Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 170-194). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 223-251). Also available online through Digital Dissertations.

Abstract Bone resorbing osteoclasts require tight attachment of their plasma membrane to the bone surface in order to retain the specific microenvironment and thus to be able to dissolve the bone matrix underneath. Cadherins are transmembrane glycoproteins usually mediating homophilic calcium-dependent cell-cell adhesion. In the present work we have studied the effects of the cadherin CAR sequence HAV-containing hexapeptide AHAVSE on osteoclasts. The primary attachment of osteoclasts to bone surface is not affected by the peptide, suggesting that it is not mediated by cadherins. Treatment of osteoclast cultures with AHAVSE decreased the number of resorption pits and the total resorbed area. Furthermore, we show rapid inactivation of osteoclasts with AHAVSE, which is seen as a decrease in the percentage of osteoclasts with actin rings. Pan-cadherin antibodies localized cadherin-like molecule in the sealing zone area of osteoclasts. These results suggest that cadherin-like molecules may mediate the tight attachment of osteoclasts in the sealing zone area and that the decrease of resorption in AHAVSE-treated osteoclast cultures is due to prevention of sealing zone formation. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy revealed that the vesicular stomatitis virus glycoprotein (VSV G) was targeted to the culture medium-facing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing the bone marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the bone substrate-facing surface of the UMR-108 cells. Electron microscopy showed that VSV particles were budding from the culture medium-facing surface, whereas Influenza viruses budded from the bone substrate-facing plasma membrane. These findings suggest the bone attaching plasma membrane of osteoblasts is apical, and the circulation or bone marrow facing plasma membrane is basolateral in nature. Gap junctions often mediate communication between different cells and cell types. In the present work, we demonstrate that rat osteoclasts show connexin-43 staining localizing in the plasma membrane of the cells in cell-cell contacts and over the basolateral membrane of osteoclasts. The effects of heptanol and Gap 27, known gap- junctional inhibitors, were studied using the well-characterized pit formation assay. The inhibitors decreased the number and activity of osteoclasts, suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. Furthermore, the total resorbed area and the number of resorption pits also decreased in the cultures. These results suggest that gap-junctional connexin-43 plays a functional role in osteoclasts, and that the blocking of gap junctions decreases both the number and the activity of osteoclasts.

by Chan Eliza. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 183-196). / ACKNOWLEDGEMENTS / ABSTRACT --- p.i / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter CHAPTER TWO: --- LITERATURE REVIEW / Chapter Section 1: --- History of Merkel cells --- p.3 / Chapter Section 2: --- Morphology and characteristic response of Merkel cell receptors in the skin --- p.5 / Chapter Section 3: --- Merkel cells and other mechanoreceptors in the mammalian sinus hair --- p.16 / Chapter Section 4: --- Functions of Merkel cells --- p.29 / Chapter Section 5: --- Review of technical approaches in the study of Merkel cell physiology --- p.39 / Chapter Section 6: --- Monitoring intracellular Ca2+ with the microfluorimetric technique --- p.42 / Chapter Section 7: --- Properties of voltage-gated and ligand-operated Ca2+ channels --- p.52 / Chapter CHAPTER THREE: --- METHODS / Chapter Section 1: --- Isolation of the rat vibrissal follicles --- p.60 / Chapter Section 2: --- Procedures for fluorimetric studies --- p.63 / Chapter Section 3: --- Procedures for electrophysiological study --- p.72 / Chapter Section 4: --- Chemicals --- p.82 / Chapter CHAPTER FOUR: --- RESULTS / Chapter Section 1: --- Electrophysiological studies in an isolated sinus hair preparation --- p.89 / Chapter Section 2: --- Electrophysiological studies on slowly adapting type I (SA I) mechanoreceptors in an isolated skin-nervein vitro preparation --- p.109 / Chapter Section 3: --- Microfluorimetric studies of Merkel cells in the isolated sinus hair preparation --- p.117

Les REMORINES du groupe 1 sont des protéines spécifiques des plantes, localisées dans la membrane plasmique. Nous avons montré que StREM1.3 (REM) constitue un marqueur des radeaux lipidiques, des domaines membranaires du plasmalemme enrichis en stérols et sphingolipides. De plus, REM se trouve enrichie dans les plasmodesmes (PD), des canaux ancrés dans la paroi qui assurent les communications intercellulaires. Nous avons mis en évidence pour la première fois le rôle physiologique de REM dans la plante, cette protéine est capable de ralentir la propagation virale du Potato Virus X (PVX) et d’autres virus. Par ailleurs, l’activité antivirale de REM est régulée par phosphorylation et conduit à une modification de la taille du pore des PD par dépôt de callose. Des candidats protéiques ont été sélectionnées et leur validation fonctionnelle a été initiée in planta par des approches de transgénèse, en expression transitoire et sur des plantes transgéniques soumises à des infections virales pour étudier la propagation des virus. Des approches de biochimie d’interaction des protéines, et d’imagerie ont également été envisagés. Le sujet de cette thèse vise à appréhender les mécanismes de l’interaction de REM avec ses partenaires dans la membrane lors de l’infection virale, en se focalisant sur les interactions protéines-protéines lors de la réponse au PVX. Nous nous intéresserons plus particulièrement aux protéines des PD et des radeaux membranaires qui sont très probablement ciblées lors de cette interaction avec les virus. / Group 1 REMORINs are plant-specific proteins located at the plasma membrane. We have shown that StREM1.3 (REM) is a marker of lipid rafts, plasma membrane domains enriched in sterols and sphingolipids. In addition, REM is enriched in plasmodesmata channels (PD) which are anchored within the cell wall and enable intercellular communication between virtually all plant cells. We have demonstrated for the first time the physiological role of REM in plants, this protein is able to reduce the viral cell-to-cell movement of Potato Virus X (PVX) and other viruses. Moreover, the antiviral activity of REM is regulated by phosphorylation and leads to a modification of the pore size of PD via the accumulation of callose, a sugar polymer, around the neck regions of PD. In order to understand how REM is able to induce the accumulation of callose in these specific regions, a large set of proteins have been selected and the deciphering of their functions have been initiated in planta by transgenic approaches, in transient expression and on transgenic plants, which will be subjected to viral infections to study the spread of viruses. Protein interaction, biochemistry and imaging approaches were also used to study this question. This thesis aims at understanding the mechanisms of the REM interaction with its membrane partners during viral infection, focusing on the protein-protein interactions during the response to PVX. We will focus more particularly on PD proteins and membrane rafts that are most likely targeted during this interaction with viruses

In order to optimize productivity of a cell culture it is necessary to understand growth and productivity and couple these features of the culture to extracellular nutrients whose profiles can be manipulated. Also, since growth and productivity are directly affected by cell death mechanisms such as apoptosis, it is imperative to understand these mechanisms. This work describes the development of a differential equation based population balance model of apoptosis in a Chinese Hamster Ovary cell culture producing Anti-RhD monoclonal antibody (mAb). The model was verified in isolation and was then coupled to a metabolic flux model. The model distinguishes between various subpopulations at normal healthy states and at various stages of apoptosis. After finding that glucose and glutamine are not limiting nutrients for this culture, different hypotheses were explored to explain growth arrest. Initially, it was hypothesized that there is some unknown nutrient in either media or serum which is depleted, thus causing growth arrest. Accordingly a first model was developed assuming depletion of this nutrient. Subsequent experiments with different additions of media and serum showed that there is no such nutrient limitation for the media and serum conditions used in most of the experiments. Additional experiments with different culture volumes showed that cell growth was actually controlled by a compound that accumulates and causes pH deviation from its optimal range of operation. Since strong correlations were found between culture volume and growth, it was hypothesized that the compound may be carbon dioxide (CO2), which is inhibitory for growth and may accumulate due to mass transfer limitations. Following this finding, a second model was proposed to take into account the accumulation of this inhibitor, although the specific inhibiting compound could not be exactly identified. This second mathematical model of cell growth was then integrated with a metabolic flux model to provide for a link between intracellular and extracellular species balances, since the latter are the ones to be manipulated for increasing productivity. This final model formulation was then used to describe mAb productivity. The model was also able to reasonably predict all cell subpopulations, nutrients, metabolites and mAb. In an attempt to mitigate the effect of CO2 accumulation and renew the cell growth, culture perfusions were performed. Although this approach resulted in some renewal of growth, the cell concentration progressively decreased after each successive perfusion event. This suggests that irreversible cell damage occurs because of CO2 accumulation. The model was used to describe the perfusion experiments. Agreement between data and model predictions were reasonable. In addition, it was shown that operation with successive perfusions results in a significant increase in productivity and therefore it can be used for further process optimization.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: IRBP expression & turnover. Includes bibliographical references (p. 176-177). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 104-120).