The liver is a vital organ that secrets waste, metabolizes drugs, and breaks down blood to provide nutrients to the rest of the body. The liver is also the sole organ in the human body capable of regenerating after transplant surgery or treatment of cirrhosis, unfortunately the human body is not always efficient in the regenerative process, especially in the case of severe damage to the tissue. My research is centered around the use of 3D bio-printers to replicate the intricate biometric structures within the liver for artificial tissue transplantation. I focus on using CAD design through the SolidWorks program to design such intricate structures while simultaneously testing the resolution of the printer and its ability to print microscale channels within the structures. Additionally, I have focused on a heat producing circuit to promote biocompatibility and the efficiency of the printer during the printing process. Heat is a natural stimulus for gene expression and regulated temperature is vital for living cells. Implementing a component such as an induction heater and controlling its applied energy may be able to provide the necessary electrical current to heat cells at livable temperatures during the printing process and promote gene expression, without denaturing the cell. If 3D printers can effectively and efficiently be used to replicate microscale structures, scientist will be able to print artifical livers at low costs and fast rates for patients who are suffering from liver diseases. 

Microphysiological systems (MPS) or Organs-on-a-chip (OOC) are novel in vitro models being used to understand human disease and support drug development. Using a Kidney MPS, we are investigating potential causal factors, Ochratoxin A (OTA) and heat stress, for a disease state termed Chronic Kidney Disease of Unknown Etiology (CKDu). Thus far, we have conducted experiments treating human primary proximal tubule cells (PTECs) with OTA +/- transient heat stress (39 degrees for 24 hours) in conventional 2D cultures and MPS to understand the synergistic nephrotoxic effects of the two treatments. We found that gene expression of CDKN1A is increased in cells treated with OTA +/- heat. CDKN1A codes for the protein p21, which inhibits cyclin dependent kinases responsible for regulating the cell cycle. Chronic cell cycle arrest in G1/S and G2/M checkpoints induced by high levels of p21 can lead to injury and subsequent development of CKD. To further probe this finding, we are developing a protocol to increase the efficiency of obtaining sections of the kidney tubule from our MPS to be used for the purpose of immunocytochemistry (ICC) staining. A reagent called Optimal Cutting Temperature (OCT) solidifies the tissue into a block so that it can be cut using a cryostat. Then, we can stain OTA and heat-treated kidney tubule cross sections for Ki67, a marker for cell proliferation, and p21 using ICC. The expected results should demonstrate a higher expression of p21 in the cytoplasm and less staining of Ki67 in OTA +/- heat treated tissue in the nucleus. We have optimized antibody concentrations for staining and observed appropriate localization of Ki67 in the nucleus and p21 in the cytoplasm in 2D cell culture samples. By understanding OTA’s mechanism of toxicity, preventative interventions can be implemented to mitigate CDKu risk in impacted regions around the world.

Genetic diseases affecting the skeletal system present with a wide range of symptoms and phenotypes that make diagnosis and treatment difficult. One such disease, Bruck Syndrome, is a rare form of osteogenesis imperfecta, or brittle bone disease, and has been associated with the PLOD2 gene in human genome-wide association studies. Due to advances in 3D phenotyping and CRISPR gene editing, zebrafish have become a good model system for assessing the genetic contributions to skeletal disease. While the phenotype of the homologous zebrafish gene (plod2) has been elucidated in the axial skeleton, the effects on the craniofacial skeleton have not yet been quantified. The objective of this study was to quantify how plod2 crispant zebrafish skulls differ from their wildtype siblings. We used the open-source 3DSlicer software to place 21 traditional landmarks and 372 pseudolandmarks on micro-CT scans of 22 zebrafish (11 crispant and 11 wildtype), and used geometric morphometric methods implemented in R to analyze the complex shape differences between the two groups. Preliminary results show that plod2 crispants have skulls that are wider and shorter than their wildtype siblings. The plod2 crispant fish as a group exhibit more variation than wildtype fish across the morphospace, which may be consistent with the broad array of phenotypes associated with PLOD2 mutations and Bruck Syndrome in clinical observations. Using a zebrafish model and 3D data pipeline could enable rapid screening to investigate the causes of other human skeletal diseases. In addition to these clinical implications, this project contributes to an understanding of the genotype to phenotype pipeline, one of the most active areas of biological inquiry.

Disability Services (DS) are the most common strategy for ensuring that students with disabilities have access to higher education. These offices provide accommodations for students that can apply to both physical (e.g., building) and digital (e.g., course content) accessibility. Prior work has shown that the access needs of disabled students are not always successfully provided, and if they are, they are not always accomplished in a timely manner. Consequently, there may be dissonance between when professors believe an accommodation has been fulfilled (e.g., when a PDF they posted has been made accessible by DS) and when it is actually completed. While prior work has documented the accessibility challenges faced on university campuses and with disability services, work has not yet focused on how to better support DS, student, and faculty interactions and communication. This study aims to do so by investigating how communication currently flows, the issues that arise within interactions and implementations, and whether technical solutions can better support interaction for the accommodation fulfillment process.

Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza virus. HA is responsible for binding to sialic acid receptors on host cells and mediating membrane fusion. Because it is involved in these essential functions, HA is targeted by neutralizing antibodies (nAb) against the influenza virus. When the antigen-binding fragment (Fab) domain of a nAb binds to HA, it can neutralize infectivity either by blocking receptor binding of HA, by inhibiting conformational changes required for membrane fusion, or by disrupting HA structure. All of these mechanisms can prohibit the virus from entering the cell. In a hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiment, we attempted to map the epitope-- the site on the antigen where the antibody binds-- of the 5E10 neutralizing antibody on the HA from the 2011 Victoria H3 influenza strain. A deuterium exchange time course was performed and HDX-MS was used to determine the rate at which deuterium is exchanged for hydrogen on specific peptide segments of the HA protein backbone. We hypothesized that the epitope would be an area that becomes more protected, meaning that it would take up less deuterium, due to local structural ordering from the bound Fab fragment. Through our research, we have identified two potential epitope sites. Identifying the 5E10 epitope on H3 HA will tell us about sites of vulnerability that can be targeted by the immune system. Furthermore, it will help us understand the way the antibody can neutralize the virus as well as predict if the antibody would be able to neutralize other strains of flu based on comparisons of amino acid conservation at the epitope. This information can be used by other scientists to design vaccines that direct an immune response to a specific epitope.

Genome Wide Association Studies enable researchers to identify which genes are associated with human diseases. Once genes are identified, gene editing of zebrafish models allows researchers to further examine the link between genotype and phenotype, with the long-term goal of treatment development. One such gene is MEOX1, which encodes for transcription factor Mesenchyme Homeobox 1. MEOX1 mutations in humans have been associated with Klippel-Feil syndrome, a congenital disorder with the most frequent symptoms being a shortened neck caused by the fusion of cervical vertebrae, leading to a decreased range of motion. Previous studies have established that zebrafish with loss-of-function meox1 mutations present with similar phenotypes in the axial skeleton. However, fusion of the vertebral column could also impact other aspects of fish morphology. The goal of this study is to test if a semi-automated screening tool can be used to quantify craniofacial variations in meox1 crispant zebrafish. Here, I utilized micro-CT scans of CRISPR-modified zebrafish with meox1 mutations (n=12) and compared them to wild type zebrafish (N=12) from the same clutch. I used 3D Slicer to manually landmark 23 major anatomical points on each individual wildtype and crispant zebrafish. I then used a semi-automated process to distribute pseudolandmarks on the surface of each zebrafish. Anatomical differences between the groups were quantified using a geometric morphometrics approach. Preliminary results show that meox1 mutations are associated with a wider posterior section of the skull and a shorter skull length. There were also differences found in the degree of asymmetry between groups. This last result in particular aligns with previous human studies of Klippel-Feil syndrome. The findings from this investigation are important for the understanding of how diseases from meox1 mutations present clinically as well as the testing of a semiautomated pipeline that will be used as a screening tool for crispant zebrafish.

Advances in molecular techniques have allowed for genome-wide studies to examine which genes are associated with many different human diseases, including those of the skeletal system. In one such study, mutations in the human SOST gene were linked to the skeletal disease Sclerosteosis 1, which presents as hyperostosis in the skull and long bones of the axial skeleton. Functional SOST genes influence the production of sclerostin, a protein which inhibits osteoblastic bone formation. The homologous gene in zebrafish is hypothesized to have a similar function based on ongoing work examining the axial skeleton in mutant zebrafish. Our goal for the current study is to test how the zebrafish cranial skeleton is affected by the SOST gene. I used the open-source 3DSlicer software to place landmarks on micro-CT scans of 27 zebrafish (9 SOST mutants, 9 SOST heterozygotes, and 9 wildtype fish) from the same clutch. I also used 3DSlicer to generate digital models of the cranial skeleton as well as to place 308 pseudolandmark points on the models. From here, I used geometric morphometric methods implemented in R to analyze the complex shape differences between the three groups. Preliminary results suggest that SOST mutants have narrower posterior cranial skeletons than heterozygous or wildtype fish, and that groups may vary in their degree of cranial asymmetry. In addition to quantifying the effect of SOST on zebrafish cranial morphology, this study is part of a larger project to establish a baseline craniofacial analysis method, and create a screening tool for examining genotype-phenotype relationships in genes associated with human skeletal diseases within zebrafish models.

Pitch perception in noise is a critical skill for children to have because it allows them to enjoy music, understand speech, communicate, and learn in noisy real-world environments like playgrounds and classrooms. To date, little is known about how well school-aged children can discriminate pitch in background noise. In this study, we evaluated pitch perception in normal hearing children between 8 and 11 years of age (n=32) and adults (n=16) by administering a remote 3-Alternative Forced-Choice (3-AFC) adaptive pitch discrimination task on MATLAB Web Apps while monitoring participants’ behavior over Zoom. The study consisted of three conditions, each with three runs: (1) 200 Hz pure tone in quiet, (2) Resolved harmonics 2 to 9 of a 200 Hz fundamental frequency (F0) in noise and (3) Unresolved harmonics 12 to 19 of a 200 F0 in noise. Musical training and tonal language experience were documented in all participants. My role in this study included participant recruitment, data acquisition, analysis, and manuscript preparation. Preliminary analyses suggest that children perform worse in the condition with the higher numbered harmonics (unresolved condition), which is also more difficult for adult listeners. Moreover, improvement in all three conditions is observed with age, with the older children discriminating pitch as well as adults for both pure and complex tones. This study provides important insight into the developmental trajectory of pitch perception. In the next phase of analysis, we will investigate the role of music experience on children’s perception of pitch in noise.