Bachelor of Arts, Capital University, 2021, Biological and Environmental Science

Fetal alcohol syndrome is a serious condition that affects the development of fetuses with irreversible effects that can impact individuals throughout their lives. The cardiovascular system is one example of an organ system in which abnormalities caused by alcohol can occur. The heart is one of the first structures to be formed, and heart development is highly conserved among amniotes. There are difficulties studying the effects of ethanol on human embryos due to ethical concerns; as a result, the use of animal models, particularly avian models, is widely used. The effects of ethanol have not been widely studied on Pekin ducks, Anas platyrhynchos, and ducks offer advantages compared to other model organisms, such as their larger size and durability. The purpose of this study was to develop a method for testing the effects of ethanol on the development of the heart and cardiovascular system in ducks. The development of the cardiovascular system occurs over several stages of development, and treatment of ethanol at different stages leads to various potential abnormalities of heart structure and function. The developed protocol determines which stages of heart development are most sensitive to ethanol effects, and what anomalies are expected to form after exposure to ethanol.

Committee: Nancy Swails (Advisor) Subjects: Biology

PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Congenital heart disease (CHD) stands as the most prevalent congenital anomaly, impacting approximately 4 per 1000 live births. Single ventricle (SV) CHD, constituting 7.7% of CHDs, represents a severe and intricate form of CHD where patients typically exhibit a single dominant left or right ventricle at birth, insufficient to sustain normal pulmonary and systemic circulation. SV CHDs encompass diverse cardiac malformations, including semilunar (SL) or atrioventricular (AV) valve hypoplasia, stenosis/atresia, and malalignment of the pulmonary artery and aorta. The varied manifestations of SV abnormalities imply a complex disease etiology intertwined with various cardiac developmental anomalies. Despite substantial progress in understanding the molecular mechanisms of various SV diseases, intrinsic defects in human cardiac endothelium and related valvular structures remain elusive. The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized our ability to precisely dissect cellular identity, functions, and interactions during cardiogenesis and CHD pathogenesis. Leveraging human induced pluripotent stem cells (iPSCs) expands our experimental repertoire, allowing the generation of various cardiac cell types to functionally validate genomic and transcriptomic data in cellular models. Herein, we combined scRNA-seq analysis of human tissues with patient-derived iPSCs to unravel the endothelial/valvular pathobiology in SV diseases, such as hypoplastic left heart syndrome (HLHS) and pulmonary stenosis (PS). The effort extended to specify iPSC-derived valve endothelial cells (VECs) and valve interstitial cells (VICs) representing the specificity of all four valves. Despite the previous knowledge of myocardial defects in HLHS pathology, we demonstrated the intrinsic defect in disease vascular arterial endothelium, including KMT2D-NOTCH mediated proliferation, angiogenesis, and EC-smooth muscle cell interactions. Our results suggested the importance o (open full item for complete abstract)

Committee: Mingxia Gu M.D. Ph.D. (Committee Chair); Susanne Wells Ph.D. (Committee Member); Mei Xin Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); Yanbo Fan Ph.D. (Committee Member) Subjects: 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

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

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

Heart failure causes millions of deaths annually and presents a large global healthcare burden. A regenerative cure for the damaged myocardium, where new muscle forms by proliferation of pre-existing cardiomyocytes, is an attractive therapeutic goal. Adult mammalian cardiomyocytes are terminally-differentiated, only capable of proliferating at a very low rate that is insufficient for a regenerative response after myocardial infarction. However, over the past decade, a transient innate capacity for cardiac regeneration during the early neonatal period has been described in both rodents and swine. Whether such a capacity exists in newborn human infants is unknown. Studying the mechanisms of regenerative potential in neonatal rodents and swine could offer greater insight into heart development in human neonates, and also facilitate discovery of novel targets for human heart disease therapy. Cardiomyocyte maturational processes occur concurrent with loss of heart regenerative potential in early neonatal mice. These maturational processes, and the transcriptional mechanisms regulating them, have been successfully manipulated to induce cardiac regenerative repair in adult mouse hearts after injury. Pigs also possess a similar period of early neonatal heart regenerative capacity as mice. However, the maturational dynamics of cardiomyocyte growth in the postnatal pig heart are not well-defined, despite popularity of swine as large mammal models for cardiac preclinical studies. In Chapter 2 of this dissertation, we describe cardiac maturation in postnatal swine from newborn to adolescent ages. Our results show discordance between time of terminal cardiomyocyte maturation and loss of heart regenerative potential in postnatal swine, dissimilar to rodents. Further, postnatal pig cardiomyocytes are distinct from rodents and humans, exhibiting extensive multinucleation of up to 16 nuclei per cardiomyocyte by 6 postnatal months. These differences hold importance for preclin (open full item for complete abstract)

Committee: Katherine Yutzey Ph.D. (Committee Member); Stacey Huppert Ph.D. (Committee Member); Joshua Waxman Ph.D. (Committee Member); Sakthivel Sadayappan Ph.D. (Committee Member); Nancy Ratner Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2021, Medicine: Molecular and Developmental Biology

Congenital heart disease (CHD) is the most common birth defect, and unexplained CHD may be secondary to undiscovered roles of gene/environment interactions. Improper placentation causes disruptions to the intrauterine environment, making the fetus more vulnerable to birth defects like CHD. In this dissertation, I first explore the involvement of placental abnormalities in human congenital heart disease. To do this, I examined placental structural changes, changes in both nutrient transporter expression and location, and gene expression in term placentas at delivery from patients born with Hypoplastic Left Heart Syndrome (HLHS) and Transposition of the Great Arteries (TGA). I discovered that vascular changes were present in both CHD subtypes, but that nutrient transporter changes were divergent. Additionally, genes important for heart development were differentially expressed in the placenta but varied by subtype. Despite placental changes in both subtypes, TGA fetuses maintained a normal growth trajectory, while HLHS patients were generally born small for gestational age. I also investigated gene expression patterns of first trimester human cell populations of placenta and heart. Substantial numbers of genes were expressed in both first trimester human placental and cardiac cell populations, demonstrating that shared gene pathways govern placental and heart development. To better characterize the impact of genetic changes on placental and heart development, I utilized a mouse model with a conditionally activated Hand1 mutation associated with HLHS. Activating the mutation in labyrinth trophoblast and heart resulted in early gestational lethality caused primarily by failure of appropriate trophoblast differentiation and labyrinth vascularization. In contrast, when I restricted the Hand1 mutation to endothelial cells, placental weight dropped, placental labyrinth vasculature remodeling was impaired, heart development disrupted, and fetal growth restriction/demise occu (open full item for complete abstract)

Committee: Helen Jones Ph.D. (Committee Chair); Elisa Boscolo Ph.D. (Committee Member); James Cnota M.D. (Committee Member); Nathan Salomonis M.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2021, Molecular, Cellular and Developmental Biology

Congenital heart disease (CHD) is the most common birth defect in live births with an estimated incidence of 1%.  Although advancements in surgical care have significantly improved patient outcomes, CHD is still a major contributor to morbidity and mortality. The link between genetics and CHD has been well established following population-based studies and single-gene knockout approaches in animal models. Although a subset of CHD can be attributed to a few key genetic contributors, the genetic underpinnings of most CHD cases remain unresolved.  Therefore, uncovering novel genetic contributors to CHD is of critical importance. Large scale sequencing approaches of patients with CHD have identified numerous potentially damaging genetic variants. However, gene prioritization and experimental validation for the majority of these candidate genes are lacking. Model organism studies, specifically those performed in mice, have been instrumental in uncovering the developmental impact that genetic contributors have on the developing heart. The growing availability of genomic technologies allows us the opportunity to exploit mouse models of CHD as a tools to facilitate gene discovery. We propose that transcriptomic profiles derived from the normal developing heart and animal models of CHD can be used to prioritize candidate genes that contribute to CHD phenotypes in patients and also identify novel genes and molecular pathways critical for heart development. To better understand the genetic contributors of tetralogy of Fallot (TOF), we designed a gene prioritization pipeline that made use of transcriptomic data derived from a highly penetrant mouse model of outflow tract (OFT) malformations and genetic variant information derived from patients with TOF. The common OFT is the anatomical precursor of the aorta, pulmonary artery, and semilunar valves. Disruption of the normal development of the OFT contributes to OFT malformations such as TOF.  We performed bulk RNA-Sequencing o (open full item for complete abstract)

Committee: Vidu Garg MD (Advisor); Federica Accornero PhD (Committee Member); Lynette Rogers PhD (Committee Member); Brenda Lilly PhD (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2020, Medicine: Toxicology (Environmental Health)

The focus of this dissertation is the characterization of the epigenetic, structural, and functional consequences of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a potent ligand of the aryl hydrocarbon receptor (AHR), in differentiated cardiomyocytes and the adult heart as a result of continuous exposure from fertilization through adulthood. Congenital heart disease (CHD) is the most common congenital abnormality and one of the leading causes of mortality worldwide. Ongoing scientific investigations show that a precise etiology remains elusive, but is likely to result from complex interactions between genetic and environmental factors during heart development, at a time when the heart adapts to diverse physiological and pathophysiological conditions. Crucial among these is the regulation of cardiomyocyte development and postnatal maturation, governed by dynamic changes in DNA methylation. Previous work from our laboratory showed that exposure to the environmental toxicant tetrachlorodibenzo-p-dioxin (TCDD) disrupts several molecular networks responsible for heart development and function. In addition, interference with endogenous developmental functions of the AHR, either by gene ablation or by in utero exposure to TCDD, was shown to cause structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. These studies demonstrated the potential role of AHR in the pathogenesis of CHD by its disruption during development. The biological half-life of TCDD is 7-10 years in humans. Therefore, it is of clinical importance to understand the consequences of continuous activation of AHR by TCDD from development through adulthood on cardiac structure and function. Chapter One summarizes the current body of knowledge surrounding TCDD, the AHR, heart development, CHD, the risk and mechanisms of heart failure, and the role of DNA methylation in cardiomyocyte maturation. Chapter Two describes the effects of TCDD on DNA me (open full item for complete abstract)

Committee: Alvaro Puga Ph.D. (Committee Chair); Katherine Burns Ph.D. (Committee Member); Jack Rubinstein M.D. (Committee Member); Ying Xia Ph.D. (Committee Member); Xiang Zhang Ph.D. (Committee Member) Subjects: Toxicology

Doctor of Philosophy, Case Western Reserve University, 2020, Biomedical Engineering

Congenital heart disease/defects (CHDs) account for approximately one-third of all birth defects globally. Many of these will require invasive treatment within the first year of life, subsequent interventions, and a lifetime of monitoring. Alcohol has been known as a teratogen for several decades, and prenatal exposure alone is a significant cause of worldwide CHDs. Optical Coherence Tomography (OCT) has been used as an imaging modality for nearly three decades and is especially suited to the non-invasive imaging of small, semi-transparent tissue structures. Doppler OCT (DOCT) adds the ability to visualize and measure fluid flow and movement of reflective components within the tissue. This work presents the development of technology to further enable DOCT as a tool in measuring the function of anatomical structures. First, an interferometer design is described, which significantly reduces phase noise by incorporating a second, narrow band, continuous-wave light source as a phase-reference. By implementing this interferometer and related processing algorithms into a DOCT system, significant frequency noise reduction is demonstrated in reflective and scattering samples. Second, a modification to a DOCT system is presented using a single sample beam that provides velocity information from multiple angles within the beam. By introducing a delay element into part of the OCT beam path, the sample beam is divided into several components, each with a different group delay and each providing a separate interferogram with its own effective Doppler angle. By combining the Doppler shift measured in each of these component interferograms, the flow velocity vector is fully determined. OCT and DOCT are then applied to an embryonic avian model for Fetal Alcohol Spectrum Disorder (FASD) in order to study the effects of alcohol on early heart development. Folic acid is administered to a test group to learn more about its role in preventing CHDs. Through these embryonic stud (open full item for complete abstract)

Committee: Andrew Rollins (Advisor); Michiko Watanabe (Committee Member); Kenneth Singer (Committee Member); Xin Yu (Committee Chair) Subjects: Biomedical Engineering; Developmental Biology; Medical Imaging; Optics; Physics; Scientific Imaging

Doctor of Philosophy, The Ohio State University, 2019, Educational Studies

The purpose of this study was to explore the relationship between Heart Rate Variability (HRV) and counselor trainees' cognitive development. More specifically, this study explored the influence of resting HRV (rMSSD) on counselor trainees' sustained attention (Continuous Performance Task), working memory (n-back Task) and cognitive performance on a practice NCMHCE exam. The study used descriptive and multivariate analyses to examine data collected from a sample of counselor trainees enrolled in a CACREP-accredited clinical mental health counseling program in the state of Ohio (N = 69). Additional exploratory analyses were conducted to enhance the understanding of the data. The analyses revealed two key findings that are the first of their kind in counselor education. First, group differences revealed that counselor trainees' HRV is connected to cognitive complexity. An Analysis of Variance (ANVOVA) identified that high HRV levels (50.8ms and higher) were connected to faster reaction time (p < 0.05) and more accurate responses on two sustained attention tasks, a Choice Reaction Time Task (p < 0.05) and a Serial Pattern Matching Task (p < 0.05). Second, group differences in counselor trainees' developmental level revealed that NCMHCE scores are reflective of counselor trainees' developmental level. A multivariate regression analysis identified that counselor trainees in the post-practicum stage of their development scored higher on a practice NCMHCE exam than did counselor trainees in the pre-internship stage of their development (p < 0.05). The results of this study provide empirical support for the relationship between HRV and sustained attention in counselor trainees, suggesting that HRV may be a mechanism for higher levels of cognitive complexity. Additionally, the results of this study provide empirical support for the relationship between developmental level and practice NCMHCE exam scores, suggesting that the NCMHCE is reflective of counselor developmental and (open full item for complete abstract)

Committee: Darcy Haag Granello (Advisor); Paul Granello (Committee Member); Colette Dollarhide (Committee Member) Subjects: Counseling Education

PhD, University of Cincinnati, 2019, Medicine: Molecular and Developmental Biology

Congenital heart diseases (CHDs) are the most common birth defects. About one-third of CHDs are caused by malformations of the outflow tract (OFT). The OFT is a portion of the ventricle derived from later differentiating cardiac progenitors called the second heart field (SHF) during heart development. Although the number of chambers among animal species may differ, regulation of SHF development is highly conserved in vertebrates. In the past few years, individual signaling pathways and transcription factors involved in SHF development have been well studied. However, coordinated transcriptional and epigenetic mechanisms that direct the development of SHF progenitors remain largely unknown. In this dissertation, we explored the requirement of the epigenetic regulator Histone deacetylase 1 (Hdac1) during the SHF development using the zebrafish mutant called cardiac really gone (crg). Although hdac1 is broadly expressed in the embryo during early development, we found that the loss of hdac1 in crg mutant zebrafish causes a specific deficit of ventricular cardiomyocytes (VCs) and smooth muscle in the OFT, which is due to decreased proliferation of cardiac progenitors in the SHF. Furthermore, we found that there is overlap between Hdac1 and retinoic acid (RA)-responsive genes, as increases in RA signaling produce similar OFT defects. Specifically, we identified that the ectopic expression of the RA-responsive gene ripply3, which acts as a transcriptional co-repressor for Tbx1, is partially responsible for the specific reduction of VCs in hdac1 mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing the expression of the RA-responsive gene ripply3 within SHF progenitors. Our study of Hdac1 exemplifies the spatiotemporal role of a commonly expressed epigenetic regulator for heart development and how it may coordinate RA signaling. Overall, the information gained from this study (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Brian Gebelein Ph.D. (Committee Member); Jerry Lingrel Ph.D. (Committee Member); S. Steven Potter Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 1977, Graduate School

Committee: Not Provided (Other) Subjects: Psychology

Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Heart valve dysfunction affects ~2.5% of the population, and this incidence increases significantly in the elderly, affecting up to 13.2% of people over the age of 75. While the pathology has been well defined, mechanisms underlying progressive valve deterioration are still unknown. The mature, healthy valve structure is composed of two main cell types, the overlying valve endothelial cells (VECs) that form an uninterrupted monolayer surrounding the valve, and valve interstitial cells (VICs) that make up the core structure and secrete specialized layers of extracellular matrix (ECM) proteins. These specialized layers are arranged in a tri-laminar fashion, providing all the necessary biomechanics to withstand hemodynamic forces. In contrast to healthy young valves, aging valves are associated with deterioration of the ECM organization, leading to alterations in biomechanical properties that can result in stiffened or stenotic valves which fail to fully open. Although the pathogenic changes in ECM organization have been well described, little is known about how age-related changes in valve cell populations contribute to the progression of degeneration in the elderly. Here we provide insight as to how changes in the phenotype and function of valve cell populations occur throughout maturation and aging, which likely contribute to age-related valvular dysfunction. We identify key cellular processes within VECs that decline with age, including metabolism, endothelial to mesenchymal potential, proliferation and barrier function. Furthermore, using a novel method to isolate murine VECs, we perform RNA-sequencing analysis and report the differential and common transcriptomes of VECs at embryonic, post-natal, young-adult and aging-adult stages, revealing the age-dependent heterogeneity of these cells. In addition to resident valve cell populations, we show that circulating, CD45+ cells incorporate into the valve structures beginning at embryonic stages and continuing into ad (open full item for complete abstract)

Committee: Joy Lincoln PhD (Advisor); Vidu Garg MD (Committee Member); Brenda Lilly PhD (Committee Member); Heithem El Hodiri PhD (Committee Member) Subjects: Biology; Molecular Biology

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

Doctor of Philosophy, Case Western Reserve University, 2015, Biomedical Engineering

Blood flow is a critical factor that regulates developmental programs during cardiogenesis. During early embryonic development, deviations from the normal blood flow pattern have been shown to lead to congenital heart defects including septal defects and outflow tract anomalies. To better understand the role flow and the resulting hemodynamic forces play during cardiovascular development precise tools are needed to rapidly calculate and monitor these forces. Optical coherence tomography (OCT) is a noninvasive imaging modality that is well suited for imaging the developing heart due to its high spatial and temporal resolution. OCT is also capable of analyzing various cardiac functions by measuring the blood flow through the developing heart via the Doppler effect. Here we present several techniques we have developed that use structural and Doppler OCT to monitor and measure hemodynamic parameters in the early embryonic heart. First, we generated 4-D (3-D volumes over time) shear stress maps from Doppler OCT data sets. These maps enabled comparisons of shear stress from the inner curvature versus the outer curvature at different regions of the looping heart tube over the duration of a heartbeat. We also developed an orientation independent technique for measuring the absolute blood flow in a vessel from individual cross sectional images. This technique utilizes a dual angle delay encoding technique to obtain instantaneous pulsatile blood flow measurements irrespective of the vessel orientation. We used this technique to image the aortic arches in control and ethanol exposed embryos. The aortic arches undergo a significant morphogenesis from a symmetrical system of paired vessels to an asymmetrical structure and hemodynamics is thought to play a critical role in this transformation. Blood flow and shear stress were both calculated as well as the cross sectional area of the pharyngeal tissue surrounding the aortic arch vessel. Finally, we developed a technique for measu (open full item for complete abstract)

Committee: Andrew Rollins PhD (Committee Chair); Michiko Watanabe PhD (Committee Member); David Wilson PhD (Committee Member); Kenneth Singer PhD (Committee Member) Subjects: Biomedical Engineering; Developmental Biology

PhD, University of Cincinnati, 2015, Medicine: Molecular and Developmental Biology

Congenital heart defects are found in over 1% of live births. The cardiovascular system is required throughout development and adulthood to efficiently transport oxygen, nutrients, and waste products through the body. The size and structure of the heart muscle plays a key role in the efficiency of the heart to pump blood throughout the body. This dissertation focuses on developmental signaling events that set up the initial cardiac cell population and subsequent organ size. We investigate the temporal requirements for the Wnt signaling pathway in cardiac development, and the role of Nr2f proteins downstream of retinoic acid signaling in cardio-pharyngeal cell specification. Previous studies indicate that heart development requires reiterative phases of canonical Wnt/ß-catenin (Wnt) signaling. Here, we find three early phases of Wnt signaling regulate cardiac cell numbers. Wnt signaling promotes mesoderm and pre-cardiac mesoderm specification, is sufficient to inhibit cardiomyocyte differentiation, and promotes the differentiation of additional atrial cells. Interestingly, the inhibition of cardiomyocyte differentiation by Wnt signaling leads to cardiac progenitor cell death through a p53/Caspase-3 independent mechanism. Together with a report for a late role for Wnt signaling in restricting ventricular cell proliferation, our results indicate four distinct phases of Wnt signaling during the first three days of zebrafish development. We also examined downstream effectors of RA signaling, specifically Nr2f1a in zebrafish, as a regulator of early cardiac progenitor specification. Our results indicate that Nr2f proteins might have an early role in specification of the cardiac progenitor field in addition to the previously described later role in atrial cell specification. Interestingly, Nr2f1a regulates the cardiac field through mediating a cardio-pharyngeal fate decision, promoting pharyngeal muscle development at the expense of cardiac progenitor specifica (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Chair); Brian Gebelein Ph.D. (Committee Member); Jerry Lingrel Ph.D. (Committee Member); Saulius Sumanas Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2015, Cell Biology

The heart is the first organ to become fully functional during embryonic development. During tissue development, proteins undergo dynamic ebbs and flows, as genes are induced and repressed. Although RNA binding proteins (RBPs) intimately interact with target transcripts and mediate their processing, their involvement in cardiac development has been largely underappreciated. CUG-BP, Elav-like family member 1 (CELF1) is a multi-functional RBP found in various tissues, including the myocardium of the developing heart, and has been implicated in both nuclear and cytoplasmic roles. Currently, little is known about the roles of CELF1 in heart tissue and less is known about its involvement in normal organogenesis. In order to address the roles of CELF1 during embryonic development, I first investigated the expression patterns of two closely related CELF proteins (CELF1 and CELF2) in mouse and chicken embryos, demonstrating the diversity of isoforms and expression patterns of these transcripts and proteins, and the evolutionary conservation of these patterns during vertebrate embryonic development. Targets of CELF1 regulation during heart development are not known. I report here the identification of MYH7B (a sarcomeric myosin heavy chain) as a novel direct target of CELF1 regulation in the embryonic chicken heart. The regulation of MYH7B by CELF1 demonstrates the versatility and regulatory complexity of RBPs. Based on these findings, as well as published data implicating CELF1 in cardiac contraction and contractility post-natally, I hypothesized that CELF1 is involved in the structure and function of the myocardial contractile apparatus in the embryonic heart. I describe investigations of roles of CELF1 during cardiac morphogenesis at the cell, tissue, and embryo levels. I report that CELF1 is important for myofibrillar organization in cultured primary chicken embryonic cardiomyocytes (the only cells in the developing heart in which CELF1 is expressed), as well as for the (open full item for complete abstract)

Committee: Andrea Ladd PhD (Advisor); Donna Driscoll PhD (Committee Chair); Alan Tartakoff PhD (Committee Member); Hua Lou PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Developmental Biology

Doctor of Philosophy, The Ohio State University, 2014, Health and Rehabilitation Sciences

Infants with complex congenital heart disease (CCHD) require palliative surgery of their heart in the first weeks of life. Long-term developmental outcomes in infants with CCHD is increasingly becoming an area of concern for medical and rehabilitation professionals as these infants are now surviving into adulthood. Recent work has focused on identifying impairments in cognition, motor, and autonomic nervous system function during preschool and school age in those with CCHD and brain injury in infancy. It is unknown if impaired cognitive, motor, and autonomic nervous system function are identifiable during early infancy in infants with CCHD. Since infants with CCHD demonstrate similarly immature brain as preterm infants, who are well known as a high-risk population for developmental impairments, a group of preterm infants and a group of typically developing (TD) infants were also included for the same tests. The first purpose of this research was to examine cognitive, motor, and autonomic function in infants with CCHD, infants with TD, and infants born preterm at 3 months of corrected age (CA). The second purpose of this research was to identify the associations between the cognitive performance, specifically learning or not learning, and the function autonomic, motor, and state organizational systems in infants with CCDH, infants with TD, and infants born preterm. The synactive theory is a model used to describe how the organization of different body systems influences neurobehaviors in preterm infants; it was used as the conceptual framework in this research. Learning and short-term memory were evaluated with the mobile paradigm. In the mobile paradigm task, infants' kicking movement causes the mobile to move, which produces visual and auditory feedback. This process allows infants to learn a cause-and-effect relationship. The mobile paradigm was used to test learning on Day 1, and short-term memory on Day 2. Learning and short-term memory were measured within (open full item for complete abstract)

Committee: Jill Heathcock (Advisor); Tondi Harrison (Committee Member); Ajit Chaudhari (Committee Member); Deborah Larsen (Committee Member) Subjects: Health Sciences; Physical Therapy

PhD, University of Cincinnati, 2013, Medicine: Molecular and Developmental Biology

Congenital heart disease (CHD) is the most common congenital anomaly occurring in 1% of live births in North America. The molecular and genetic mechanisms of CHD are largely unknown. Mutations in genes important for left-right (LR) patterning have been identified in patients with laterality disorders associated with CHD as well as in patients with isolated CHD. X-linked heterotaxy-1 (HTX1) is a developmental condition of laterality caused by mutations in the zinc finger transcription factor ZIC3 (OMIM #300625) located on Xq26.2. Mutations in ZIC3 exhibit variable expressivity and result in a wide spectrum of congenital anomalies. Randomization in the placement of visceral organs in addition to midline and central nervous system (CNS) defects results in a complex phenotype with high rates of morbidity and mortality typically from complex congenital heart malformations. The requirement of ZIC3 in LR patterning appears to be conserved with all vertebrate model organisms displaying molecular and morphological patterning defects similar to humans. While data suggest that ZIC3 plays multiple roles in development, the exact mechanism(s) by which ZIC3 affects LR patterning and other developmental processes has been unknown. The regulators and downstream targets of ZIC3 are largely unknown making it problematic to place ZIC3 within an established signaling pathway. In this dissertation, conditional loss-of function studies in mouse reveal a critical early role for Zic3 in node morphogenesis, the vertebrate LR organizer, which results in LR patterning and heart looping defects. These experiments establish, for the first time, the temporal and tissue-specific requirement for Zic3 for normal LR development. Studies in mouse, Xenopus and zebrafish are beginning to uncover multiple roles of ZIC3 in various developmental processes. While studies in human continue to reveal complex phenotypic variability involved with ZIC3 mutations. To gain a better understanding of the molecular (open full item for complete abstract)

Committee: Stephanie Ware M.D., Ph.D. (Committee Chair); Steven Potter Ph.D. (Committee Member); Robert Hinton M.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2001, Medicine : Molecular and Developmental Biology

Understanding the molecular signals involved in cell lineage decisions is becoming increasingly important for clinical applications. Recruitment of multipotential embryonic stem cells to the cardiomyogenic lineage is being pursued to repair cardiovascular injury in vivo. The current studies were aimed at unraveling the molecular pathways active in the lateral plate mesoderm that establish the cardiac cell fate in the anterior of primitive streak stage chicken embryos. Experiments were focused on the chicken model system because of the ease of accessibility and experimental manipulation during early development. Initially, explantation studies were undertaken to clarify inconsistencies among classical fate maps of the cardiac progenitors in the gastrulating avian embryo and to reconcile this position with the expression of genes implicated in early cardiogenesis. Previous evidence indicates the existence of a positive cardiac inductive pathway in the anterior regulated by Bone Morphogenetic Proteins (BMPs) that activates the expression of the earliest known cardiac marker, cNkx-2.5. Coincidence of the expression of these genes with the redefined lateral position of the cardiac progenitors in the anterior mesoderm indicates for the first time that prospective heart cells are in contact with a positive cardiac inductive signal as soon as they migrate to the lateral plate. Additionally, expression of the activin type IIa receptor in regions responsive to BMP-2 suggests a role in the BMP-2/cNkx-2.5 cardiac inductive pathway. Further experiments indicate that cardiogenic cell fates are negatively regulated in the posterior lateral plate mesoderm by a pathway activated by the caudal-related transcription factor, cCdx-B. cCdx-B is expressed throughout the posterior non-cardiogenic region in the gastrulating avian embryo, and activates the expression of posterior Hox genes and the Wnt signaling family member, cWnt-8c. Recent evidence indicates that cWnt-8c, a promoter of non (open full item for complete abstract)

Committee: Katherine Yutzey (Advisor) Subjects: Biology, Molecular