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Dissertations / Theses on the topic 'Epithelial cells'
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1
Liu, Ke. "Role of second messengers in controlling growth patterns of corneal epithelial cells /." View thesis, 2002. http://library.uws.edu.au/adt-NUWS/public/adt-NUWS20030718.102224/index.html.
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Thesis (Ph. D.)--University of Western Sydney, 2002."This thesis is submitted in fulfilment of the requirements of the degree of Doctor of Philosophy to the University of Western Sydney School of Biological Sciences."t.p. Includes bibliographical references (leaves 138-150).
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Wu, Ka-kei. "Effects of polysaccharides on gastric epithelial cells." Click to view the E-thesis via HKUTO, 2003. http://sunzi.lib.hku.hk/hkuto/record/B31971386.
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胡嘉麒 and Ka-kei Wu. "Effects of polysaccharides on gastric epithelial cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31971386.
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Samadikuchaksaraei, Ali. "Derivation of pulmonary epithelial cells from stem cells." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422341.
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Hedengran, Faulds Malin. "Estrogen receptor signalling in mammary epithelial cells /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-936-6/.
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Robson, Ewan John Douglas. "Characterisation of epithelial-mesenchymal transition in murine mammary epithelial cells." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616130.
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Kalair, Waseem. "Isolation and transplantation of murine intestinal stem cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ59179.pdf.
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Tan, Chong Da. "Metabolic regulation of epithelial sodium channels in human airway epithelial cells." Thesis, St George's, University of London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546781.
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Liu, Mengfei, and 刘梦菲. "Epithelial morphogenesis in three-dimensional cell culture system." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208611.
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In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue.
In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix.
In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure.
To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis.
The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion.
Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
10
Ellis, Steven E. "Xenogeneic transplantation of immortalized bovine mammary epithelial cells." Thesis, Virginia Tech, 1994. http://hdl.handle.net/10919/46164.
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The focus of this research was to investigate the use of an immortalized bovine mammary epithelial cell line as a starting material for xenogeneic transplantation into the mammary glands of immunocompetent recipients. PSG-5 cells (a clonal derivative of the MAC-T cell line engineered to express ovine IGF-I ) were transplanted into the cleared mammary fat pads of recipient mice. Following transplantation, spheroidal cell structures were observed in the cleared mammary fat pads of immunocompetent control mice and in mice exposed to PSG-5 cells during fetal development. Spheroidal cell structures were not observed in glands that had not received cell transplants. The success of this xenogeneic transplantation prompted the development of a MAC-T cell clone expressing a bacterial β-galactosidase (βMAC-T's) for use as a histological marker protein. Studies were then performed to determine the most appropriate age and location for cell transplantation into ovine recipients. Between three and 12 weeks of age, parenchymal volume in the mammary glands collected from e\ves in this study (n=4/age group: 3,6,9, and 12 weeks) increased nearly lO-fold (3.8cm3 to 34.5cm3 ). Total gland volume increased approximately 5-fold (67.3cm3 to 316.2cm3 ). Based on these determinations of parenchymal and glandular volumes, we determined that transplantation should begin with lambs at about three weeks of age. This data provides a starting point to begin trials using βMAC-T cells, which have been engineered to express a histological marker protein, for transplantation into intact ovine and murine mammary glands.
Master of Science
11
Bellett, Gemma Louise. "Microtubule deployment in polarised epithelial cells." Thesis, University of East Anglia, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426441.
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Lane, Carol. "Transplantation of retinal pigment epithelial cells." Thesis, University of Southampton, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316114.
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Nobbs, Angela Helen. "Interactions of streptococci with epithelial cells." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268782.
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Verma, Vandana. "Ryanodine receptors in secretory epithelial cells." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621856.
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Amineh, Abbadi. "Hyaluronan Rafts on Airway Epithelial Cells." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1407316041.
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Sakaram, Suraj. "Delineating ΔNp63α's function in epithelial cells." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1484411625682248.
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Vladar, Eszter Katalin. "Centriole assembly in ciliated epithelial cells /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Hong, Hee Ling. "Regulation of neutral proteinase and plasminogen activator secretion by epithelial cells in vitro." Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/24687.
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The aim of this thesis was to study the regulation of proteinase secretion by epithelial cells (E-cells) derived from the epithelial cell rests of Malassez. Since these epithelial cell rests are present only in small numbers in-vivo, E-cells derived from porcine cell rests were cultured according to Brunette et al. (1976) and conditions chosen so that detectable amounts of the proteinases, neutral proteinase and plasminogen activator, could be obtained. The regulation of the secretion of these enzymes was investigated by varying the cell population density, adding E.Coli lipopolysaccharide to the cultures and altering the shape of the E-cells by both chemical and physical means.
Cell population density modulated both neutral proteinase and plasminogen activator secretion. Neutral proteinase secretion was highest at low cell population densities and the activity decreased with increasing cell population density. Plasminogen activator secretion followed a similar pattern. Escherichia coli lipopolysaccharide (E.coli LPS) stimulated both neutral proteinase and plasminogen activator secretion. LPS extracted by the phenol method and LPS extracted by the trichloroacetic acid method caused similar increases in neutral proteinase activity but the increase in plasminogen activator activity was greater when the trichloroacetic acid extracted LPS was used. These findings support the proposal that bacterial LPS in contact with periapical tissues could stimulate the epithelial cell rests into increased production of proteinases, thereby contributing to the degradation of connective tissue associated with dental cyst formation. E-cell shape was altered by physical and chemical means. Addition of cholera toxin and dibutyryl cAMP caused E-cells to flatten. Phorbol myristate acetate, however, caused the cells to retract slightly. Mechanical stretching was applied to the cells to cause cell flattening, and cell rounding was effected by mechanical relaxation. Another method made use of E-cells grown on a substrate with V-shaped grooves which caused the cells to adopt a rounder shape more frequently than cells grown on a flat substrate. In addition, dishes coated with increasing concentrations of poly(HEMA) solution, which altered dish adhesivity to the cell, caused the cells to become less well-spread. In all experiments, a more flattened cell shape correlated with a reduced level of neutral proteinase and plasminogen activator secretion while a more rounded shape correlated with increased amounts of neutral proteinase and plasminogen activator secretion.Dentistry, Faculty of
Graduate
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Qiao, Bin. "Epithelial-Mesenchymal Transition and Mesenchymal-Epithelial Transition in Oral Stem Cell Carcinogenesis." Thesis, Griffith University, 2011. http://hdl.handle.net/10072/367467.
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Oral squamous cell carcinoma (OSCC), derived from normal oral epithelium transformation, remains a major public health problem world-wide. The prognosis of OSCCs that occur on lips is good, while other sites of oral mucosa where OSCC appears are more progressive, invasive and metastatic. A small subset of cells within a malignant neoplasm, named cancer stem cells (CSCs) or tumour initiating cells are thought to be capable of initiating the neoplasm itself, and of driving its growth and recurrance after treatment. The precise origin of CSCs is an ambiguous issue at present. The first proposal of the origin of CSCs is that CSCs develop from tumour cells themselves via cellular dedifferentiation. The secondary hypothesis for the origin of CSCs proposes that CSCs are the product of malignant transformation of adult stem cells. In this Ph.D thesis, we tried to demonstrate that CSCs in OSCC may be produced from those pathways.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
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Neil, Gillian Walker. "Lymphoepithelial interactions : inhibition of T cell activation by epithelial cells." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426785.
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Norman, M. J. "Loss of scribble causes cell competition in mammalian epithelial cells." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302397/.
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Cancer is a disease caused by transformation of cells by the activation or over-expression of oncogenes such as Ras and c-myc, and the loss of tumour suppressor genes such as E-cadherin and scribble. The initial stage of tumourigenesis is the transformation of a single cell in an otherwise normal epithelium. What occurs at this stage is largely unknown - do the transformed cells and normal cells co-exist or is there an antagonism between them? This thesis examines the fate of epithelial cells that lose the tumour suppressor scribble when in an otherwise normal epithelium. The fate of scribble knockout clones has been studied in Drosophila melanogaster larval imaginal discs. It has been observed that scribble knockout clones are removed from the larval tissues by c-Jun N-terminal kinase (JNK) dependent apoptosis. It is though that this is an innate tumour suppressive mechanism. It is therefore of great interest and importance to understand if a similar phenomenon can be seen in mammalian cells. Scribble knockdown Madin-Darby canine kidney (MDCK) epithelial cells die only when surrounded by normal MDCK cells. Dead scribble short-hairpin RNA (shRNA) cells are apically extruded from the epithelium after cell death and exhibit classical apoptotic markers such as cytoplasmic condensation, caspase 3 activation and DNA fragmentation. Extrusion of dead scribble knockout cells occurs after initiation of apoptosis as blocking myosin activation results in many dead scribble knockout cells staying in the epithelial monolayer. Prior to cell death they maintain normal cell-cell adhesion with their normal MDCK neighbours and activate the stress induced protein kinase p38, but not c-‐Jun N‐- terminal kinase (JNK).
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Offiah, I. "Cross-talk between human T cells, mast cells and conjunctival epithelial cells." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1348498/.
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The ocular surface is continually exposed to the outside environment and is a common site of inflammation. Conjunctival epithelial cells are thought to play a role in innate responses at the ocular surface. The hypothesis of my study is that conjunctival epithelial cells also contribute to T cell and mast cell effector mechanisms in chronic allergic eye disease via secretion of cytokines. In this study we initially demonstrate that the conjunctiva expresses TLRs, and that the TLR3 ligand (poly I:C) activates conjunctival epithelial cells in vitro to secrete inflammatory mediators as part of the innate immune response. Conjunctival tissues were also shown to express the Th2 associated cytokine, IL-13 as well as TSLP – a cytokine thought to be involved in Th2 differentiation. Conjunctival tissues from chronic allergic eye disease subjects were found to have increased IL-13 and TSLP expression compared to normal controls. Using a human conjunctival epithelial cell line, cells could be induced to express increased levels of TSLP following exposure to poly I:C or pro-inflammatory cytokines. Th17 cells, identified by coexpression of CD4 and IL-17, were also detected in CAED tissues and a high level of expression of IL-17A was localised to the epithelium. However, although capable of secreting IL- 25, IL-17A was not secreted by conjunctival epithelial cells, indicating that the IL-17 observed histologically may have been IL-17 binding to the surface of the epithelium. IL-17 receptor C (IL-17RC) expression was found to be increased in CAED tissues whilst IL-17RA was upregulated when conjunctival epithelial cells were stimulated with pro-inflammatory cytokines together with poly I:C. Blockade of IL-17RA and subsequent stimulation with IL-17 led to increased IL-8 and decreased TGF-β secretion. Although being implicated in the immunopathogenesis of certain diseases, IL-17 and its other family members may potentially serve to play an immunoregulatory role in immunity at the ocular surface.
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Huynh, The Hung. "Establishment of bovine mammary epithelial cell lines : an in vitro model for lactation." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60426.
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Clonal cell lines were isolated from mammary gland tissue epithelial cell cultures of lactating cows. Early passage clonal bovine mammary epithelial cells (clone LMH17) gave rise to several established cell lines (MAC-T lines) after being cotransfected with plasmids containing the temperature sensitive mutant SV40 large T antigen gene (pBAPSV40TtsA58) and the bacterial phosphotransferase gene (pSV2-neo). Unlike other cell types which were transformed after being transfected with SV40, MAC-T cells maintained many characteristics of non-transformed cells: MAC-T cells were serum and anchorage dependent, showed contact inhibition, and were not tumorigenic in immunodeficient mice. However, Southern transfer analysis revealed an integrated SV40 gene and cells showed no senescence after 50 passages. These cells are morphologically indistinguishable from parental LMH17 cells and retain the typical morphology of mammary epithelial cells. Positive cytokeratin immunostaining and the absence of vimentin staining indicated that these cells were epithelial in origin.MAC-T cells grew rapidly on plastic substratum with a doubling time of approximately 17 hours and became differentiated when grown on floating collagen gels in the presence of prolactin. The differentiated phenotype was characterized to include (1) the ability to form secretory domes with a lumen from a pavement of columnar cells; (2) increased casein mRNA abundance; (3) increased alpha S and beta casein secretion; (4) increased number and size of casein secretory vesicles; and (5) increased lactose synthesis and secretion.
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Liu, Ke. "Role of second messengers in controlling growth patterns of corneal epithelial cells." Thesis, View thesis, 2002. http://handle.uws.edu.au:8081/1959.7/387.
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The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells.
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Barry, Megan M. Crockett Robert S. "Three-dimensional scaffolds for mammary epithelial cell growth : a thesis /." [San Luis Obispo, Calif. : California Polytechnic State University], 2008. http://digitalcommons.calpoly.edu/theses/12/.
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Thesis (M.S.)--California Polytechnic State University, 2008.Major professor: Robert S. Crockett, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering." "May 2008." Includes bibliographical references (leaves 38-45). Also available on microfiche (1 sheet).
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Fahlgren, Anna. "Defence capabilities of human intestinal epithelial cells." Doctoral thesis, Umeå : Univ, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-151.
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Chivers, Joanna Elizabeth. "Mechanisms of glucocortical action in epithelial cells." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415076.
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Klingner, Christoph. "Cortical actomyosin network organization in epithelial cells." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-179783.
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Epithelzellen, die Modell-Zelllinien dieser Dissertation, sind für die Aufteilung und Abtrennung verschiedener Kompartimente eines Organismus zuständig, indem sie sich zu Grenzflächen zusammenschliessen, welche häufig hohen physikalischen Spannungen und Kräften
ausgesetzt sind. Um diese physikalischen Kräfte zu verarbeiten oder sie selbst zu produzieren,
verwenden Epithelzellen, wie alle anderen Zelltypen auch, das Zytoskelett, das sich im Allgemeinen aus den Komponenten Mikrotubuli, Intermediär-Filamenten und Aktin sowie den
damit korrespondierenden Motorproteinen Dynein, Kinesin sowie Myosin zusammensetzt.
In dieser Dissertation wird das Zusammenspiel von Aktin und Myosin auf der apikalen Seite
von Epithelzellen untersucht. Im Falle von konfluenten Zellen mit vollständig ausgebildeten Zell-Zell-Kontakten sind auf der apikalen Seite der Zellen Mikrovilli zu finden, kleine, mit Aktin-Bündeln gefüllte Ausstülpungen aus der Zelloberfläche, welche für die optimierte Nahrungsaufnahme sowie als Antennen für Signalverarbeitung zuständig sind. Im Zuge der
Arbeit konnten wir feststellen, dass sich der Aktin-Myosin-Aufbau auf der apikalen Seite
von Einzelzellen ohne Zell-Zell-Kontakte, sogenannten nicht-konfluenten Zellen, grundsätzlich ändert. Mittels Fluoreszenz-Mikroskopie und anderen experimentellen Methoden zeigen wir, dass zwar ähnliche Ausstülpungen auf der apikalen Oberfläche von Einzelzellen zu finden, diese jedoch häufig verlängert, gebogen, hoch-dynamisch und oft parallel zur Zellmembran
orientiert sind. Wir zeigen mittels molekularbiologischer Methoden, dass ein zusätzliches, innerhalb der apikalen Zellmembran liegendes isotropes Akto-Myosin-Netzwerk für die dynamische Reorganisation der Mikrovilli-Ausstülpungen verantwortlich ist.
Der Identifzierung des isotropen Akto-Myosin-Netzwerkes, welches eine der Hauptaussagen
dieser Dissertation ist, wird eine detaillierte Analyse der dynamischen Netzwerkreorganisation
angefügt, die mittels temporaler und örtlicher Bild-Korrelationsanalysen charakteristische
Zeiten und Längen der Dynamik definiert. Des Weiteren entwickeln wir mehrere Bild-
Analyseverfahren, allen voran die Methode der iterativen temporalen Bildkorellation sowie des
optischen Flusses, wodurch wir eine Oszillation der Netzwerk-Reorganisationsgeschwindigkeit
identifizieren und parametrisieren können. Verschiedene, auf Fluoreszenzmikroskopie und automatisierter optischer Fluss-Bildanalyse basierende Experimente geben Hinweise auf zwei
mögliche Erklärungen für die identifizierten Oszillationen. Sowohl Myosin aktivitätsregulierende Proteine als auch spontan auftretende Spannungsfluktuationen im unter Zugspannung liegenden Netzwerk können mögliche Ursachen für die identifizierten Netzwerkoszillationen sein. Obwohl eine eindeutige zelluläre Funktion des apikalen Akto-Myosin-Netzwerkes im Rahmen dieser Doktorarbeit noch nicht identifiziert werden konnte, so können wir aufgrund von verschiedenen Resultaten dennoch postulieren, dass das hier identifizierte Netzwerk eine entscheidende Rolle bei der Zellmigration und Signaltransduktion einnimmt. Unabhängig davon repräsentiert das hier gefundene Netzwerk die faszinierende Möglichkeit, ein aktives, zweidimensionales Akto-Myosin-Netzwerk nicht nur in vitro, sondern in seiner natürlichen Umgebung studieren und biophysikalische Eigenschaften analysieren zu können.The cytoskeleton plays a central role in cellular morphogenesis by generating, sensing and transmitting physical forces. Actin filaments are key cytoskeletal elements that are mostly located close to the cell cortex. They can generate protrusive or contractile forces in combination with myosin motor proteins. We have identified a novel, highly dynamic actin structure at the apical side of non-polarized epithelial cells that is driven by an underlying non muscle myosin II network. By using various image analysis techniques, such as maximum intensity tracking, optical flow and correlation analysis, we observe contractile actomyosin activity within subregions of the cell cortex. The resulting spatially restricted mechanical forces differ in directionality which leads to shear stress and friction within the apical cell cortex. Additionally, we identified a global oscillatory behavior using autocorrelation analysis methods. The actomyosin network oscillates between states of low and high activity, as confirmed by iterative temporal image correlation (ITIC), a newly developed method for global feature extraction from image sequences, and highest intensity tracking. These remarkable features of subcellular cortex regulation give important insights into how mechanical force generation and propagation control cell shape and migration in non-polarized epithelial cells.
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Rakhit, Soma. "Signalling pathways in cultured equine epithelial cells." Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245291.
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Tarran, Robert. "Chloride currents in murine airway epithelial cells." Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362418.
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McCoubrey, Amanda. "Adhesion of lactobacilli to porcine epithelial cells." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240879.
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Zhan, Hong. "The immunological role of conjunctival epithelial cells." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405640.
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Hanson, Amanda Marie. "TORC2 Mediated Chemotaxis in Mammary Epithelial Cells." Thesis, The University of Arizona, 2016. http://hdl.handle.net/10150/613561.
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Chemotaxis is the directional movement of cells in response to an extracellular chemical gradient. It is thought to be involved in cancer cell metastasis by recognizing chemokines and growth factors; therefore, deregulation of chemotactic pathways can result in increased tumor cell metastasis. The Target of Rapamycin Complex 2 (TORC2) regulates chemotaxis downstream of these signaling molecules, or chemoattractants. Examining the role of TORC2 in chemotaxis of cancer cells could provide insight into the deregulation of signals leading to cancer cell metastasis. The non-tumorgenic cell line MCF10A and the two breast cancer cell lines MCF7 and MDA-MB-231 were employed in this study. Epidermal Growth Factor (EGF) and Insulin-like Growth Factor 1 (IGF-1) showed potential as chemoattractants by stimulating TORC2. Wound healing assays were performed on MCF10A, MCF7, and MDA-MB-231 cells exposed to TOR inhibitors, as well as MCF10A cells with Rictor knocked down. Cells with Rictor knocked down and MCF10A cells exposed to Torin 2 showed a decrease in cell migration. Ibidiμ-Slide chemotaxis slides were used to perform a chemotaxis assay with MCF10A cells in response the EGF. Cells showed greater directionality toward EGF in the experimental well as compared to the control with EGF on both sides of the cell chamber. Future examination of other potential chemoattractants as well as chemotaxis assays with other chemoattractants will give more insight into the goals of this research.
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Perrett, Charlotte Averil. "Factors affecting Salmonella invasion of epithelial cells." Thesis, University of Bristol, 2007. http://hdl.handle.net/1983/a06abf7d-cf06-43f3-bd89-5ff76988c637.
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Spiteri, Cornish Daniella. "Nasal epithelial cells in different wheezing conditions." Thesis, University of Aberdeen, 2017. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=232239.
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Background: Wheezing disorders have increased worldwide. The respiratory epithelium plays an important role in the pathogenesis of wheeze. Nasal epithelial cells (NEC) are a valid surrogate for bronchial airway epithelial cells, are accessible and could be a valuable tool in translational epidemiological studies. A better understanding of this layer may decrease the burden of wheezing disorders. Objectives: To determine the feasibility of sampling and culturing NEC from children and adults with and without different wheezing conditions in epidemiological studies. To study NEC mediator release in these individuals following different environmental exposures. Methods: NEC were sampled from unsedated children and adults with and without a wheezing condition by brushing. NEC were cultured in media and also exposed to interleukin-1 (IL-1) & tumour necrosis factor alpha (TNF-), house dust mite (HDM) extract, lipopolysaccharide (LPS) and extracted tobacco smoke (ETS) for 24 hours. Resulting supernatants were analysed via Enzyme – linked immunosorbent assay (ELISA) and cytometric bead array (CBA) for mediator release. Results: 287 individuals including 164 children and 123 adults where phenotyped and brushed. 81 samples reached tertiary passage. Decreased release of vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein -1 (MCP-1) was observed in wheezing individuals when compared to healthy controls. These cytokines were increased in individuals with chronic obstructive pulmonary disease (COPD) relative to both asthmatic and healthy adults. Individuals with/without allergic rhinitis demonstrated different mediator release. Conclusions: It is feasible to obtain NEC in adults and children for both epidemiological and translational research, although the presence of allergic rhinitis may act as a potential confounder. Differences are present in adults and children with asthma compared to healthy controls. Contrasting differences between COPD and asthma suggest that these are different conditions.
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Ribeiro, Ana Sofia dos Santos Marques. "Lipidomics of mammary epithelial cells throughout differentiation." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/9683.
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Mestrado em BioquímicaA glândula mamária desenvolve-se maioritariamente após o nascimento. O seu desenvolvimento é regulado por alterações hormonais que ocorrem em diferentes etapas da vida reprodutiva adulta como puberdade, gravidez, lactação e involução. A necessidade de renovação do tecido epitelial devido a contínua remodelação do tecido sugere a existência de células estaminais mamárias (MSCs), que podem suportar ciclos contínuos de proliferação e diferenciação e apoptose. As MSCs demonstraram várias semelhanças com os tipos de cancro da mama com pior prognóstico e têm sido alvo de vários estudos, uma vez que o estudo do seu programa de diferenciação pode contribuir para um melhor entendimento do desenvolvimento e progressão tumoral. Várias proteínas envolvidas no metabolismo e sinalização lipídica parecem estar altamente reguladas durante a diferenciação das MSCs em diferentes linhas celulares. As proteínas e os fosfolípidos (PLs) estão ambos presentes na membrana celular. PLs são um grupo bastante diverso de biomoléculas essenciais para a manutenção da integridade estrutural celular e sinalização celular. Alterações em lípidos particulares pode refletir alterações na atividade metabólica e/ou ambiente, o que afeta a sinalização celular. Assim, o objectivo deste trabalho foi utilizar uma abordagem lipidómica para analisar o perfil fosfolipídico de células epiteliais mamárias em diferentes estágios de diferenciação (MSC, pré-diferenciação e diferenciação funcional) para identificar espécies moleculares associadas a alterações no metabolismo dos PLs. Para atingir este objectivo foi usada uma abordagem lipidómica que combinou cromatografia de camada fina (TLC) e cromatografia líquida de alta resolução (HPLC) com espectrometria de massa (MS). Esta abordagem permitiu a identificação e quantificação de uma grande variedade de espécies de PLs. Os resultados obtidos neste trabalho demonstraram que a fosfatidiletanolamina (PE) apresenta um aumento em conteúdo relativo com a progressão para a diferenciação, enquanto que para as outras classes de PLs não se observaram alterações significativas quando comparados os três estágios de diferenciação. Além disso, apesar de as espécies moleculares observadas serem as mesmas para os 3 tipos de células, foram encontradas diferenças nas abundâncias relativas de algumas espécies moleculares de fosfatidilcolinas, PEs, fosfatidilinositois e fosfatidilgliceróis entre os estados de diferenciação. Esta análise pode contribuir para uma melhor compreensão do processo de diferenciação de células epiteliais mamárias e da sua susceptibilidade ao desenvolvimento de cancro da mama.
The mammary gland develops mainly after birth. It’s development is regulated by hormonal changes that occur at different stages of the adult reproductive life such as puberty, pregnancy, lactation and involution. The need of renovation of the epithelial tissue due to continuous tissue remodeling, suggest the existence of mammary stem cells (MSCs), which can support continuous cycles of proliferation, differentiation, and apoptosis. MSCs show several similarities with breast cancer types with worse prognosis and have been a target of several studies, as study of their differentiation program may provide better understanding of tumor development and progression. Several proteins involved in lipid metabolism and lipid signaling seem to be highly regulated throughout differentiation of MSCs into different epithelial lineages. Proteins and phospholipids (PLs) are both present in the cellular membrane. PLs are very diverse group of biomolecules essential for the maintenance of cellular structure integrity and cellular signaling. Changes in particular lipids may reflect alterations in metabolic activity and/or environment, which affect cellular signaling. Thus, the aim of this work was to use a lipidomic approach to analyze the phospholipid profile of mammary epithelial cells in distinct differentiation stage (MSC, predifferentiation and functional differentiation) in order to identify molecular species associated to changes in PL metabolism. To achieve this goal we used a lipidomic approach that combined thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) with mass spectrometry (MS). This approach allows the identification and quantification of a great variety of PL species. The results obtained in this work indicate that phosphatidylethanolamine (PE) show an increase in relative content with the progression to differentiation, while in the case of the other PL classes no significant changes were observed, when comparing the three types of cell. Besides, though the molecular species observed were the same for the 3 cell types, differences in the relative abundance of some molecular species of phosphatidylcholine, PE, phosphatidylinositol and phosphatidylglycerol, presented between differentiation states, were detected. This analysis can contribute to a better understanding of the process of differentiation of mammary epithelial cells and their susceptibility to the development of breast cancer.
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Capra, J. (Janne). "Differentiation and malignant transformation of epithelial cells:3D cell culture models." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526218236.
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Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosisTiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta
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Ouertani, A. "Determinants of cell cycle progression in human mammary epithelial MCF12 cells." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1362848/.
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Cancer of the mammary gland is the most common type of cancer in women worldwide, and the vast majority of breast cancers originate from a cluster of malignant cells in the epithelial tissue of the breast, which initially confines the ductal carcinoma in situ. Research has shown that the signalling pathways that increase differentiation and maintain proliferation in normal epithelial cells are of utmost importance for sustaining this barrier against malignant cells. As a model for normal mammary epithelial cells, the MCF-12A cell line was used to determine factors that are required for cell cycle progression of these cells. A discontinuous treatment assay was developed in which the MCF-12A cells were treated with epidermal growth factor (EGF) and insulin at two distinct times to induce cell cycle re-entry. The use of these chemically defined growth factors enabled us to determine that continuous stimulation with mitogenic factors is not required for these cells to re-enter the cell cycle. An initial activation of the MAP kinase pathway and an up-regulation of the transcription factor c-Myc, followed by activation of the PI3K pathway, resulted in full competence to progress into S phase. The order in which the growth factors were applied, and thus the sequence in which the subsequent proteins were triggered, was of great importance for successful S phase entry. We found that estradiol (E2) was unable to induce the factors necessary for cell cycle progression. Furthermore, we report for the first time that E2 did not affect estrogen-regulated genes which normally are under the control of a ligand-bound estrogen receptor (ER). We suggest that the mechanism by which the ligand-activated ER usually interferes with the estrogen responsive element in the promoter region of the target genes is defective in the MCF-12A cell line. The results presented here may contribute to new approaches in chemotherapy, taking advantage of the diverse molecular mechanism in place for cell cycle progression and proliferation in malignant cells compared to normal mammary epithelial cells.
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Woodfield, Richard John. "Investigation of the association of P13K with the cadherin-catenin adhesion complex." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368435.
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Binti, Kamarudin Taty Anna. "Differentiation of human pluripotent stem cells into corneal epithelial like cells." Thesis, University of Newcastle upon Tyne, 2018. http://hdl.handle.net/10443/4182.
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Cornea is the clear outermost protective layer of the eye which enables transmission of light onto the retina. The corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in limbal stem cell deficiency (LSCD). Transplantations of ex vivo expanded autologous LSCs from patient's healthy eye onto the affected eye have provided a successful treatment for unilateral LSCD. This however is not applicable to patient with total bilateral LSCD, whose both eyes are affected. This thesis investigated the potential of human induced-pluripotent stem cell (hiPSCs) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD, and tested the engraftment of the differentiated cells in LSCD mouse model. Combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid (RA) and epidermal growth factor (EGF) for the first nine days of differentiation followed by cell-replating on collagen-IV coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESCs) to corneal epithelial progenitors and mature corneal epithelial-like cells. Differences in the ability of hiPSCs lines to undergo differentiation to corneal epithelial-like cells were observed. These were dependent on the level of endogenous BMP signalling and could be restored via activation of this signalling pathway by a specific TGFβ inhibitor (SB431542). The hESC and hiPSCs-derived corneal epithelial cells were transplanted into a LSCD mouse model where they survived up to 14 days, but failed to provide long term engraftment and corneal surface regeneration. The findings showed a differential ability of hESCs and hiPSCs lines to generate corneal epithelial cells which is underlined by the endogenous BMP signalling pathway activity. However, the engraftment and functionality of the differentiated cells in the LSCD animal model has yet to be improved.
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Zhang, Hao. "Cytogenetic and molecular alterations in immortalization of normal esophageal epithelial cells." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B32047010.
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Stewart, Alasdair Gwilym. "Studies of focal adhesion kinase in epithelial cells : involvement in cell-cell adhesion." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446839/.
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Epithelial cell-cell adhesion is mediated by tight junctions, adherens junctions and desmosomes. Epithelial cell-matrix adhesion is mediated by hemidesmosomes and focal contacts. These complexes exhibit great plasticity, and each contains molecular components which are able to participate in one or more of the other adhesive complexes. Focal adhesion kinase (FAK/pl25FAK) is a non-receptor tyrosine kinase which transduces signals from integrins at sites of focal contact to promote adhesion, spreading and migration. FAK possesses a central kinase domain which is flanked by large, non-catalytic, amino- and carboxy-terminal domains. Whereas the functions of the carboxy-terminal and kinase domains of FAK are well understood, the role of the amino-terminal domain remains unclear. FAK expression was examined in the human epithelial cell line, HEK 293. Amino-terminal FAK immunoreactivity was noted at sites of cell-cell contacts and in the nucleus, in contrast to carboxy-terminal immunoreactivity, which was largely cytoplasmic and perinuclear. Western blot analysis of endogenous FAK revealed expression of a presumptive proteolytic cleavage fragment corresponding to the amino- terminal domain. A series of FAK constructs was generated to test the hypothesis that the observed amino-terminal FAK localisation was due to this proteolytic fragment. Epitope- tagged Amino-Terminal FAK (ATF) constructs localised primarily at areas of cell-cell contact and in the nucleus in HEK 293 cells. This localisation was independent of Tyrosine 397, the major FAK autophosphorylation site. This sub-cellular distribution was confirmed in another epithelial cell line, MDCK, in which transiently transfected ATF constructs also localised primarily to the nucleus and at cell-cell contacts. HEK 293 cells were characterised with respect to expression of adhesive proteins, and ATF was found to co- localise with the tight junction protein occludin, with cortical actin and with junctional ?1 integrin. Immunoprecipitation data suggests that none of these proteins forms a precipitable complex with ATF. These findings indicate that the amino-terminal domain of FAK is capable of localising at epithelial cell-cell contacts and suggest a novel role for FAK in mediating cross-talk between focal contacts and cell-cell contacts through endogenously expressed amino-terminal FAK fragments.
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Relova, Anne-Jacqueline. "Mechanisms for and Effects of Airway Epithelial Damage in Asthma." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5312-0/.
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Holman, Brena Baze. "Cripto-₁ and amphiregulin production in transformed mammary epithelial cells grown on hydroxyapatite scaffolding." Online access for everyone, 2007. http://www.dissertations.wsu.edu/Thesis/Spring2007/B_Holman_042307.pdf.
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Barnard, Zeke. "Analyses of Cytokeratins and p63 Isoforms Expressed by Human Limbal Epithelial Cells." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/367809.
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Cultivated autologous limbal epithelial grafts have shown promise as a valuable treatment to treat ocular surface injuries. The rationale for this therapy is that the limbus contains progenitor cells of the corneal epithelium and that when expanded and transplanted these cells help to restore a healthy epithelium. Nevertheless, this approach is still experimental and the optimal procedure for producing, grafting and assessing the presence of limbal stem cells in these grafts remains under development.
The first results chapter examines the differences in cytokeratin expression between donor corneoslceral rims and cultivated limbal epithelial cultures derived from these donor rims. The expression of keratins 3 and 14 is more stable between the in situ and in vitro environments, while keratins 6 and 19 appear to be upregulated in limbal epithelial cells in response to their isolation and culture. This highlights the role of keratins in limbal epithelial cell function and provides valuable knowledge for assessing the phenotype of cultivated limbal epithelial grafts. Cytokeratin expression is more important in identifying the transient amplifying population than the progenitor population, indicating the need for more specific markers to identify the progenitor cells.
The second results chapter investigated the potential of p63 in identifying the progenitor population of the limbal epithelium. The use of RT-PCR enabled a more detailed examination of the RNA expression of the p63 isoforms. The experiments performed assessed p63 in situ and in vitro, including conditions designed to expand the progenitor population in culture. This work confirms the value of p63 as a marker for immature limbal epithelium and also demonstrates for the first time the p63 isoforms produced by human limbal epithelial cells in vitro.
The final results chapter describes the application of techniques developed and utilised in the previous 2 chapters upon autologous cell samples and constructed grafts used to treat 5 patients clinically. The phenotypic analyses demonstrate that both the cultivated epithelium and the grafts created from the different donor biopsies exhibit similar properties in the areas examined. In addition, these results allowed comparison between clinically used autologous cultures and the methods employed to cultivate limbal epithelium examined in the previous chapter.
The use of both p63 and the various cytokeratins in assessing the level of differentiation hold merit, however more specific markers for the stem cell population of the limbal epithelium remain elusive. The results present a deeper analysis of the material used to treat limbal stem cell deficiency and give insight into further evolution of these treatments.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Lehman, Teresa Ann. "Studies in human skin epithelial cell carcinogenesis /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487332636474889.
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Gillard, Geoffrey Oliver. "Developmental mechanisms regulate the generation and maintenance of mTEC heterogeneity and peripheral antigen expression /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8318.
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Beckstead, Benjamin L. "Control of epithelial differentiation by cell-instructive scaffolds /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8096.
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Geada, Maria do Rosario Moreno Cruz Colaco. "Molecular mechanism of magnesium transport in epithelial cells." Thesis, University of Central Lancashire, 1998. http://clok.uclan.ac.uk/20300/.
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Gastrointestinal secretions are controlled mainly by the gut hormones and by the neurotransmitters released by the autonomic nervous system. The hormones and neurotransmitters (secretagogues) utilise different intracellular mediators to elicit enzyme and fluid secretion. One particular mediator is the second messenger calcium (Ca2+) and there is now much evidence that the abundant divalent cation magnesium (Mg 25 may play an important physiological role in regulating the mobilisation of cellular Ca 2t Thus, the present study was designed to investigate (a) the effect of a modification of extracellular Mg2 on secretagogue-evoked enzyme and hydrochloric acid (HCI) secretion and Ca 2 homeostasis in the pancreatic acinar cells and parietal cells and (b) to characterise the molecular mechanism of Mg 2 transport using fluorimetric and spectroscopic studies. The results have shown that application of either cholecystokinin-octapeptide (CCK-8) or acetylcholine (ACh) to rat pancreatic segments can result in marked increases in amylase and trypsinogen output in normal (1.1 mlvi) extracellular magnesium [Mg 2 0. When [Mg2 0 was elevated to 10 mlvi, there was a significant (P C 0.05) decrease in secretagogue-evoked pancreatic enzyme secretion. On the other hand, in low (0 mM) [Mg2 0 both CCK-8 and ACh elicited marked increases in enzyme secretion similar to the responses obtained in the presence of normal [Mg 2 o . The effects closely correlate with the concurrent reductions and increases in intracellular free calcium concentrations 2 +-. [Ca j ifrom studies performed with fljra-2-AM loaded pancreatic acmi dunng perturbation of [Mg2 0 These findings indicate that Mg 24 can influence enzyme secretion by regulating Ca24 mobilisation. The possible site of action of Mg2 in controlling Ca24 appears to be at the level of Ca 24 influx, since experiments, using thapsigargin or ionomycin, agents which release Ca2+ from intracellular Ca2+ stores, were not affected by a variation in [Mg 2 0 . This study also employed the technique of microspectrofluoritnetry to fi.irther characterise the mechanism of Mg2 transport using mouse pancreatic acinar cells loaded with magfisra-2-AM. Stimulation of acini with CCK-8 evoked an initial sharp rise in [Mg 2 1 followed by a decrease to a new steady-state level (Mg 24 efflux). On removal of CCK-8 [Mg2 1 returned to the pre-stimulated basal level (Mg 24 reuptake). In contrast, CCK-8 gave rise to Ca24 oscillations. When acinar cells were co-loaded with 1 ,2-bis (2- aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 piM) and either magflira-2-AM or fijra-2-AM, CCK-8 evoked only the normal decrease in [Mg 2 j and a slight [Ca2 j+ ,e levation. (10 nM). This . results suggests that the in. i.t ial ns. e in. [Mg 2+-. ji seen with CCK-8 in normal conditions may be due to the Ca 24 interfering with the magfura-2-AM signal. Both thapsigargin (0.5 pM) and ionomycin (5 piM) evoked a marked decrease in [Mg 2+,. . . . 2-i-,jj in magfiira-2-AM loaded acmar cells and an elevation in [Ca j' from fi.ira-2- VAM loaded acinar cells. The results indicate that cytosolic Ca 2 is associated with secretagogue-induced decrease in [Mg 24]1. When acinar cells were pre-treated with either forskolin (10 pM), N Nitro-L-arginine (N-NLA; 2 mM), 8 -Bromo guanosine guanosine cyclic mono-phosphate (Br cGMP; 100 pM) or staurosporine (1 pM), CCK-8 elicited only a small decrease in [Mg24]j compared to a much larger response with CCK-8 alone. In contrast, genistein (10 pM) and 12-0-tetradecanoyl phorbol 13 a acetate (TPA; 1 pM) augmented the decrease in [Mg 24]1 evoked by CCK-8. The results indicate that Ca2 mobilising secretagogues can stimulate Mg 2 efflux which is also mediated by a number of intracellular mediators. This study also investigated the mechanism of Mg 2 transport from the rat pancreas. Permeabilised pancreatic acini were loaded with Mg 2 ' by employing a high Mg2 (12 mM) buffer containing the tonophore A23 187 (6 .dv1). Net Mg 2+ efflux was measured by using the technique of atomic absorption spectrophotometry. Incubation of pre-loaded acini in a buffer deficient in Mg2 resulted in a large and time-dependent release of Mg 2 with maximal efflux occuning within 40 min. Pre-treatment of loaded acini with either bumetadine, SITS or ouabain had no significant effect on Mg2 efflux. In contrast, when acini were pre-treated with either 10 mM dinitrophenol (DNP), io M amiloride, 1 mM lidocaine or I mM quinidine there were significant (P < 0.05) decreases in net Mg 2 efflux. Replacement of extracellular sodium [Na4]0 with either N -methyl-D-glucamine (NMDCI), or choline chloride resulted in a significant (P C 0.05) inhibition of Mg2 effiux. The results of this study indicate that Mg 2 transport (efflux) in rat pancreatic acinar cells may not be associated with the N atKtATPase, the NatKtCl cotransporter or the anion exchanger, but with a Na+ -sensitive Mg2+ transport system. However, this Na+ sensitivity was found to be species dependent as in mouse pancreatic acinar cells Mg2 transport occurred in the absence of [NC] 0 . Studies performed on rat gastric panetal cells have demonstrated that elevated [Mg 2 10 has the same inhibitory effects on secretagogue-evoked acid secretion and cellular Ca 2 transport as that observed with pancreatic acinar cells whereas low [Mg 2 0 had the opposite effect. In conclusion, the results of this study have demonstrated marked interactions between the two divalent cations Mg2 and Ca2 in epithelial secretory cells of the gastrointestinal tract. Mg2+ seems to regulate secretagogue-evoked secretion by controlling cellular Ca2+ mobilisation.
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Bird, Sarah Anne. "Spatial Regulation of MT1-MMP in Epithelial Cells." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487203.
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Tube structures are fundamental in multicellular organisms and are found in many epithelial organs. Madine-Darby Canine Kidney (MOCK) epithelial cells are used as a model to study tubulogenesis as they form branching tubules in a 3-D type-l collagen gel upon stimulation with Hepatocyte Growth Factor (HGF). During this process, cells need to degrade the surrounding collagen matrix and they have been shown to primarily utilise the collagenase membrane-type I metalloproteinase (MTI-MMP, MMP-14) to achieve this. The aim of this thesis is to investigate the mechanisms regulating MTl-MMP during this process. We observed that MTlMMP preferentially localised to the apical surface of polarised MOCK cells cultured on type-I collagen. However, upon stimulation with HGF, a significant amount of MTI-MMP was detected on the basal surface. This localisation has a functional impact as cells degrade type-I collagen only upon HGF stimulation in 2-D culture. We hypothesise that MTl-MMP activity is spatially regulated in polarised MOCK cells, enabling enzyme activity to be higher at the growing tip of the tubule than atthe base. We also observed that MOCK cell attachment to collagen is required for MTl-MMP to localise to the basal surface. By analysing domain deletion mutants of MTI-MMP, it became clear that HGF-dependent localisation to the basal surface is catalytic domain/linker-I and hemopexin domain-dependent. Specifically, the MT-Loop C63pYAYlREG170 ), a unique sequence in the catalytic domain of all;ransmembrane-type MT-MMPs, is essential for basal localisation. Further characterisation of the MT-Loop region indicated that it also plays a critical role in the localisation of MTl-MMP in non-epithelial cells. COS-7 cells expressing the MT-Loop deletion mutant showed a significant reduction in collagen and gelatin degradation in 2-0 culture, even though the cell surface expression level and in vitro catalytic activity of this mutant was similar to full-length enzyme. Taken together, my research has revealed a novel mechanism that regulates MTl-MMP and hence tube formation of MOCK cells, which may be applicable to tubulogenesis in vivo.