Bone Morphogenetic Protein (BMP) signalling is an evolutionary conserved pathway, which functions to regulate numerous developmental processes such as cell fate determination and cellular proliferation. In Drosophila melanogaster, the ortholog of the vertebrate BMP2/4 is Decapentaplegic (Dpp). The most extensively characterised role of Dpp signalling in Drosophila is embryonic Dorsal-Ventral patterning. In this developmental environment, the Dpp morphogen acts as a step gradient to specify different concentration thresholds for target gene activation. The resulting nested domains of target gene expression in the embryo cooperate to induce the formation and subsequent maintenance of a simple extra-embryonic tissue, the amnioserosa. The amnioserosa tissue acts as an ideal model tissue to study Dpp-regulated differentiation. This study aims to identify and validate new targets of Dpp signalling, which are required for determining cell fate and differentiation of the amnioserosa tissue during embryogenesis. Additionally, this study aims to identify new regulators of the core signalling pathway. The work presented here was performed using a two tiered approach to understand in more detail the processes that regulate BMP-responsive transcription and the downstream effects. Firstly, RNA-Sequencing was performed on embryos with ectopic Dpp signalling in the early embryo. BMP-responsive target genes were idenitifed as differentially expressed when compared to control embryos. Expression studies have validated novel Dpp target genes and the list of genes that are regulated by BMP signalling has now been expanded. It can be invoked that these genes are involved in specification and/or mainentance of the amnioserosa tissue. Furthermore, I have uncovered a putative multi-tiered mechanism that exists between the Dpp and EGF signalling pathways to thus ensure correct cell fate specification and fine tuning of the Dpp signal in the Drosophila embryo. To further investigate how BMP signalling mediates such transcriptional regulation, a genome-wide RNAi screen was designed and performed to identify novel regulators of BMP transcription. Analysis of the screen data has identified a putative link between BMP-regulated transcription and transcriptional effectors of the Hippo signalling pathway, Scalloped and Yorkie. The data presented here suggests a co-regulatory requirement of these transcription factors to mediate Smad-dependent transcription.

Diese Dissertation analysiert BMP2 und BMP2-Derivate als neue therapeutische Strategien für die Behandlung des Multiplen Myeloms (MM). Das MM ist eine maligne neoplastische Erkrankung des Knochenmarks mit Plasmazellvermehrung und erhöhten Leveln an Aktivin A im Blutserum, wobei eines der Hauptsymptome das Auftreten von schmerzvollen Osteolysen ist. In den letzten Jahren rückte Aktivin-A als interessantes Target zur Behandlung des Multiplen Myeloms in den Vordergrund. Die Reduzierung der Aktivin-A Level durch decoy-Rezeptoren führte zu einer signifikanten Verbesserung der Osteolysen und einem reduzierten Proliferationsverhalten der neoplastischen B-Zellen, sowohl im Tierexperiment als auch in Studien der klinischen Phase II. Die Aktivin-A-Antagonisierung ist somit ein neuer und vielversprechender Ansatz in der Therapie des Multiplen Myeloms. Das Bone Morphogenetic Protein 2 ist aufgrund seiner molekularen und biologischen Eigenschaften ein interessantes Target für die Therapie des Multiplen Myeloms. Es ist auf molekularer Ebene ein Aktivin-A-Antagonist, besitzt aber auch osteoinduktives Potential und apoptotische bzw. anti-proliferative Eigenschaften auf neoplastische B-Zellen. Da die in der Literatur bereits beschriebenen, durch Mitglieder der TGF-β-Familie induzierten Apoptosemechanismen, noch nicht genauer untersucht waren, wurde in dieser Arbeit die BMP2-induzierte Apoptose in 10 unterschiedlichen humanen MM-Zellen analysiert. Erstens konnte dabei nachgewiesen werden, dass 7 von 10 Zelllinien nicht BMP2-responsiv waren. Eine genauere Untersuchung ergab, dass neben der Expression spezifischer BMP-Rezeptoren auch die Expression von inhibitorischen Smad-Proteinen über die BMP2-Responsivität entscheidet. Zweitens zeigte die genauere Analyse der Apoptosemechanismen, dass entgegen der in der Literatur publizierten Ergebnisse, BMP2 keine apoptotische Wirkung auf die von uns untersuchten Zelllinien hat. Mehrere verschieden durchgeführte Experimente, u.a. die Verwendung von spezifischen Inhibitoren des programmierten Zelltodes, unterstützen dieses Ergebnis und klassifizieren BMP2 als einen rein anti-proliferativen Faktor. Der letzte Teil der Arbeit befasst sich mit der Analyse von potentiellen Aktivin-A-Antagonisten in Form verschiedener BMP2- und GDF5-Derivate und inwiefern sie sich zum Einsatz in der Therapie des Multiplen Myeloms eignen. Die unterschiedlichen Eigenschaften der einzelnen Mutanten wurden in verschiedenen Zellsystemen getestet. So konnte aufgezeigt werden, dass neben einer erhöhten biologischen Aktivität in Form eines gesteigerten osteoinduktiven und anti-proliferativen Potentials auf neoplastische B-Zellen (Superagonisten), sich die verschiedenen Derivate als Super-Antagonisten zu Aktivin A eignen und damit unterschiedlichen Ansprüchen der adjuvanten Therapie im Multiplen Myelom gerecht werden. / This dissertation analyses BMP2 and BMP2 derivatives as new therapeutic agents in multiple myeloma (MM). MM is a malignant neoplastic disease of bone marrow with plasmatic cell proliferation and increased Activin-A-level in blood serum. A particular symptom of this disease is the occurrence of painful osteolysis. In the last few years Activin A has become an important target for the treatment of multiple myeloma. Reducing Activin A levels by decoy receptors led to a significant improvement in osteolysis and reduced proliferation behavior of neoplastic B cells, both in animal experiments and clinical phase II trials. Activin A antagonization is thus a new and promising approach in the treatment of multiple myeloma. Bone Morphogenetic Protein 2 is a promising prospect for the treatment of multiple myeloma due to its molecular and biological properties. It is an Activin-A-antagonist at the molecular level, but also has osteoinductive potential and apoptotic or anti-proliferative properties on neoplastic B cells as already described in literature. Since the apoptotic mechanisms, which members of the TGF-β family induced in MM-cells, have not yet been investigated in detail, the BMP2-induced apoptosis was analyzed in 10 different human MM cells. Firstly it was shown that 7 out of 10 cell lines were not responsive to BMP2. A more detailed analysis revealed that, besides the expression of specific BMP receptors, the expression of inhibitory Smad proteins determines BMP responsiveness. Secondly the more precise analysis of the apoptotic mechanisms revealed that, contrary to the results published in the literature, BMP2 has no apoptotic effect on the cell lines we have examined. Several different experiments, e.g. the use of specific inhibitors of programmed cell death, support this result and classify BMP2 as a purely anti-proliferative factor. The last part of this research deals with the analysis of potential Activin-A-antagonists in form of different BMP2 and GDF5 derivatives and how they are suitable for use in the therapy of multiple myeloma. The different properties of the individual mutants were tested in diverse cell systems. The results demonstrate that in addition to increased biological activity in form of increased osteoinductive and anti-proliferative potential on neoplastic B cells (superagonist), the various derivatives are suitable as super-antagonist to Activin A and cope with different requirements of adjuvant therapy in context of multiple myeloma.

In vertebrate embryonic development, correct specification of tissue fates along the dorsoventral (D/V) axis is known to require the secreted signaling ligand Wnt8. Wnt8 signaling promotes ventral fates and antagonizes the expansion of the dorsal domain known as the organizer. Maintenance of the organizer is critical for proper development as this tissue is known to produce inhibitors of Wnt and BMP (Bone Morphogenetic Protein) family ligands; BMPs are also known to play a major role in promoting ventral fates. In order to understand how Wnt8 antagonizes the organizer, we analyzed the epistatic relationship between Wnt8 and the transcriptional repressors Vent and Vox using zebrafish as a model organism. We found that Wnt8/β-catenin signaling directly regulates the transcriptional levels of vent and vox so that they can repress the transcription of dorsal genes on the ventral side of the embryo. To understand the contribution of Wnt8 towards ventral fate specification, we carefully analyzed its relationship with BMP signaling during gastrula stages. We found that bmp expression in the mesoderm is under the control of Wnt8 at mid-gastrulation and that regulation of bmp explains many of the ventral defects observed in wnt8 mutants. Antagonism of the expression of organizer-derived BMP inhibitors by Wnt8 also indirectly allows timely BMP signaling. Analysis of wnt8; bmp double mutants revealed an early unsuspected function of BMP in the antagonism of the organizer. Further, we uncovered a mechanism through which regulation of vent, vox and a related-gene ved expression by both Wnt8 and BMP antagonizes dorsal/axial mesoderm identity to preserve the integrity of ventral/non-axial tissues. In summary, we have revealed some of the mechanisms of Wnt8 function in D/V mesoderm patterning: it restricts the organizer domain by regulating vent and vox, it allows BMP induced differentiation through its inhibition of BMP antagonists derived from the organizer and it co-regulates vent, vox, and ved with BMP signaling to allow maintenance of the non-axial domain.

The performance of an urban stormwater bioretention management practice in the Kingsbrooke Subdivision of Prince William County, Virginia was examined over a one-year period. Bioretention is a relatively new urban stormwater best management practice (BMP) intended to mimic the pollutant-removal characteristics of an upland forest habitat. Typical bioretention areas utilize shallow ponding and highly-infiltrative sandy soils to treat the stormwater runoff from small commercial or residential drainage sites. The Kingsbrooke bioretention area was found to be atypical in several ways, including its relatively large, 14 acre, drainage area and the high clay content of its topsoil. Hydrologic and chemical data were collected by Virginia Tech staff for a total of 8 months in 2003 and 2004. Analysis of pollutant loading data was complicated by the presence of three unmeasured water flows: overland inflow bypassing the inflow gage, and groundwater flows both entering and exiting the bioretention soils. The BMP did reduce peak runoff rates for some storms, but did not significantly reduce total storm volumes because of the combined effects of the large drainage area to BMP area ratio and the poor infiltration capacity of the soil. Pollutant load calculations determined that the site removed about 28% of total suspended solids, 32% of total phosphorus, and about 15% of total nitrogen. Removals of approximately 16% and 7% were observed for lead and zinc, respectively. Although the Kingsbrooke bioretention area did improve water quality, the pollutant removal efficiencies were lower than those reported in the literature from more conventional bioretention areas. / Master of Science

Ashby Pond in the City of Fairfax, Virginia was retrofitted to treat runoff from 54.7 hectares of urban land of mixed use. The pond discharges into Accotink Creek, a highly urbanized tributary of the Potomac River and Chesapeake Bay that is listed on the State of Virginia 303(d) list for multiple impairments. The entire multi-state Chesapeake Bay Watershed is subject to Total Maximum Daily Load (TMDL) restrictions on sediment, phosphorus and nitrogen. Virginia and local municipalities assign pollutant reduction credits to retention ponds that meet certain design requirements. However, to actually meet existing and future water quality goals set by TMDLs, it must be proven that such ponds truly provide the water quality benefits for which they have been credited. The inflow and outflow water quality of Ashby Pond was examined over 7 months from fall 2012 to spring 2013. During that period, the pond provided statistically significant reductions of phosphorus, nitrogen and suspended sediment, but not organic carbon or oxygen demand. Ashby Pond had non-significant export of sodium, chloride and calcium. The pond underperformed when compared to state reduction credits for phosphorus load and concentration, but met and exceeded the credits for nitrogen load and concentration, respectively. The pond was under-sized compared to state design standards, and some underperformance should be expected. / Master of Science

Η Οστεοαρθρίτιδα ή αλλιώς η εκφυλιστική νόσος των αρθρώσεων δεν είναι μια μόνο νόσος αλλά μάλλον το τελικό αποτέλεσμα διαφόρων παθήσεων των αρθρώσεων. Σε μεγαλύτερη ή μικρότερη έκταση χαρακτηρίζεται πάντοτε από εκφύλιση του αρθρικού χόνδρου και ταυτόχρονη ανάπτυξη νέου οστού , χόνδρου και συνδετικού ιστού . Η εν λόγω ανάπτυξη έχει ως αποτέλεσμα την αναδιαμόρφωση του περιγράμματος της άρθρωσης . Οι φλεγμονώδεις αλλοιώσεις στην αρθρική μεμβράνη είναι συνήθως μικρές και δευτεροπαθείς . Πρόκειται για μια νόσο η οποία εμφανίζεται στο 80% των ατόμων ηλικίας πάνω από 65 έτη , ως ακτινολογικό εύρημα , ενώ στο 25% αυτών θα παρουσιάζει συμπτώματα . Διάφοροι παράγοντες επηρεάζουν την πορεία της νόσου . Εκτιμάται ότι σε λίγα χρόνια η επίπτωση της νόσου θα είναι τόσο μεγάλη έτσι ώστε δε θα επαρκούν οι ορθοπαιδικοί να χειρουργούν τους ασθενείς !!! Λόγω αυτών επιχειρείται η κατανόηση των μοριακών παθογενετικών μηχανισμών οι οποίοι λαμβάνουν χώρα στην οστεοαρθρίτιδα (ΟΑ) . Ο χόνδρος της άρθρωσης είναι αυτός που εκφυλίζεται με αποτέλεσμα την παραμόρφωση της άρθρωσης . Για την διατήρηση της ακεραιότητας του αρθρικού χόνδρου υπεύθυνη είναι μια λεπτή ισορροπία μεταξύ αναβολικών και καταβολικών διεργασιών που αφορούν τόσο στα χονδροκύτταρα όσο στην εξωκυττάρια θεμέλια ουσία . Η εκφύλιση του εκτελείται από αυξημένη δραστηριότητα των μεταλλοπρωτεασών του στρώματος (Αγκρεκανάσες) καθώς και των υπολοίπων πρωτεολυτικών ενζύμων τα οποία επάγονται από κυτοκίνες ( Il-1 , TNF-a) ή/και προϊόντα αποικοδόμησης της θεμέλιας ουσίας .Αν η εκφύλιση και η καταστροφή του χόνδρου είναι μεγαλύτερη από την παραγωγή νέας θεμέλιας ουσίας από τα χονδροκύτταρα τότε οδηγούμαστε στην ΟΑ . Νέα δεδομένα και απόψεις έρχονται συνεχώς στην επιφάνεια τα οποία υποστηρίζουν την συμμετοχή τόσο του αρθρικού υμένα όσο και του υποχόνδριου οστού στην ανάπτυξη της οστεοαρθρίτιδας . Ο κύριος στόχος αυτής της βιβλιογραφικής εργασίας είναι η λεπτομερής περιγραφή όλων των μορίων και των μονοπατιών που οδηγούν στην ενεργοποίηση των αγγρεκανασών , συνεπώς στην ανάπτυξη της οστεοαρθρίτιδας , την παράθεση των στοιχείων που εμπλέκουν , εκτός του αρθρικού χόνδρου , τον αρθρικό υμένα και το υποχόνδριο οστό στην παθογένεια της , καθώς επίσης τα διάφορα cross talks τα οποία υπάρχουν ανάμεσα στην αναβολική (σύνθεση) και καταβολική (καταστροφή) πορεία του χόνδρου , παρέχοντας έτσι μια καινούρια βάση για μελλοντική ερεύνα και ανακάλυψη νέων φαρμάκων τόσο προληπτικών όσο και θεραπευτικών της οστεοαρθρίτιδας . / -

Precise spatial and temporal control of cellular adhesion and signal transduction events are necessary for accurate animal development. Given the necessity for cell communication in carrying out processes like cell fate specification, growth, cell migration and differentiation, it is not surprising that signaling transduction pathways, such as EGFR, BMP, Notch, Wingless and Hippo, are intimately involved. All these pathways encompass a cascade of molecular events over which there is exquisite spatial and temporal control. A wide array of mechanisms, involving a diverse set of molecules, acts to provide this regulatory control. One such molecule implicated in the BMP signaling pathway in Drosophila development is Kek5, a Leucine rich repeat and Immunoglobulin domain (LIG) family member. Here I show that Kek5 modulates both BMP signaling and adherens junctions. For these functions, I further demonstrate that structural elements in both extracellular and intracellular region of Kek5 are critical, providing new insight into the LIG family and their roles in signaling pathways.

Bone morphogenetic protein-2 (BMP-2) plays a critical role in bone formation. The aim of this study was to compare the activity of the mammalian cell BMP-2 to the E-coli cell BMP-2. In vitro, the potency of mammalian and E-coli BMP-2 was compared by adding BMP-2 to C2C12 cells and measuring the level of alkaline phosphatase activity. In vivo, the activity was evaluated by placing the bioimplants in the thigh muscle of mice, and measuring the amount of bone induced. The in vitro assay clearly showed that mammalian BMP was significantly more potent than E-coli BMP. In vivo, on the calcium phosphate carrier, mammalian BMP produced more bone than E-coli BMP, but E-coli BMP produced higher density tissue than mammalian BMP. On both mammalian and E-coli BMP, the calcium phosphate carrier had a significant effect on the density but not the quantity of bone produced versus the absorbable collagen sponge carrier.

Bone morphogenetic protein-2 (BMP-2) plays a critical role in bone formation. The aim of this study was to compare the activity of the mammalian cell BMP-2 to the E-coli cell BMP-2. In vitro, the potency of mammalian and E-coli BMP-2 was compared by adding BMP-2 to C2C12 cells and measuring the level of alkaline phosphatase activity. In vivo, the activity was evaluated by placing the bioimplants in the thigh muscle of mice, and measuring the amount of bone induced. The in vitro assay clearly showed that mammalian BMP was significantly more potent than E-coli BMP. In vivo, on the calcium phosphate carrier, mammalian BMP produced more bone than E-coli BMP, but E-coli BMP produced higher density tissue than mammalian BMP. On both mammalian and E-coli BMP, the calcium phosphate carrier had a significant effect on the density but not the quantity of bone produced versus the absorbable collagen sponge carrier.

No description available.