PhD, University of Cincinnati, 2007, Medicine : Molecular and Developmental Biology

The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus, the endoderm is specified by a vegetally localized transcription factor, VegT, and releases nodal signals that instruct the adjacent marginal zone of the blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this study, we show that the Forkhead-box protein FoxI1e is required at the blastula stage for normal formation of the two early derivatives of the ectoderm, the central nervous system and epidermis. FoxI1e is also required to maintain the regional identity of the animal cells of the blastula, the precursors of ectodermal structures. We have also studied the control of FoxI1e expression. In situ hybridization for FoxI1e shows a mosaic expression pattern at the blastula stage, with expressing cells interspersed with non-expressing cells. It is expressed in a wave initiated on the dorsal side of the animal hemisphere, and extends across to the ventral side by the mid-gastrula stage, confined to the inner layers of cells in the animal cap, and expressed in a mosaic fashion throughout. Finally, we show that this pattern of expression is controlled by both short and long range signals. Notch signaling controls both the mosaic, and dorsal/ventral progression in expression. Notch signaling is controlled, in turn, by the vegetally localized TGFβ ligand Vg1. FoxI1e expression is also regulated by nodal signaling downstream of VegT. Canonical Wnt signaling contributes only to late changes in the FoxI1e expression pattern. Overall, these results provide an initial framework for the activation of ectoderm formation in the Xenopus embryo, and provide new insights into the roles of vegetally localized mRNAs in controlling zygotic genes expressed in the animal hemisphere by long range signaling.

Committee: Dr. Christopher Wylie MD (Committee Chair); John Bissler MD (Other); Masato Nakafuku MD, PhD (Other); Iain Cartwright PhD (Other); Aaron Zorn PhD (Other) Subjects:

PhD, University of Cincinnati, 2004, Medicine : Neuroscience/Medical Science Scholars Interdisiplinary

The study of oligodendrogenesis and of stem cells in general has become a burgeoning field in recent years. The ability to replace specific cells of the body as things begin to go awry, to lessen the suffering of humanity is the holy grail in the eyes of many researchers. In order to successfully manipulate endogenous or cultured stem cells or more restricted precursors to replace those damaged in disease required specific knowledge of the complexities of developmental influences. Many factors influencing the specification and fate of oligodendrocyte progenitors have been discovered in recent years, but what links the various pathways is largely unknown. Some factors, including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF2), are involved at many stages of oligodendrocyte development. We chose to utilize a mouse model of neurofibromatosis type 1 (NF1) to study the influence of neurofibromin and ras signaling on oligodendrocyte development. Patients with NF1 exhibit a broad spectrum of CNS abnormalities that altered glia could play a part in. In our first study we demonstrate increased numbers of oligodendrocyte progenitor cells (OPCs) in vivo and in vitro embryonically and in adult mice. The accumulation of OPCs in vitro results in part from a farnesyl-transferase inhibitor (FTI; an H-ras inhibitor) -sensitive FGF2 hypersensitivity. This is the first linking of neurofibromin and ras signaling to FGF2 hypersensitivity. In our second study we begin to focus on the ability of Nf1 mutant OPCs to differentiate normally. Nf1 mutant cells have an FTI-sensitive tendency to abnormally coexpress GalC and GFAP, markers of oligodendrocytes and astrocytes respectively. We also begin to explore a link between Nf1 mutation and increased activation of another factor that influences oligodendrocyte development, Notch-1.

Committee: Nancy Ratner (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Congenital heart disease (CHD) impacts approximately 1% of live births in the United States and is the primary cause of birth-defect related infant mortality. The etiology of CHD involves both genetic and environmental risk factors. Pathogenic variation is known to contribute to ~30% of all CHD cases, encompassing chromosomal aneuploidies, chromosomal copy number variations (CNVs), and single nucleotide variants (SNVs). Conversely, ~10% of CHD cases stem from gestational exposure to environmental teratogens, which range from extrinsic factors such as teratogenic drugs or chemicals to intrinsic factors such as maternal illnesses. However, over half of CHD cases lack a definite etiology, raising the possibility that complex gene-gene or gene-environment interactions may be contributing to CHD pathogenesis.  Maternal diabetes is among the most prevalent environmental risk factors for CHD. Extensive research has identified dysregulation of several cardiac developmental processes with exposure to maternal diabetes mellitus (DM). Despite progress in understanding the molecular impacts of maternal diabetes on cardiac development, there remains a substantial gap in understanding how these effects interact with susceptible genetic backgrounds to contribute to a gene-environment etiology that is hypothesized in human CHD. Previously, we reported maternal diabetes interacts with Notch1 haploinsufficiency in mice to increase the incidence of membranous ventricular septal defects (VSD), reporting a novel Gene-Environment (GxE) interaction between Notch1 and maternal diabetes. To better understand the role of GxE interactions in CHD, we performed phenotypic and molecular characterization of the Notch1-maternal diabetes interaction in the context of cardiac development using in vivo and in vitro approaches. We demonstrate a cell lineage specific effect of Notch1 haploinsufficiency in maternal diabetes, implicating endothelial/endocardial derived structures in the developing hea (open full item for complete abstract)

Committee: Vidu Garg (Advisor); Kedryn Baskin (Committee Member); Aaron Trask (Committee Member); Brenda Lilly (Committee Member) Subjects: Developmental Biology; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2024, Biological Sciences (Arts and Sciences)

The neurovascular unit (NVU) is an anatomical and functional feature of the mammalian brain that encompasses the brain microvasculature and is pivotal for maintaining healthy nutrient, waste, and ion exchange between the blood supply and parenchymal tissue. Specializations of the NVU tightly regulate this exchange, thereby restricting the entry/exit of solutes to/from brain tissue and forming a functional blood-brain barrier (BBB). This complex network is composed of tightly interlinked cellular components – including vascular endothelial cells (ECs), pericytes, neurons, astrocytes, and microglia – making the brain vulnerable to pathologies affecting any single NVU element. Disruption in one part can lead to wider dysfunction, exacerbating neurological disease pathogenesis and symptoms. Cells of the NVU interact closely to maintain overall brain health. Pericytes are vital for vascular development and homeostasis, regulating cerebral blood flow and establishing the BBB. Astrocytes are also critical to the development and maintenance of the BBB and allow for vessel-parenchyma crosstalk through perivascular astrocytic end-feet. Microglia serve as regulators of immunogenic function and vascular development and are key players in maintenance of vessel integrity and diameter. Brain arteriovenous malformations (AVMs) present as high-flow vascular anomalies, distinguished by dilated vascular channels that forego a capillary bed, resulting in direct arterial-to-venous connections. This abnormality predisposes patients to spontaneous intracerebral hemorrhages. Currently, therapeutic options are restricted to invasive procedures, including stereotactic radiosurgery, endovascular embolization, and surgical excision. Such limitations underscore the urgent demand for the development of preventive strategies and less invasive treatment approaches. This dissertation reports on the molecular and cellular impacts to the NVU during pathogenesis of brain AVMs. Our lab uses a genetic m (open full item for complete abstract)

Committee: Corinne Nielsen (Advisor); Shiyong Wu (Committee Member); Daewoo Lee (Committee Member); Janet Duerr (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology; Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2022, Chemistry

Predicting the effect of a mutation in a protein on its function is still a complex unsolved problem. Analysis of a large number of protein mutants based on their activity, affinity and stability is an excellent method to understand this sequence-function relationship. Alanine scanning attempts it rationally, where the individual mutant is cloned, purified, and assayed to evaluate the consequence of a mutation. Large protein libraries coupled with a functional screen use a more comprehensive way to tackle this problem by saving time and costs of cloning and purifying individual variants. Here we have employed both approaches for two different protein systems to understand their sequence-function relationship. Since library studies are easier to perform on model proteins with a robust structure, we used Rop for our research. It is a small homodimer that controls the replication of the ColE1 plasmid by enhancing the interaction of RNAs that initiate plasmid replication. To probe the sequence fitness landscape of Rop and understand its binding interface at the molecular level, we developed a deep mutational scanning approach and verified it using a model experiment. We generated sixty-two individual point libraries spanning its whole sequence and enriched them using a growth-based assay. We used three environmentally different positions, i.e., core, loop, and surface and validated the functionality of the approach. We also identified unique functional variants at surface and core positions. The comprehensive sequence-function map showed that helix H1/H1' is more important for binding than H2/H2'. We found that layers 5,6 and 7 of the hydrophobic core are less tolerant to mutations, positions 57-63 are highly tolerant, and the stretch of positions from 10-15 is entirely intolerant to mutations. We also identified interesting multiple-point mutants with strong selection and thermal stability from high throughput thermal scanning, which need further analysis. Since Rop (open full item for complete abstract)

Committee: Karin Musier-Forsyth (Committee Member); Thomas Magliery (Advisor) Subjects: Chemistry

Doctor of Philosophy (PhD), Ohio University, 2022, Biological Sciences (Arts and Sciences)

The mammalian vasculature caters to tissue specific gaseous exchange and metabolic needs. The brain accounts for the largest consumption of oxygen and glucose, for which a structurally organized and functional cerebrovasculature is essential. Brain arteriovenous malformations (BAVM) are characterized by abnormally enlarged blood vessels, which direct blood through arteriovenous (AV) shunts, bypassing the normal artery-capillary-vein network. High-pressure, low-resistance AV shunts disrupt healthy blood flow and can result in cerebrovascular hemorrhage. BAVM is the leading cause of intracerebral hemorrhage in children, and accounts for 50% of stroke incidences in children and young adults. Clinically, BAVM treatments are invasive and not applicable to all cases; thus, there is critical need to understand BAVM mechanisms and develop targeted therapeutics. Using a mouse model of BAVM, that is deficient in Notch effector Rbpj from endothelial cells, from birth – we show that isolated Rbpj-deficient (RbpjiΔEC) brain endothelial cells (BECs) elicit altered whole-genome transcriptomic profile, early at Postanal day (P)7 when expansion of AV diameters – the most prominent BAVM phenotype is not observed, suggesting contribution of Rbpj regulated effector molecules in triggering onset of BAVM pathogenesis. Cellular studies over the course of characterized developmental time-periods at P7, P10, and P14 revealed that AV expansion in RbpjiΔEC mice do not originate from hyperplastic or hypertrophic mechanisms; but RbpjiΔEC BECs acquired atypical morphology and increased BEC density along AV shunts, as compared to controls, in postnatal mice. RbpjiΔEC mice also showed reduced regression of BECs over AV connections when studied through empty basement membrane collagen sleeve (EBMS) dynamics, suggesting lack of remodeling and accumulation of BECs over capillary like vessels in vivo. Using isolated postnatal mouse BECs, we found altered small GTPase activity in (open full item for complete abstract)

Committee: Corinne Nielsen (Advisor); Mark Berryman (Committee Member); Fabian Benencia (Committee Chair); Monica Burdick (Committee Co-Chair); Soichi Tanda (Committee Member) Subjects: Biochemistry; Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2022, Molecular, Cellular and Developmental Biology

Notch signaling is a highly conserved pathway that mediates communication between adjacent cells during development. Notch signaling plays a major role in the segmentation clock. The segmentation clock regulates the oscillatory gene expression that times the periodic patterning of undifferentiated posterior mesoderm into skeletal tissue precursors. These precursors then develop into the spinal elements of the axial skeleton. Across evolution, the cycling of Notch activation is timed by the interaction between the Notch receptor and two Notch ligands. An activating Notch ligand on the surface of a cell interacts with a Notch receptor on the surface of an adjacent cell initiating trans-activation and driving the expression of a self-repressing core oscillator in the signal-receiving cell. Expression of an additional ligand that does not appreciably activate the receptor in trans but is competent to interact with the receptor provides additional regulation that alters the level of Notch activation. In the mammalian lineage, interactions between the activating Notch ligand Deltalike1 (DLL1), Notch receptor NOTCH1, and the cis-only acting Notch ligand Deltalike3 (DLL3) that mediate Notch activation are additionally regulated through glycosylation by a glycosyltransferase Lunatic Fringe (LFNG). The involvement of DLL3 in regulating DLL1-NOTCH1 interaction, the role of LFNG in mediating these iii interactions, and how DLL3 and LFNG function to alter the ticking of the segmentation clock are not well understood. To begin to understand these mechanisms, I utilized a mammalian cell system to determine how Notch signaling is regulated in this complex environment. I determined that in the absence of DLL3, cells co-expressing DLL1 and NOTCH1 do not appreciably send signal to receiving cells. Induction of DLL3 expression leads to increased loss of NOTCH1 protein through a Notch activation-dependent pathway. The loss of NOTCH1 recep (open full item for complete abstract)

Committee: Susan Cole (Advisor); Marcos Sotomayor (Committee Member); Helen Chamberlin (Committee Member); Sharon Amacher (Committee Member) Subjects: Developmental Biology; Molecular Biology

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

Gene-pairs represent at least 50% of E.coli and 10% of human genes. The gene pairing phenomenon is likely to have appeared during genome gene-duplication events. However, it is not entirely understood whether there is an evolutionary advantage of gene pairing. We do not have clear evidence to differentiate between the following scenarios: Is gene pairing simply a side effect of gene duplication or does gene paring ensure robust outcomes during embryonic development? In my thesis, I will discuss how gene-pairing can lead to co-expression of genes. For fast dynamic systems, such as somitogenesis, co-expression of regulatory genes results in robust pattern formation, increasing overall fitness of species.

Committee: Ertugrul Ozbudak Ph.D. (Committee Chair); Brian Gebelein Ph.D. (Committee Member); Christian Hong Ph.D. (Committee Member); Raphael Kopan Ph.D. (Committee Member); Tongli Zhang Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Human innate lymphoid cells (ILCs) comprise a diverse population of lymphocytes with various roles in tissue homeostasis, inflammation, host defense, and malignancy. ILCs collectively include the cytotoxic natural killer (NK) cells, which participate in direct lysis of infected or malignant cells, as well as distinct helper ILC populations (ILC1s, ILC2s, and ILC3s), which mediate their immunomodulatory roles via production of cytokines. Many physiologic pathways rely on the effector functions of NK cells and other ILC subsets; yet, the mechanisms that regulate their differentiation and maturation are not fully known. The overall goal of this research is to further characterize the pathways of human ILC development and to identify the mechanisms that regulate the differentiation and function of mature ILC populations. The results of these studies elucidate the roles of signal pathways and cellular interactions in the regulation of ILC biology. Activation of the Notch signaling pathway promoted the differentiation of non-NK helper ILC subsets at specific stages of development. While direct co-culture of ILC precursor cells with stromal feeder cells (and exogenous IL-7) was sufficient for the acquisition of NK cell markers (CD94 and NKG2A), the simultaneous presence of Notch ligand was required for the differentiation of functional ILC2s and ILC3s under these conditions. Although Notch was not required for NK cell differentiation, it did have an impact on the phenotype of in vitro-derived NK cells. Activation of Notch in the presence of stromal cells promoted the expression of the activating receptor NKp80, suggesting a role for Notch in the later stages of NK cell maturation. Based on the expression patterns of individual Notch receptors, it was hypothesized that there are stage-specific differences in the signaling mechanism. In support of this, it was shown that both NOTCH1 and NOTCH2 were involved in ILC2 and ILC3 differentiation from ILC precursor cells. Alternati (open full item for complete abstract)

Committee: Aharon Freud MD PhD (Advisor); Robert Baiocchi MD PhD (Committee Member); Michael Caligiuri MD (Committee Member); Timothy Cripe MD PhD (Committee Member); Mikhail Dikov PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), Ohio University, 2020, Molecular and Cellular Biology (Arts and Sciences)

Breast cancer cells (BCCs) potentiate hematogenous metastasis by expressing specialized glycosylated proteins and lipids that act as ligands to E-selectin, a cell adhesion molecule expressed on cytokine-activated endothelial cells that line blood vessel walls, under hemodynamic shear stresses. Recently, the functional E-selectin ligand activities of BCCs were shown to be modulated by Snail and Twist transcription factors (TFs) that regulate the epithelial-to-mesenchymal transition (EMT) and the mesenchymal-to-epithelial transition (MET), key processes in metastasis. However, the influence on the functional E-selectin ligand activities of BCCs by physiologically important molecules reported to initiate EMT or MET signaling cascades has yet to be determined. Consequently, the potential for microRNA-200c (miR-200c), an miR overexpressed in the blood of metastatic BC patients, to modify the functional E-selectin ligand activities of BCCs via the MET was determined. Transient overexpression of miR-200c in MDA-MB-231 BCCs induced the MET and flow cytometry analysis revealed that the expression of binding epitopes recognized by E-selectin was significantly higher in these cells compared to cells treated with a negative control miR. Furthermore, cells overexpressing miR-200c had higher functional E-selectin ligand activities, as these cells had significantly lower rolling velocities on E-selectin, significantly higher levels of firm adhesion to E-selectin, and significantly lower detachment from E-selectin in shear flow adhesion assays. Consistent with these findings, gene expression of fucosyltransferase 3 (FUT3) and FUT6, the primary enzymes responsible for the synthesis of functional E-selectin ligands on epithelial BCCs, was significantly higher in cells following miR-200c-induced MET. MiR-200c-induced MET is caused by Zeb1 and Zeb2 TFs, suggesting a novel pathway by which the MET regulates the functional E-selectin ligand activities of BCCs. Over-activation of Notch si (open full item for complete abstract)

Committee: Monica Burdick (Advisor); Douglas Goetz (Committee Member); Fabian Benencia (Committee Member); Amir Farnoud (Committee Member) Subjects: Bioinformatics; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2019, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Notch is a highly conserved signaling pathway in multicellular organisms that regulates fundamental cellular processes such as proliferation, differentiation, and cell fate determination. Notch has a litany of roles in human biology and pathologies associated with aberrant Notch include cancers, cardiovascular diseases and developmental disorders. The Notch transcription factor CSL mediates either transcriptional activation or repression of Notch target genes, depending on the context, by forming coactivator or corepressor protein complexes that reside on Notch target promoters and enhancers. Notch pathway components remain very attractive as therapeutic targets due to the widespread downstream effects of the pathway and the limited success of current treatments. However, the pursuit of small molecule modulators of Notch is complicated by the dual role of CSL as an activator and a repressor, and even moreso by the realization that coactivators and corepressors often bind to CSL via conserved motifs. Still, any hope of developing small molecules with complex specificity relies on a growing body of molecular insight provided by crystal structures of the CSL-coregulator complexes and detailed understanding of the binding determinants of the complexes. A recently identified putative CSL binding protein called L3MBTL3 belongs to the malignant brain tumor (MBT) family of proteins that use a variable number of MBT structural domains to recognize mono- and di-methylated lysine residues on histone tails and facilitate chromatin compaction and transcriptional repression. In this dissertation, I will describe the work done to determine this poorly understood protein's involvement in the Notch pathway. Chapter 1 will be an introduction to the Notch pathway, from the protein machinery to the biological impact and human health implications. Chapter 2 is a thorough structural and binding analysis of CSL protein complexes. This will be an extended version of a review that we have r (open full item for complete abstract)

Committee: Rhett Koval Ph.D. (Committee Chair); Andrew Herr Ph.D. (Committee Member); Carolyn Price Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Biochemistry

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

The Notch signaling pathway has long been intricately linked with the development and function of the vasculature. In vascular smooth muscle cells (VSMCs), Notch signaling has a great influence on phenotype and is a strong promoter of differentiation and expression of contractile genes necessary to produce a functional vessel wall. However, the role of Notch signaling in VSMC proliferation and survival is less well defined, and some cases contradictory reports are given. Also, the contributions of each individual Notch receptor have not been clearly described. Thus, to better understand Notch signaling in VSMC phenotype, we investigated the specific roles of the predominant Notch receptors in VSMCs as they relate to differentiation, proliferation, and survival. We found that Notch3 promotes Platelet-Derived Growth Factor (PDGF)-induced proliferation in VSMCs, while Notch2 inhibits it. We also found that Notch3 was able to promote cell survival in response to apoptosis cues, while Notch2 had no discernible effect. Interestingly, we also found the expression of Notch2 and Notch3 were changed in response to proliferation and apoptosis inducers. Notch2 mRNA was significantly decreased after PDGF-BB treatment, a proliferation inducer, and Notch3 protein was degraded rapidly in response to induction of apoptosis. Additionally, we demonstrated that Notch3's induction of cell survival genes required MEK/ERK signaling and Notch3 was capable of increasing levels of phosphorylated ERK. Altogether, these findings demonstrate that Notch2 and Notch3 have unique functions in regulating VSMC phenotype. In a mouse model devoid of Notch2 and Notch3 in smooth muscle cells, we were able to show that Notch2 and Notch3 are required for normal closure of the ductus arteriosus. Animals without Notch2 in VSMCs presented with patent ductus arteriosus with increasing incidence combined with the loss of Notch3. These mice died within one day of birth and also presented with aortic dilation. (open full item for complete abstract)

Committee: Brenda Lilly PhD (Advisor); Joy Lincoln PhD (Committee Member); Andrea Doseff PhD (Committee Member); Aaron Trask PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology

PhD, University of Cincinnati, 2016, Medicine: Molecular Genetics, Biochemistry, and Microbiology

The Notch pathway is an essential component of metazoan development and tissue homeostasis. Dysfunctional Notch signaling has been linked with cardiovascular disease, birth defects, and various cancers. Notch signaling ultimately results in changes in gene expression, which is regulated by the DNA binding protein CSL. CSL functions as both a repressor and activator of transcription from Notch target genes by interacting with transcriptional corepressors and coactivators, respectively. A new transcriptional coregulator has been identified, termed RITA, which binds CSL and facilitates its export out of the nucleus. RITA is thought to function as a corepressor by preventing the assembly of active transcription complexes at Notch target genes. However, the molecular details of the CSL-RITA complex are unknown. In this work, using a combination of biophysical, biochemical/cellular, and structural techniques, we characterize the interaction between CSL and RITA. Chapter 1 is a brief overview of the Notch signaling pathway, from pathway discovery to present day. Chapter 2 contains the structural, biophysical, and functional characterization of the CSL-RITA complex, in which the high affinity interaction between CSL and RITA is demonstrated. Chapter 3 consists of comparative binding analysis of known CSL binding partners using CSL point mutations shown to disrupt CSL activation. Finally, Chapter 4 is a summary of this work as it pertains to future directions in the Notch field.

Committee: Rhett Kovall Ph.D. (Committee Chair); Kenneth Greis Ph.D. (Committee Member); Andrew Herr Ph.D. (Committee Member); Anil Menon Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2014, Molecular Genetics

The Notch signaling pathway is a cell-to-cell signal transduction mechanism utilized throughout the kingdom metazoa. While we have a strong understanding of the mechanism by which Notch mediates intercellular communication, a growing roster of molecules able to modulate the activity of the Notch pathway represents an area of ongoing investigation. The Delta-like 1 homologue (Dlk1) is a maternally imprinted, alternatively spliced, atypical Notch ligand thought to have important roles in embryonic growth and differentiation. Despite numerous investigations examining the mechanisms by which Dlk1 mediates differentiation in vitro, the in vivo roles for Dlk1 have proven difficult to study. One proposed explanation for this is a requirement for multiple Dlk1 isoforms in DLK1-mediated Notch signaling. To this end, we undertook an examination of the early embryonic expression pattern of all known Dlk1 transcript slice variants. We identified novel expression domains at early developmental stages that suggest a more complex role for multiple Dlk1 splice variants than previously suggested. The Notch pathway is used reiteratively throughout cardiovascular development and maintenance. Perturbed Notch signaling leads to arterial/venous malformations, cardiac disorders, and adult-onset stroke disorder. The reiterative use and complex expression of various components of the Notch pathway in cardiovascular development and maintenance indicates a need for tight regulation of Notch activity throughout the vascular system. A lack of studies directly examining regulation of ligand-mediated Notch activation represents a significant gap in our understanding of the role of Notch signaling in this context. We identify a novel role for the Radical fringe (Rfng) gene, a known modulator of Notch activity, in vascular smooth muscle cell differentiation. Our qPCR analysis shows that Rfng is expressed multiple vascular support cell types. Loss of Rfng function leads to perturbed vascular smo (open full item for complete abstract)

Committee: Susan Cole Ph.D. (Advisor); Mark Seeger Ph.D. (Committee Member); Brenda Lilly Ph.D. (Committee Member); Michael Ostrowski Ph.D. (Committee Member) Subjects: Developmental Biology; Genetics; Molecular Biology

PhD, University of Cincinnati, 2014, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Collectively, the ubiquitousness and necessity of Notch signaling throughout embryonic development as well as homeostasis of adult tissues highlight the importance of this signaling pathway. Disrupting mutations in any one of the Notch signaling components can result in serious diseases, such as cancer, cardiovascular disease, and developmental malformities. Therefore, understanding on the molecular level how each Notch signaling component, (i.e. DSL, the Notch receptor, and CSL), contributes to the tight transcriptional regulation of Notch target genes is necessary in effectively therapeutically targeting any one of these components. The current focus of our lab is to understand how CSL-coactivator and CSL-corepressor complexes assemble at Notch target genes, and furthermore, how CSL transitions between coactivator and corepressor complexes. The specific contribution of my dissertation was to structurally and thermodynamically characterize two distinct CSL-corepressor complexes—the CSL-KyoT2 complex, and the CSL-MINT-NCoR complex. The comprehensive analysis I have performed on each of these corepressor complexes has shed valuable insight into how CSL interacts with these corepressor proteins to further recruit chromatin remodeling machinery, ultimately resulting in the repression of Notch target genes.

Committee: Rhett Kovall Ph.D. (Committee Chair); Nadean Brown Ph.D. (Committee Member); Gary Dean Ph.D. (Committee Member); Andrew Herr Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2014, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Collectively, the ubiquitousness and necessity of Notch signaling throughout embryonic development as well as homeostasis of adult tissues highlight the importance of this signaling pathway. Disrupting mutations in any one of the Notch signaling components can result in serious diseases, such as cancer, cardiovascular disease, and developmental malformities. Therefore, understanding on the molecular level how each Notch signaling component, (i.e. DSL, the Notch receptor, and CSL), contributes to the tight transcriptional regulation of Notch target genes is necessary in effectively therapeutically targeting any one of these components. The current focus of our lab is to understand how CSL-coactivator and CSL-corepressor complexes assemble at Notch target genes, and furthermore, how CSL transitions between coactivator and corepressor complexes. The specific contribution of my dissertation was to structurally and thermodynamically characterize two distinct CSL-corepressor complexes—the CSL-KyoT2 complex, and the CSL-MINT-NCoR complex. The comprehensive analysis I have performed on each of these corepressor complexes has shed valuable insight into how CSL interacts with these corepressor proteins to further recruit chromatin remodeling machinery, ultimately resulting in the repression of Notch target genes.

Committee: Rhett Kovall Ph.D. (Committee Chair); Nadean Brown Ph.D. (Committee Member); Gary Dean Ph.D. (Committee Member); Andrew Herr Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member) Subjects: Biology

PhD, University of Cincinnati, 2013, Medicine: Molecular Genetics, Biochemistry, and Microbiology

The Notch pathway is a cell-to-cell signaling mechanism conserved in multicellular organisms that plays a vital role in cellular development, differentiation and homeostasis. Biological and experimental evidence has shown that improper regulation leads to disease states such as kidney disease, stroke, and cancer. A properly regulated Notch signal effects transcriptional regulation of target genes through coregulator interactions with the nuclear effector CSL (CBF-1, Su(H), Lag-1). CSL functions as an activator when the intracellular domain of Notch is bound; however, in the absence of Notch, corepressors that mediate transcriptional repression bind CSL. This opposing regulatory role of the nuclear effector underscores the importance of understanding the molecular details of the protein-protein interactions that mediate both activation and repression. The work described in the following chapters characterizes the molecular details of CSL interacting with the corepressor MINT. Chapter 1 reviews Notch signaling. In Chapter 2, the quantified thermodynamic details of the MINT:CSL interaction is described. Based on these findings we determine the minimum binding constructs of MINT required to bind to CSL as well as the domains of CSL required to bind to MINT. In Chapter 3, the X-ray crystallographic structure of MINT bound to CSL is determined. Here I use mutational studies to validate the structure. In Chapter 4, I summarize the work presented here on the MINT:CSL interaction and postulate about further experiments to fully delineate the molecular mechanisms of Notch signaling transcriptional regulation.

Committee: Rhett Kovall Ph.D. (Committee Chair); Gary Dean Ph.D. (Committee Member); Andrew Herr Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); Pearl Tsang Ph.D. (Committee Member) Subjects: Biophysics

PhD, University of Cincinnati, 2010, Medicine : Molecular Genetics, Biochemistry, and Microbiology

The Notch pathway is an indispensable signaling mechanism, in which contacts between neighboring cells result in changes in gene regulation that dictate cellular development, proliferation, and differentiation. The necessity for proper signaling is self-evident, as mutation of Notch pathway components has been shown to result in cancer, development defects, and congenital diseases. Notch signals are transduced into transcriptional outputs by the pathway's sole transcription factor, CSL (CBF-1, Su(H), Lag-1), which regulates expression from Notch responsive genes. In the absence of a Notch signal, CSL functions as a repressor by forming complexes with transcriptional corepressor proteins. This dual functionality of CSL highlights its importance in signaling and the need to understand at the molecular level its interactions with coactivators and corepressors. The work described herein aims to characterize the thermodynamic, structural, and functional details of CSL mediated transcription complexes. In Chapter II, using ITC and X-ray crystallography, we provide a quantitative description of the Notch RAM domain and its interaction with CSL. Based on these findings, we propose an allosteric model, in which RAM binding facilitates ternary complex formation. In Chapter III, ITC and X-ray crystallography are also used to investigate the molecular details of CSL recognizing consensus and nonconsensus DNA binding sites. These experiments give a detailed thermodynamic and structural explanation for CSL binding to two known in vivo binding sites, highlighting the differences between the two sites and the overall moderate affinity of CSL for DNA. In Chapter IV, we investigated the binding of the corepressor MINT to CSL, which is the first quantitative study of a CSL-corepressor complex. This study provided molecular insights into the proposed competition between coactivators and corepressors for binding sites on CSL. Finally, in Chapter V, knowledge gleaned from our structural (open full item for complete abstract)

Committee: Rhett Kovall PhD (Committee Chair); Andrew Herr PhD (Committee Member); Gary Dean PhD (Committee Member); Carolyn Price PhD (Committee Member); Carol Caperelli PhD (Committee Member) Subjects: Molecular Biology

PhD, University of Cincinnati, 2005, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Disruption of signal transduction pathways is common during cellular transformation and tumor development. The evolutionarily conserved Notch signal transduction pathway is one example, as its components are misregulated or overexpressed in many developmental diseases and cancer. Misexpression or mutation of the Delta/Serrate/Lag-2 (DSL) protein JAGGED1 occurs in the developmental disorder Alagille syndrome, which affects the a number of organs including the liver and heart, and in specific tumors types such as colon and cervical cancers. In addition, Jagged1 knockout mice are associated with embryonic lethalality due to a number of developmental deficiencies including vascular defects. The mechanism by which JAGGED1 contributes to these disorders is unknown. DSL proteins such as JAGGED1 are single-pass transmembrane proteins that are ligands for Notch receptors. While much is known about the molecular mechanisms of Notch signaling, there is less understanding of the biological role of DSL proteins. This work tested the balance between DSL and Notch proteins and demonstrated that overexpression of JAGGED1 in vivo leads to defects in thymus development and T-cell differentiation in JAGGED1 transgenic mice. Misexpression of JAGGED1 in vitro leads to cellular transformation dependent on the C-terminal PDZ ligand of JAGGED1. Therefore, we have identified a novel PDZ- dependent signaling mechanism intrinsic to JAGGED1 that provides a link between misexpression of JAGGED1 and tumorigenesis. We propose a bi-directional signaling model in which DSL proteins have two distinct functions: to initiate Notch signaling in a neighboring cell and to initiate a PDZ-dependent signaling mechanism in the DSL-expressing cell.

Committee: Anthony Capobianco PhD (Advisor); Thomas Doetschman PhD (Other); Joanna Groden PhD (Other); Carolyn Price PhD (Other); David Robbins PhD (Other); Robert Brackenbury PhD (Other) Subjects: Biology, Molecular

Doctor of Philosophy, The Ohio State University, 2004, Molecular, Cellular, and Developmental Biology

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to develop into neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to one or two cells within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein has previously been shown to be a negative regulator of the EGFR pathway during eye development. Work from our lab has shown that Ed has a role in restricting neurogenic potential during embryonic neurogenesis. I have extended the functional and genetic analysis of Ed. Loss of ed suppresses the lack of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature sensitive allele of ed, I show that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of Enhancer of split m7 {E(spl)}and enhanced by overexpression of Delta. Thus Ed interacts synergistically with the Notch signaling pathway. I have identified a paralog of Ed, Friend-of-echinoid (Fred). fred function was examined in transgenic flies using inducible RNAi. Suppression of fred in the developing wing disc results in specification of ectopic SOPs, additional microchaeta and cell death. These phenotypes can be suppressed by increasing the activity of the Notch signaling pathway. Dosage-sensitive genetic interaction suggests a close functional relationship between fred and ed. Microarray analysis of fred RNAi discs revealed a number of genes that are misregulated in the absence of Fred activity. Changes in th (open full item for complete abstract)

Committee: Harald Vaessin (Advisor) Subjects: