Master of Science (MS), University of Toledo, 2020, Pharmaceutical Sciences (Pharmacology/Toxicology)

The zebrafish, Danio rerio, was used to develop a colon cancer model in order to show the therapeutic effects of various chemotherapeutic drugs. Microinjections were used to inject the three-day post fertilization, 3dpf, zebrafish with colon cancer cells, HCT116. The success of this experiment was tracked by using fluorescent GFP tagged wild type cancer cells and taking photos of the translucent zebrafish with fluorescent imaging. Recently, we identified three thienopyridine analogs namely TPH104, TPH104c and TPH104m that have shown beneficial activity against colon cancer cells HCT116 in in vitro settings. These promising lead compounds were set to test their safety in vivo setting in zebrafish larvae five-day post fertilization, 5dpf. An acridine orange staining test was done to analyze the zebrafish embryos after being exposed to the various chemotherapeutic drug concentrations. The results of the toxicity assays allowed us to determine a therapeutic dosage for the zebrafish larvae and identified changes in morphology, heart rate and behavior. Please note that due to COVID-19 crisis this work was disrupted and was not able to be replicated to draw a meaningful statistical significance, hence the work presented here should be considered preliminary findings only.

Committee: Amit Tiwari (Committee Chair); Frederick Williams (Committee Co-Chair); Jerry Nesamony (Committee Member) Subjects: Pharmacology; Pharmacy Sciences; Toxicology

Master of Science, University of Akron, 2023, Biology

In this thesis I investigated the relationship between a high-calorie diet and bone turnover in zebrafish scales using alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) assays as well as a calcium stain, calcein, to visualize resorption lacunae. I also modified current ALP and TRAP scale assay protocols to have an increased sensitivity through use of the fluorescent substrate 4-methylumbelliferyl phosphate. Finally, I reported my scale assay data in a repeatable way through the conversion of absorbance data to activity per area of scale. I found that a high-calorie diet does not alter bone formation activity. The bone formation assay results were not significantly different between the fish in the high-calorie diet group and the fish in the low-calorie diet group. However, bone resorption activity had a near significant increase in the low-calorie diet group fish. When the interaction term was removed, there was a significant increase in bone resorption activity in the low-calorie diet group. Paired with the increased number of resorption lacunae in this group, our lab believes that a low-calorie diet alters the bone resorption activity of zebrafish scales.

Committee: Rich Londraville (Advisor); Qin Liu (Committee Member); Robert Duff (Committee Member) Subjects: Biology

Bachelor of Science, Wittenberg University, 2021, Biology

Insecticide use is steadily increasing in the United States. Of these, the insecticide Sevin™ has the active ingredient carbaryl, which is a xenobiotic. This is a reverse inhibitor of acetylcholinesterase, which prevents nerve excitation causing paralysis and death in insects. Though insects are the target species, carbaryl has many routes of exposure such as run-off, inhalation, and physical contact making it easy to encounter for non-target species like aquatic life and humans. Carbaryl has been studied within in vitro models to test its effects on zebrafish. The insecticide has already been shown to act on the acetylcholinesterase (AChE) pathway, but it also has potential to act on a second pathway: the aryl hydrocarbon receptor (AhR) pathway. Zebrafish genes and genetic pathways are homologous to humans, allowing for insight on how carbaryl could be affecting humans as well. This study set out to use an in vivo model to show how carbaryl is inducing cytochrome P450 or other AhR pathway gene expression. Gene expression of carbaryl treated embryos were compared to untreated embryos to examine this possible relationship. Cyp1a and cyp1b gene expression was increased with an increase in carbaryl concentration suggesting that carbaryl may be causing induction of the AhR pathway.

Committee: Michelle McWhorter (Advisor); Matthew Collier (Committee Member); Daniel Marous (Committee Member) Subjects: Agricultural Chemicals; Biochemistry; Biology; Developmental Biology; Environmental Science; Genetics; Molecular Biology; Toxicology

Master of Science, University of Akron, 2012, Biology

Embryonic ethanol exposure is known to cause birth defects and neurodevelopmental disorders in many vertebrates, including humans. In this study, zebrafish (D. rerio) were used to investigate the affects of embryonic ethanol exposure on embryo/larval development and the subsequent adult behavior. Although zebrafish have become a widely used model for prenatal alcohol exposure, in many cases the concentrations used are high and the period of observation is short. In order to record both larval and adult measures, this study utilized a low concentration of ethanol (0.4%) throughout the embryonic period of development (0-72 days post fertilization). Anatomical and physiological data was collected from larvae (including eye and heart measures) to investigate the affects of ethanol on embryonic development and an aggression/avoidance assay was used to determine if embryonic ethanol exposure influenced adult behavioral phenotypes. Exposure to ethanol throughout embryonic development resulted in decreased eye size, increased ventricle area, increased cardiovascular function, and reduced size in the larvae. In the adult fish, neither measures of size nor behavior of the treatment group were found to be significantly different from that of the control. In conclusion, low levels of embryonic ethanol exposure can result in changes in both form and function of larval anatomy and physiology. However, this affect does not seem to carry into adulthood through size or behavioral aggression/avoidance behaviors.

Committee: Brian Bagatto Dr. (Advisor); Francisco Moore Dr. (Committee Member); Richard Londraville Dr. (Committee Member) Subjects: Anatomy and Physiology; Animals; Behavioral Sciences; Biology; Developmental Biology; Physiology

Master of Science, University of Akron, 2024, Biology

I investigated hypocretin's signaling role in appetite in zebrafish (Danio rerio). Hypocretin is a small neuropeptide known for its effects on circadian rhythm and appetite. I tested hypocretin receptor knockout mutants from ZIRC (hu2098) with a premature stop codon for the receptor. Wild-type and heterozygous hu2098 zebrafish were raised to adulthood (3-4 months post fertilization) and genotyped. Fish were food restricted for 19-29 hours before a feeding session, then feeding rate was determined by the regression of brine shrimp consumed/min over an eight-minute feeding period. Fish feeding was measured directly using a novel technique that analyzed images from fish feeding continuously on brine shrimp, in which all individual brine shrimp were identified in a tank with a feeding fish over nine images in an eight-minute trial. Utilizing a mixed-effects model and accounting for weight as a covariate, heterozygous fish (hcrtr2 + / hcrtr2 -) ate brine shrimp at a significantly faster rate (mean 23.4 +/- 12.6 shrimp/min, n = 12) than wildtype fish (hcrtr2 + / hcrtr2 +) (20.5 +/- 13.8 shrimp/min, p = .033, n = 11). Weight had a significant impact on the feeding rate of wild-type and heterozygous fish (+/- 29.1 mg, p = .028, n =23). These results support a role for hcrtr2 in appetite regulation.

Committee: Dr. Richard Londraville (Advisor); Dr. Qin Liu (Committee Member); Dr. Jordan Renna (Committee Member) Subjects: Biology; Neurobiology; Neurosciences

Master of Sciences, Case Western Reserve University, 2024, Biology

The zebrafish is a valuable model for exploring the mechanisms of water motion detection, vestibular function, and hearing, as well as the molecules responsible for the proper functioning of hair cells. Hair cells are responsible for transducing the deflection of a series of stereocilia at their apex into signal for associated nerves to carry stimuli to the brain. The zebrafish lateral line consists of neuromasts, containing hair cells that face either anteriorly or posteriorly in anterior-posterior neuromasts, which are responsible for detecting water flow in the anterior-posterior axis. Hair cells in neuromasts may also face dorsally or ventrally in dorsal-ventral neuromasts, which are responsible for detecting water flow in the dorsal-ventral axis. The mechanoelectrical transduction (MET) channel candidate proteins transmembrane channel-like 2a (Tmc2a) and transmembrane channel-like 2b (Tmc2b), localize at the tips of stereocilia in these hair cells. Tmc2a in particular has been shown to only express in certain polarity hair cells in zebrafish posterior lateral line neuromasts. We used RNA in situ hybridization to visualize the mRNA transcripts of tmc2a and tmc2b in zebrafish anterior and posterior lateral line neuromasts. First, we established the percentage of neuromast hair cells with tmc2a or tmc2b transcripts in wild-type zebrafish. Next, we explored the percentage of neuromast hair cells with tmc2a or tmc2b transcripts in emx2-/- mutant fish and in their sibling control fish. The transcription factor emx2 is necessary for reversing the polarity of hair cells. However, lack of emx2 in mutant fish did not result in a difference in the percentage of neuromast hair cells with either tmc2a or tmc2b transcripts.

Committee: Brian McDermott (Advisor); Ruben Stepanyan (Committee Member); Deborah Harris (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutation of the Dystrophin gene resulting in complete absence of the dystrophin protein. Loss of dystrophin destabilizes the muscle membrane and increases muscle's susceptibility to injury. Over time, continuous rounds of muscle insult, muscle degeneration, and regeneration ultimately exhaust muscle's regenerative and reparative capacity and leads to loss of muscle function. There is currently no cure for DMD and there are many significant hurdles in the field for dystrophin-restoration based therapies. Thus, there is a critical need to develop new therapeutics. Genome-wide association studies (GWAS) have uncovered putative genetic modifiers of DMD. One identified class of modifiers are regulators of TGFb signaling. In Chapter 2, we use pharmacological and genetic means to investigate TGFb signaling as a modifier of DMD. Here I show that inhibition of TGFb using TGFb receptor inhibitors LY364947 and SB431542 leads to significant improvement in birefringence intensity, a read out of muscle organization, but causes developmental defects. Modifiers uncovered by GWAS include allelic variants that decrease expression of TGFb regulators latent TGFb binding protein 4 (LTBP4, ltbp4 in zebrafish) and thrombospondin-1 (THBS1, thbs1a and thbs1b in zebrafish) and correlate with decreased TGFb bioavailability and a less severe DMD phenotype. To model the decreased expression alleles of LTBP4 and THBS1 we used zebrafish mutants and CRISPR/Cas-9 F0 'crispants' of ltbp4, thbs1a, and thbs1b and show that mutation of thbs1b or ltbp4 improves survivorship in dmd mutant embryos and combinatorial mutation of thbs1a and ltbp4 leads to improved birefringence intensity, an indiciator of muscle organization. Together, these findings demonstrate that inhibition of TGFb signaling is protective in dmd mutant zebrafish. GWAS for DMD modifiers have also identified SNPS that lie in non-coding putative regulato (open full item for complete abstract)

Committee: Sharon Amacher (Advisor); Martin Haesemeyer (Committee Member); Harold Fisk (Committee Member); Susan Cole (Committee Member) Subjects: Biology; Genetics

Doctor of Philosophy, University of Akron, 2024, Chemistry

Organic sensors emitting in the near-infrared region (600-900 nm) are desirable for biological applications. Through the combination of different functional groups with Excited State Intramolecular Proton Transfer (ESIPT) segments, novel probes have been crafted to attain large Stokes shift, improved sensitivity, selectivity, and the ability to tune emission towards the NIR region. Chapter I summarizes recent progress in this field, by including the photophysical properties inherent in ESIPT dyes, and investigated the impact of structural and environmental factors to their fluorescence. Furthermore, the potential applications of these probes as imaging reagents are exemplified such as labeling intracellular membranes, mitochondria, lysosomes, and detecting biomolecules. In Chapter II, four NIR-emitting ESIPT dyes, each featuring distinct cyanine terminal groups, were scrutinized to assess their fluorescence lifetime characteristics in the polar aprotic solvent. By using time–correlated single–photon counting (TCSPC) method, these ESIPT-based dyes revealed a two-component exponential decay in about 2-4 nanoseconds (ns) which was proved to exclusively from keto tautomer. The model compounds (1.10 and 2.2) as well as low-temperature fluorescence spectroscopy (at -189 ℃) identified intramolecular charge transfer (ICT) as a prominent factor influencing the lifetime values. ESIPT simplified lifetime components. Chapter III includes ESIPT probes with benzoindolium terminal group, which shows large Stokes shift (Δλ≈250 nm) and good quantum yield. The probe also exhibited a minor absorption (~580 nm in DCM) and emission (∼ 610 nm in DCM), attributed to a zwitterionic structure formed through deprotonation of phenolic proton. Fluorescence confocal microscopy investigations highlighted the probe's excellent selectivity for mitochondria, and unusually strong emission in the 595 nm channel rather than the expected 700 nm channel. The study thus illustrated a reaction-based pro (open full item for complete abstract)

Committee: Yi Pang (Advisor); Christopher Ziegler (Committee Member); Chrys Wesdemiotis (Committee Member); Chunming Liu (Committee Member); Tianbo Liu (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Chemistry; Organic Chemistry; Physical Chemistry

PhD, University of Cincinnati, 2024, Medicine: Molecular and Developmental Biology

The vertebrate heart is a multichambered structure requiring precise coordination of many signals and transcriptional regulators to properly develop. Disrupting this regulation can result in congenital heart defects (CHDs), which constitute the largest subset of congenital malformations. Many CHDs are subclinical until later in adult life, but it has been historically difficult to model severe heart defects in mature animals and because the molecular etiology of the majority of CHDs remains unknown. Nr2fs comprise a family of conserved orphan nuclear receptors with documented roles in cardiovascular development. Murine Nr2f2 is indispensable for specification and maintenance of atrial identity and human mutations in NR2F2 cause a variety of CHDs. However, elucidating the regulation imparted by Nr2f2 has been hindered by embryonic lethality in current mouse models. We bypassed this impediment utilizing a hypomorphic allele for a loss of nr2f1a (the zebrafish equivalent of mammalian Nr2f2), which survives to adulthood despite lacking an atrial chamber. We showed that an absence of the atrium causes morphological adaptations of the sinus venosus (SV) including increased cellularity and thickness of the elastic sinus walls. This remodeling was induced by increased hemodynamic stress and was rescuable upon treatment with vasodilators. RNA sequencing uncovered an unanticipated similarity between the SV and the bulbus arteriosus (BA) and showed that the mutant SV takes on an arterial-like identity. Additionally, comparing our SV and BA datasets to sequencing from the blood sinuses of the Ciona heart illuminated their shared evolutionary heritage. Since the focus of our developmental work spanned the entire lifetime of the fish, we also endeavored to explore cardiac regeneration. Although adult mammals cannot replace heart muscle lost in the event of myocardial infarction (MI), mature zebrafish can. However, investigations of myocardial regeneration in fish have f (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Tony De Falco Ph.D. (Committee Member); Stacey Huppert Ph.D. (Committee Member); Joshua Gross Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Congenital heart defects (CHDs) are the most common types of birth defects, but despite their high prevalence the molecular etiology underlying CHDs are not well understood. Vertebrate hearts are formed with the temporal differentiation of cardiac progenitors that occurs in two continuous waves. Early differentiating cardiac progenitors within the anterior lateral plate mesoderm (ALPM) give rise to the first heart field (FHF), while later differentiating progenitors comprise the second heart field (SHF). Tight regulation of retinoic acid (RA) is critical for normal heart development in all vertebrates. RA signaling has a conserved requirement restricting the size of the cardiac progenitor field within the ALPM, but how RA signaling affects the populations of earlier-differentiating FHF and the later-differentiating SHF progenitors remains poorly understood. Here, to elucidate how loss of RA signaling affects these cardiac progenitors within the ALPM, we first quantified the number of cardiac progenitors and earlier-differentiating cardiomyocytes (CMs) in RA-deficient zebrafish mutants. We found there was a significant expansion in the relative proportion of differentiating FHF CMs compared to SHF progenitors in mutant embryos within these cells. Using photoconversion-mediated lineage tracing, we see that fewer SHF-derived CMs differentiate at the arterial pole. We also observed a lack of SHF-derived smooth muscle within the outflow tract (OFT) at later stages in RA-deficient embryos. We find that at the venous pole, pacemaker CMs also fail to differentiate in RA-deficient embryos. Our data indicate that in zebrafish, early RA signaling restricts the size of the earlier differentiating FHF and is required to promote differentiation of the SHF later at the arterial and venous poles of the developing heart. A screen for RA-responsive genes within the ALPM of 8 somite (s) stage zebrafish embryos shows that RA loss results in an expansion of six1b and six2a expre (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Lindsey Barske Ph.D. (Committee Member); David Wieczorek Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Christina Gross Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2023, Molecular, Cellular and Developmental Biology

Cardiovascular disease is the leading cause of death worldwide, partly because humans lack appreciable capacity to repair or replenish lost or damaged cardiac tissue. Unlike mammals, adult zebrafish have a remarkable ability to regenerate large portions of cardiac tissues lost following injury. Most studies on zebrafish heart regeneration have focused on determining transcriptional changes occurring during this process. However, comparison of protein levels verses mRNA abundance shows that mRNA expression accounts for just 40% of the protein product. Only a minority of proteins, changing in abundance during heart regeneration, correlate with the amount of mRNA suggesting an important role for post-transcriptional regulation. Here, we describe a new family of translation factors called Eif4e1c which are related to canonical Eif4e proteins that initiate the rate limiting step of mRNA translation by binding to the 5' mRNA cap. Zebrafish mutants for eif4e1c have fewer cardiomyocytes and have reduced proliferation of heart muscle after injury. Additionally, eif4e1c mutants show widespread translational dysregulation of mRNA and metabolic changes in their hearts. Eif4e1c is more conserved throughout evolution than the canonical Eif4e proteins with a core group of 23 amino acids retained from ray-finned fish to sharks across ~480 million years. Interestingly, the Eif4e1c family is lost in amphibians and is absent from all terrestrial species. To our knowledge this makes eif4e1c the only fish specific gene with described roles in heart regeneration. In sum, we show that eif4e1c is critical for development and regeneration of zebrafish hearts revealing context-dependent requirements by translation initiation regulators during heart regeneration.

Committee: Joseph Goldman (Advisor); Amanda Bird (Committee Member); Jill Rafael-Fortney (Committee Member); Sharon Amacher (Committee Member) Subjects: Cellular Biology; Developmental Biology; Molecular Biology

Master of Science, University of Toledo, 2023, Pharmaceutical Science

In recent years, there has been an increase in the number, potency, and availability of novel psychoactive drugs that duplicate the effects of many well-established illicit drugs. Many of these drugs are synthetic derivatives of cathinone, the naturally occurring Β - keytone analogue of amphetamine found in Khat (Catha edulis) (Valente, 2014). Some synthetic psychoactive cathinones (SPCs) like methylone, dimethylone, and others can mimic methylenedioxymethamphetamine (MDMA) and produce unfavorable sympathomimetic effects such as tachycardia and hypertension. These unfavorable effects are often exacerbated by elevated ambient temperatures (Cappon, 1997), and this has been shown in mice (Chen, 2021). As a higher throughput approach to evaluating SPC toxicity, the Hall laboratory has been using larval zebrafish. It is not known whether higher ambient temperatures exacerbate lethal toxicity in larval zebrafish. This question was addressed in experiments examining MDMA and a series of structurally similar MDMA-like SPCs. Zebrafish were exposed to each different drug in various concentrations for 5 hours. Post-exposure, each zebrafish was evaluated for cardiac activity to assess lethality. Firstly, these studies showed that high ambient temperatures exacerbate MDMA toxicity in larval zebrafish. Moreover, the same was observed for all SPCs. There was some variability in the magnitude of this effect. The largest magnitude seen was a 14.18 mM fold difference between methylone values at a high ambient temperature and a normal ambient temperature. The weakest magnitude seen was a 2.77 mM fold difference between pentylone values. Varying magnitudes of ambient temperature affects may be used to identify structure activity relationships as a wider range of analogues are studied.

Committee: F. Scott Hall (Committee Chair); Isaac Schiefer (Committee Member); Frederick Williams (Committee Member) Subjects: Pharmacology; Pharmacy Sciences; Toxicology

Doctor of Philosophy, University of Toledo, 2023, Experimental Therapeutics

Phelan-McDermid syndrome is a genetic disorder that stems from the deletion of a segment of chromosome 22 in the 22q13 region or a defect in the Shank3 gene. Behavioral symptoms of autism spectrum disorder (ASD) are reported in half of cases of Phelan-McDermid syndrome. ASD is a neurodevelopmental disorder characterized by early-onset social, motor, and cognitive deficits. ASD has two sets of core symptoms – 1) communication difficulties and social challenges and 2) restricted and repetitive behaviors. There are currently no pharmacological treatments approved by the Food and Drug Administration (FDA) that target the core symptoms. A behavioral screen was developed to monitor repetitive behaviors in 5 dpf wild-type (WT) and Shank3B mutant zebrafish and identify compounds that modulate levels of repetitive behaviors. Zebrafish were exposed to various concentrations of compounds in a behavioral assay in alternating light and dark periods for a total of 90 minutes. A maximum tolerated concentration (MTC) assay was performed to provide a preliminary assessment of in vivo toxicity and establish a range of concentrations to use in these assays. Video tracking software (Noldus DanioVision) was used to measure locomotor activity and other parameters including angular velocity and variance of turn angle – measures of repetitive behaviors. In the preliminary studies, the effects of BQCA, a positive allosteric modulator (PAM) of acetylcholine potency at M1 muscarinic receptors were examined in WT larval zebrafish. BQCA at doses of 3 and 10 µM resulted in a significant decrease in locomotor activity, accompanied by an increase in angular velocity and turn angle. Additionally, several novel PAMs of acetylcholine efficacy at M1 muscarinic receptors also significantly reduced swimming while promoting turning behaviors and increasing the variance of turn angle. Comparatively, Shank3B+/- zebrafish exhibited lower levels of locomotor activity during the initial spontaneous swimming (open full item for complete abstract)

Committee: William Messer Jr. (Committee Co-Chair); Isaac Schiefer (Committee Member); F. Scott Hall (Committee Member); Frederick Williams (Committee Co-Chair) Subjects: Pharmacology

Master of Science, University of Akron, 2023, Biology

The zebrafish (Danio rerio) is an important model organism in human related research. Although commonly used in lab settings, there is a lack of consistency in diets fed to cohorts. This inconsistency is amplified by an incomplete understanding of the impact on offspring because of parental diets. Due to the importance of this animal model in human related studies, we aimed to explore the potential impacts from diet on adult behavior and offspring development through evaluation of feeding preference and the amount of yolk provided to developing embryos. Utilizing a 3D printed arena and machine learning, a spatial preference was seen and was further linked to specific food items. This provided encouragement for the use of machine learning and updated technology to further understand zebrafish behavior. During extended feeding, diets fed to adult female zebrafish resulted in weight gain, variation in standard length, and differences in the yolk to chorion ratios for each of the treatments. High carbohydrate diets impacted the ability for females to gain weight at the same rate of the control, high protein, and high lipid diets. However, the ratio of yolk to the chorion of the eggs for the high carbohydrate diet, high protein diet, and the control diet were significantly higher than the high lipid diet, regardless of the production of eggs following spawning events.

Committee: Brian Bagatto (Advisor); Todd Blackledge (Committee Member); Richard Londraville (Committee Member) Subjects: Animals; Aquatic Sciences; Biology; Physiology

Master of Science, The Ohio State University, 2023, Environment and Natural Resources

A better understanding of the physiology, ecology, genetics, and nutrition of fish can enhance sustainable fishery management and aquaculture production. The use of model organisms can help develop scientific knowledge and thoroughly investigate the biology of over 25,000 described fish species and over 212 farmed species. The Zebrafish (Danio rerio) is a well-established model organism in the scientific experimental biology community to study genetics, toxicology, medical sciences, and aquaculture. Triploidy (3n), the condition of having three sets of chromosomes and can be induced through physical shocks during meiotic cell division. Previous studies on triploid Zebrafish have indicated male-biased sterility and abnormal gonad development, with the exception of one study where females produced functional gametes. Sterile triploids are the desired sex for domesticated fish species production to avoid backcrossing to wild populations to reduce risks of exotic fish introductions. Therefore, the first experiment of this study investigated the reproductive ability of triploid males and females by crossing offspring from triploid fish. Triploid progenies were produced by applying cold shock using different strains of Zebrafish in four trials. Triploid females were produced in 12% of first and 10% of the fourth trials out of four trials. In most cases, successful spawning occurred when triploid females were crossed with triploid and diploid males, and the larvae survived until 10 days post-fertilization (dpf). However, embryos obtained from crosses between DT and TT fish did not survive beyond one dpf. The increase in aquaculture production has produced substantial fish waste, with approximately two-thirds of the catch being discarded. Improper disposal of fish waste can lead to environmental challenges. Like fish waste, acid whey waste is a by-product of the dairy industry, and removal of this product has become an environmental burden due to its high organic load (open full item for complete abstract)

Committee: Konrad Dabrowski (Advisor); Macdonald Wick (Committee Member); Robert Gates (Committee Member) Subjects: Biology; Environmental Science; Natural Resource Management; Nutrition; Sustainability

PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Congenital heart defects (CHDs) are the most common type of congenital birth defect and can be caused by mutations in genes that are involved in the specification or maintenance of cardiomyocyte identity. There are multiple subpopulations of cardiomyocytes within the heart: those that populate the atrium, ventricle, atrioventricular canal (AVC), and pacemaker/sinoatrial node (SAN), each possessing distinct properties that allow the heart to function properly. It has been shown that cardiomyocytes possess a certain amount of plasticity that allow them to change identity based on the expression of different transcription factors. However, our understanding of this plasticity, particularly with regard to the atrium, remains incomplete. The Nr2f (Coup-tf) family of transcription factors are known conserved regulators of atrial differentiation and maintenance. Mutations in NR2F2 are associated with CHDs in humans and work in mice has shown that Nr2f2 is required to maintain atrial identity at the expense of ventricular identity. Previous work from the Waxman lab has shown that zebrafish Nr2f1a, the functional equivalent of mammalian Nr2f2 with respect to heart development, is required for differentiation of atrial cardiomyocytes at the venous pole and restriction of the AVC. Yet the mechanisms by which Nr2f proteins function within the atrium are still not completely understood. In this work, we investigated the mechanisms by which Nr2f transcription factors function within the atrium to maintain atrial identity. Using nr2f1a mutant zebrafish, we found that loss of Nr2f1a leads to a progressive acquisition of ventricular identity within the atrium, consistent with previous mouse data; however, we found that this occurs specifically within the cardiomyocytes of the expanded AVC of nr2f1a mutants. At the venous pole of the atrium, we found a progressive expansion of SAN identity. We show that Nr2f1a represses pacemaker identity in part by maintaining atrial (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Brian Gebelein Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); David Wieczorek Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2023, Molecular, Cellular and Developmental Biology

During early vertebrate embryogenesis, muscle and skeletal stem cells are grouped into reiterated segments, called somites, in a process called somitogenesis. Somite formation is established by a genetic oscillator called the segmentation clock, comprised of a network of genes expressed periodically in the presomitic mesoderm. Precise control of segmentation clock oscillations is driven by robust temporal regulation of mRNA production, translation, and mRNA decay, and my work explores post-transcriptional mechanisms that regulate oscillatory expression. proline-rich nuclear receptor coactivator 2 (pnrc2) regulates oscillatory mRNA decay in zebrafish embryos and loss of pnrc2 results in stabilization and accumulation of segmentation clock transcripts. Despite an increase in segmentation clock mRNA abundance, pnrc2 mutant embryos exhibit normal segmentation clock protein expression and somite patterning is not disrupted. I discovered a closely related gene, pnrc1, is upregulated in MZpnrc2-/- mutants and can rescue the her1 misexpression phenotype when overexpressed in pnrc2 mutant embryos. I hypothesized that pnrc1 may partially compensate for loss of function of pnrc2, resulting in normal somitogenesis in MZpnrc2-/- embryos. However, I found that pnrc1-/-;pnrc2-/-double mutant embryos are morphologically indistinguishable from wild-type embryos and are molecularly indistinguishable from pnrc2-/- single mutants, with respect to her1 expression (Chapter 2). To determine the translation status of accumulated transcripts, I performed polysome profiling analysis on wild-type and MZpnrc2-/- mutant embryos at mid-segmentation stage. This revealed segmentation clock gene transcripts that are upregulated upon loss of pnrc2, including her1, her7, and rhov, are significantly increased in the ribosome-unbound (non-translating) fractions, indicating that accumulated transcripts are not efficiently translated, thus explaining the lack of significant protein accumulation (Chapte (open full item for complete abstract)

Committee: Sharon Amacher (Advisor); Guramrit Singh (Committee Member); Aaron Goldman (Committee Member); Susan Cole (Committee Member) Subjects: Cellular Biology; Developmental Biology; Molecular Biology

MS, University of Cincinnati, 2022, Medicine: Biomedical Research Technology

Although decades of genetic and developmental biology research have revealed many genes controlling the formation of craniofacial structures, the genes that confer the unique properties and shapes of the upper jaw bones remain largely unknown. Here we report a novel requirement for PAX9 in upper jaw bone development in zebrafish. PAX9 is a member of the paired box family of transcription factors. In mammals, PAX9 plays an important role in tooth development and is required for secondary palate closure. However, its expression pattern in zebrafish suggested that it may play additional, previously unappreciated roles in upper jaw development. During embryonic stages (24-36 hpf), pax9 is expressed in the pharyngeal arches and somites, as previously reported, but it persists around the mouth from 10-14 dpf, when the intramembranous bones of the jaw are developing. This suggests that pax9 may not just play an early patterning function but rather may be involved in later stages of jaw formation. We created new pax9 mutant lines and evaluated craniofacial development in mutants and sibling controls from larval through adult stages by Alcian Blue and Alizarin red staining. No major differences were observed at 5-6 dpf. Control larvae show mineralization of the maxilla by 7 dpf and premaxilla by 14 dpf; however, the pax9 mutant presents a shortened maxilla at 7 dpf and no apparent premaxilla at 14 dpf. Adult pax9 mutants similarly show an abnormally truncated maxilla, absence of premaxilla, loss of barbels, and reduction of the dentary bone at the mandibular symphysis. Despite these severe jaw defects, mutants are viable, fertile, and have normal pharyngeal teeth. To test whether the reduction of mineralized bone in the upper jaw reflects altered numbers or differentiation of resident osteoblasts, we crossed the pax9 mutant to two transgenic osteoblast reporter lines, RUNX2:mCherry and sp7:GFP. RUNX2 is a master regulator of osteoblast differentiation that is expressed in ea (open full item for complete abstract)

Committee: Lindsey Barske Ph.D. (Committee Member); Ertugrul Ozbudak Ph.D. (Committee Member); Samantha Brugmann Ph.D. (Committee Member) Subjects: Genetics

Doctor of Philosophy, University of Akron, 2022, Integrated Bioscience

Cadherins are cell-adhesion molecules that play important roles in animal development, maintenance and/or regeneration of adult animal tissues. In order to understand cadherins' functions in adult vertebrate visual structures, one must study their distribution in those structures. First, I examined expression of cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 in the visual structures of normal adult zebrafish using RNA in situ hybridization, followed by studying expression of two Kruppel-like transcription factors (klf6a and klf7), that are known markers for regenerating adult zebrafish retinas and optic nerves, in normal adult zebrafish brain, normal and regenerating adult zebrafish retinas. Then, I investigated expression of these cadherins in regenerating adult zebrafish retinas using both RNA in situ hybridization and quantitative PCR. Finally, as the first step in elucidating molecular mechanisms underlying protocadherin-17 (one of the cadherins that I studied) function in zebrafish visual system development, I used DNA microarray analysis to study gene expression of zebrafish embryos with their protocadherin-17 expression blocked by morpholino antisense oligonucleotides (these embryos are called protocadherin- 17 morphants). The major findings include: 1) cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 were differently expressed in the retina and major visual structures of normal adult zebrafish brain. 2) klf6a and klf7 showed similar expression patterns in most visual structures in the adult fish brain, and in regenerating retinas, but klf6a appeared to be a superior regeneration marker based on RNA in situ hybridization. 3) These four cadherin molecules showed distinct expression patterns in the regenerating zebrafish retinas. 4) Several genes involved in vision and/or visual development were significantly downregulated in the protocadherin-17 morphants compared to control embryos. My results suggest that (open full item for complete abstract)

Committee: Qin Liu (Advisor); Richard Londraville (Committee Member); Rolando Ramirez (Committee Member); Brian Bagatto (Committee Member); Zhong-Hui Duan (Committee Member) Subjects: Anatomy and Physiology; Bioinformatics; Biology; Developmental Biology; Molecular Biology

PhD, University of Cincinnati, 2022, Medicine: Molecular and Developmental Biology

Soluble N-ethylmaleimide-sensitive factor protein receptors (SNAREs) comprise a universally conserved complex of proteins that are key components of the cellular machinery required for intracellular membrane trafficking. Although the mechanism by which SNAREs mediate neurotransmitter release at the synaptic membrane is well-studied, their requirements during normal vertebrate development have only recently become appreciated. Indeed, mutations in SNAREs are now known to result in developmental syndromes, primarily synaptopathies—these disorders have been collectively termed “SNAREopathies”. However, SNAREs ubiquitously regulate vesicle fusion in virtually all cell lineages; therefore, extra-neuronal disease pathologies may also plausibly manifest as “SNAREopathies”. Cardiomyocytes are among the most specialized cell types, owing to their substantial organization and the dynamic requirements for their diverse function. While it is known that cardiomyocytes heavily rely on intracellular membrane trafficking, to date remarkably few bona fide trafficking proteins have been identified as having a specific function in cardiac tissues. SNAREs are a promising candidate for elucidating how cardiac intracellular trafficking is regulated. Consequently, herein, we endeavored to understand the in vivo requirement for Syntaxin 4 (STX4), a target-SNARE, during normal vertebrate development, cardiac conduction, and cardiomyocyte vesicular transport. This work was initiated upon the identification of two patients with damaging variants in the STX4 locus: One patient, homozygous for a R240W missense variant, presented with sensorineural hearing loss, global developmental delay, hypotonia, and biventricular dilated cardiomyopathy; ectopy; and runs of non-sustained ventricular tachycardia, requiring an orthotopic heart transplant, while a second patient with compound heterozygous truncating alleles presented with severe pleiotropic abnormalities that resulted in perinatal letha (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Member); Steve Danzer Ph.D. (Committee Member); Juan Sanchez-Gurmaches Ph.D. (Committee Member); Douglas Millay Ph.D. (Committee Member); Carlos Prada M.D. (Committee Member); Raphael Kopan Ph.D. (Committee Member) Subjects: Developmental Biology