The Role of B Cells and Autoantibody Production in Atherosclerotic Cardiovascular Disease

Understanding the formation of atherosclerotic plaques is a dire concern to human health and mitigating its impact is a large focus of cardiovascular research. These plaques form within the major arteries, occluding the flow of blood, and putting individuals at risk for major life-altering and life-threatening events. Arterial plaques are primarily composed of lipid-rich cellular deposits and emerge due to a failure of the routine immune system to foster proper clearance of lipids and debris. Recently, the role of the humoral immune system in plaque development has become better appreciated, with systemic antibody responses having both positive and negative implications for disease progression. These studies however did not determine the antigenic target of these antibodies. The work presented here answers this question to better determine the role and clinical potential of antibodies in cardiovascular disease. Over the course of this investigation, the repertoires of germinal center (GC) B cells from atherosclerotic ApoE-/- mice and IgD-CD38+ activated B cells from humans were analyzed. Heavy mutation of their V-gene segments was observed, suggesting antibody selection. B cell receptors were cloned from single cell isolates and antigen identification showed reactivity to various self-proteins. Many of these proteins are expressed within the diseased tissue. Serum antibody ELISA measurements highlighted 7 antigens with consistent antibody responses in multiple ApoE-/- mice. In humans, 5 antigens were identified that correlated with Framingham risk scores in males. Further study of two of these human antigens showed decades-long persistent antibody responses, suggesting individuals can become immunologically predisposed to cardiovascular disease. Furthermore, immunization with these antigens resulted in increased plaque burden in ApoE-/- mice, illustrating the potential for autoantibodies to promote plaque development. Together, this work identifies the production of autoreactive antibodies as a key target for understanding the development of cardiovascular disease

PORT RISK ASSESSMENT

Since the collapse of the Francis Scott Key Bridge in Baltimore, Maryland by the open ocean 984-foot container ship, Dali, Maryland and United States experienced a major disruption in commercial trade, commercial and personal traffic, and most importantly a lack of incident preparedness. This is not the first time a vessel has damaged or destroyed key infrastructure. But this devastating experience reinvigorated research about crucial infrastructure protection, risk assessment, and critical contingency planning in the path of major shipping ports. This research this will provide insight for indications, warning, and risk analysis to prepare the United States for further incidents such as the one most recently experienced

Integrating Tactile Sensing with Soft Robotics for Sensory Feedback in Upper-Limb Prosthesis

This research is motivated by the need for natural and bio-inspired prosthetic hands that integrate tactile sensing and compliant grasping, offering individuals with upper limb loss enhanced sensory feedback for activities of daily living. Current prosthetic technologies lack the combined dexterity, compliance, and sensory capabilities required to mimic the human hand effectively. By leveraging the inherent compliance, safety, and biomimicry potential of soft robotics, this work seeks to create transformative solutions for upper-limb prosthesis users. Soft robots provide safer and more dexterous handling of delicate objects, directly interface with human bodies, and reduce costs while improving human-robot interaction. Tactile sensing, proven critical for object manipulation, delivers sensory feedback to improve functionality in prosthetic applications. This dissertation introduces two innovations: a hybrid robotic hand and a smart fingertip, both integrating tactile sensing and soft robotics. The hybrid robotic hand combines soft robotic joints with a rigid endoskeleton and multilayered neuromorphic tactile sensing inspired by human physiology. This biomimetic prosthetic enables compliant grasping of objects with varied surface textures, weights, and compliance, achieving 99.69% object differentiation accuracy and 98.38% texture discrimination accuracy. Controlled via electromyography (EMG), it offers individuals with upper-limb loss the ability to manipulate objects with precision and detect surface textures during daily tasks. The second contribution, the Opti-Jam fingertip integrates granular jamming-based soft robotics with optical tactile sensing to achieve simultaneous sensing and grasping. This compliant fingertip manipulates small, delicate objects while reconstructing their 3D shapes with high accuracy. Using a photometric stereo imaging algorithm and a high-resolution camera, the Opti-Jam fingertip achieved 89% classification accuracy across 10 delicate objects, demonstrating its potential for tactile feedback in robotic and prosthetic applications. These innovations redefine the boundaries of prosthetic design, providing safer, more dexterous, and sensory-enabled solutions. They hold promise for improving the quality of life for individuals with upper-limb loss and advancing robotic manipulation capabilities in challenging environments

CHARACTERIZATION OF RWPE-1 PROSTATE ORGANOIDS: EFFECTS OF CULTURE MEDIUM COMPOSITION, SEEDING DENSITY, AND STROMAL CELL CO-CULTURE

As the most commonly diagnosed cancer in men in the United States, prostate cancer imposes a substantial public health burden. Despite the generally favorable prognosis following primary treatment, a subset of patients initially present with or eventually progress to the metastatic form of prostate cancer, which is associated with poor prognosis and remains therapeutically challenging. Although substantial progress has been made in prostate cancer research to date, several key aspects of the cancer’s life cycle, including tumorigenesis, progression, and therapeutic responses, remain to be fully elucidated. In order to study the earlier stages of tumorigenesis, an in vitro model of the healthy prostate epithelium is needed. Such a model has traditionally been challenging to construct, as normal prostate luminal cells are non-proliferative and thus exhibit poor growth in flask-based cell culture systems. Organoid cultures, in which cells with stem cell properties are seeded on bioactive hydrogels under appropriate conditions to induce differentiation into the desirable lineages and the formation of physiologically relevant structures, serve as a potential solution to this problem. In this work, we provide a comprehensive characterization of prostate organoids derived from the immortalized prostate epithelial cell line RWPE-1. We characterize RWPE-1 organoids grown under different culture conditions, such as the inclusion of serum and androgen in the medium, the initial seeding density, and co-culture with stromal cells. This study evaluates the potential of RWPE-1 prostate organoids to serve as an in vitro normal prostate epithelium model for studies aimed at determining factors that contribute to early prostate cancer development. This study also evaluates the physiological relevance of the RWPE-1 organoid model, describing key behaviors of the organoids, including acinar morphogenesis, branching morphogenesis, and extracellular matrix deposition under various culture conditions

Development of Neuronal Connectivity in the Murine Neocortex

In this thesis, I explore a fundamental question in developmental neurobiology: how is ordered neuronal connectivity established in the brain? I use the murine somatosensory cortex as a model to first explore the coordinated emergence of neuronal subtype-diversity and spatial organization. Then I address the molecular and cellular mechanisms underlying the development of cortical neuron-subtype specific axonal projection patterns. In the cerebral cortex, glutamatergic projection neurons are organized into layers based on their time of birth, as well as into areal domains in the plane perpendicular to the radial axis. In Chapter 2, I describe how the transcription factor Mef2c controls the acquisition of laminar and areal identities in post-mitotic cortical neurons during embryonic development. Chapter 3 focuses on postnatal functions of Mef2c, identifying it as one of the first regulators of long-range intracortical axonal projection targeting. I then describe functional manipulations that demonstrate a role for EphA-EphrinA signaling downstream of Mef2c in mediating homotopic targeting of callosal projections. These observations offer a first glimpse into the molecular logic of interhemispheric projection targeting in the mammalian neocortex. Chapters 4 and 5 focus on a critical aspect of specific intracortical connectivity: the formation of neuron-type specific patterns of intracortical axon collateral arbors. In Chapter 4, I introduce inducible sparse-labeling and genetic manipulation strategies developed in the Kolodkin laboratory to target developing neurons in cortical Layer 2/3, permitting the quantitative analysis of axonal collateral arbors at single neuron resolution. I then discuss their utility in uncovering novel cytoskeletal and cell-surface determinants of laminar specific Layer 2/3 cortical neuron axon branching. In Chapter 5, I detail how sparse-labeling of Layer 6 corticothalamic neurons, combined with brain clearing and lightsheet microscopy, reveals the developmental dynamics of intracortical arbor elaboration by this understudied neuron subtype. This work sets the stage for future studies of molecular mechanisms that dictate divergent patterns of axonal elaboration, within the same target region, by distinct classes of cortical neurons. My thesis work highlights the invariably pleiotropic nature of key regulators of animal development. This work also underscores the importance of temporally controlled, cell-type specific genetic access towards a comprehensive understanding of gene-function in development

SIGNIFICANT BARRIERS TO INCORPORATING ADVANCED NUCLEAR ENERGY INTO THE U.S. ENERGY PORTFOLIO

Advanced nuclear reactors, particularly small modular reactors and micro-reactors are a recognized aspect of meeting future global energy demand. To better understand the advanced nuclear industry in the United States, this paper seeks to define the top barriers facing the industry today and summarize the current state of each of the top three barriers in the United States. Through initial AI-supported research, literature review, and interviews with industry experts, significant barriers to advanced nuclear technology deployment in the United States are identified. With high levels of financial risk realized in new and near-memory nuclear power plant construction as well as new risks introduced through first-of-a-kind designs, identifying and securing sufficient funding for advanced nuclear technologies is a challenge. The infrastructure required to streamline construction and operation of advanced nuclear reactors, including built structures, experienced workforce, and a materials supply chain can only grow relative to the deployment of new designs. Despite efforts to streamline regulations and licensing, domestic policy continues to be a complicated and slow process for new technologies to comply with. The United States is poised to lead the world into a future of reliable, accessible, and clean energy through the new nuclear technologies developed by American scientists and engineers – IF barriers to deployment are addressed in time

MODELING ARTERIAL DISEASE AND INJURY USING ENGINEERED HUMAN ARTERIES-ON-A-CHIP

Arterial diseases affect the mechanical properties of blood vessels, which then impair their function via complex mechanisms. To develop and test effective treatments, microphysiological systems replicating the mechanics and function of human arteries are needed. Here, we establish an artery-on-a-chip (ARTOC) using vascular derivatives of human induced pluripotent stem cells (iPSCs) cultured with pulsatile flow on an electrospun fibrin hydrogel. ARTOCs have mature, laminated smooth muscle that expresses robust extracellular matrix and contractile proteins, contracts in response to pressure and vasoagonists, and exhibits tissue mechanics comparable to human small arteries. We monitor real-time distention and luminal pressure to inform computational fluidic modeling, and we can easily tune biomechanical cues using scaffold thickness and flow rate to promote survival and function of endothelial and smooth muscle cells in the ARTOC. To test the ARTOC as a disease modeling platform, we first use non-isogenic iPSC-derived smooth muscle cells (iSMCs) from a polycythemia patient, showing significantly altered cell phenotype and increased vessel wall stiffness compared to controls. We then test a novel isogenic disease model in ARTOCs using iPSCs CRISPR-edited with a Hutchinson-Gilford Progeria Syndrome LMNA mutation (LMNA G608G; LMNA-HGPS). LMNA-HGPS ARTOCs show extracellular matrix accumulation, medial layer loss, premature senescence, and loss of tissue elasticity and ductility. Finally, we use ionizing radiation as an injury model and show that exposure of ARTOCs to gamma rays and heavy ions induces a Senescence- Associated Secretory Phenotype that dysregulates transcript and protein expression, cytokine secretion, and tissue mechanics. We then partially rescue this phenotype in ARTOCs by targeting the AP-1 transcription factor with small molecule therapeutics. With this work, we establish the ARTOC as a translational platform to link cell phenotype and protein dysregulation to tissue mechanics and dysfunction in arterial diseases

Tuning Post-Translational Modifications in Mammalian Cells: Exploring Media Optimization and Gene Editing Strategies

The production of biologics has revolutionized the treatment of numerous diseases, offering targeted high-efficacy therapeutics for conditions ranging from cancer to autoimmune disorders. These biologics are typically produced using mammalian cells, requiring carefully optimized manufacturing processes to ensure high yields and product quality attributes that meet regulatory standards. However, the manufacturing of these complex therapeutics is challenged by the need for finely tuned cell culture conditions to ensure optimal product yield and quality. Small changes in cell culture conditions can significantly impact cell metabolism and protein quality attributes, potentially rendering the final product unsuitable for therapeutic use. This thesis looks to address the challenge of modulating product quality attributes by employing gene editing techniques to develop cell lines tailored for producing recombinant proteins with desirable quality attributes and refining cell culture media formulations to control the availability of key components in media. First, as a model case, we demonstrate the impact of product quality on therapeutic efficacy using butyrylcholinesterase (HuBChE), a human enzyme of significant value as a medical countermeasure against organophosphate poisoning. Given the scarcity of HuBChE in human plasma, recombinant production in mammalian cells is necessary to meet therapeutic demands. Through gene editing, we established a mammalian production platform that generates recombinant HuBChE with product quality attributes closely resembling the native human enzyme. Next, we explored the regulation of metal availability in cell culture media, focusing on copper as a critical trace element that affects cell growth and protein PTMs. Using copper-specific chelating agents, we developed copper-buffered systems to maintain stable and bioavailable copper levels throughout the culture process. This controlled environment mitigates the adverse effects of copper fluctuations, supporting consistent cell performance and ensuring predictable quality attributes in the produced therapeutics. Next, we seek to elucidate solubility interactions between different amino acids in aqueous solutions. By studying combinations of two amino acids in water, we identified negative, neutral, and positive interactions between amino acids. These experiments were also extended to see how amino acid solubilities were affected as a function of salt concentrations in solution as well, to ultimately guide more efficient cell growth media design

EXPLORATORY ANALYSIS OF COLLECTED BAT AND TREE DATA FROM DATE PALM SAP HARVESTING IN BANGLADESH FROM DECEMBER 2023 TO MARCH 2024

In Bangladesh, the primary route of human exposure to zoonotic Nipah virus is through the consumption of date palm sap that has been contaminated with saliva, urine, or feces from Pteropus bats. Since the first outbreak in 2001, there have been almost annual human infections, with more outbreaks occurring in colder winters. Identifying a relationship between sap sweetness and quantity may provide insight into the factors driving the rise in Nipah cases during colder months. Additionally, understanding the timing of Pteropus visitations is critical, as increased visitation rates create more opportunities for date palm sap contamination and, consequently, a higher risk of spillover events. In this study, we aimed to better understand what factors, including weather and the ecology of bats and date palm trees, affect the frequency of sap contamination by bats. Between December 2023 to May 2024, infrared cameras were set up on 20 date palm trees for eight nights of observation in Bangladesh. For both Pteropus and non-Pteropus bats, there were more visitations in colder months, December (np = 155, nnp = 954) and January (np = 521, nnp = 1289). Overall, non-Pteropus bats visited more frequently than Pteropus bats. Measurements of date palm sap yield and sweetness showed that more, but less sweet sap was produced at lower temperatures. A slight negative trend (p = 0.664) was observed between sap quantity and sweetness, while a slight positive trend was found between sap quantity and total bat visitations for both Pteropus (p < 0.001) and non-Pteropus (p < 0.001) species. Among non-Pteropus bats, there was a slight positive trend between visitations and sap sweetness, but no such trend was observed for Pteropus bats. Based on our study findings, colder weather produced more (p-value = 0.003) but less sweet (p = 0.35) sap. Additionally, the increase in bat visitations to date palm trees seem to be more driven by colder temperature rather than sap quantity or sweetness

SUBZERO PRESERVATION OF KIDNEY CELLS USING AN ANTIFREEZE PEPTOID-BASED PRESERVATION MEDIA

Background: Kidney transplant is an important procedure for end-stage renal failure. However, the high organ discard rate has contributed to the organ shortage crisis. This is partially driven by the limitation of the current organ preservation method involving storage in the University of Wisconsin (UW) preservation solution at 4°C. To address this gap, one possible solution is the usage of XT-ViVo (XTV), a preservation solution that utilizes antifreeze proteins to overcome the damaging effects of ice crystal formation of subzero preservation (below 0°C). Such low temperatures can increase organ preservation times by decreasing metabolic activity, though further investigation is needed to determine the efficacy of this new solution. Methods: Digested mouse kidney cells were stored at 4°C or –5°C in either complete media (CM), UW, or XTV for one of the following durations: 24H, 48H, 72H, 96H, or 120H. Cells were rewarmed following storage, and recovery of metabolic activity was assessed through the measurement of total ATP levels. All statistical analysis was performed at the significance level of 0.05. Results: Relative to the baseline, there was a significant decrease in metabolic activity recovery following storage at 4°C for 24H in CM, 96H in UW, and 72H in XTV, as well as at –5°C for 24H in CM, 96H in UW, and 96H in XTV. Cells stored in CM showed statistically significant lower relative metabolic activity than UW or XTV cells during all timepoints of storage at both 4°C and –5°C. UW cells showed significantly lower metabolic activity than XTV cells at –5°C at 48H (p < 0.0013), 72H (p < 0.0006), and 120H (0.013), but not for the 24H and 96H timepoints. No significant difference was found when comparing cells stored in UW at 4°C to XTV at 4°C, UW at 4°C to UW at –5°C, or UW at 4°C to XTV at –5°C. Conclusions: Following storage, cells showed a delay in the decrease of metabolic activity recovery in UW or XTV than compared to CM. Storage in XTV at –5°C showed significantly higher recovery of metabolic activity than UW at –5°C, and similar recovery to UW at 4°C