Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 137-159). Also available via the Internet.

Thesis (Ph. D.)--University of Queensland, 2001. / Includes bibliographical references.

Theses (M.S.)--Marshall University, 2006. / Title from document title page. Includes abstract. Includes vitae. Document formatted into pages: contains xi, 85 pages. Bibliography: p. 44-47.

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy

Retinoic acid (RA) exerts important effects in the processes of vertebrate development, cellular growth and differentiation, and homeostasis. However, the mechanisms of action of RA in the control of cellular and developmental processes are incompletely understood, as the retinoid target genes have not been fully characterized. The goal of these studies described herein was to contribute towards a greater understanding of the cellular effects of retinoids through the identification and characterization of an RA-induced gene from mouse P19 embryonal carcinoma cells. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may function in cell signaling pathways. Towards the goal of identifying which signaling pathways GRASP may participate in, a yeast two-hybrid screen was performed using GRASP as a bait to identify protein interaction partners. The general receptor for phosphinositides 1 (GRP1), a guanine nucleotide exchange factor for the ADP-ribosylation factor 6 (ARF6) GTPase, was identified as a GRASP interaction partner. GRASP was shown to colocalize with endogenous ARFs in cells and enhance GRP1 association with the plasma membrane, suggesting that GRASP may function as a scaffold protein in the recruitment of GRP1 and ARF6 to plasma membrane loci. Overexpression of GRASP was observed to induce accumulation of GRASP in the endosomal compartment where GTP-binding deficient mutants of ARF6 reside, suggesting that GRASP induced a block in an ARF6 plasma membrane recycling pathway. Coexpression of GRP1, but not a catalytically inactive mutant, dramatically reduced the accumulation of GRASP in this compartment. Furthermore, GRP1 mutants that lack the region of interaction with GRASP failed to prevent accumulation of GRASP in the endosomal compartment, suggesting that GRASP recruits GRP1 to the endosomal compartment where GRP1 stimulates nucleotide exchange on ARF6 and recycling. Results described herein demonstrate that GRASP functions in the ARF6 regulated plasma membrane recycling pathway, and that upon overexpression, induces a block in recycling. Our results suggest a role for GRASP as an adapter or scaffold protein that may link cell surface receptors to the ARF6 recycling pathway, resulting in modulation of signal transduction events at the cell surface. / Graduation date: 2002

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Feb. 1, 2010). Includes bibliographical references (p. 19-24).

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

A potent gene transcription regulator, retinoic acid (RA) is known to influence more than 500 genes, whose functions include regulation of neuronal differentiation and patterning of the developing nervous system. Furthermore, its ability to stimulate neurite outgrowth has been documented in primary neurons such as postnatal cerebellar granule neurons, adult cortical neurons, or embryonic and adult dorsal root ganglia neurons (DRGs) (Corcoran et al. 2000, Wong et al. 2006, Yip et al. 2006, Puttagunta et al. 2011). It has been previously proposed that RA synthesis takes place only in the cytoplasm mediated by three retinaldehyde dehydrogenases (Raldhs) namely Raldh1, Raldh2 and Raldh3. From this cytoplasmic location RA is then transported to the nucleus where RA receptors (RAR) α, β and γ initiate gene transcription. RA is then removed from the nucleus and degraded by microsomal cytochrome 450 (Cyp26) A1, B1 or C1 enzymes. New studies have pointed to additional roles for RARs in the cytoplasm to regulate kinase function. Thus, as well as being a highly potent gene regulator, RA might act on several different levels involving separate mechanisms all converging towards its pro-regenerative abilities. The starting hypothesis for this thesis was that neurons in culture that re-grow dendrites and axons following their removal from their previously interconnected state in the brain activate intrinsic RA signalling mechanisms. Neurons that regenerate in culture following dissection of CNS were termed throughout the thesis as “regenerating neurons” by extrapolation from a similar terminology used for neurons cultured from the dissected CNS of Lymnea stagnalis (Farrar et al. 2009). The aim of this thesis was to determine if the synthesis of RA occurs within these neurons and, if so, where such synthesis is localised within sub-regions of the regenerating neuron. Such localisation of RA signalling can be genomic (RAR control of nuclear transcription) or non-genomic (RAR control of cytoplasmic kinases). Three types of neurons from hippocampus, cortex and retina, as well as the tissue of origin, were analysed by means of immunochemistry for the expression of Raldh1 and Raldh2 as well as RAR α, β and γ. Since many genes can be regulated by RA, a possible candidate gene, and effects of its inhibition on neurite outgrowth with or without RA, was investigated. Considering the promising results that appear on the horizon concerning the potential therapeutic effects that RA may have on neuronal regeneration, various retinoids were tested for genomic and non-genomic activity. The genomic activity was determined using a reporter cell line that has lac-Z driven by a RA response element while the non-genomic activity was assessed though an assay performed with MCF-7 cells that measured the phosphorylation of Erk1/2 as one of the potential RA-regulated downstream kinases. The main finding of this thesis was that in-vivo, neurons in postnatal hippocampus, cortex and retina showed very little expression of the components for RA synthesis while expression of RARs seemed to decrease dramatically within days of postnatal life. Nevertheless neurons from all these regions, when regenerating in culture, possessed the ability to synthesize RA as well as responding to it due to the up regulation of the necessary receptors. This thesis also suggested that the Raldhs, the main enzymes responsible for RA synthesis, are much more dynamic than previously considered and their location as well as intensity can change over time, or as a response to typical neuronal plasticity manipulation. Since all regenerating neurons switch on RA signalling within a couple of days of culture, this suggests that RA could be therefore the decisive factor that tips the balance between the two possible outcomes for a neuron that has its neurites cut away: to live and regenerate or to die. In the attempt to identify potential retinoids with greater effect in stimulating the ability of neurons to regenerate, it was found that some retinoids appeared to have a substantial influence on neurite outgrowth while different retinoids had unique combinations of genomic and non-genomic potencies.

During embryogenesis, vitamin A metabolism to retinoic acid (RA) is tightly regulated in a precise spatiotemporal pattern. Aberrant retinoid signalling in the wrong place or at the wrong time has devastating consequences for development. Much is understood about the mechanisms by which at the level of specific tissues, RA synthesis from vitamin A is able to occur and activate target genes necessary for the developmental programme at any given stage. However, little is known about how retinoid transport and synthesis at the subcellular level occurs. Chapter 3 of this thesis investigates the subcellular compartments with which the vitamin A metabolising enzyme RALDH2 associates in embryonic neuronal cell lines. It is found that not only is RA synthesis tightly controlled by tissues, but that RA signalling within cells themselves may also be segmented. Aside from the importance of vitamin A during early development, it is now known that this dietary component is also vital in the maintenance of homeostasis during adulthood. This is particularly important for the adult CNS, where it is proposed that RA mediates hippocampal neurogenesis; hypothalamic neurogenesis; and physiological responses to seasonal changes in day length. The nocturnal hormone melatonin is a key signalling molecule relaying the length of the night to the rest of the brain and periphery, and by this means conveying information about the time of year. Vitamin A deficiency is known to severely reduce the amount of melatonin produced by the Quail pineal gland at night. Chapter 4 of this thesis investigates the circadian and circannual rhythms of retinoid genes such as the RALDH enzymes and retinoic acid receptors in both the SCN, and the pineal gland. RA is sufficient to increase the expression of the melatonin synthesising enzyme AANAT, and that this gene expression may be under the control of RALDH1.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.