Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

Selective cell-to-cell adhesion is essential for normal development of the vertebrate brain, contributing to coordinated cell movements, regional partitioning and synapse formation. Members of the cadherin superfamily mediate calcium-dependent cell adhesion, and selective adhesion by various family members is thought to contribute to the development of neural circuitry. Members of the δ-protocadherin subfamily of cadherins are differentially expressed in the vertebrate nervous system and have been implicated in a range of neurodevelopmental disorders: schizophrenia, mental retardation, and epilepsy. However, little is known about how the δ-protocadherins contribute to the development of the nervous system, nor how this development is disrupted in the disease state. Here I focus on one member of the δ-protocadherin family, protocadherin-19 (pcdh19), since it has the clearest link to a neurodevelopmental disease, being the second most clinically relevant gene in epilepsy. Using pcdh19 transgenic zebrafish, we observed columnar modules of pcdh19-expresing cells in the optic tectum. In the absence of Pcdh19, the columnar organization is disrupted and visually guided behaviors are impaired. Furthermore, similar columns were observed in pcdh1a transgenic zebrafish, located both in the tectum and in other brain regions. This suggests protocadherin defined columns may be a theme of neural development. Our X-ray crystal structure of Pcdh19 reveals the adhesion interface for Pcdh19 and infers the molecular consequences of epilepsy causing mutations. We found several epilepsy causing mutations were located at the interface and disrupted adhesion, which further validated the interface and revealed a possible biochemical cause of Pcdh19 dysfunction. Furthermore, sequence alignments of other δ-protocadherins with Pcdh19 suggest that this interface may be relevant to the entire δ-protocadherin subfamily. We used the information gained about Pcdh19 to design PCDH19-FE mut (open full item for complete abstract)

Committee: James Jontes (Advisor); Marcos Sotomayor (Advisor); Heithem El-Hodiri (Committee Member); Sharon Amacher (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Biophysics; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences

PhD, University of Cincinnati, 0, Medicine: Molecular and Developmental Biology

Endocardial cells form a single cell-layer that lines the heart lumen and play several critical roles in directing major morphogenetic changes during cardiogenesis including valvulogenesis, septation, establishment of the conduction system, and ventricle trabeculation. Though important, little is known about the developmental mechanisms and origins of endocardial cells. Previous studies have suggested that embryonic vascular endothelial, endocardial and myocardial lineages originate from multipotential cardiovascular progenitors. However, their existence in vivo has been debated and molecular mechanisms that regulate specification of different cardiovascular lineages are poorly understood. Endocardial cells develop in close association with the adjacent myocardial layer but sparse data exists describing any developmental crosstalk between these populations prior to valvulogenesis.

Committee: Saulius Sumanas Ph.D. (Committee Chair); Joshua Waxman Ph.D. (Committee Member); Daria Narmoneva Ph.D. (Committee Member); Stephanie Ware M.D. Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology

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

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

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

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

Doctor of Philosophy, University of Akron, 2021, Integrated Bioscience

Leptin is a peptide hormone with established roles in metabolic regulation, satiation, and many other regulatory functions. This hormone has been extensively studied in mammals beginning in 1994, with comparative models emerging as useful in studying evolutionarily conserved functions of leptin across species. Here I focus on leptin signaling in the central nervous system of zebrafish. I used multiple techniques to investigate leptin signaling in zebrafish: Western Blot to measure STAT3 and phosphorylated STAT3, a plus-maze to examine associative learning, and an enzyme-linked immunosorbent assay capable of measuring leptinA titers. My results indicate that when leptin production is dysregulated through diet-induced obesity, STAT3 phosphorylation and leptinA expression do not change in the brain, but general activity and reward-seeking behavior decreases. However, food-restricted zebrafish do show an increase in leptinA in brain tissue. Additionally, I describe an outreach experience created to promote understanding of authentic science practices in elementary-aged students, with the goal of increasing interest in biology among young children. Overall, this dissertation describes contributions to basic science in leptin signaling, and in stimulating the public's interest in science.

Committee: Richard Londraville (Advisor); Nidaa Makki (Committee Member); R. Joel Duff (Committee Member); Jordan Renna (Committee Member); Qin Liu (Committee Member) Subjects: Biology

Doctor of Philosophy, University of Toledo, 2020, Medicinal Chemistry

Alzheimer's disease (AD) is a progressive, neurodegenerative disease resulting in cognitive decline, dementia, and eventually death. This work investigates furoxans (1,2,5 oxadiazole N-oxides) for their utility in the treatment of AD. Furoxans are thiol dependent nitric oxide (NO) mimetics, capable of releasing NO in a cellular environment. This work focuses on attenuated furoxans, designed to release NO in a slow, controlled manner to engage the NO/sGC pathway, resulting in in the phosphorylation of CREB, increasing the production of pro-survival gene products such as BDNF. IS-1-41 was identified as a hit compound, possessing promising biological activity such as: neuroprotection, procognitive effects, and a long systemic half-life. Efforts to explore attenuated furoxans resulted in a library of more than 60 novel attenuated furoxans. Several novel attenuated furoxans were found to be neuroprotective; however, when incubated with cysteine, none were found to breakdown to release NO despite evidence showing neuroprotection was NO/sGC dependent. We investigated the bioactivation of attenuated furoxans via selenocysteine and found that, while attenuated furoxans breakdown in the presence of selenocysteine, the resulting breakdown product has not released NO. This supports the hypothesis that attenuated furoxans interact with unknown protein target(s) to release NO, providing the observed neuroprotection, which can be further explored via photolytic target ID studies. After extensive pre-clinical development, four lead candidates, IS-1-41, AH-1-91, AH-2-36, and AH-2-87 were identified and are being investigated for their utility in the treatment of AD. Tangentially, work was undertaken to establish a platform to accomplish in vivo photoaffinity target ID studies to explore the mechanism of action of β-phenethylamines with a non-biased approach. Casper zebrafish were employed as the in vivo model, as casper zebrafish lack melanin and therefore cannot block t (open full item for complete abstract)

Committee: Isaac Schiefer (Committee Chair); William Messer (Committee Member); Zahoor Shah (Committee Member); Erin Prestwich (Committee Member); Wei Li (Committee Member) Subjects: Analytical Chemistry; Chemistry; Neurosciences; Pharmaceuticals; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2019, Molecular Genetics

Post-transcriptional control of gene expression is essential for proper development and is achieved largely by RNA-binding proteins. My graduate research has focused on understanding the role of one such RNA binding protein complex, the Exon Junction Complex (EJC) in development. EJC is deposited about 24 nts upstream of exon-exon junctions during splicing. EJC influences many aspects of post-transcriptional regulation such as mRNA splicing, export, localization and nonsense mediated mRNA decay (NMD). EJC-dependent NMD is recognized as one mode of rapidly regulating gene expression of normal mRNAs that contain NMD-inducing features such as 3′UTR intron or an upstream ORF. How regulation of gene expression via EJC-dependent NMD influences development of specific tissues is unknown. My work utilizes a strong developmental and genetic model system, zebrafish, to understand how EJC-dependent NMD shapes development. In order to address my scientific questions, I utilized the RNA-Seq technique. Our lab has a custom RNA-Seq library preparation protocol which I wanted to improve in order to increase the efficiency of isolating sample cDNA after reverse transcription of RNA fragments (Chapter 2). To improve the protocol, I incorporated biotinylated dNTPs in the RT reaction so that cDNA generated from RNA fragments could be extracted using streptavidin beads. This amendment to the library preparation protocol efficiently selects for extended RT product and avoids ligation of the unextended adapter and generation of insert-lacking cDNAs in the library. To study EJC function during development (Chapter 3), I generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants (EJC mutants) are paralyzed and have muscle and neural defects. As expected, RNA profiling revealed that annotated aberrant and normal NMD targets are significantly upregulated in EJC mutants. An mRNA is targeted for NMD by the key NMD-regulator Upf1 when an exon-ex (open full item for complete abstract)

Committee: Sharon Amacher (Advisor); Guramrit Singh (Advisor); Robin Wharton (Committee Member); Anita Hopper (Committee Member); Beattie Christine (Committee Member) Subjects: Bioinformatics; Developmental Biology; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2019, Pharmaceutical Sciences

With an estimated 425 million adults with diabetes world-wide,1 type 2 diabetes is increasingly becoming associated with the words “epidemic” and “pandemic” in research and medical fields.1–9 Unfortunately, the therapeutic options for type 2 diabetes include lifestyle changes that meet with significant patient resistance, expensive drugs with undesirable administration routes, and often severe side effects. It is thus critical to find new agents with possible antidiabetic and hypoglycemic effects with fewer adverse reactions. Natural products research historically has been a successful approach for the discovery of new drugs and lead molecules for medicinal chemistry optimization efforts. This high rate of success may be due to the structural complexity and diversity of natural products. Additionally, as secondary metabolites that serve some biological purpose for their source organism, naturally products frequently possess a biologically interactive conformation or orientation for a receptor, protein, or other target of different organisms. The power of natural product extracts and compounds was thus an enticing source of potential antidiabetic leads for investigation toward drug discovery and development. A library of edible spices and a library of plant and fungal natural product compounds were evaluated for antidiabetic effects in a panel of in vitro assays associated with anti-inflammatory, hypoglycemic, and anti-glucogenic activity. Through bioactivity-guided isolation, four known compounds were isolated from mace (a spice from the same species as nutmeg, Myristica fragrans), and one compound from each library was selected for derivatization. The isolates, derivatives, and numerous fractions were evaluated through the panel of in vitro assays. However, type 2 diabetes is characterized by a lack of insulin sensitivity, and since hormone regulation in cell culture models of diabetes are extremely variable and limited, cell culture is not an ideal model of (open full item for complete abstract)

Committee: Esperanza Carcache de Blanco (Advisor) Subjects: Biochemistry; Chemistry; Pharmacy Sciences

PhD, University of Cincinnati, 2018, Medicine: Molecular and Developmental Biology

            The cardiovascular system is one of the earliest organ systems to form in the developing embryo. Development of blood vessels is a strictly regulated process that is crucial for embryonic development as well as for organ functionality throughout the life of an organism. The circulatory system is necessary for the transportation of fluid, oxygen, metabolites, waste products, hormones, and immune cells and cytokines throughout the body. These functions cannot be carried out without proper formation of blood and lymphatic vessels. The focus of this dissertation is increasing the understanding of the mechanisms and pathways necessary for blood and lymphatic vessel development.             Previous studies have implicated mixed models of de novo vasculogenesis and angiogenesis in the development of blood vessels in organs. These studies, carried out in mouse and chick embryos, lack the ability to track vessel development in live embryos and therefore identify the source of cells forming these vessels. Here we identify a novel mechanism of organ vascularization in which endothelial cells in existing vessels migrate to form new vessels. We utilize zebrafish due to the rapid development, transparent embryos, and amenability to genetic manipulation to study vascularization of the embryonic gut tube. Vegfaa expression in the presumptive endoderm drives ventral migration of endothelial cells out of the posterior cardinal vein (PCV), which then coalesce to form the sub-intestinal vein (SIV) and supra-intestinal artery (SIA). These results represent a novel mechanism of organs vascularization that is likely to occur in other organs and in other vertebrate organisms.             We also investigate the role of ETS transcription factor, Etv2, in lymphatic development/lymphangiogenesis. Prior studies have demonstrated the requirement of Vegfc/Vegfr3 signaling in lymphatic development, however questions about the process remain, including how this signaling is regulate (open full item for complete abstract)

Committee: Saulius Sumanas Ph.D. (Committee Chair); Elisa Boscolo Ph.D. (Committee Member); Tony J. DeFalco Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Chunyue Yin Ph.D. (Committee Member) Subjects: Developmental Biology

Master of Science, University of Akron, 2017, Biology

Mammalian leptin (LEP) is a pleiotropic peptide hormone best characterized for its roles related to obesity and diabetes. However, the molecular function of the leptin signal transduction pathway in non-mammals is less clear. Comparative studies that address leptin signaling in non-model organisms are integral components of the leptin phylogenetic history, and there is little evidence addressing the functional disparities between the teleost leptin paralogues and mammalian leptins. To demarcate genes and biochemical pathways regulated by leptin signaling in developing zebrafish, microarray gene expression data were generated with total RNA isolated at 48 hours post fertilization from leptin-a morpholino oligonucleotide “knockdown”, recombinant leptin-a “rescue”, and wild type embryos. Expression estimates were computed for 26,046 genes across 16 microarray samples. Differentially expressed genes (DEG), (KEGG) pathways, and Gene Ontologies (GO) were evaluated for three contrasts (Morphant:Control, Rescue:Morphant, Rescue:Control). Signaling pathways that respond to leptin-a knockdown and rescue are analogous to gene targets of the mammalian LEP system (“GnRH”, “MAPK”, “Adipocytokine”, “Phosphatidylinositol”, “mTOR”, “ErbB”, “FoxO”, and “Notch”). A subset of differentially expressed transcription factors in leptin-a morphants are homologous to putative regulators of LEP expression in mammals (cebpb, creb5, fosl1a, mybl1, pax5, pou3f1, pparg, stat1a). “Neuroactive ligand-receptor interaction” as well as cAMP-responsive hormone signaling pathways responded to leptin-a. Consistent with leptin-a as an endocrine regulator, agouti-related peptide-2 (agrp2), cocaine-and-amphetamine-related-transcript (LOC557301), gonadotropin-releasing hormone 2 (gnrh2), and melanocortin receptor 5a (mc5ra) were dysregulated in rescue embryos. Further, “Notch signaling” and “Spinal cord/CNS development” were enriched in morphants whereas rescue arrays were comparable to wild type e (open full item for complete abstract)

Committee: Richard Londraville (Advisor) Subjects: Bioinformatics; Biology; Comparative; Developmental Biology; Endocrinology

PhD, University of Cincinnati, 2016, Medicine: Molecular and Developmental Biology

Congenital heart defects are the most common birth defect in the US affecting 1% of all live births. Normal heart development requires proper regulation of retinoic acid (RA) signaling levels. One way the developing embryo limits RA levels is through the Cyp26 enzymes, which metabolize RA into easily degraded derivatives. Loss of Cyp26 enzymes, and subsequently increased RA signaling, has been implicated in several human diseases with heart malformations, however the mechanisms underlying these cardiac defects are not understood. This dissertation focuses on the mechanisms through which perturbations in the RA signaling levels result in congenital heart defects and understanding the feedback relationship between RA signaling and Cyp26 enzymes. Previous studies have shown that loss of Cyp26a1 or Cyp26a1 and Cyp26c1 together (referred to as Cyp26 deficient) results in cardiovascular defects in vertebrate embryos, however these defects have not been characterized. Here, we find that loss of Cyp26 enzymes leads to an anterior shift in patterning that results in an increase in atrial progenitors at the expense of anterior endothelial progenitors. Furthermore, we found that, while Cyp26 expression partially overlaps with the cardiovascular progenitors, the effects of Cyp26 on cardiovascular specification are mainly non-autonomous. Therefore, our findings suggest Cyp26 enzymes are required to balance the cardiac and vascular lineages during early development. Excess RA signaling during human development results in RA embryopathies, which commonly have outflow tract defects. We found that excess RA, due to loss Cyp26 enzymes, results in failure of second heart field addition and loss of first heart field integrity. Interestingly, we found that the primary cause of these heart defects was increased MMP9 expression, suggesting the requirement for extracellular matrix in both first and second heart field development. Altogether, this work suggests that the etiology of RA- (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Burns Blaxall Ph.D. (Committee Member); Kenneth Campbell Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology; Obstetrics

PhD, University of Cincinnati, 2016, Medicine: Molecular and Developmental Biology

Retinoic acid (RA) and canonical Wnt/ß-catenin (Wnt) signaling are two major pathways that direct early embryonic development. Abnormalities in either pathway lead to congenital birth defects of the limb, heart and central nervous systems. This thesis is comprised of three studies that provide novel insights into the roles of RA and Wnt signaling during zebrafish development. Utilizing a ligand trap method, we created new zebrafish transgenic RA reporters, which act as a direct sensor for RA. We have shown these novel transgenic lines are responsive to RA and report previously unknown areas of activity. These reporters will be valuable tools to aid in studying early embryonic RA signaling and responses. Crossregulation of RA and Wnt signaling has been reported in different cell types and physiological conditions with clinical and physiological relevance. Both RA and Wnt regulate Dact (Dapper antagonist of catenin) proteins, thus this family of proteins might act as a linker between these two pathways in various developmental processes. With that aim we have examined the dynamic expression pattern and regulation of dact3 genes in zebrafish. Our analysis has shown dact3b but not dact3a is negatively regulated by RA in the hindbrain. Multiple congenital defects include both cardiac and craniofacial abnormalities, which suggest there is close relationship between adjacent cardiac and pharyngeal field. Signaling pathways that regulate progenitor specification in the cardiopharyngeal field (CPF) are not yet well understood. Previous studies have shown that Wnt promotes cardiac specification and inhibits pharyngeal muscle (PM) development during somitogenesis. Our work demonstrates that contrary to previous studies, Wnt signaling promotes PM loss only prior to gastrulation, and interestingly this timing correlates with increases in cardiac specification. We also find that excess Wnt leads to an increase of differentiated first heart f (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Louis Muglia M.D. Ph.D. M (Committee Member); Brian Gebelein Ph.D. (Committee Member); Jerry Lingrel Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Biology

Master of Science, University of Akron, 2015, Biology

The stimulant effects of methamphetamine are known to affect cardiovascular function and behavior in rats and adult humans, but little is known about cardiovascular development in the presence of methamphetamine. The purpose of this study was to determine if methamphetamine exposure (acute and chronic) affects the survivability and cardiovascular development of zebrafish (Danio rerio) embryos. Zebrafish embryos were either injected with a single dose of methamphetamine (acute exposure) or soaked in a methamphetamine solution for 24 hours (chronic exposure). Survival, morphological development, and cardiovascular development were recorded at 48 hours post-fertilization. Embryonic methamphetamine exposure resulted in increased eye size, cardiac output, and red blood cell velocity and a decreased yolk size. Behavior was also assessed in juvenile zebrafish to determine methamphetamine's role in aggression and avoidance behaviors. Four month old zebrafish were injected with either methamphetamine or a saline control and subjected to mirror image stimulation to quantify aggression. No differences were found in the amount of aggression or avoidance between methamphetamine-exposed fish and control fish. Methamphetamine exposure during development causes select cardiovascular and morphological alterations, while exposure to juvenile fish has no effect on certain behaviors such as aggression and avoidance.

Committee: Brian Bagatto Dr. (Advisor); Qin Liu Dr. (Committee Member); Jordan Renna Dr. (Committee Member) Subjects: Biology; Developmental Biology; Physiology

Doctor of Philosophy, Case Western Reserve University, 2014, Genetics

Epigenetic regulation of the genome is essential for regulating gene expression to ensure proper tissue differentiation and patterning during embryonic development. The importance of epigenetic regulation is best highlighted by the large number of diseases that arises from its misregulation. One particular epigenetic regulator that signifies this importance is the chromatin remodeling protein, CHD7. Changes in CHD7 expression during development results in a congenital disorder known as CHARGE syndrome comprising a complex constellation of developmental abnormalities in humans affecting multiple organ systems such as the eyes, ears, and heart. The CHD7 protein functions to regulate both nucleoplasmic and nucleolar gene expression; however, the respective contributions of each of these functions to proper embryonic development and of their misregulation to CHARGE syndrome is not clear. To this end, we sought out to model changes in CHD7 using the zebrafish as a model for early embryonic development. Targeting of the zebrafish chd7 homolog resulted in developmental abnormalities in multiple organ systems overlapping with those affected in CHARGE syndrome patients. Further investigation in to the chd7 morphant zebrafish revealed global decreases in cellular proliferation accompanied by increased expression of cell-cycle regulator genes. By targeting the expression of fbxl10, a known negative regulator of both rRNA expression and cellular proliferation, we successfully modulated the levels of cellular proliferation and significantly restored the majority of morphological abnormalities. Collectively, these studies indicate that CHD7 plays a significant role in regulating cellular proliferation during embryonic growth and development and provides a novel explanation for the pathogenesis of CHARGE syndrome. To further investigate this possibility, we next sought to test this hypothesis in a human model of embryonic development, or rather, human induced pluripotent stem ( (open full item for complete abstract)

Committee: Peter Scacheri PhD (Advisor); Ronald Conlon PhD (Committee Chair); Peter Harte PhD (Committee Member); Brian McDermott Jr., PhD (Committee Member) Subjects: Genetics

PhD, University of Cincinnati, 2011, Medicine: Toxicology (Environmental Health)

Dioxin-like compounds (DLCs) are potent teratogens that persist in the environment and pose significant risk to human and ecological health. Variability in risk of congenital heart defects (CHDs) during development caused by DLCs has been demonstrated within and among several vertebrate species. Beyond our knowledge of the aryl hydrocarbon receptor (AHR) and its role in mediating toxicity for this class of compounds, little else is known concerning the precise downstream targets influencing this vulnerability. In the present study, six lines of zebrafish (Danio rerio) with divergent genetic backgrounds were screened for susceptibility to developmental cardiotoxicity caused by the prototypical DLC, 3,3',4,4',5-pentachlorobiphenyl (PCB126); a range of up to ~40-fold differences was observed. The most susceptible and the most resistant strains were then chosen for quantitative trait loci (QTL) analysis (F2-intercross). High-resolution microsatellite molecular markers spanning the entire genome were used to determine association between the phenotype (risk of PCB126-induced cardiac dysregulation) and genotype (chromosomal loci) in the recombinant generation. Multiple QTL were identified–some acting alone, others additively, and others via epistatic interaction. Together, these QTLs account for 23.48% of the phenotypic variance observed in heart defects resulting from PCB126 exposure (LOD score = 13.14; P = 2.52 x 10–7). The transcription factor E2F1 (e2f1) and the tropomyosin 4 (tmp4) genes were identified as potential candidates involved in susceptibility to PCB126-induced developmental cardiotoxicity. The consequences that a loss-of-function of either gene has on the developing zebrafish heart were investigated relative to what is observed following PCB126 exposure. The phenotypes of zebrafish injected with morpholinos targeting E2F1 or TPM4 are highly similar to that of PCB126 cardiotoxicity. Loss of e2f1, tpm4, or exposure to PCB126 diminishes heart function, inhibi (open full item for complete abstract)

Committee: Daniel Nebert MD (Committee Chair); Daniel Prows PhD (Committee Member); Thomas Bartman MD,PhD (Committee Member); Mary Beth Genter PhD (Committee Member); Alvaro Puga PhD (Committee Member) Subjects: Genetics

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Cytosolic sulfotransferases are phase II drug metabolizing enzymes responsible for carrying out a sulfonation reaction which adds a sulfonyl group to a hydroxyl or amino group of its substrate. These enzymes are believed to be involved with the regulation of endogenous hormones such as neurosteroids as well as xenobiotics. Considering the increasing rates of environmental estrogen exposures, it is important to understand the role that sulfotransferases may play in the early stages of vertebrate development. SULT2 ST1 is a sulfotransferase identified in zebrafish which resembles the SULT2 A1 gene in humans. To establish a link between sulfotransferase expression and developmental toxicity, the SULT2 ST1 gene was knocked down in the zebrafish model. This was accomplished by microinjection of translation blocking morpholino oligonucleotides into <4 cell stage zebrafish embryos. The phenotypic changes observed in the SULT2 ST1 knockdown fish indicate that the absence of the enzyme may be responsible for underdeveloped fin and systemic failure in embryo development phenotypes. Western Blot was used to confirm a reduction in expressed protein in knockdown animals.

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science in Pharmaceutical Sciences, University of Toledo, 2010, College of Pharmacy

Sulfonation is an important reaction in regulating the biological activities of a variety of endogenous and environmental compounds and is catalyzed by cytosolic sulfotransferases that use PAPS (3'-phosphoadenosine-5'-phosphosulfate) as the sulfonate (SO3-) donor. SULTs are present in mammals and other vertebrates and play an important role in the detoxification of xenobiotics especially environmental estrogens. SULTs are also involved in the biotransformation of endogenous compounds (hormones, steroids) which might be a mechanism for maintaining the homeostasis of these compounds in-vivo. In this study we have attempted to assess the role of the hydroxysteroid sulfotransferase SULT2 ST2 in zebrafish (Danio rerio) development by knocking down the expression of the enzyme using a morpholino. Zebrafish embryos were microinjected with a morpholino which had a sequence complementary to the sequence of the SULT2 ST2 gene. The injections were done when the embryos were in the 1-4 cell stage with three concentrations of the morpholino: 0.5 ng/nL, 1.0 ng/nL and 2.0 ng/nL. The embryos were then observed for survival rates and abnormal phenotypes up to 144 hours post fertilization (hpf). The phenotypes observed were cardiovascular abnormalities such as cardiac edema and irregular heartbeat, abdominal edema, lordosis, notochord deformities, tail deformities and very few cases of craniofacial malformations. At the highest concentration of the morpholino, almost all knockdowns displayed mild to severe cardiac edema which was the specific phenotype at that concentration after the hatching period (48-55 hpf). The extent of knockdown was also determined by western blot experiment and it was found that the knockdown was partial and there was some enzyme present at measurable levels in the embryos. In conclusion, morpholino knockdown of the SULT2 ST2 gene in zebrafish embryos caused several non-specific phenotypes. At the 2.0 ng/nL concentration mild to severe cardiac edema was obser (open full item for complete abstract)

Committee: Frederick Williams PhD (Committee Chair); Ming-Cheh Liu PhD (Committee Member); Steven Peseckis PhD (Committee Member) Subjects: Pharmacology; Toxicology