction

2. Varying coordination number at a given junction

3. Making custom paths

Changing any of these factors have varying impacts on the nyquist plots for a given pore network. Fine tuning a multitude of geometric parameters with a pore network geometry can optimize characteristic charging time and charge density. The project is significant because it theoretically predicts key characteristics of a specific pore network geometry without having to experimentally create and test them and provides insight on what geometric features make an ideal pore network.