Bone Morphogenetic Proteins (BMPs) bilden die größte Untergruppe der Transforming Growth Factor-β (TGF-β) Superfamilie sekretierter Wachstumsfaktoren. Sie haben Schlüsselfunktionen während der frühen Embryogenese inne und regulieren darüber hinaus die Organogenese sowie die Homöostase zahlreicher Organe und Gewebe. BMPs vermitteln ihre Signale über zwei Typen transmembranärer Serin-/Threoninkinaserezeptoren, die als Typ I und Typ II Rezeptoren bezeichnet werden. Den etwa zwanzig BMP-Liganden stehen dabei nach aktuellem Kenntnisstand nur fünf Typ I und drei Typ II Rezeptorkinasen gegenüber, wodurch sich insbesondere die BMP-Familie durch eine hohe Promiskuität der Ligand-Rezeptor-Interaktion auszeichnet. Damit dennoch Liganden-spezifische Signale vermittelt werden können, müssen die Signaleigenschaften dieser Faktoren komplex reguliert werden. Die Mehrzahl der Regulationsmechanismen beeinflusst die Signaltransduktion negativ. Kürzlich wurden jedoch die ersten, spezifisch auf die BMP-Familie wirkenden membranassoziierten Agonisten beschrieben – die Corezeptoren der Repulsive Guidance Molecule (RGM) Familie bestehend aus RGMa, RGMb und RGMc. Für das Familienmitglied RGMb werden neben pro-BMP-Prozessen allerdings auch hemmende Wirkungen auf die BMP-abhängige Signaltransduktion diskutiert. Um diese teils widersprüchlichen Funktionen zu beleuchten, wurde RGMb im Rahmen dieser Arbeit umfangreich biochemisch und biophysikalisch charakterisiert. Zunächst konnte erfolgreich ein Verfahren zur Herstellung und Isolierung von hochreinem rekombinanten RGMb Corezeptorprotein etabliert werden. Dies ermöglichte die Entwicklung umfangreicher in vitro Interaktionsstudien mit verschiedenen Liganden sowie Rezeptorektodomänen der TGF-β Superfamilie basierend auf dem Verfahren der Oberflächen-Plasmonresonanz (Surface Plasmon Resonance, SPR). Dadurch konnte gezeigt werden, dass RGMb spezifisch und hochaffin mit Wachstumsfaktoren der BMP-Familie, nicht aber mit Vertretern anderer Untergruppen der TGF-β Superfamilie wechselwirkt. Im Widerspruch zu Literaturdaten konnten darüber hinaus keine direkten Interaktionen zwischen RGMb und den analysierten Typ I und Typ II Rezeptorektodomänen nachgewiesen werden. Zellbasierte Kompetitionsanalysen ergaben, dass der lösliche RGMb Corezeptor BMP-induzierte Signale dosisabhängig inhibiert, während die membranverankerte RGMb-Variante eine Verstärkung des BMP-Signals durch eine Erniedrigung der halbmaximalen Ligandenkonzentration hervorruft. Mittels Oberflächen-Plasmonresonanz konnte im Rahmen von Coinjektionsstudien außerdem beobachtet werden, dass RGMb die Bindung der untersuchten BMP-Liganden an die Typ I Rezeptorektodomänen hemmt. Daraus kann geschlossen werden, dass RGMb an das Typ I Rezeptorbindeepitop der BMP-Liganden bindet und dadurch deren Signalaktivität neutralisiert. Ein abweichendes Bild zeigt sich für die Beeinflussung der BMP/Typ II Rezeptorinteraktion durch RGMb. So wurde im Rahmen von SPR-basierten Coinjektionsstudien beobachtet, dass BMP-Liganden in Gegenwart des Corezeptors RGMb ausschließlich mit der Ektodomäne des Typ II Rezeptors ActR IIB, nicht aber mit den Rezeptoren ActR-II oder BMPR-II interagieren können. Diese Ergebnisse deuten darauf hin, dass zwar der Kernbereich des Typ II Rezeptorepitops durch die Interaktion des Liganden mit RGMb unbeeinflusst bleibt, jedoch eine partielle periphere Überlagerung bei gleichzeitiger Bindung von RGMb und den Typ II Rezeptoren für die Ausbildung der beobachteten Selektivität verantwortlich sein muss. Um diese Wechselwirkungen auch auf zellulärer Ebene analysieren zu können, wurden fluoreszenzbasierte Fusionskonstrukte für BMP-Rezeptoren sowie für RGMb synthetisiert und ein funktionelles Färbeprotokoll für die konfokale Mikroskopie etabliert. Die biochemischen Analysen sowie die in dieser Arbeit präsentierten umfassenden Charakterisierungen der Corezeptorinteraktionen mit einer Vielzahl an BMP-Liganden sowie deren Rezeptoren grenzen das RGMb-Bindeepitop ein und bilden so einen idealen Ausgangspunkt für die genaue Identifizierung und Charakterisierung dieses Epitops mittels gerichteter Mutagenese. Darüber hinaus weisen die vorliegenden in vitro Bindungsstudien auf einen deutlich komplexeren als bisher in der Literatur angenommenen, möglicherweise völlig neuartigen Modulationsmechanismus des BMP-Signalweges durch den Corezeptor RGMb hin. So wird in Anwesenheit von RGMb die Typ II Rezeptorspezifität und vermutlich auch die Lokalisierung der BMP-Liganden in bestimmten Membrankompartimenten - etwa Lipid Rafts - selektiv reguliert, wodurch BMP-induzierte Signale fein moduliert werden könnten. Die in dieser Arbeit synthetisierten fluoreszenzbasierten Fusionskonstrukte stellen zudem zusammen mit den etablierten Protokollen zur konfokalen Mikroskopie effektive Werkzeuge für eine zukünftige detaillierte Aufklärung (z. B. durch FRET-Studien) der komplexen RGMb-abhängigen Regulation des BMP-Signalweges auf zellulärer Ebene dar. / Bone Morphogenetic Proteins (BMPs) comprise the largest subgroup of the Transforming Growth Factor-β (TGF-β) superfamily of secreted growth factors. They are key regulators of early embryogenesis and orchestrate the development as well as the homeostasis of several tissues and organs in adult organisms. BMPs transduce their signals through two types of transmembrane serine/threonine kinase receptors termed type I and type II receptors. An imbalance in BMP-ligand and receptor numbers - about twenty different BMP-ligands face only five type I and three type II receptors - causes a pronounced promiscuity in ligand/receptor interactions. Therefore, a complex regulatory network is necessary to enable specific signaling of each BMP ligand despite a temperospatial expression overlap. Most of the discovered regulatory processes inhibit BMP-signal transduction. Recently, the first membrane-associated BMP-specific agonists - the Repulsive Guidance Molecule (RGM) family of coreceptors comprised of RGMa, RGMb and RGMc - have been discovered. Aside from stimulatory effects, evidence for a potential inhibitory activity of the RGM family member RGMb on BMP-induced signal transduction has previously been described. To clarify these contradictory RGMb functions, extensive biochemical and biophysical characterizations were carried out. In the course of this work, an efficient expression and purification strategy was established resulting in great yields of highly pure recombinant RGMb coreceptor protein. Subsequent in vitro binding assays based on surface plasmon resonance (SPR) showed that RGMb specifically interacts with BMP ligands, while no binding to other subfamily members could be detected. Contradicting published data, no direct interactions of the RGMb coreceptor with type I and type II receptor ectodomains were determined. Furthermore, cell-based competition assays revealed that soluble RGMb protein lacking the glycosylphosphatidylinositol anchor dose-dependently inhibits BMP-induced signal transduction. Characterizing the influence of the membrane anchored RGMb coreceptor on BMP signaling pointed to a more complex situation. Hence, only minor deviations of the BMP-dependent signal transduction in RGMb-transfected compared to control cells could be detected. These differences could possibly indicate a sensitizing activity of the RGMb coreceptor on BMP-signaling as it has been described before. SPR-based co-injection experiments revealed that BMP ligands bound to RGMb lose their ability to interact with type I receptor ectodomains. Thus, the coreceptor blocks the type I receptor binding epitope, thereby neutralizing the signaling activity of BMP ligands. However, comparative experiments using BMP variants pointed out that the coreceptor/ligand interface involves other binding hot spot residues than the type I receptor/ligand interaction. Analyzing the influence of RGMb on the BMP/type II receptor interaction based on SPR measurements led to a different outcome: In the presence of the RGMb coreceptor, BMP ligands were solely able to interact with the extracellular domain of the type II receptor ActR IIB, while no binding could be detected with neither ActR II nor BMPR II ectodomains. Hence, binding of the coreceptor does not directly interfere with the BMP ligand/type II receptor core interface, whereas a partial peripheral overlap of RGMb with the type II receptor epitope may evoke the observed selectivity for ActR IIB. In order to clarify the observed interactions in the cellular context, RGMb and BMP receptor fusion constructs based on the SNAP-/CLIP-tag® technology as well as efficient labeling protocols and confocal microscopic procedures were established. The biochemical characterization of RGMb along with the extensive interaction studies of this coreceptor with a multiplicity of BMP-ligands and their receptors presented in this work, narrow down the location of the RGMb binding epitope, thus providing an ideal starting point for the subsequent determination of the coreceptor binding domain by site directed mutagenesis. Moreover, the presented in vitro binding studies point to a more complex and possibly novel regulatory mechanism of the BMP signal transduction by RGMb than previously assumed. Thus, the presence of the coreceptor selectively regulates the type II receptor specificity and potentially also the recruitment of BMP-ligands into certain membrane compartments (e.g. lipid raft-domains), herewith fine-tuning BMP-induced signals. The SNAP- and CLIP tag® fusion constructs produced in this work together with the established protocols for confocal microscopy analyses provide efficient tools for subsequent extensive examinations of theses regulatory processes in living cells (e.g. by FRET measurements).

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is engaged in regulating versatile cellular processes that are pivotal for development and homeostasis of most tissues in multicellular organisms. TGF-ß signal transduction is initially propagated by binding of TGF-ß to transmembrane serine/threonine kinase receptors, designated TßRI and TßRII. Upon activation, the receptors phosphorylate Smad proteins which serve as downstream mediators that enter the nucleus and finally trigger transcriptional responses of specific genes. During the past years, it became evident that signaling cascades do not proceed in a linear fashion but rather represent a complex network of numerous pathways that mutually influence each other. Along these lines, members of the TGF-ß superfamily are attributed to synergize with neurotrophins. Together, they mediate neurotrophic effects in different populations of the nervous system, suggesting that an interdependence exists between TGF-ßs on the one hand and neurotrophins on the other. In the present work, the crosstalk of NGF and TGF-ß/Smad signaling pathways is characterized in rat pheochromocytoma cells (PC12) which are frequently used as a model system for neuronal differentiation. PC12 cells were found to be unresponsive to TGF-ß due to limiting levels of TßRII. However, stimulation with NGF results in initiation of Smad-mediated transcription independent of TGF-ß. Binding of NGF to functional TrkA receptors triggers activation of Smad3. This NGF-dependent Smad activation occurs by a mechanism which is different from being induced by TGF-ß receptors in that it provokes a different phosphorylation pattern of R-Smads. Together with an inferior role of TßRI, Smad3 is proposed to serve as a substrate for cellular kinases other than TßRI. Based on the presented involvement of components of both, the MAPK/Erk and the TAK1/MKK6 cascade, signal mediators of these pathways rank as candidates to mediate direct activation of Smad3. Smad3 is subsequently translocated to the nucleus and activates transcription in a Smad4-dependent manner. Negative regulation is provided by Smad7 which was found to act as a potent inhibitor of Smad signaling not only in TGF-ß- but also in NGF-mediated cascades. The potential of NGF to activate the Smad pathway independent of TGF-ß might be of special importance in regulating expression of genes that are essential for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those which are TGF-ß-resistant. / Das multifunktionelle Zytokin TGF-ß ist an der Regulation vielfältiger zellulärer Prozesse beteiligt. Diese sind für die Entwicklung und die Homöostase der meisten Gewebe vielzelliger Organismen essenziell. Die TGF-ß Signaltransduktionskaskade wird durch die Bindung des Zytokins an spezifische transmembrane Serin/Threonin-Kinase Rezeptoren (TßRI und TßRII) initiiert. Eine solche Rezeptoraktivierung führt zur Phosphorylierung von Smad Proteinen. Diese dienen der Signalweiterleitung, indem sie anschließend in den Zellkern translozieren und dort die Transkription spezifischer Zielgene modulieren. In den letzten Jahren wurde deutlich, dass Signalkaskaden nicht nur linear weitergeleitet werden, sondern dass vielmehr ein komplexes Netzwerk aus zahlreichen, sich gegenseitig regulierenden, Signalwegen existiert. In diesem Zusammenhang wird auch den Mitgliedern der TGF-ß Superfamilie zugeschrieben, dass sie mit Neurotrophinen kooperieren und somit deren Effekte in unterschiedlichen neuronalen Zellpopulationen unterstützen. In der vorliegenden Arbeit wurde der "crosstalk" von NGF- und TGF-ß/Smad-Signalwegen charakterisiert. Als Zellsystem dienten dazu Ratten Pheochromocytoma Zellen (PC12), die weithin als Modellsystem für neuronale Differenzierung verwendet werden. Basierend auf der Expression limitierender Mengen an TßRII, zeigen PC12 Zellen keine Responsivität gegenüber TGF-ß. Stimulation mit NGF hingegen resultiert - unabhängig von TGF-ß - in der Initiation von Smad-vermittelter Transkription. Die initiale Bindung von NGF an TrkA Rezeptoren führt zur Aktivierung von Smad3. Diese NGF-induzierte Smad-Aktivierung unterscheidet sich von der durch TGF-ß-Rezeptoren initiierten Aktivierung hinsichtlich des Phosphorylierungsmusters der R-Smads. Da weiterhin gezeigt werden konnte, dass die TGF-ß Rezeptoren für NGF-induzierte Ereignisse eine untergeordnete Rolle spielen, wird angenommen, dass Smad3 ein Substrat für andere zelluläre Kinasen als TßRI ist. Die hier nachgewiesene Beteiligung der MAPK/Erk Kaskade sowie des TAK1/MKK6 Signalwegs an der Weiterleitung des NGF-Signals machen deren Signalmoleküle zu potenziellen Kinasen für die direkte Aktivierung von Smad3. Im Anschluß daran erfolgt die nukleäre Translokation des Smad3 und spezifische Promotoraktivierungen unter Beteiligung von Smad4. Abschließend konnte gezeigt werden, dass das Smad7 Protein, nicht nur nach TGF-ß- sondern auch nach NGF-Stimulation als effektiver Inhibitor der Smad Signalkaskade wirkt. Die bislang unbekannte Fähigkeit, den Smad-Signaltransduktionsweg unabhängig von TGF-ß zu aktivieren, schreibt NGF eine besondere Bedeutung für die Genregulation in neuronalen Zellpopulationen oder anderen NGF-sensitiven - insbesondere TGF-ß-resistenten - Zellen zu.

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Doctor of Philosophy (PhD) / Background: The development of the blood supply in the primate retina has been extensively studied; however the relationship of the differentiating retina to the choroidal blood supply is less well known. The interaction of astrocytes and vascular endothelial cells promotes the development of the retinal vasculature from 14 weeks’ gestation (WG). Initially, astrocytes lead the developing capillaries from the optic nerve towards the macular area. However, neither astrocytes nor endothelial cells enter a prescribed avascular area, within which the fovea later forms. This may be attributed to expression of a factor that inhibits astrocyte and endothelial cell proliferation in the fovea. A factor found in the CNS that is already known to have these effects is transforming growth factor-β (TGF-β). Aims: This thesis investigated the relationship between retinal maturation and choroidal blood vessel supply and the possible role for TGF-β as an antiangiogenic factor in maintaining an avascular fovea during primate retinal development. Methods: Human eyes between 11 WG and 40 years were obtained with ethical approval from Prince of Wales Hospital and the NSW Lions Eye Bank and fixed and sectioned for histological procedures or prepared for polymerase chain reaction (PCR). Macaque eyes from foetal day (fd) 64 to postnatal 11years (p11y) were obtained from Bogor Agriculture University, Indonesia with the approval of the Ethics Committee of the University of Washington, Seattle, USA. Macaque eyes were also fixed and sectioned for immunohistochemistry and in situ hybridisation. RNA was extracted from human foetal retinas and used for RTPCR (Reverse Transcriptase PCR), QPCR (Quantitative PCR) and preparation of riboprobes. PCR products were analysed using both restriction digest and sequencing. RTPCR was used to identify TGF-β1, TGF-β2 and TGF-β3 in the developing human and in the developing and adult macaque retinas whilst QPCR was used to quantify the TGF-β isoforms in central compared to peripheral retina and in foetal compared to adult retina. In situ hybridisation was performed according to a standard protocol and visualised using Roche HNPP Fast Red detection set with designed riboprobes for TGF-β1, TGF-β2 and TGF-β3 (DIG RNA labelling kit). Some sections were counterstained with vimentin antibody. Immunohistochemistry was performed on human retina and choroid sections using antibodies to CD34 and Ki67 and on human and macaque retina using antibodies to synaptophysin, vimentin, GFAP, calbindin, S-opsin, RG-opsin, rhodopsin, TGF-β1, TGF-β2, TGF-β3 and their receptors TβRI and TβRII. Sections of the retina were imaged and analysed using either a Leica Confocal microscope and TCSNT software or Zeiss Confocal microscope and LSM 5 Pascal software. Data from the human retina and choroid sections corresponding to different regions (foveal, parafoveal nasal, parafoveal temporal, nasal and temporal) was collected to measure the number of Ki-67 immunolabelled mitotic endothelial cells and the area of CD34 immunolabelled choriocapillaris using Adobe Photoshop version 5.0.2, NIH software version 1.62 (measurement macros) and Excel. In the human and macaque sections the intensity of TGF-β protein and mRNA expression was captured from different regions of the retina (foveal, parafoveal nasal, parafoveal temporal, nasal, temporal, nasal to disc) to compile montages. Montages were then re-imported into LSM 5 Pascal software to measure the optical density across each montage along the ganglion cell layer, outer neuroblastic zone and photoreceptor layer collecting data in Excel for graphical representation. In addition to the montages, individual sections were assessed for co-localisation of TGF-β and TβR to various retinal cell types. Results: Analyses of choriocapillaris area and endothelial cell (EC) proliferation were able to demonstrate that the area of choriocapillaris endothelium is greater in the foveal region at all ages (14-18.5WG), that the rate of choriocapillaris EC proliferation declines dramatically over this same period and that the lowest rates of EC proliferation are at the incipient fovea. Most importantly these findings indicate that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons which are more developed with higher oxygen and nutrient demands, which is the mechanism widely thought to regulate development of the retinal vasculature. PCR showed all TGF-β isoforms to be present in the human developing and adult retina. QPCR revealed that TGF-β2 was the most predominant isoform, followed by TGF-β3 with very small amounts of TGF-β1 seen. The isoforms were more abundant in developing rather than adult retina and in central rather than peripheral retina. Studies of the distribution of TGF-β protein and mRNA using immunohistochemistry and in situ hybridisation confirmed the low levels of TGF-β1 protein and mRNA observed in QPCR and demonstrated distinct centroperipheral gradients in the photoreceptor layer for TGF-β2 and TGF-β3. Relative high amounts of TGF-β in the fovea could affect vascular patterning due to TβRI seen in astrocytes which lead the blood vessels at the foveal rim at the level of the ganglion cell plexus. TGF-β2 and TGF-β3 expression is detected before formation of the foveal avascular zone (FAZ) at fd64 (~15WG) - fd73 (~17WG) with levels peaking in the foveal region at fd105 (~25WG) by the time the FAZ forms. Conclusions: This thesis has shown that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons as reduced rates of EC proliferation in the ‘foveal’ chorioretinal location were observed at all ages studied between 14 and 18.5WG. The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina and are therefore different to those governing the formation of the retinal vasculature. All TGF-β isoforms are expressed in developing and adult human and macaque retina with TGF-β2 being the predominant isoform. TGF-β isoforms are more abundant in central compared to peripheral retina and in developing compared to adult retina. Centro-peripheral gradients of TGF-β2 and TGF-β3 across the photoreceptor layer and TβRI on astrocytes support the presence of TGF-β in the fovea as an antiproliferative and antiangiogenic factor by helping to define the FAZ early in development, well before 23-25 WG in humans and before fd100 in macaques.

The transforming growth factor (TGF)-β superfamily mediates a wide range of differentiation and developmental processes across many genera. GDF9 and BMP15 are expressed exclusively in the mammalian ovary and are the only TGF-β ligands that lack the conserved cysteine residue used for dimerisation. As a platform for studying the interactions between GDF9 and BMP15 and their receptors, BMPRII and BMPRIb, a variety of strategies were attempted to produce soluble and active proteins from recombinant systems. Both ligands and receptors showed a tendency to form insoluble aggregates when expressed in prokaryotic systems; however after extensive screening, quantities of biologically active GDF9 were produced using in vitro refolding. When expressed alone, either containing a histidine tag or as an untagged protein, the BMPRII ectodomain was deposited as insoluble inclusion bodies. This protein, subjected to in vitro refolding procedures, exhibited multiple species following anion exchange chromatography and size exclusion chromatography, as visualised on native PAGE. Separation of these species could be achieved using a MonoP matrix. One of these separated fractions, representing about 5% of the starting material, was amenable to crystallisation, and furthermore exhibited activity in a rat granulosa cell thymidine incorporation assay. Two different crystals forms of the extracellular domain of BMPRII were grown from the same protein batch under similar crystallisation conditions. Notably, the tetragonal form that grew more slowly possessed several disordered finger regions, while electron density for the entire molecule was clear in the orthorhombic form. The hydrophobic core of the ligand binding surface of BMPRII , as seen in both structures, resembles that of ActRII bound to BMP2. The A-loop of BMPRII, which is involved in ligand binding, lies in two different conformations in the two structures of BMPRII, mediated by a rearrangement in disulfide Cys94-Cys117. It is proposed here that the tetragonal form represents the ligand-bound receptor structure. Although the majority of the hydrophobic binding surface is shared with ActRII(b), it is likely that His87 and Tyr40 are unique residues that confer specificity in BMPRII ligand binding.

Elastic fibres, a major component of many connective tissues, are composed of an amorphous elastin core surrounded by fibrillin - containing microfibrils. The function of these microfibrils appears to require the co - ordinated interactions of fibrillins with a range of extracellular matrix ( ECM ) macromolecules including, latent transforming growth factor - β ( TGF - β ) binding proteins ( LTBPs ). LTBPs share a high degree of structural similarity to fibrillins, since they both contain unique 8 - cysteine motifs. Of the four members of the LTBP family, LTBPs - 1, - 3 and - 4 covalently bind to latent forms of TGF - β. LTBP - 1 has been shown to interact with the N - terminal domains of fibrillin - 1 and - 2 and LTBP - 4 interacts with the N - terminal domains of fibrillin - 1, suggesting that fibrillin - containing microfibrils may act as TGF - β stores and localise latent TGF - β complexes to the ECM. LTBP - 2 differs from other members of the LTBP family since it does not covalently bind latent TGF - β. However, LTBP - 2 strongly co - localises with fibrillin - containing microfibrils in a number of tissues suggesting that LTBP - 2 could have a structural role associated with these elements presumably independent of TGF - β storage, or could act to mediate specific microfibril - ECM interactions. To understand more about the function of LTBP - 2, this study involved screening for potentially important molecular interactions of LTBP - 2 with fibrillins and a variety of ECM proteins. Human recombinant LTBP - 2 ( r - LTBP - 2 ) was cloned, expressed and purified using a mammalian cell culture system. Solid phase binding assays were used to screen for interactions between r - LTBP - 2 and continguous fragments of fibrillin - 1 and - 2 as well as MAGPs, tropoelastin, collagens and proteoglycans. A cation dependant interaction was found between the C - terminal domains of LTBP - 2 and the N - terminal domains of fibrillin - 1, but not with the analogous region of fibrillin - 2. Thus, LTBP - 2 seems to have an exclusive role associated with fibrillin - 1 - containing microfibrils. Further studies found that the C - terminal region of LTBP - 2 competes with LTBP - 1 for binding to fibrillin - 1, suggesting that the binding site for LTBP - 2 on fibrillin - 1 is the same or in close proximity to that for LTBP - 1. Immunohistochemical analysis of LTBP - 1 and - 2 within developing human aorta indicated that both LTBPs co - localised with fibrillin - 1. However, the two LTBPs did have distinct distribution patterns in relation to each other, in that LTBP - 2 was found throughout the medial layer whereas LTBP - 1 was mainly located in patches of the outer medial layer. No regions of strong co - localisation of the two LTBPs were found. Thus, these findings suggest that LTBP - 2 could indirectly modulate the presence of TGF - β upon the fibrillin - containing microfibrils by competing for binding with the LTBP- 1 / TGF - β complex to these structures. Other binding studies showed a cation independent interaction between r - LTBP - 2 and an as yet unidentified component of a crude bovine collagen - IV extract. Since collagen - IV is a major component of basement membranes, an interaction between r - LTBP - 2 and a protein within this bovine collagen - IV preparation suggests LTBP - 2 may have a further function involving a basement membrane component. It will be interesting to determine if LTBP - 2 acts as a bridging molecule between basement membrane structures and fibrillin - containing microfibrils or if it has another function independent of these microfibrils. / Thesis (Ph.D.)--School of Medical Sciences, 2006.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 137-153).

In pigs and other mammals, embryonic losses often occur during implantation when the conceptus (embryo plus its extra-embryonic membranes) attaches to the maternal uterine epithelium. Mechanisms controlling this process are not completely understood. Integrins and growth factors are among many molecules likely involved in controlling implantation. Numerous integrins (ITG), including subunits ITGAV (alpha v), ITGB1 (beta 1), ITGB3 (beta 3), and ITGB5 (beta 5), and transforming growth factor betas (TGFBs), in both latent and active forms, are present at the porcine conceptus-maternal interface. TGFBs are released as latent precursors which cannot interact with TGFBRs prior to their activation. Latency associated peptide (LAP), part of the TGFB latent complex, contains an amino acid sequence Arg-Gly-Asp (RGD) that is found in other extracellular matrix molecules and may interact with and signal through integrins. We hypothesize that LAP will bind to and activate ITGAV-containing heterodimers at the conceptus-maternal interface and that these interactions are a functional component of implantation. We also hypothesize that TGFB acting via TGFBRs has critical roles during peri-implantation, and such roles may include promoting conceptus development, survival, and adhesion. Immunofluorescence was used to colocalize TGFB, LAP, and integrins in porcine peri-implantation uterus and conceptus; immunohistochemistry of phosphorylated SMAD2/3 provided evidence of TGFB activity. Affinity chromatography identified cell surface integrins on porcine trophectoderm that are capable of binding LAP. In vivo, intrauterine infusions of LAP with its native RGD site (LAP-RGD) resulted in inhibition of conceptus elongation; LAP-RGE infusions yielded normal-appearing filamentous conceptuses at d13 of pregnancy. At d24, allantois length and fetal weights were greater in gilts which received LAP-RGE infusions compared to controls which received vehicle only. Results provide evidence for 1) active and latent TGFB in porcine conceptus and uterus; 2) receptor-ligand interactions of integrins and LAP; 3) integrin aggregation and potential focal adhesion formation at the conceptus-maternal interface; and 4) TGFB- and/or integrin-associated mechanisms which regulate conceptus elongation and placental and fetal size. Collectively, results suggest that TGFB and integrins are extensively involved in communication at the porcine conceptus-maternal interface, particularly regulating conceptus development, adhesion, and placental and fetal development.