Biology Department Theses and Dissertations

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Understanding Actin Dynamics and Its Regulatory Mechanisms in Developing Stereocilia
(2025-12) Liao, Xiayi; Perrin, Benjamin; Balakrishnan, Lata; Mastracci, Teresa; Cummins, Theodore; Zhao, Bo
Stereocilia, the actin-based protrusions of auditory hair cells, are essential for converting mechanical stimuli into electrical signals. Their formation and maintenance require tightly regulated actin dynamics, including elongation and widening through filamentous actin (F-actin) incorporation. Here, we show that newly expressed actin first incorporates at stereocilia tips and then extends along the shaft to promote widening, with additional filaments assembling at the periphery of a stable actin core. Notably, we detected signals of both barbed and pointed ends present at stereocilia tips, revealing a previously unrecognized population of short actin filaments that contribute to stereocilia growth. Overexpression of actin further enriched short filaments along the shaft, linking their abundance to widening. Short filament levels correlated with the presence of the unconventional myosins MYO3A/B and MYO15A at stereocilia tips, implicating these motors in generating or stabilizing filaments required for elongation and widening. Focusing on class III myosins, we demonstrate that MYO3A is the predominant paralog in cochlear inner hair cells, indispensable for determining bundle dimensions and preventing overgrowth. Although MYO3B accumulates at tips in the absence of MYO3A, it provides only partial compensation, failing to maintain short filament populations or transport cargo such as ESPN-L1 to stereocilia tips. Strikingly, simultaneous deletion of MYO3A and ESPN1 eliminated MYO3B from stereocilia tips and sharply reduced tip width, suggesting that MYO3 complexes regulate actin incorporation specifically at the upper shaft. Moreover, kinase-deficient MYO3 mutants produced excessively long stereocilia and disrupted the distribution of MYO7A and EPS8L2, uncovering an unexpected role for the kinase domain in controlling tip-localized actin dynamics. Together, these findings establish a mechanism by which stereocilia widen and uncover MYO3A as a central regulator of tip widening in cochlear hair cells, providing mechanistic insights into how its dysfunction leads to DFNB30-associated hearing loss.
Fluorescence Recovery Kinetics Uncover Two Distinct Actin Population During Stereocilia Development
(2025-12) Joshi, Priyanka; Perrin, Benjamin; Marrs, James; Meyer, Jason; Berbari, Nicholas; Balakrishnan, Lata
Inner ear hair cells are essential for hearing, and this function is supported by specialized rod-like projections called stereocilia located on their apical surface. Each stereocilium contains long, unbranched actin filaments bundled together by actin crosslinking proteins, with their barbed ends oriented toward the tip and pointed ends toward the base. During development, stereocilia increase in both length and width, largely due to the elongation and addition of actin filaments. When actin is overexpressed in hair cells, EGFP-actin monomers first incorporate at the stereocilia tips and later along the shaft, supporting a tip-down model where actin added at the tip moves downward along the shaft. To examine actin dynamics, we used Fluorescence Recovery After Photobleaching (FRAP) on transgenic mice expressing EGFP-actin. Our results showed actin turnover occurring at both the tip and shaft in P2 and P4 mice, but by P6, turnover was restricted mainly to the tips. Quantitative analysis revealed that the size of the tip and shaft regions remains constant despite increases in fluorescence intensity, which challenges the simple tip-down model predicting expansion of the tip zone to fill the shaft. Additionally, the differing recovery rates between tip and shaft suggest distinct regulatory mechanisms for actin in these regions. Further investigations involved treating explants with the actin-capping drug Cytochalasin D (CytoD), followed by FRAP analysis after drug removal. EGFP-actin incorporation was uniform along the stereocilia, indicating reduced turnover at the tips and suggesting that actin addition along the shaft occurs independently of tip assembly. CytoD treatment caused a reduction in stereocilia width at P4 but not at P6, highlighting the increased stability of the shaft actin at later developmental stages. This stabilization depends on the actin crosslinker protein ESPN, as stereocilia in jerker mice lacking ESPN displayed highly dynamic actin that recovered rapidly after photobleaching. The two main conclusions from this study are that actin along the stereocilia shaft becomes progressively more stable during development due to ESPN-mediated crosslinking, and that actin incorporation along the shaft proceeds independently of assembly at the stereocilia tips.
Msh2-Msh3 Mediated Replication Defects Promote Genome Instability
(2025-12) Sridharan, Madhumita; Balakrishnan, Lata; Berbari, Nicolas F.; Perrin, Benjamin; Georgiadis , Millie; Turchi, John
High fidelity DNA replication is vital for the maintenance of a stable genome and efficient cell functioning. DNA replication fidelity is governed by numerous mechanisms within the cell with a major factor being the DNA mismatch repair pathway (MMR). The indispensability of MMR for accurate DNA replication and genomic stability is evident in multiple genetic disorders including several cancers associated with MMR deficiency. Paradoxically, the mismatch binding protein, Msh2-Msh3 has been implicated in the promotion of trinucleotide repeat (TNR) expansion events which are the leading cause of several neurological disorders. Research into underlying mechanisms reveal that Msh2-Msh3 may interfere with the Okazaki fragment maturation (OFM) process, specifically FEN1 and Lig I to enhance TNR expansions. In addition to TNR expansions, this finding also exposes a path to genomic instability through interference with DNA replication. In the current study, we explore Msh2-Msh3 mediated OFM interference with a focus on mechanistically understanding the nature of this interference. It is important to note that Msh2-Msh3’s accurate repair function is crucial for the maintenance of genomic stability and the overall efficiency of MMR suggests that pathogenic functioning by Msh2-Msh3 only occurs under specific conditions. Hence, we also aim to study specific factors that trigger aberrant repair by Msh2-Msh3 including various DNA structures, Msh2-Msh3’s ability to bind and hydrolyze ATP, protein-protein interactions, and the lysine acetylation status of Msh2-Msh3. So far, our findings suggest that Msh2-Msh3 has a detrimental effect on OFM proteins, which opens a path for genomic instability. We also establish that binding to various DNA structures and the conformational shift within Msh2-Msh3 as a consequence of ATP binding are important factors underlying this effect. This study additionally reveals that Msh2-Msh3 physically interacts with DNA polymerase (Pol δ) and that its transactions with Pol δ are conserved between S. cerevisiae and humans. Finally, we elucidate the role of lysine acetylation in Msh2-Msh3’s interaction with OFM proteins and suggest that lysine acetylation may be a means by which Msh2-Msh3 abundance is regulated within the nucleus. Understanding the factors responsible for abnormal repair by Msh2-Msh3 brings us one step closer to therapeutics targeting pathogenic repair by Msh2-Msh3 specifically and preserving accurate repair.
Exploring the Effects of RNase H2 Regulation and Protein Lysine Acetylation on Genomic Stability
(2025-12) Battapadi, Tripthi; Balakrishnan, Lata; Berbari, Nicolas F.; Perrin, Benjamin J.; Turchi, John J.
Maintenance of genomic integrity during DNA replication requires precise coordination of multiple enzymatic processes. The antiparallel nature of the duplex DNA necessitates asymmetric replication, where the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously as Okazaki fragments. In human cells, approximately 50 million Okazaki fragments are generated per replication cycle and must be matured and seamlessly joined to produce an intact lagging strand. Defects in this process contribute to genomic instability and have been implicated in cancer development. The Okazaki fragment maturation (OFM) process involves a coordinated action of several key enzymes: DNA Polymerase δ (Pol δ) synthesizes and displaces the preceding fragment creating a flap, which is then processed by flap endonuclease 1 (FEN1) and the remaining nick is sealed by DNA LigI. Additionally, despite their high fidelity, replicative polymerases can occasionally incorporate ribonucleotides during synthesis, which is processed by Ribonuclease H2 (RNase H2) in the ribonucleotide excision repair (RER) pathway. Central to both pathways is proliferating cell nuclear antigen (PCNA), a sliding clamp that mediates these activities through protein-protein interactions via PCNA-interaction peptide (PIP) domains. The fidelity and efficiency of lagging strand synthesis therefore depend critically on the sequential handoff of DNA substrate between these coordinated enzymes. This study investigates two regulatory mechanisms that modulate protein function and influence pathway efficiency and overall genome stability. First, we examined RNase H2’s role as a potential coordinator linking OFM and RER pathways. Our results reveal bidirectional stimulatory interactions between RNase H2 and key downstream enzymes - Pol δ, FEN1, and DNA LigI, suggesting a coordinated regulatory network. Second, we investigated how lysine acetylation, a dynamic post-translational modification, affects the enzymatic activity of RNase H2, Pol δ, and RPA. In vitro acetylation studies demonstrate enhanced enzymatic activity for all three proteins, indicating that this modification may serve as a governing mechanism controlling OFM efficiency. Collectively, our findings suggest that these protein interaction networks, and regulatory mechanisms offer potential therapeutic targets for cancer and other diseases associated with genomic instability.
Calliphorid Hygrosensation Perception and the Effect of Humidity on Oviposition Behavior
(2025-12) Moctar, Khadija; Picard, Christine; Balakrishnan, Lata; Roper, Randall; Benbow, Eric
Forensic entomology relies on the predictable development of insects under specific environmental conditions to estimate the postmortem interval. However, the accuracy of these estimates can be affected by the time between death and insect colonization, the pre-appearance interval. In cases where insect evidence is absent, it is often unclear whether colonization has yet to occur or if environmental conditions, such as humidity, have inhibited oviposition. While the effects of temperature on blow fly behavior are well established, humidity remains less understood. This research examined the oviposition behavior of Phormia regina (Diptera: Calliphoridae) under varying humidity levels across different seasons, using populations collected from Indiana and Arizona. Results indicate that humidity alone did not significantly impact oviposition timing or egg output, although temperature and season were significant drivers of both outcomes (both p <0.001). Wild-caught flies were more sensitive to 70 % rh than lab-adapted flies, laying significantly more eggs (p = 0.04). Additionally, females reared under low humidity conditions laid the majority of their eggs in the evening, suggesting that developmental or maternal effects may influence oviposition behavior. Flies native to Arizona showed delayed oviposition at high humidity (85 %) compared to low (30 %) (p < 0.001), while Indiana flies produced more eggs across conditions. To explore the physiological mechanisms underlying humidity perception in P. regina, we analyzed the expression of odorant-binding protein 59a and ionotropic receptor 40a, genes associated with hygrosensation in Drosophila. Ir40a expression differed significantly by location (p = 0.003) and sex (p = 0.0007), with higher levels in males and in Arizona flies, but showed no response to humidity. In contrast, Obp59a expression differed significantly by both location (p = 0.031) and humidity (p = 0.046), with approximately 2.5-fold higher expression in flies from Arizona and those reared under low humidity conditions. Together, these patterns suggest distinct roles of these genes in humidity sensing. Collectively, these findings collectively demonstrate how humidity, temperature and geographic origin influence Phormia regina oviposition behavior and gene expression related to hygrosensation, providing critical insights to refine the accuracy and reliability of entomological evidence in postmortem interval estimation.
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.

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