Doctor of Philosophy, The Ohio State University, 2009, Molecular, Cellular, and Developmental Biology

Pofut1 (Protein O-fucosyltransferase 1) is an enzyme which specifically transfers fucose to serine or threonine of EGF domains on proteins. The role of O-fucosylation has been shown to be necessary for Notch receptor ligand binding and signaling. However, EGF domain proteins remain to be tested for the O-fucose modification. In this study, we identify functional roles for O-fucosylation in muscle agrin activity and in skeletal muscle and brain. In chapter 2, we show that agrin is O-fucosylated by Pofut1. Agrin is an extracellular matrix protein which stabilizes AChR (Acetylcholine receptor) clusters in postsynaptic membrane of skeletal muscle in vivo and induces AChR clustering in myofibers in vitro. Agrin is produced by motor neurons and muscles as different isoforms which are distinguished by their ability to induce AChR clustering: neural agrin is active but muscle agrin is inactive. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. O-fucosylation has a negative regulatory role over the activity of muscle isoform. Muscle agrin produced in the absence of Pofut1 or mutant agrin defective in O-fucosylation acquires AChR clustering activity. In vivo, deletion of Pofut1 induces ectopic AChR clustering in primary myotubes and in skeletal muscle fibers. In chapter 3, the role of Pofut1 in skeletal muscle was investigated by conditionally knocking out Pofut1 postnatally in skeletal muscle fibers. Deletion of Pofut1 resulted in progressive dystrophic phenotypes including reduced myofiber size, increased variability of myofiber diameter, and increased central nuclei. These phenotypes, together with down regulation of cell cycle genes, suggest that satellite cell proliferation and activation may be affected in Pofut1 deficient skeletal muscles. In chapter 4, the role of Pofut1 in adult neurogenesis was investigated by specific knockout of Pofut1 in neural progenitor cells. We show that dele (open full item for complete abstract)

Committee: Paul Martin PhD (Advisor); Christine Beattie PhD (Committee Member); Susan Cole PhD (Committee Member); Brian Kaspar PhD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Biological Sciences

Reports have described cells with stem-like protein expression in the hypothalamic suprachiasmatic nucleus (SCN), which contains the principal circadian pacemaker of the body. Additionally, there are oligodendrocyte progenitor cells (OPCs) scattered throughout the SCN and other brain areas with reported abilities to differentiate into neurons and glia. The SCN is a particularly good structure for studying adult neurogenesis because its cellular manipulation has known quantifiable effects on specific parameters of circadian rhythms. The objectives of this study were to characterize stem and progenitor cells in the SCN and to study neurogenesis from SCN OPCs in vitro. We first performed a meta-analysis to identify the expression of stem cell-related genes in the SCN and then used defined serum-free media for inducing stem and progenitor cell proliferation in SCN explant cultures, identified by immunocytochemistry and confocal microscopy. In the meta-analysis, we analyzed 25 genes associated with stem cell maintenance and increased motility, out of which over 90% were expressed at higher levels in the SCN than in other brain areas. In explant cultures maintained in stem and progenitor cell medium (SPM), cells expressed stem cell proteins: SOX2, nestin, MSI2 and OCT4. Explant cultures had ongoing mitotic activity and extensive cell loss. Despite neuronal loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging. The circadian rhythm in SCN gene expression persisted in brain slice cultures in SPM. SCN explants maintained in NeuralX medium supporting OPC proliferation, formed a cell monolayer and a suspended cell culture that included 87% OPCs. These cells were then induced to differentiate into neurons, which were identified by immunocytochemistry and electrical impulses recorded with microelectrode arrays. In differentiating cultures, a subset of OPCs formed oligodendrocytes that myelinated nascent neurons. These results provide evid (open full item for complete abstract)

Committee: Michael Geusz PhD (Advisor); George Bullerjahn PhD (Committee Member); Howard Cromwell PhD (Committee Member); Paul Morris PhD (Committee Member); Pascal Bizarro PhD (Other) Subjects: Biology; Biomedical Research; Cellular Biology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2017, Pharmaceutical Sciences

Circadian rhythms are defined as oscillations in biological processes with a period similar to the 24 hour period of the earth's rotation. In mammals, circadian rhythms are controlled by the suprachiasmatic nucleus (SCN), a hypothalamic structure working as a central pacemaker that drives the circadian rhythms that are expressed in other brain regions, as well as throughout the rest of the organism. The manifestation of circadian rhythms is structured by the presence in nearly all cells of a molecular clock, a transcriptional and translational feedback loop that directs the timing of numerous cellular processes. Although endogenous circadian rhythms are self-sustained, entrainment of the SCN circadian clock is induced by diverse environmental factors in order to maintain a synchrony with external geophysical cycles, with light being recognized as one of the most robust entrainment signals. Here, I first present a study aimed at describing genome-wide expression changes in the SCN triggered by light inputs in the SCN (Chapter2). In this respect, previous studies have shown that the effects of light in the SCN are time-of-day specific, resulting from differential responses in specific neuronal signaling pathways. In particular, activation of the mitogen-activated protein kinases/ extracellular signal–regulated kinases (MAPK/ERK) signaling pathway in the SCN couples photic stimuli with circadian entrainment through changes in gene expression. Using microarray analysis and pharmacological interventions, I describe here the number and nature of transcripts induced by light at multiple points of the circadian cycle, with a particular emphasis on the subgroup of light-induced genes whose expression is modulated by the activation of the MAPK/ERK signaling pathway. This examination is followed by studies aimed to determine the influence of circadian rhythms on the function of the hippocampus. First, I assess the role of circadian rhythms in the formation and retrieval of co (open full item for complete abstract)

Committee: Kari Hoyt PhD (Advisor); Karl Obrietan PhD (Other) Subjects: Neurobiology; Neurosciences; Pharmacology; Pharmacy Sciences