Matrix Extracellular Phosphoglycoprotein (MEPE) is a member of a family of proteins called small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) which play key roles in biomineralisation. Altered MEPE expression is associated with several phosphate and bone-mineral metabolic disorders such as oncogenic osteomalacia and hypophosphatemic rickets. Despite this, it remains undetermined what impact MEPE has on the growth plate; the cartilage anlagen from which endochondral ossification, the process responsible for linear bone growth, occurs. The work of this thesis has characterised the ATDC5 cell line and the metatarsal organ culture as useful in vitro models of endochondral ossification. These will prove vital in the pursuit of underpinning the molecular mechanisms involved in endochondral bone growth. These models form the basis of the further studies in this thesis examining the role of MEPE within this highly orchestrated process. Before such role can be defined, this thesis details the spatial and temporal localisation patterns of MEPE in 10-day- and 4-week-old murine growth plates. More specifically, MEPE protein and mRNA were preferentially expressed by the hypertrophic chondrocytes as shown by immunohistochemistry and in situ hybridisation respectively. Microdissection of the murine growth plate confirmed this. Localisation of the cleavage product of MEPE, a 2.2kDa acidic serine- and aspirate-rich motif (ASARM) peptide, followed a similar pattern of expression. The localisation of MEPE to sites of mineralisation serves to strengthen its potential role in chondrocyte matrix mineralisation. This thesis identified this role in both mineralising ATDC5 cells and the metatarsal organ culture. The ASARM peptide was found to be the functional component of MEPE and this function was dependent upon its post-translational phosphorylation. Phosphorylated (p)ASARM peptides significantly inhibited chondrocyte matrix mineralisation without altering the proliferation or differentiation of the chondrocyte cells, or their ability to produce an extracellular matrix. mRNA analysis by qPCR indicted a feedback system by which the pASARM peptide functions to allow the release of further ASARM peptides. Moreover, the pASARM peptide inhibited mRNA expression of markers of vascular angiogenesis highlighting a novel mechanism by which they may inhibit chondrocyte matrix mineralisation. This thesis also determines the regulatory cross-talk between the chondrocytes of the murine growth plate, with the most abundant bone cell type, the osteocyte. This cross-talk inhibits chondrocyte matrix mineralisation and is attributed to sclerostin, an osteocyte-specific secretory protein. Furthermore, it is shown that sclerostin acts through the MEPE-ASARM axis to regulate chondrocyte matrix mineralisation and thus endochondral ossification. The work described herein has characterised and validated in vitro models of growth plate chondrocyte matrix mineralisation and has used these to identify the role of MEPE within chondrocyte matrix mineralisation.

Seidenfibroin findet hauptsächlich als Zellträgermatrix im Bereich Tissue engineering Anwendung. In Kombination mit verschiedenen Calciumphosphatphasen kann es als Material zur Knochenregeneration verwendet werden. In dieser Arbeit stelle ich mineralisierte Seidenfibroin-Scaffolds mit kontrollierter Makroporosität vor. Im Vergleich zu anderen Studien lag das Ziel auf der simultanen Gelierung und Mineralisation von Seidenfibroin-Scaffolds durch Einlegen von gefrorenen Seidenfibroin Monolithen in angesäuerte Calciumphophosphat Lösung, was zu einer Präzipitation von Monocalciumphosphat in der Seidenfibroinmatrix führt. Im zweiten Teil wurde eine Umsetzung von eingearbeiteten ß-Tricalciumphosphat-Partikeln erreicht. Des weiteren führte ein kontrollierter Cryostrukturierungsprozess von Seidenfibroin-Scaffolds zu parallel ausgerichteten Poren mit Durchmesser zwischen 30 und 50 µm. / Silk fibroin is commonly used as scaffold material for tissue engineering applications. In combination with mineralization with different calcium phosphate phases, it can also be applied as material for bone regeneration. Here, we present a study which was performed to produce mineralized silk fibroin scaffolds with controlled macroporosity. In contrast to former studies, our Approach focused on a simultaneous gelation and mineralization of silk fibroin by Immersion of frozen silk fibroin Monoliths in acidic calcium Phosphate Solutions. This was achieved by thawing frozen silk fibroin Monoliths in acidic calcium Phosphate solution, leading to the precipitation of monocalcium phosphate within the silk fibroin matrix. In the second approach, a conversion of incorporated ß-tricalcium Phosphate particles into brushite was successfully achieved. Furthermore, a controlled cryostructuring process of silk fibroin scaffolds was carried out of leading to the Formation of parallel oriented pores with Diameters of 30-50 µm.

Riparian buffer zones, adjacent to waterways, may protect water quality by intercepting and removing nitrogen in runoff from agricultural land. This research comprised four parts: (1) a field study of nitrogen buffering by differently vegetated riparian zones in a United Kingdom (UK) sheep-grazed pastoral catchment; (2) a field study of surface and subsurface runoff hydrology, and nitrogen flux, in a UK riparian wetland; (3) a laboratory study ((^15)N tracer-isotope dilution) of microbial inorganic nitrogen production and removal processes in the UK riparian wetland soil; and (4) a laboratory microcosm study ((^15)tracer) of nitrate removal processes in bare and plant-inhabited (Glyceria declinata) New Zealand (NZ) riparian wetland soil. Dissolved organic nitrogen and ammonium were generally more important components of subsurface runoff than nitrate in the three UK riparian zones. All riparian zones were poor buffers having minimal effect on the nitrogen concentration of subsurface runoff. In the UK riparian wetland site subsurface (saturated zone) and surface 'preferential flow paths' typically conveyed large quantities of catchment runoff rapidly into, and across the site, and hindered nitrogen buffering. However, under low flow conditions, runoff-riparian soil contact increased and the wetland decreased the catchment nitrogen flux by 27%. In the UK riparian wetland soil most nitrate removal was attributable to denitrification (87- 100%) as opposed to dissimilatory nitrate reduction to ammonium (DNRA) (0-13%) and immobilisation (0-10%). Total ((^14)N+(^15)N) transformation rates for these processes were 1.3-47, 0.5-1.5 and 0.6-2.5 μg N g soil(^-1) hr(^-1) respectively. In the NZ riparian wetland soil Glyceria declinata assimilated 11-15% of nitrate but, more importantly, increased soil oxidation and altered the proportions of nitrate removal attributable to denitrification (from 29% to 61-63%) and DNRA (from 49 to <1%), but not immobilisation (22-26%). Denitrification and, thus, nitrogen buffering might be enhanced, in some riparian zones by increasing the extent of moderately anoxic soil with plants that release oxygen from their roots or with water table management.

Regensburg, Univ., Diss., 2008

Würzburg, Univ., Diss., 2004. / Dateien in unterschiedlichen Formaten

Osteozyten stehen vermehrt im Fokus als wesentliche Regulatoren der Knochenmineralisierung. Das ähnlich einem neuronalen Netzwerk aufgebaute lakunokanalikuläre Netzwerk der Osteozyten breitet sich im Knochen in drei Ebenen aus. Es wurde in dieser Arbeit ein 3D-Kollagengel-Modell verwendet und dort die Osteoblasten- bzw. Osteozytenzelllinien MLO-A5 und MLO-Y4, sowie humane mesenchymale Stammzellen aus Hüftköpfen eingebettet. Es wurden die optimalen Kulturbedingungen entwickelt und die Zellen über mehrere Wochen kultiviert, beobachtet und mit dem herkömmlichen 2D-Kulturmodell verglichen. MLO-A5 und MLO-Y4 bilden die zelltypischen Zellfortsätze. Die Gele kontrahieren, wenn hMSC und MLO-A5 eingebettet sind, mit MLO-Y4 zeigt sich über den gesamten Kultivierungszeitraum keinerlei Kontraktion der Kollagengele. Die Zellen wurden zudem osteogen differenziert und mit FGF23 und Klotho stimuliert. Es ergaben sich erste Hinweise auf eine FGF23 / Klotho-abhängige Inhibierung der lokalen Mineralisierung in osteogen differenzierten MLO-A5. Es konnten einige osteogene Marker durch PCR und in den histologischen Schnitten mittels Antikörperfärbungen nachgewiesen werden, eindeutige Expressionsmuster und deren zeitliche Verläufe im Vergleich der osteogenen Differenzierungen und Zugabe von FGF23 und Klotho sind allerdings noch nicht identifizierbar und bedürfen womöglich höherer Fallzahlen und weiterer Untersuchungsmethoden. Insgesamt gesehen erweist sich das System aber als einfach und mit niederschwellig erreichbaren Methoden und Materialien durchzuführen. / Osteocytes increasingly establish as the key regulators of bone mineralisation. The network of osteocytes - similar to the neuronal network - is spreaded in three dimensions in bone. In this work the osteoblast/osteocyte cell lines MLO-A5 and MLO-Y4 were embedded in a 3D collagen gel model and compared to human MSCs. Optimal culture conditions were developed, cells cultivated and observed over several weeks and compared to 2D cell culture models. MLO-A5 and MLO-Y4 build the typical cell dendrites. Gels contract with MSCs and MLO-A5 whereas MLO-Y4 do not contract the gels. Cells were differentiated in osteogenetic pathway and stimulated with FGF23 and Klotho. First results suggest that FGF23 and Klotho can inhibit local mineralisation in MLO-A5 but more reproductions need to be made. Osteogenetic markers were identified by mRNA-PCR and antibody stainings of paraffin slices. Clear demarcation throughout osteogenetic differentiation and stimulation with FGF23 and Klotho could not be identified. Reproductions of the model prove to be easy and with low-threshold resources.

Proper management of soil organic matter (SOM) contributes to increasing plant productivity and reducing dependency on mineral fertilizers. Organic matter is widely regarded as a vital component of a healthy soil as it plays an important role in soil physical, chemical and biological fertility. Plant residues are the primary source of SOM. Therefore, proper SOM management requires a better understanding of plant residue decomposition kinetics in order to synchronize nutrient release during decomposition and plant uptake and prevent nutrient losses. In natural and managed ecosystems, residues are added frequently to soil, in the form of dead roots and litter fall of plant species with different C/N ratios. However, in most studies on residue decomposition, residues with different C/N ratios are added once and the effect of the presence of plants on residue decomposition is rarely investigated. In this project, four experiments were carried out with different objectives in order to close these knowledge gaps. The aim of the first experiment was to investigate the effect of frequent wheat residue addition on C mineralization and N dynamics. The experiment consisted of five treatments with different frequency of residue addition (2% w/w of wheat residues in total): once (100%W), every 16 days (25%), every 8 days (12.5%) or every 4 days (6.25%) and noresidue addition (control) with four replicates. The results showed that increasing frequency of low-N wheat residue addition increased C mineralization. Compared to 100%W, cumulative respiration per g residue at the end of the incubation (day 80) was increased by 57, 82 and 92% at 25%W, 12.5%W and 6.25%W, respectively. Despite large increases in cumulative respiration, frequent residue addition did not affect inorganic N or available N concentrations, microbial biomass C and N or soil pH. It is concluded that experiments with single residue additions may underestimate residue decomposition rates in the field because with several additions, soil microbes respire more of the added C (and possibly native soil C) per unit biomass but that this does not change their N requirements or the microbial community composition. In the second experiment, the effect of mixing of high and low C/N residues at different times during incubation was investigated. There were 4 addition times; 25% of a total of 2% (w/w) residue was added either as wheat (high C/N) or lupin (low C/N) residue. Wheat residue was added to lupin residues on days 16 (LW-16), 32 (LW-32) or 48 (LW-48). Additional treatments were 100%L (added 25% of lupin residues on days 0, 16, 32 and 48) and 100%W (added 25% of wheat residues on days 0, 16, 32 and 48) and 0% (the control) with four replicates. Adding high C/N residues into decomposing low C/N ratio residue strongly decreased the respiration rate compared to the addition of low C/N residues, and lowered the availability of inorganic N, but significantly increased soil pH and altered microbial community composition. By the end of the incubation on day 64, the cumulative respiration of LW-16, LW-32 and LW-48 was similar and approximately 30% lower than in the treatment with only lupin residue addition. The third experiment studied the effect of spatial separation of high and low C/N residues on decomposition and N mineralization. Each microcosm consisted of two PVC caps of 70 mm diameter and 20 mm height with the open end facing each other separated by a 30μm mesh. The caps were filled with soil mixed with either low or high C/N residue with three replicates. Contact of high and low C/N residues led to an increase in the decomposition rate of the high C/N residues at the interface whereas it decreased it in the low C/N residues. The results showed that N and soluble C compounds moved from the easily decomposable residues into the surrounding soil, thereby enhancing microbial activity, increasing inorganic N and significantly changing soil pH in the layer 0-5 mm from the interface compared to the 5-10 mm layer of the high C/N residues, whereas the movement of soluble C and N to high C/N residues decreased the decomposition of the low C/N residues. The final experiment investigated the effect of living plants on decomposition of high and low C/N residues. Wheat was grown in pots with a 30 μm mesh at the bottom. After a root mat had formed (>50% root coverage), a PVC cap with soil with high and low C/N residues (2% w/w) was placed against the mesh. The presence of plant roots significantly increased the respiration rate, N immobilization and increased the soil pH in the 0-5 mm layer in the first 4 days compared to the 5-10 mm layer. This enhanced microbial activity (and probably microbial biomass) can be explained by root exudates. The microbial community composition of plant treatments differed significantly from treatments without plants and the effect was greater in the immediate vicinity of the roots. / Thesis (M.Ag.Sc.) -- University of Adelaide, School of Earth and Environmental Sciences, 2009

The cement industry contributes around 5-7% of man-made CO2 emissions globally because of the Portland Cement (PC) production. Therefore, innovative reactive magnesia cement, with significant sustainable and technical advantages, has been proposed by blending reactive MgO and hydraulic binders in various proportions. MgO is currently produced from the calcination of magnesite (MgCO3), emitting more CO2 than the production of PC, or from seawater/brine which is also extremely energy intensive. Hence this research aims to investigate an innovative method to produce MgO from reject brine, a waste Mg source, through carbon sequestration, by its reaction with CO2, to provide a comparable low carbon manufacturing process due to the recycling of CO2. The produced deposits are then calcined to oxides with potential usage in construction industry. The entire system is a closed loop to achieve both environmental optimisation and good productivity. This research focuses on the chemical manufacturing process, integrated with material science knowledge and advancements, instead of concentrating purely on chemistry evaluations. Six series of studies were conducted, utilising MgCl2, CaCl2, MgCl2-CaCl2, MgCl2-CaCl2-NaCl, and MgCl2-CaCl2-NaCl-KCl to react with CO2 under alkaline conditions. The precipitates include hydrated magnesium carbonates, calcium carbonates and magnesian calcite. Generated carbonates were then calcined in a furnace to obtain MgO, CaO or dolime (CaO•MgO). All six series of carbonation processes were carried out under a controlled pH level, to study the constant pH’s effect on the process and resulting precipitates. Other controllable factors include pH, temperature, initial concentration, stirring speed, and CO2 flux rate. In conclusion, the optimum parameters for the production of the carbonated precipitates are: 0.25MgCl2 + 0.05CaCl2 + 2.35NaCl + 0.05KCl, 700rpm stirring speed, 25oC room temperature, pH=10.5, and 500cm3/min CO2 infusion rate. Reaction time is within a day. These parameters are chosen based on the sequestration level, particle performance morphology and the operational convenience. The optimum calcination parameters are at 800oC heating temperature with a 4h retention time.

Globally agricultural production is facing serious challenges to provide adequate food supply to meet a growing population. However, the reduced capacity of soil to support and sustain agricultural production as a result of soil fertility decline is impacting negatively on agricultural growth. Increase in the price of inorganic fertilisers and limited availability of nutrients from organic amendments has reduced progress in improving soil fertility. This research therefore aims at contributing knowledge towards evaluating the maximisation/optimisation of nutrients in compost and secondary treated sewage effluent (STSE) amended soils to meet the nutritional requirements of crops for sustainable crop production and environmental protection. STSE was irrigated on soils (sandy loam and clay loam) amended with greenwaste compost in soil incubation, glasshouse/pot and lysimeter studies. Perennial ryegrass (Lolium perenne) was grown in the pots and lysimeter studies. The incubation experiment showed that for a clay loam, N mineralisation in treatments with STSE alone and combinations of compost and STSE was higher than the applied N. Increasing compost quantity in compost and STSE nutrient integration resulted in reduced net N mineralisation in the clay loam soil. In the sandy loam, increasing compost contribution in compost and STSE nutrient integration resulted in an increase in net N mineralisation. Cation exchange capacity, microbial diversity, quality of available carbon and drying and rewetting cycles influenced the net nitrogen mineralisation dynamics in both soil types. Increasing the contribution of STSE while reducing compost quantity resulted in increased nitrogen use efficiency and ryegrass dry matter yield. The environmental threat to ground and surface water pollution through NO3 --N leaching may be enhanced by the inclusion of STSE in integrated compost and STSE nutrient supply to plants. Similarly, the threat to eutrophication due to phosphorous leaching is likely to be higher with integration of compost and STSE. Ryegrass dry matter yield reduced with increasing compost contribution while the concentration of N in ryegrass herbage for the combinations of compost and STSE was above the minimum requirement for N in herbage for productive grazing and dairy cattle in the pot experiment. Using compost and STSE of similar characteristics, the ideal approach to maximise nutrient potential from compost through irrigation with STSE is when 25% compost is integrated with 75% STSE with respect to nitrogen supply.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.