Biology Department Theses and Dissertations

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Effect of Elf3 Knockout on Zebrafish Early Development
(2025-10) Manikandan, Priyadharshini; Marrs, James A.; Balakrishnan, Lata; Perrin, Benjamin J.; Li, Jiliang; Leung, Yuk Fai
Elf3 (E74 like ETS transcription factor 3) is a member of the ETS (erythroblast 26 transformation-specific) family of transcription factors that regulate gene expression through a conserved by DNA-binding domain. Disruptions in elf3 in zebrafish have been linked to abnormalities such as craniofacial cartilage malformation and impaired notochord development, particularly in the context of prenatal alcohol exposure, recapitulating effects of fetal alcohol spectrum disorder (FASD). Previous morpholino oligonucleotide (MO) studies showed that elf3 is required for epidermal, mesenchymal, and neural tissue development. This study hypothesizes that knockout of elf3 in zebrafish embryos disrupts embryogenesis through dysregulation of extracellular matrix (ECM) remodeling proteins. To test this, a zebrafish elf3 mutant line was generated using CRISPR-Cas9 mutagenesis. Heterozygous adult zebrafish displayed skeletal abnormalities indicating that even partial loss of elf3 function produces noticeable structural abnormalities. Homozygous elf3-/- embryos displayed morphogenesis defects and cell death beginning around 10 hours post fertilization (hpf), most prominently within the head anlagen or anterior neural plate, resulting in embryonic lethality. Molecular analysis revealed significant dysregulation of ECM-related transcripts in elf3-/- embryos. elf3+/- embryos also showed phenotypic consequences, including delayed growth during early development and the emergence of a chordoma-like phenotype in later stages. Chordomas are rare tumors characterized by the invasion of notochordal tissue into surrounding structures. Tissue architecture and cellular changes associated with the chordoma-like phenotype were examined to assess tissue organization and adhesion properties. Similar craniofacial and notochordal defects have been reported previously elf3 morpholino knockdown experiments, supporting the validity of the mutant phenotypes. Together, these results suggest that reduced elf3 expression alters the regulation of ECM proteins during epithelial to mesenchymal (EMT) transition, including matrix metalloproteases (Mmps) and Snail2, which are critical for morphogenesis and maintenance of tissue structure. These findings highlight elf3 as a critical regulator of zebrafish embryogenesis, neural crest cell migration, and tissue morphogenesis, with implications for congenital disorders such as FASD and notochordrelated diseases, including cancer. This elf3 mutant line thus provides a valuable model to further investigate molecular pathways regulated by elf3 and its relevance to vertebrate development and disease.
Understanding the Role of Espin-Like (ESPNL) in Auditory Stereocilia Development and Maintenance
(2025-08) Hawbaker, Katelin M.; Perrin, Benjamin; Cummins, Theodore; Corson, Timothy; Mastracci, Teresa; Walsh, Susan
Hair cells are the fundamental unit of hearing, as they transform the mechanical force of sound waves into electrical signals through a process called mechanotransduction (MET). Stereocilia are large, actin based, microvilli-like “hairs” that arise from the apical surface of the cell. Approximately 50-150 stereocilia are organized into a bundle made by three rows of graded heights, resembling a staircase, the tallest being called row 1 and the shortest row 3. The stereocilia are connected from the tip of the shorter stereocilium to the shaft of the taller stereocilium by a connection known as a tip-link. When acted upon by sound, stereocilia deflect toward row 1, allowing tip-links to pull open the gated MET channels at the tips of row 2 and 3. The dimensions of stereocilia are tightly regulated by actin binding proteins to operate in this highly precise manner. When stereocilia dimensions become dysregulated, the bundle cannot be efficiently stimulated by sound and hearing loss occurs. Dysregulation is exacerbated by age, as damage accumulates over the lifespan. A problem humans face is that mammalian hair cells are terminally differentiated and must last many decades. As the world’s population is living longer and industrialization is increasing the sound levels of daily life, further research is necessary to develop stereocilia loss prevention interventions and/or methods for hair cell regeneration. Mechanotransduction is not only the primary function of hair cells but also an influence on actin regulation in stereocilia development, maintenance, and seemingly hair cell viability. In this work we study two similar actin binding protein families, ESPN and ESPNL, to understand their roles in stereocilia development. Our EspnlD20 /D20 mutant exhibited a common age-related hearing loss phenotype characterized by progressive row 2 dysregulation and subsequent loss. We also identified a novel isoform of ESPNL that responds to MET. Lastly, we attempted to further study progressive transducing stereocilia degeneration and its effects on hair cell health through a harmonin mutant which exhibits weakened tip-link complexes. This work furthers our understanding of stereocilia development and moves us closer to a model to analyze the critical role MET plays in auditory cell health.
Keap1/Nrf2 Signaling in Aged Bone
(2025-08) Sandy, Brandon; Li, Jiliang; Dai, Guoli; Roper, Randall
Bone health is a critical element in mammalian quality of life. The Keap1/Nrf2 signaling pathway has recently been observed to be instrumental in a multitude of regulatory processes pertaining to cytoprotective gene expression, with growing evidence of its role in bone metabolism. Nrf2, activated through Keap1 deletion, is hypothesized to enhance bone quality by modulating osteoblastic, osteoclastic, and osteocytic activity. In this study, impact on bone metabolism is observed via osteoblast and osteocyte activity managed by Keap1/Nrf2 inactivation and activation respectively. Here we aimed to test if Nrf2 activation positively affects bone strength and quality by way of increased bone metabolism and as a consequence of mechanical loading. The experimental results show that osteocyte and, subsequently, osteoblast activity and operation are driven by Nrf2 actuation and reaction to the mechanical loading of bone. It is found here that the Keap1/Nrf2 pathway executes an important function in bone metabolism and increasing bone mass, strength, and quality. It is expected that this study can and will be used in further experiments of other cell types to examine the impact of the Keap1/Nrf2 pathway. Though there is more research to be performed, the Keap1/Nrf2 axis and its ability to influence bone metabolism shows promise for the potential therapeutic care and forestallment of osteoporosis in a clinical setting.
Sensory Neuron Development and Skeletal Deficits in Down Syndrome
(2025-07) Thomas, Jared R.; Roper, Randall J.; Wallace, Joseph M.; Li, Jiliang; Hardy, Tabitha M.; Allen, Matthew R.
All individuals with Down syndrome (DS) present with low bone mineral density (BMD) and neurodevelopmental delays. Altered skeletal development and homeostasis result in skeletal abnormalities observed in humans with DS and DS mouse models increasing the risk of early onset osteoporosis. Exercise or physical activity increases BMD and decreases the risk of osteoporosis. Reduced sensory nerve function and decreased innervation leads to impaired skeletal development and decreased skeletal adaptation to mechanical loads. We studied the impact of triplicated Hsa-21 orthologues, particularly Dyrk1a, on sensory neuron development, function and skeletal innervation, and skeletal adaptation to mechanical loads in DS-related skeletal phenotypes. We hypothesized that i) triplication of Dyrk1a disrupts communication between bone and peripheral sensory neurons during development leading in impaired skeletal development and adaptation and ii) normalization of Dyrk1a will rescue skeletal deficits associated with DS by restoring morphological and functional deficits of sensory neurons. Neuropeptide calcitonin gene-related peptide (CGRP), found in sensory nerve fibers innervating bone and the periosteum, is released in response to mechanical stimulation and promotes bone formation. Ts65Dn male mice (at 6-weeks) released significantly more CGRP in response to capsaicin stimulation compared to controls. Both Ts65Dn and Dp1Tyb male mice (16-weeks) had decreased sensory neurons in L4 dorsal root ganglion (DRG). There were no differences in sensory or sympathetic innervation in Ts65Dn, while Dp1Tyb male mice had slightly increased sympathetic innervation of the distal femur. Mechanical stimulation was performed in male and female Dp1Tyb and control mice with expected and reduced Dyrk1a copy number in sensory neurons. Loading the left tibiae at 1500 με, but not 1800 με for 4 weeks improved trabecular and cortical architecture in male and female mice across all genotypes. These data suggest that triplicated Hsa-21 orthologues impair sensory neuron development and function, potentially disrupting communication between bone and the sensory nervous system during skeletal development and adaptation. Loading may improve bone architecture in DS mice, but the limited response highlights the need to find optimal strain levels to produce an anabolic response and identify Hsa-21 genes that affect skeletal development.
Behavioral and Developmental Effects of Lead Acetate Exposure in Embryonic Zebrafish
(2025-07) Gyamfi, Angela; Marrs , James; Cummins, Theodore R.; Belecky-Adams, Teri
Despite lead’s ban several decades ago, it remains a public health threat due to its persistent nature. Zebrafish are highly effective for modeling several disorders, particularly those affecting neurological and cognitive functions, and are well-suited for assessing the impact of environmental toxins like lead. This study aimed to investigate the behavioral and developmental effects of embryonic lead exposure using the zebrafish (AB strain) model system. Embryos were exposed to lead acetate (PbAc) at concentrations ranging from 0.3 to 0.7 µg/ml using an exposure window of 6 to 48 hours postfertilization. Lead acetate exposure induced dose-dependent teratogenic effects, such as spinal kinks, spinal curvatures, beaky mouths and uninflated swim bladder. Behavioral assessments conducted at 3- and 7-days post fertilization (dpf) revealed significant behavioral alterations. At 3 dpf, spontaneous circle swimming behavior suspected to be seizure-like was observed in lead-exposed group and was more pronounced under light conditions in a dose-dependent manner. A calcium biosensor assay validated this seizure-like behavior, revealing a general increase in fluorescence intensities in lead-treated groups compared to controls. Electrophysiological recordings confirmed that larvae exhibiting circle swimming behavior had heightened neural activity, indicating a potential seizure-like phenotype driven by lead exposure. At 7 dpf optomotor response assay, suggested altered response in the optomotor response of lead-exposed larvae and light/ dark locomotor testing showed reduced motor activity levels, particularly in the dark. Thigmotaxis assays did not show any significance in anxiety-like behaviors between control and lead treated groups. Overall, our findings suggest that early-life lead exposure leads to morphological defects, behavioral alterations and seizure susceptibility, enhancing our understanding of lead’s neurotoxic potential during early development.
Low Intensity UVB as an Immunostimulant in the Yellow Mealworm Tenebrio molitor (Coleoptera: Tenebrionidae)
(2025-03) Smith, Michael Bartlett; Picard, Christine J.; Barrett, Meghan R.; Tomberlin, Jeffery K.
As the demand for sustainable proteins grows, insect farming has emerged as a promising solution. The yellow mealworm (Tenebrio molitor), among other commercially farmed species, presents a variety of environmental and nutritional advantages over conventional livestock. However, mealworm colonies are vulnerable to pathogens, necessitating novel strategies for enhancing their immune resilience. Here, we employed a transcriptomic approach to investigate the immunostimulatory effects of low-intensity ultraviolet B (UVB) irradiation on mealworm larvae. Approximately 3-week-old mealworms were exposed to UVB light for 14 days, followed by fungal challenge (Beauveria bassiana). UVB irradiation did not confer significant differences in survival at 14 days post-challenge and had a negative effect on larval weight. Genome-wide differential gene expression analysis revealed significant dysregulation of Toll pathway transcripts, including those encoding three antimicrobial peptides (AMPs), immediately after UVB exposure. Targeted gene expression analysis validated the upregulation of tenecin 4 via a response in the fat body at one and 14 days after the initiation of UVB irradiation. This study indicates that while UVB irradiation positively modulates AMP production in mealworms, its impact on enhancing resistance to pathogens remains uncertain. These observations lay the groundwork for additional studies exploring the effects of UVB irradiation on insect immune systems and its potential implications for insect farming.
Behavioral and Molecular Analysis of a Dyrk1a Functional Reduction in the Ts65Dn Mouse Model of Down Syndrome
(2025-05) Duerst, Alyssa Nicole; Roper, Randall; Marrs, Kathleen; Perrin, Benjamin
Children with Down syndrome (DS) experience learning, physical, and motor delays early in life. Overexpression of Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a gene located on human chromosome 21 (Hsa 21), is in triplicate in DS and is thought to be a contributing cause of some neurodevelopmental delays seen in DS. The Ts65Dn mouse model of DS similarly contains triplication of Dyrk1a, along with 90 other orthologous Hsa 21 genes. Ts65Dn pups have shown to have significant DYRK1A protein overexpression on postnatal day 6 (P6) in the hippocampus, cerebral cortex, and cerebellum. We hypothesize that reduction of DYRK1A prior to this timepoint of overexpression will improve neurodevelopmental phenotypes in these pups. Dyrk1a was reduced from three to two copies in Ts65Dn pups at P0 to correspond with a second trimester DYRK1A reduction in developing fetuses with DS. Physical and behavioral assessments were performed on these pups from P3-P21. Ts65Dn pups showed physical, motor, and behavioral deficits as compared to euploid pups. However, Dyrk1a reduction in Ts65Dn pups did not result in vast behavioral improvements. This may be correlated with the failure of Dyrk1a functional reduction to normalize DYRK1A protein and mRNA levels seen in the P21 hippocampus, cerebral cortex, and cerebellum of these animals. Although no overall behavioral improvements were seen in the neonatal period analyzed, improvements in phenotypes may emerge later in life, as Dyrk1a functional reduction did improve Ts65Dn male pups’ emergence of running. A combination of different overexpressed protein targets in DS or an earlier Dyrk1a reduction may be ways to better explore DYRK1A reduction effects in early life.
Covariate and Co-Structural Influences on Human Facial Morphology: Decoding the Structural Blueprint Behind Facial Shape
(2025-05) Wilke, Franziska; Walsh, Susan; Roper, Randall; Balakrishnan, Lata; Wilson, Jeremy; Wetherill, Leah; Lapish, Christopher
The human face is one of the most intricate yet informative structures, serving as a key identifier in forensic investigations, an indicator of medical conditions, and a crucial factor in surgical planning. Over the past few decades, significant effort has been dedicated to understanding the genetic architecture underlying facial morphology. However, this focus often overlooks the substantial influence of covariates, such as biogeographic ancestry, and structural components like the skull. While these factors are acknowledged, their anthropological is frequently reduced to statistical models that bypass anatomical considerations. Furthermore, many of the complex models developed to reconstruct facial shape are not yet practically applicable. This dissertation addresses these gaps by investigating how regional, rather than just global, biogeographic ancestry influences facial morphology and whether genetic models of biogeographic ancestry align with phenotypic expression. Our findings indicate that broad categorizations such as “European” do not fully capture ancestral variation, yet incorporating too many genetic principal components risks overcorrection. To address this, we introduce a novel standardized, phenotype-based approach using consensus faces. Additionally, we present a validated, standardized method for efficiently masking and analyzing the human skull using over 6,000 quasi-landmarks. This methodology is further expanded to include a facial mask, where both the skull and face are intrinsically linked through anatomically corresponding quasi-landmarks. This innovation enables the simultaneous study of facial soft tissue thickness (FSTT), cranial shape, and facial morphology in a computationally efficient manner that has not been previously achieved. The use of correspondence masks permits modeling of the relationship between the skull and face, facilitating craniofacial reconstruction and laying the foundation for an open-source FSTT and facial measurement database. Ultimately, this dissertation explores standardization, global applicability, with the aim of facilitating real-world applications of a scientifically transparent computational approach to facial image projection from skeletal remains. By integrating genetic, anthropological, and statistical approaches, it describes a streamlined methodology that can harness structural knowledge of facial variation to develop practical tools useful in forensic and medical applications. Moreover, it highlights the need for global large-scale collaborative research to further advance this field on both fundamental science and applied levels.
Understanding The Role Of Transforming Growth Factor-β Activated Kinase-1 During Inflammation in Diabetic Retinopathy
(2024-12) Poudel, Umanga; Belecky-Adams, Teri; Slayback-Barry, Denise; Dai, Guoli
The role of microglial TAK1 in inflammation during diabetic retinopathy is not well understood but is known to affect the health of neurovascular units through the activation of inflammatory factors and cytokines. Loss in barrier activity in the neurovascular unit leads to leukocyte infiltration leading to the activation of microglia. The role of TAK1 was investigated in murine retinal microglial cultures in vitro by looking into the activation of TAK1 and downstream MAP kinase p38 activated by the treatment of pro-inflammatory factors; LPS and TNF-α, through western blotting. Treatment with LPS showed negative regulation of p38 by TAK1 in vitro in cells treated in non-diabetic conditions. Under conditions that mimicked hyperglycemia, TAK1 activation was not seen in murine retinal microglia treated with pro-inflammatory factor; LPS 20ng/ml or TNF-α 50pg/ml for 1hr. Moreover, there were no significant changes in murine retinal microglia treated with TAK1i subsequently after LPS 20ng/ml treatment for 1hr. On the contrary, the downstream MAPK p38 activation was not seen in murine retinal microglia treated in conditions that mimicked hyperglycemia while it showed significant response to changes in osmolarity. The in vivo study in retinal flat mounts showed TMEM119 as a potential candidate that could drive the td-Tomato expression in murine retinal microglia as 95% of the cells were colabelled with murine retinal microglia td-Tomato driven by TMEM119 and IBA1 which was further supported by the conditional TAK1 knockout experiment which showed 95% of the cells had TAK1 knocked out from the murine retinal microglia, confirming TMEM119 as excellent candidate for protein expression in murine retinal microglia.
Utility and Efficacy of Human Tissue Xenograft in Bone Healing
(2024-12) Ganguly, Upasana; Kacena , Melissa A.; Li, Jiliang; Roper, Randall J.
Connective Tissue Matrix (CTM) allografts are structural implants intended to supplement or replace damaged or inadequate tissues. Generated from human amnion, chorion, and umbilical elements, these allografts contain structural proteins as well as tissue-specific and –agnostic growth factors. In these studies, we examined the efficacy of CTM allografts to improve bone healing and whether CTM alters injury-associated pain behaviors in a pre-clinical animal model. The study involved two surgery models: a surgically induced femoral fracture model and a segmental bone defect (SBD) model. In the femoral fracture model, 12-week-old male C57BL/6J mice were divided into four groups: Saline Control, CTM Membrane, CTM Paste, and CTM Membrane + Paste. For the SBD model, the same age strain of male mice was divided into five groups: Saline Control, BMP-2, CTM Paste, CTM Membrane, and CTM Membrane + Paste. Complete blood count analysis demonstrated no significant alterations in blood cell numbers due to CTM treatments in the femoral fracture model. Despite the presence of various pro-inflammatory cytokines and growth factors in CTM, treated mice showed no significant increase in pain-associated behaviors post-fracture. Bioluminescence imaging demonstrated a significant reduction in inflammation for the CTM Membrane + Paste group on day 14 post-surgery. µCT analysis indicated significant improvement in the mineralized callus area for the CTM Membrane + Paste group when compared to all the other groups in the surgically induced femoral fracture model. Histomorphometric analysis further supported these findings, revealing an increased bone percentage in the callus area for CTM Membrane + Paste group compared to Saline controls in the surgical fracture model. However, biomechanical testing indicated no significant differences between Saline Control and the CTM treated groups. Interestingly, an increasing trend was observed in stiffness and toughness for the CTM Membrane group. Surgical fracture, being a simpler model of fracture, and heal without intervention with time. Hence, that led us to test our hypothesis that CTM products can promote bone healing in a more difficult model, of SBD mice. Long-term evaluations at 13 weeks for the SBD model showed that the BMP-2 group significantly increased the callus area compared to saline controls, with increasing trends observed in CTM-treated groups. Biomechanical testing demonstrated an increasing trend in the ultimate torque in all the groups when compared to Saline Control. Overall, the CTM Membrane + Paste group demonstrated promising potential in enhancing bone healing and reducing inflammation in the surgical fracture model, without triggering additional pain behaviors. The findings from the SBD model revealed that CTM Membrane + Paste has similar impact on bone healing, as observed in the surgical fracture model. Specifically, having a larger callus area as compared to Saline Control group and a significantly higher bone volume / tissue volume (BV/TV) percentage compared to all the other groups. These preclinical findings suggest that CTM allografts may promote fracture repair, providing rationale for further investigations, including a randomized controlled trial for human fractures in weightbearing bones.

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