Direct-ink writing (DIW) 3D printing as a means of fabrication allows for the deposit of viscous fluids to create three-dimensional structures along a computer-guided print path. In this work, 3D-printed anisotropic structures were fabricated by customizing the direction of extruded filament, and the mechanical properties were studied. A polydimethylsiloxane (PDMS) ink mixture was prepared with optimal rheological properties for DIW printing. We tune the printing parameters, such as pressure and print height to reduce various defects such as stretching and overhanging of the extruded filament. A MATLAB script was developed to orient the print direction at any given angle within any convex polygon; print instructions were converted to G-Code using a Python script with the Mecode module. Planar rectangular structures were printed with filaments aligned along the horizontal, vertical, and diagonal directions. Printed structures were subjected to uniaxial tensile tests and stress-strain curves were obtained for quasistatic loading and unloading. Results suggest that the stiffness has a strong dependence on the print angle. Methods used here, specifically G-Code creation, will facilitate the fabrication of active morphing structures by DIW printing of Liquid Crystal Elastomers (LCEs) in the future.

Prostate cancer is the most commonly diagnosed cancer among men and one of the leading causes of cancer related death. While localized prostate cancer treatment involves surgical removal of the prostate, with high success, recurrent prostate tumors can lead to patient lethality. Current treatment of recurrent prostate cancer involves targeting the androgen receptor signaling axis. Unfortunately, some tumors develop resistance to these therapies demonstrating the need for new treatment strategies. Long-term treatment with the clinical anti-androgen drug enzalutamide often leads to treatment-resistance. As tumor cells adapt to anti-androgen therapy, they rewire their metabolism. This suggests that metabolic pathways may be critical for treatment-resistance and thus, they may be potential druggable targets. Therefore, we will assess alterations in cellular metabolism that accompany gain of treatment-resistance. Ultimately, we aim to identify potential pathways of exploitation to re-sensitize treatment-resistant prostate cancer cells to anti-androgens. Using metabolic profiling and western blotting, we are investigating the role of key metabolic pathways in prostate cancer treatment-resistance. Previously, we have identified altered metabolites as well as candidate proteins associated with treatment-resistance and are investigating their dynamics following exposure to anti-androgens. Identifying targetable metabolic pathways will lead to a deeper understanding of how cancer cells alter metabolism and help identify novel treatment strategies for treatment-resistant prostate cancer.

The COVID-19 pandemic has led to an influx of telehealth appointments, causing difficulty in assessing vital signs virtually. Our work focuses on remote photoplethysmography (R-PPG), the concept of measuring heart rate using color fluctuations in the face. Many different algorithms already exist with varying levels of success; however, one major gap is the lack of performance of these existing algorithms on darker skin tones, which, given the prevalence of cardiovascular disease in African American communities, creates a pressing issue. We have employed a VGG-style convolutional network known as DeepPhys to learn different spatial masks and increase robustness across the board. My work has focused on improving this existing network to increase medical accuracy by manipulating the structure of the network along with training parameters.

Microglia are the primary immune cells of the central nervous system (CNS). They play a critical role in responding to CNS injury/disease, inflammatory factor signaling, and removing damaged cells or debris through phagocytosis. In the deep brain, microglia vary across brain regions in morphology, cell density, and gene expression profiles. These differences contribute to region-specific immune response and vulnerability to aging/disease. Two regions – the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) – are particularly vulnerable to disease and exhibit changes in microglia early in the aging process. Gene expression profiles revealed that during this microglial response mitochondrial genes are altered, suggesting microglial mitochondria may play an important role in overall response to aging/disease in these vulnerable regions. This research project aims to understand how changes in microglial mitochondria regulate microglial function and why this is important for region-specific microglial immune response. We mined two RNA transcriptome datasets from published papers for known mitochondria-related genes to gain an understanding of microglial gene expression. Analysis of the first dataset of multiple cell types in the CNS provides information about how microglial mitochondria are functionally different from mitochondria in other cell types. Analysis of the second dataset of an Alzheimer’s disease mouse model compared to wildtype mice provides insight into how microglial mitochondria respond to disease progression. These analyses will build foundational information necessary to further research how microglial mitochondria influence response to aging and neurodegeneration.

Obesity is one of the most prevalent diseases worldwide. Increasing sedentary lifestyles exacerbate the obesity epidemic, thereby highlighting the need to understand its driving factors in order to develop viable treatments. The gut microbiome constitutes one contributing factor, specifically the production of short chain fatty acids by bacterial species upon digestion of complex carbohydrates. Increased production of short chain fatty acids can contribute to the development of excess adipose tissue, thus increasing the likelihood of obesity. Here we investigated how the Bacteroides species B. theta was able to digest inulin into the fatty acids acetate and propionate. We propose to examine whether inhibiting the rate limiting enzymes for each pathway significantly affects fatty acid production in vivo as well as overexpressing these enzymes in vitro. Currently, I have successfully cloned pyruvate carboxylase, the rate limiting enzyme in the propionate pathways; and have successfully conjugated the pyruvate carboxylase knockout gene into B. theta. Some steps that can be done moving forward would be to investigate these two fatty acid production pathways in the more genetically complicated Bacteroides species, B. ovatus, as it also plays a role in carbohydrate digestion within the intestine; and also to overexpress the two rate limiting enzymes with the intent of purifying the enzyme and conducting ATP assays to measure their kinetic activity. All of this work seeks to further understand the mechanism behind gut microbacterial metabolism which can have significant implications in further understanding and treating obesity.

DNA methylation, the process where a methyl (CH3) group is attached to cytosine, plays a key role in regulating gene expression. While the mechanism does not change the DNA sequence, it is highly involved in cellular processes like gene suppression and X-chromosome inactivation. DNA methylation is a highly regulated process, changing in response to environmental factors like exercise and eating habits. Thus, it can function as a biomarker to assess the condition or state of the body or bodily functions. However, it is still unclear if and how methylation patterns are connected to age. I utilized bisulfite sequencing data to determine methylation patterns associated with age by generating an epigenetic clock using the Least Absolute Shrinkage and Selection Operator Cross-Validation (LASSOCV) regression model. This estimates the relationship between a dependent variable (methylation) and an independent variable (age). This specific model adds a “penalty” term that lessens the variance between the tested data. This allows us to estimate the conditional expectation of the dependent variable. In other words, we can predict age using methylation data. I expect that the regression model will generate a strong linear correlation between age and methylation since aging results in the accumulation of a wide variety of molecular and cellular damage over time. I will validate the regression model using the Pearson correlation coefficient, which measures the relationship between two datasets. If the correlation test results prove to be statistically significant, this epigenetic clock can serve as a model for analyzing aging using methylation data.

Electron beam therapy (EBT) utilizes electrons to kill cancer cells with up to 60% less radiation affecting surrounding healthy tissue compared to photon-based radiation therapies. EBT typically uses cm-scale beams; this project focuses on using Panofsky quadrupoles to guide sub-millimeter beams in a flexible and changeable trajectory so that beam placement, and therefore treatment outcomes, are improved. Flexible Printed Circuit Boards (PCBs) were designed in a Panofsky quadrupole-like geometry, which consists of parallel copper traces that generate a quadrupolar magnetic field. The flexible material of the PCB allows for manipulation of electron beams in hard-to-reach areas for deeper tissue treatment. Joule heating of the PCBs was simulated in COMSOL Multiphysics, and the limiting current density extracted. The limiting current density was used in magnetostatic simulations to find the magnetic characteristics of these devices. Particle tracing simulations were then performed to investigate efficiency of guiding electrons at different curvatures of the flex-PCB. Flex PCBs were fabricated for testing and the thermal response of the PCBs was experimentally measured using a FLIR OnePro thermal camera.

Gaining a deeper understanding of adipocyte differentiation adds to our overall understanding of metabolic diseases and reveals new potential therapeutic targets for insulin resistance and diabetes. Epigenomic analysis of human and murine adipogenesis using 3T3-L1 and hASCs models identified an increase in expression of four mitochondrial transporters with unknown substrates. We chose SLC25a35 for further study because it’s an oxoacetate homolog with liver-specific expression pattern (Mikkelsen et.al 2010). Although Slc25a35 was induced during differentiation, the function impact of Slc25a35 in adipogenesis in white or brown adipose tissues remains to be explored. In our current study, we aim at validating previous observations of upregulation of Slc25a35 expression during adipogenesis and we plan to perform gain and loss of function assays to understand the role of Slc25a35 in white adipocyte differentiation. To better visualize trends in SLC25a35 upregulation, we optimized and later analyzed 3T3-L1 differentiation at different stages of adipogenesis. RT-PCR and Oil red staining were used to quantify and visualize differentiation. The data showed that SLC25a35 upregulation was consistent throughout the different stages of adipogenesis. This not only confirmed SLC25a35's potential role in adipogenesis, but also opened the way to starting a novel investigation of SLC25a35’s potential role in white adipocyte development and metabolic processes. Going forward, these findings are to be further investigated using gene manipulation assays in both the cell and animal models.

Acute lymphoblastic leukemia (ALL) affects more than 5,500 children in the U.S each year. Commonly, children that are obese are more likely to relapse after chemotherapy. Chimeric antigen receptor (CAR)-T cell therapy is a treatment that is being used with children who relapse from chemotherapy. In this treatment, the patient's own T-cells are engineered to recognize and kill ALL cells. However, we don’t know whether obesity impacts CAR-T treatment. In this study, we aim to understand whether obesity can affect the activation and metabolism of CAR-T cells, and if this might impair CAR-T therapy. We are exploring three potential ways in which obesity could impair CAR-T cells: fat cells release adenosine, which might inhibit CAR-T cells; fat cells could reduce the metabolic rate of nearby CAR-T cells; and high insulin levels might slow CAR-T metabolism. Our pilot experiment aimed to quantify CAR-T cells metabolism rate. When bound to CD-19 antigens on the ALL cells, CAR-T cells have a high metabolism rate. To measure metabolism, we used a SeaHorse cellular metabolism analyzer assay and substituted the activator CD-3 to CD-19 beads in order to mimic CAR-T treatment for ALL. We plated CAR-T cells with adenosine and insulin, and introduced the CD-19 beads at the same time to compare the metabolism rates. Although the data has not been analyzed yet, we did see activation with the CD-19 beads and CAR-T cells. In the future, we plan to study how adipocyte conditioned media can impact CAR-T activation and antigen escape.

Two forms of behavioral control our brain uses are goal-directed action, involving thinking about potential outcomes, and habit which are repetitive practices not involving such forethought. Overdependence on habits is characteristic of psychiatric disorders and can lead to maladaptive behaviors. While studies have found that stress is one factor for overreliance on habits, information regarding the neuronal mechanisms by which it influences habit formation is lacking. This limitation may serve as a possible barrier to the development of treatment options for substance use disorders. Thus, our overarching project goal is to uncover the neuronal mechanisms by which stress influences habit formation. The central amygdala (CeA) is a major nucleus for stress responsivity and the dorsomedial striatum (DMS) controls goal-directed learning. Recent evidence suggests there is a direct CeA→DMS inhibitory projection. We hypothesize that stress information is transported by this pathway to prevent goal-directed learning and bias towards habit. To test our hypothesis, we will monitor neural activity in CeA→DMS projections using fiber photometry calcium imaging in stress and unstressed mice as they learn an instrumental action and form habits. Our findings will help us comprehend habit formation in mice in both normal instances and stressful circumstances. Future extensions of our work involve understanding molecular and cellular mechanisms of stress-induced habit formation that will likely help with treatment development for substance use and stress-related disorders.

Cellular senescence is an indefinite cell cycle arrest that not only occurs as a result of differentiation or aging, but also can be induced through improper cell division. Cells that are induced into a senescent state may exhibit delayed phenotypes that encourage tumorigenic behavior. Since the extended presence of both oncogene activation and chemotherapeutic regimens may activate cell senescence, reverting cells from senescence can prevent cancer relapse, thus increasing the efficacy of common cancer therapies. To further investigate the relationship between senescence and cancer, our project plans to conduct a chemical screen to identify novel drugs within the FDA panel that will direct senescent cells to a proliferative, apoptotic, or quiescent state. We have formed a stable HeLa cell line which recognizes quiescence through nuclear fluorescence of a mVenus-p27k marker, and identifies senescence through increased nuclear size and lack of mVenus-p27k fluorescence.