MS, University of Cincinnati, 2019, Medicine: Genetic Counseling

Objective: To describe U.S. Society for Assisted Reproductive Technology (SART) member clinical policies and provider opinions on aneuploid embryo transfer (ET). Design: Internet surveys. Materials and Methods: Two surveys about aneuploid ET were developed by the authors and issued via the SART listserv. A 7-question survey on clinical policies was sent to laboratory directors of 343 SART clinics. A 32-question survey about personal opinions was sent to all U.S. SART members. Clinical policy variables and outcomes were compared using Pearson's Chi Squared analysis. Demographic variables and outcomes were tested for association using Kruskal-Wallis tests. Open-ended questions were analyzed by performing inductive framework analysis to identify common themes in the data. Results: 49 laboratory directors responded to the clinical policies survey. 232 respondents completed the provider opinion survey. Clinics who more frequently performed PGT-A (100+ cycles/year) were more likely to have a policy in place regarding aneuploid ET (p=0.03). Providers who worked primarily in patient-facing roles held more agreeable (p=0.048) opinions towards aneuploid embryo transfer when requested by the patient. Across respondents, 50% expressed that they would agree to transfer embryo with a positive PGT result for Trisomy 21, whereas the majority would not agree to transfer embryos with positive results for the other conditions, with ranges from 13% of respondents for Trisomy 18 to 42% for Monosomy X. Across all respondents, 97% agreed that genetic counseling should be required by clinics prior to the transfer of an aneuploid or mosaic embryo. . Common themes identified included patient autonomy, limitations of preimplantation genetic testing for aneuploidy (PGT-A) testing, mosaicism, a need for documentation and informed consent, and whether PGT-A should be performed if a patient would transfer an aneuploid embryo. Conclusions: Among employees at SART me (open full item for complete abstract)

Committee: Melanie Myers Ph.D. (Committee Chair); Valentina Pilipenko Ph.D. (Committee Member); Teresa Smolarek Ph.D. (Committee Member); Leandra Tolusso M.S. (Committee Member) Subjects: Genetics

Master of Science, The Ohio State University, 2013, Comparative and Veterinary Medicine

Alpacas have a gestation length of nearly one year, and therefore females can only produce one offspring per year. In order to accelerate the genetic gain of a herd, superovulation and embryo transfer techniques can be used to produce multiple embryos from genetically superior females. We hypothesized that the use of superovulation and dual siring results in the production of multiple embryos sired by both of the males. Our objectives were: 1)to determine the parentage of embryos produced from multiple ovulations on females bred by two males, and 2) to determine the effects of the time of breeding (first vs second) on the frequency of paternity. Females received an ovulation induction agent (hCG; 1000 IU, IV or GnRH; 100ug, IM) when a preovulatory follicle >7 mm was present to induce ovulation and emergence of a new follicular wave. Starting at 60 h post-hCG/GnRH, females received twice daily intramuscular injections of FSH at decreasing doses (i.e. Day 1 = 50mg; Day 2 = 40 mg; Day 3 = 30; Days 4 to 7 = 20 mg). Administration of FSH was discontinued when half of the follicles in the growing cohort were = 7 mm in diameter, or after seven days. On the last day of FSH administration, females received cloprostenol (187 µg, IM, twice) to induce luteolysis. Receptive females were bred to proven fertile males, once in the AM by one male and once in the evening by the other male and received hCG or GnRH at time of the first breeding. Embryo collections were performed 8 to 9 days after breeding by transcervical uterine lavage without manipulating the reproductive tract per rectum. Statistical analysis was performed on data using t-test and Fisher's exact test. Significance was set at P < 0.05 and data are presented as mean ± SD. Growth of multiple dominant follicles was successfully achieved in 20/21 cycles. The mean number of days of FSH administration was 5.5±1.4. Sixteen females (80%) were receptive after FSH treatment and were bred by two males (A and B). Embryo collect (open full item for complete abstract)

Committee: Marco Courinho da Silva (Advisor); Carlos Pinto (Committee Member); Jeffrey Lakritz (Committee Member) Subjects: Animal Sciences; Veterinary Services

Master of Arts, The Ohio State University, 2024, Bioethics

The growing number of “leftover” embryos frozen and abandoned after IVF procedures is a debated problem for Catholic bioethics, which affirms the human dignity and full moral status of each of these persons. Catholics who oppose or affirm the moral liceity of a married couple adopting and gestating these embryos, embryo transfer for adoption (ETFA), typically do so by way of analogy. Four of the most discussed analogies are here presented and evaluated, namely, infidelity, surrogacy, wet-nursing, and adoption. The analogy to traditional post-natal adoption is shown to be more than fit; prenatal adoption is identified as the same type of moral act as post- natal adoption. A further three-pronged defense of the moral liceity of ETFA is made by using (1) Thomistic moral reasoning to show that the moral object of ETFA is to gestate one's child, (2) St. John Paul II's Personalism to show how ETFA conforms to the logic of gift and receptivity, and (3) a theological reflection on the Holy Family as a model for adoptive couples.

Committee: Ashley Fernandes (Advisor); Matthew Vest (Committee Member); Nicole Shirilla (Committee Member) Subjects: Ethics

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, 2022, Animal Sciences

This thesis focuses on the assessment of strategies directed at the optimization of ovum pick-up (OPU) and in vitro embryo production (IVEP) in pregnant cattle. The primary determinants for the success of OPU/IVEP are the number and quality of oocytes recovered after OPU, which in turn are affected by the number and developmental stage in the follicle population from which oocytes are collected. The objectives, therefore, were to: 1) evaluate the effect of use and dose of follicle stimulating hormone (FSH) on ovarian response and IVEP; 2) investigate the effect of synchronization of timing of follicle wave emergence (FWE) among females before ovarian superstimulation on ovarian response and IVEP; and 3) determine the efficacy of various methods for synchronizing FWE. In the first experiment, presented in Chapter 3, pregnant heifers (n = 48) were assigned to receive 0, 160 or 300 mg of porcine FSH (p-FSH) administered in four decreasing doses beginning at time of FWE. Ultrasonography, to evaluate ovarian response preceded OPU, which was performed 40 h after the last p-FSH administration and cumulus oocyte complexes (COC) that were collected were subjected to IVEP procedures. Total number of follicles at OPU and number of both total and viable COCs increased linearly (P < 0.0001) with increasing p-FSH dose. Cleavage percentage, number of cleaved oocytes, blastocyst percentage and number of blastocysts increased linearly (P < 0.0001) with increasing p-FSH dose. In the second experiment, presented in Chapter 4, pregnant heifers (n = 64) were randomly assigned to initiate superstimulatory treatments, consisting of administration of 160 mg of p-FSH, after synchronization of timing of FWE or at random stages of the follicular wave. Synchronization of timing of FWE resulted in a greater (P < 0.01) percentage of medium sized follicles, as well as greater (P < 0.01) cleavage and blastocyst percentage, which subsequently led to an increased (P < 0.01) number of blastocysts pro (open full item for complete abstract)

Committee: Alvaro Garcia Guerra (Advisor); Marco Coutinho da Silva (Committee Member); James Kinder (Committee Member) Subjects: Animal Sciences

Doctor of Philosophy (PhD), Ohio University, 2022, Molecular and Cellular Biology (Arts and Sciences)

Arabinogalactan-proteins (AGPs) are a family of structurally complex hydroxyproline-rich cell wall glycoproteins found throughout the plant kingdom. Arabinogalactan (AG) sugar chains rich in arabinose and galactose are attached to hydroxyproline (Hyp) residues in the AGP protein cores and constitute 90-98% of the overall weight of AGPs. Although much is known about AGP structure and function, little is known about the contributions of the AG sugar moieties to AGP function. To date, eight Hyp-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to initiate AGP glycosylation by catalyzing the addition of galactose sugars to Hyp residues in AGP protein core and thereby enable subsequent sugar additions. The extent of Hyp-GALT genetic redundancy, however, remains to be elucidated. To address this issue, I generated various multiple (T-DNA) galt gene knockouts, including a triple mutant (galt5galt8galt9), two quadruple mutants (galt2galt5galt7galt8, galt2galt5galt7galt9), and one quintuple mutant (galt2galt5galt7galt8galt9, referred to as galt25789), and comprehensively examined their biochemical and physiological phenotypes. The mutants had reduced AGP precipitations with β-Yariv reagent in leaves, stems, siliques, and flowers. Monosaccharide composition analysis of the silique and root AGPs in all mutants showed a decreased galactose and arabinose content. The transmission electron microscopy (TEM) analysis of the galt25789 quintuple mutant stems indicated cell wall defects coincident with observed growth impairment. As more and more Hyp-GALT genes were knocked out, the quadruple and quintuple mutants displayed additive effects on insensitivity to β-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, GALT7, GALT8, and GALT9 contributed more to primary root growth and root tip swelling under salt stress, whereas GALT2 and GALT5 played more of a role in seed morphology, germination, and seed set. Since the gal (open full item for complete abstract)

Committee: Allan Showalter Dr. (Advisor); Sarah Wyatt Dr. (Committee Member); Morgan Vis-Chiasson Dr. (Committee Member); Michael Held Dr. (Committee Member) Subjects: Biochemistry; Cellular Biology; Genetics; Molecular Biology; Plant Biology

PHD, Kent State University, 2020, College of Arts and Sciences / Department of Biological Sciences

Mammalian reproductive success depends on gonadotropin-releasing hormone (GnRH) neurons to stimulate gonadotropin secretion from the anterior pituitary and activate gonadal steroidogenesis and gametogenesis. Genetic screening studies in patients diagnosed with Kallmann syndrome (KS), a congenital form of hypogonadotropic hypogonadism (CHH) discovered several causal mutations, including those in the fibroblast growth factor (FGF) system. This signaling pathway regulate neuroendocrine progenitor cell proliferation, fate-specification, and cell survival. Indeed, the GnRH neuron system was absent or abrogated in transgenic mice with reduced (i.e., hypomorphic) Fgf8 and/or Fgf receptor (Fgfr) 1 expression, respectively. Moreover, we found that GnRH neurons could not be detected in the embryonic olfactory placode (OP) of Fgf8 hypomorphic mice, the putative birthplace of GnRH neurons. These observations and together with those in human KS/CHH patients indicate that FGF8/FGFR1 signaling system is a requirement for the ontogenesis of the GnRH neuronal system and function. In this manuscript, we will focus on both transcription and epigenetic factors which control the expression of genes, such as Fgf8 that are known to be critical for GnRH neuron ontogenesis, fate-specification, and the pathogenesis of KS/CHH.

Committee: Wilson Chung PHD (Advisor); Kristy Welshhans PHD (Committee Member); Jennifer McDonough PHD (Committee Member); Bansidhar Datta PHD (Committee Member); Mary Ann Raghanti PHD (Committee Member) Subjects: Biology; Biomedical Research; Chemistry; Developmental Biology; Endocrinology; Molecular Biology; Molecular Chemistry; Neurobiology; Neurosciences

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

Master of Science (MS), Ohio University, 2019, Plant Biology (Arts and Sciences)

Under future climate change, plant species are expected to shift their ranges in response to increasing temperatures and altered precipitation patterns. As seeds represent the single opportunity for plants to move, predicting potential range shifts requires an understanding about the relationship between the environment and reproduction. While total seed production is clearly important, seed quality is equally as critical and often overlooked. If a seed is low quality the likelihood of germination and successful recruitment decreases. Thus, to quantify how environmental variation affects seed quality and quantity, the reproductive output of red maple (Acer rubrum) was measured along an elevation gradient in the Monongahela National Forest, WV. In spring of 2018, 44 seeding trees were identified, and another 44 non-seeding trees were randomly selected for comparison. For each tree, a variety of individual-level characteristics were measured (i.e., DBH, canopy area, tree cores). Seed traps were placed under seed-bearing trees to collect samaras and quantify total seed production. Then, a random subsample of the collected seeds from each tree was micro-CT scanned to determine embryo volume, photographed for morphology measurements, and used for germination trials. The results showed that trees with larger canopy volumes were the most likely to produce seed. However, the number of seeds produced was negatively affected by frost events during flower and stand density. The trees with the most seeds also showed reduced growth (i.e., basal area increments) in the past 3 years. The likelihood of embryo presence increased with lower growth rates in the past 5 years, while embryo size increased with height, smaller DBH, and in areas dominated by hemlock. Both larger embryo volume and larger overall seed size increased the likelihood of germination. The results of my thesis highlight the importance of examining seed quality in addition to seed quantity for a more complete repre (open full item for complete abstract)

Committee: Rebecca Snell Dr. (Advisor); Jared DeForest Dr. (Committee Member); Brian McCarthy Dr. (Committee Member); Melissa Thomas-Van Gundy Dr. (Committee Member) Subjects: Botany; Conservation; Ecology; Plant Biology

Master of Sciences, Case Western Reserve University, 2018, Biology

The Drosophila blastoderm embryo is patterned by morphogens, molecules that inform cells to adopt specific fates in a threshold-dependent fashion. The morphogen Bicoid subdivides the antero-posterior axis into head, thorax and abdomen, whereas the morphogens Dorsal/NFk-B and Decapentaplegic/BMP-4 regulate cell fates along the dorso-ventral axis and subdivide the embryo into three germ layers (mesoderm, neuroectoderm and ectoderm). Together, the concentration gradients produced by these morphogens form a coordinate system along the main embryonic axes and cell fate is influenced depending on where a cell falls inside this system. For several years, the blastoderm was considered to be a static stage due to the lack of mitotic divisions or any major gastrulation movements. However, recent work demonstrated that a stereotyped movement of cells towards the dorsal midline from the lateral regions and the poles of the embryo takes place. As a result, by the end of the blastoderm stage, there is a higher density of cells in the dorsal region of the embryo and a lower density of cells in the ventral region. Results from our lab and others suggest that Decapentaplegic and Dorsal are required for these stereotyped cell movements. In this work, we tested whether the candidate gene frazzled (fra) we identified in a bioinformatics screening is a downstream regulator responsible for directing cells towards the dorsal region of the embryo. fra encodes a transmembrane receptor previously implicated in glial cell migration during late embryonic stages. The function of fra in the early blastoderm is unknown. In this work, we show that in embryos mutant for fra, cells lose their migration direction towards the dorsal midline and display an increased density within the ventral side compared to wild type embryos. We have also observed that FRA protein is located on the dorsal side of the Drosophila embryo and is localized on the apical surface of the cell vertices. Our findings suggest (open full item for complete abstract)

Committee: Claudia Mizutani (Advisor); Sarah Diamond (Committee Chair); Dianne Kube (Committee Member); Emmitt Jolly (Committee Member) Subjects: Biology; Developmental Biology; Genetics

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, 2013, Medicine: Molecular and Developmental Biology

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions with overlapping function during development, also play crucial roles in embryo implantation. Msx1 is transiently expressed in the pregnant uterus during the window of receptivity from day 3 of pregnancy until the morning of day 4 with rapid downregulation with impending implantation. Uterine inactivation of Msx genes leads to infertility due to implantation failure, which correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the transcriptional regulation of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth by cultured uterine epithelial cells.

Committee: Sudhansu K. Dey Ph.D. (Committee Chair); Stuart Handwerger M.D. (Committee Member); Sanjoy K. Das Ph.D. (Committee Member); Gurjit Hershey M.D. Ph.D. (Committee Member); Alan Jobe M.D. Ph.D. (Committee Member); Jeffrey Whitsett M.D. (Committee Member) Subjects: Developmental Biology

Master of Science (MS), Wright State University, 2012, Chemistry

Platinum group metals (PGMs), i.e., Pd, Pt and Rh, are found at pollutant levels in the environment. PGMs accumulate in plant and animal tissues, and little is known about their toxicity. Our previous studies have shown that chick embryos exposed to 1.0 mL PGM solutions of 5.0 ppm (LD50) and higher exhibited severe skeletal deformities. This work hypothesized that 1.0 ppm sublethal doses of PGMs would negatively impact the mineralization process in tibiotarsi. One milliliter of 1.0 ppm of Pd(II), Pt(IV), Rh(III) aqueous salt solutions and a PGM-mixture were injected into the air sac of the eggs on the 7th and 14th day of incubation. Control groups were also included. On the 20th day, embryos were sacrificed to analyze the PGM effects on tibiotarsi using micro-Raman imaging. Hyperspectral data analysis was performed using custom-written MatLab codes. The univariate Raman images that were created from the ν1(PO43-) integrated areas of hydroxyapatite revealed anomalous mineral inclusions within the bone marrow cavity for the PGM-mixture treatment, and explained the observed pathological changes. The age of the mineral crystals (ν(CO32-)/ν1(PO43-)) was found to be statistically lower for all treatments when compared to controls (p ¿¿¿¿¿¿¿ 0.05). The abnormal mineral distribution and the younger tibiotarsi may be explained by a delay in the endochondral ossification process as a result of PGM exposure.

Committee: Ioana Sizemore PhD (Advisor); Paul Seybold PhD (Committee Member); David Dolson PhD (Committee Member) Subjects: Biology; Chemistry; Toxicology

Master of Science (MS), Wright State University, 2010, Chemistry

Platinum group metals (PGMs) have been shown to accumulate in various tissues of organisms but their toxicity is not well-known. Raman spectroscopy is a powerful analytical technique that enables direct and non-destructive chemical characterization of bone tissues. PGMs were injected into chick embryos in the following concentrations: 0.1, 1.0, 5.0, or 10.0 ppm solutions of Pt(IV), Rh(III), Pd(II) or PGMs mixtures (0.1 ppm or 1.0 ppm of each metal). The micro-Raman maps recorded on the paraffin embedded tibiotarsus cross-sections showed significant changes in the chemical composition and structure of the bone tissue as a result of PGMs exposure of 1 ppm (i.e., anomalous calcium inclusions impeding circulation in cartilage matrix). The following bone properties were analyzed and quantified via micro-Raman spectroscopy: a) age of mineral crystals and carbonate content, b) degree of mineralization of the collagen matrix and possible losses in the organic/inorganic bone components and c) mineral crystallinity. X-Ray fluorescence showed significant changes in the distribution and quantity of calcium and phosphorus due to the PGM treatments. Flame atomic absorption spectroscopy also revealed changes in the amount of calcium found in the tibiotarsi. All three analytical techniques correlated well with each other showing the calcium inclusions negatively affecting the bone structure of the chick embryo tibiotarsi.

Committee: Ioana Pavel Ph.D. (Advisor); David Dolson Ph.D. (Committee Member); Dawn Wooley Ph.D. (Committee Member) Subjects: Biophysics; Chemistry; Toxicology

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

Doctor of Philosophy, The Ohio State University, 2009, Molecular Genetics

The novel E2F7 and E2F8 family members are thought to function as transcriptional repressors important for the control of cell proliferation in vitro. However, as the most recently indentified and least studied E2F members, their biological functions in vivo remain unknown. Here we have analyzed the consequences of inactivating E2f7 and E2f8 in mice. While loss of either E2f7 or E2f8 did not significantly affect mouse development, their combined ablation resulted in massive apoptosis, dilated blood vessels and severe placental defects, culminating in embryonic lethality by day 11.5. E2F7 and E2F8 formed homo-dimers and hetero-dimers that could recruit various co-repressor complexes to E2F binding sites of target promoters, including E2f1. Consistent with their important role in transcriptional repression, mouse embryonic fibroblasts (MEFs) deficient for E2f7 and E2f8 expressed abnormally high levels of E2f1 and other E2F-target mRNAs. These double knockout MEFs proliferated surprisingly well, but accumulated high levels of p53 protein and were hypersensitive to DNA damage-induced cell death. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis observed in double mutant embryos but failed to rescue their embryonic lethality.In order to identify the leading cause of fetal death, the critical tissues, related cellular processes and molecular mechanisms of E2F7 and E2F8 function, we utilized conditional knockout strategies to show that extra-embryonic function of E2F7 and E2F8 is both necessary and sufficient for embryo development, and thus define the placental abnormalities as the leading cause of embryonic lethality in E2f7-/-E2f8-/- embryos. Consistent with this genetic finding, cellular examination of double mutant placentas revealed ectopic DNA replication and inappropriate mitosis in certain cell lineages of placenta. We also provided two distinct molecular mechanisms of E2F7 and E2F8 function to explain these cellular phenotypes. On one hand, (open full item for complete abstract)

Committee: Gustavo Leone PhD (Advisor); Susan Cole PhD (Committee Member); Paul Herman PhD (Committee Member); Tim Huang PhD (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy, Miami University, 2008, Chemistry and Biochemistry

Mutations in the ETHE1 gene result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized by symmetric brain lesions, lactic academia, elevated excretion of ethylmalonic acid, and death in the first decade of life. ETHE1-like genes are found in a wide range of organisms; however, to date, a detailed characterization of ETHE1 has not been performed. Therefore, neither the structure nor the function has been established for the enzyme in any organism. In this dissertation, a full structural characterization of the Arabidopsishomolog of ETHE1 as well as information on its functional role in plants is presented. We have obtained the first crystal structure of an ETHE1-like protein as well as performed metal and preliminary substrate analyses providing new insights into the possible role and substrate of ETHE1. In addition, we demonstrate that ETHE1 is essential for both plant growth and development.

Committee: Christopher Makaroff (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 1993, Molecular Biology and Microbiology

Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of a family of v-src, c-src and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to this oncogene's fibroblast transforming abilities. The tumorigenic potentials detected by direct oncogene transfection faithfully reproduced the findings of similar studies using infectious, oncogenic retroviruses. Our studies establish the utility of CEF transformation by direct DNA transfection. Deregulated expression of the TGFα and EGF-R genes has been implicated in the development of a range of mammalian malignancies, most notably those of human origin. While introduction of the TGFα/EGF genes into immortalized rodent cell lines has frequently resulted in cellular transformation, attempts to generate similar results following simple, exogeneous TGFα/EGF treatment have proven largely unsuccessful. The potential role(s) played by these growth factors in the transformation of normal cells, derived from primary cultures has, heretofore, not been closely examined. In chapter 3 it is reported that both normal chicken embryo fibroblasts (CEF) and normal, diploid, human foreskin fibroblasts (HFF) can be efficiently transformed, in an apparent single-step fashion, following exogeneous TGFα/EGF treatment. CEF cells infected with a retrovirus carrying the TGFα gene generated unusually large, aggressively growing soft agar colonies. The ligand-induced transformation of CEF and HFF is affected by culture age. Cultures consistently responded less efficiently as they underwent increasing numbers of population doublings, yet there was no detectable, concomitant, diminution in the number of receptors per cell. This is the first report of (open full item for complete abstract)

Committee: Hsing-Jien Kung (Advisor) Subjects:

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

Doctor of Philosophy, Case Western Reserve University, 2013, Biology

Dermis is required for structural integrity of the skin, and provides inductive cues for hair follicle initiation. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. In my studies, I show that epidermal Wnt ligands are required for uniform dermal Wnt signaling/¿¿¿-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/¿¿¿-catenin activity, hair follicle formation is lost. Conversely, forced activation of ¿¿¿-catenin signaling leads to formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/¿¿¿-catenin in fibroblast proliferation and in the hair follicle initiation program in the epidermis. Moreover, a variety of secreted factors were identified that are differentially expressed in the mutant dermal fibroblasts using RNA-seq technology, suggesting these factors might be required for hair follicle initiation. This study lays the groundwork for developing future treatments of hair loss diseases and generating functional skin equivalents for wounded patients.

Committee: Radhika Atit (Advisor); Mark Wills (Committee Chair); Christopher Cullis (Committee Member); Sarah Millar (Committee Member); Emmitt Jolly (Committee Member) Subjects: Developmental Biology