Connective tissue growth factor (CTGF/CCN2) and bone morphogenetic protein (BMP)-2 are both produced and secreted by osteoblasts. Both proteins have been shown to have independent effects in regulating osteoblast proliferation, maturation and mineralization. However, how these two proteins interact during osteoblast differentiation remains unknown. In Chapters 2 and 3, we utilized two cell culture model systems, osteoblasts derived from CTGF knockout (KO) mice and osteoblasts infected with an adenovirus, which over-expresses CTGF (Ad-CTGF), to investigate the effects of CTGF and BMP-2 on osteoblast development and function in vitro. To observe differences in osteoblast maturation and mineralization, we performed alkaline phosphatase (ALP) staining and activity and alizarin red staining, respectively. Contrary to a previously published report, osteoblast maturation and mineralization were similar in osteogenic cultures derived from KO and wild type (WT) calvaria in the absence of BMP-2 stimulation. Interestingly, in KO and WT osteoblast cultures stimulated with BMP-2, the KO osteoblast cultures exhibited increased alkaline phosphatase staining and activity and had larger, fused nodules stained with alizarin red than WT osteoblast cultures. This increase in osteoblast differentiation was accompanied by increased protein levels of phosphorylated Smad 1/5/8 and mRNA expression levels of bone morphogenetic protein receptor Ib. These data confirm enhanced osteoblast maturation and mineralization in BMP-2 induced KO osteoblast cultures. We also examined osteoblast differentiation in cultures that were infected with Ad-CTGF and in control cultures. Continuous over-expression of CTGF resulted in decreased ALP staining and activity, alizarin red staining, and mRNA expression of osteoblast markers in both unstimulated and BMP-2 stimulated cultures. Impaired osteoblast differentiation in cultures over-expressing CTGF was accompanied by decreased protein levels of phosphorylated Smad 1/5/8. In addition to the functional assays that we performed on WT and KO osteoblast cultures, we performed ChIP assays to investigate differences in binding occupancy of transcription factors on the Runx2 and Osteocalcin promoters in BMP-2 induced WT and KO osteoblast cultures. We demonstrate that in BMP-2 induced WT and KO osteoblast cultures, there was greater Smad 1 and JunB occupancy on the Runx2 promoter and Runx2 occupancy on the Osteocalcin promoter in BMP-2 induced KO osteoblast cultures compared to WT cultures. Collectively, the data demonstrate that CTGF acts to negatively regulate BMP-2 induced signaling and osteoblast differentiation. In Chapter 4, we synthesized an active His-tagged BMP-2 recombinant protein to track surface binding of BMP-2 in CTGF WT and KO osteoblasts. We amplified mature BMP-2 in genomic DNA, which was inserted correctly into a pET-28b(+) vector. We ran a SDS-PAGE gel and stained with Coomassie blue to show that we successfully induced BMP-2 in bacteria cells, extracted the protein using urea, and purified and eluted the protein using Nickel charged agarose beads and imidazole elution buffer. Furthermore, by Western blot analysis using anti-His antibody, we confirmed the presence of the His-tag on the BMP-2 protein. Lastly, ALP staining on osteoblast cultures stimulated with our synthesized BMP-2 exhibited increased staining compared to the unstimulated osteoblast cultures, which confirmed the activity of our His-tagged BMP-2 protein. Future studies utilizing this protein will demonstrate that CTGF acts as an extracellular antagonist by limiting the amount of BMP-2 available for receptor binding. / Cell Biology

Application of biosolids as an alternative source of Nitrogen (N) is becoming a common silviculture practices on loblolly pine forest. However, little is known about how biosolids type, application rate, and timing affect forest floor and soil N availability in pine plantations. The objectives of this study were to determine the effect of different types, rates, and season of application of biosolids on forest floor and soil N. The study was established in a 17-year-old loblolly pine plantation in Amelia County, VA. Anaerobically digested (AD225), lime stabilized (LS225), and pelletized (Pellet225) biosolids and a conventional inorganic urea plus diammonium phosphate fertilizer (U+DAP225) were surface applied at a rate of 225 kg ha-1 based on Plant Available Nitrogen (PAN) between March 5th and 10th, 2006. Anaerobically digested biosolids were also surface applied at the rates of 900 kg PAN ha-1 and 1800 kg PAN ha<sup>-</sup¹ (AD900 and AD1800). Anaerobically digested biosolids at the rate of 900 kg PAN ha⁻¹ were also applied on November 5th, 2005 (AD900F). Surface application of different type of biosolids in a loblolly pine plantation increased soil N availability and mineralization when biosolids were applied at the permitted rate of 225 kg PAN ha⁻¹. Surface soil NH₄-N and NO₃-N availability and N mineralization was significantly different among biosolids type over time. N release from different type of biosolids depends on the initial inorganic N content, and N mineralization in biosolids. The average soil N availability and mineralization was significantly greater in the Pellet225 treatments than in all the other treatments. Soil N availability decreased in winter in all the treatments but remained generally higher than the control until the end of the second growing season. Nitrate-N concentrations in lysimeters were below water quality standard limits in all the treatments applied at the rate of 225 kg PAN ha⁻¹. Accumulation of N, C, and Ca in the forest floor was well correlated with the amount of biosolids applied on each treatment. The surface application of different type of biosolids had minimal impact upon total N and C in the mineral soil. Increasing application rates of anaerobically digested biosolids directly increased soil N availability and mineralization. Nitrate-N concentrations in lysimeters were above water quality standards limits during several months in the AD900 and AD1800 treatments. Significant differences in the forest floor total N, C and Ca were observed with increasing application rates of biosolids. Total C accumulation was significantly higher in the forest floor in the AD1800 treatment. However, we observed no effect on soil total C with increasing application rates of biosolids. We found that biosolids application during spring significantly increased soil extractable N, N mineralization, NO₃-N leaching, and total C in the mineral soil in comparison to the fall application. Fall application significantly increased NH₄-N leaching and soil extractable Ca. We observed no significant effect on ion exchangeable N measured on membranes, total N, C, Ca, and pH measured in the forest floor, and soil total N and pH in the mineral soil. Our results demonstrated that permitted surface application of biosolids at the rate of 225 kg PAN ha⁻¹ in a loblolly pine plantation increased surface soil N availability without increasing the potential for NO₃-N groundwater pollution. / Ph. D.

Switchgrass (Panicum virgatum L.) has potential as a biofuels feedstock. Major management questions include cultivar selection, cutting management, and optimizing N fertilization. Four cultivars of switchgrass were evaluated under two cutting regimes at eight locations within KY, NC, TN, VA, and WV in 2000 and 2001. Harvests were made once (in early November) or twice (midsummer and early November). Biomass yields averaged 15 Mg ha-1 and ranged from 10 to 22 Mg ha-1 across locations and years. There was no yield advantage to taking two harvests of the lowland cultivars ("Alamo" and "Kanlow"). If harvested twice, the upland cultivars ("Cave-in-Rock" and "Shelter") provided yields equivalent to the lowland ecotypes. A closer look at Alamo revealed much higher N removal in the midsummer harvests, late-season N translocation out of tillers, and fewer tillers developing under one-cut management. Switchgrass appears to be capable of truly perennial productivity in the upper Southeast USA with 50 kg N ha-1 yr-1 and a single harvest. A second field study was conducted on "Cave-in-Rock" switchgrass at Orange and Blacksburg, VA to examine N dynamics. For the 3-yr study, N fertilizer was applied once in May 2001 at 0, 90, 180, or 270 kg N ha-1. Switchgrass was harvested once (early November) in 2001 and twice (early July and early November) in 2002 and 2003. Tissue, root, and soil samples were collected in May, July, September, and November each year. Nitrogen fertilization had no effect on yield in 2001 and small residual effects in 2002 and 2003. Higher N removal was observed with two-cut management, where a high-yielding July cut had high shoot N concentrations. The amount of N removed as biomass from the 0 N treatments over 3 yr was 227 kg N ha-1; obviously significant amounts of N can be made available by these soils without any fertilizer applied. During the growing season, higher mineral N in soil was observed in July and September, when warmer temperatures increase microbial activity and N mineralization. Nitrogen use efficiency declined with increasing N rates. The low N response could be due to "native" N, to microbiological interactions, and/or to the ability of the plants to create internal N reserves. Proper N management of switchgrass must take into account the dynamics of several N pools. Greenhouse studies were conducted to establish switchgrass' responses to N and P under well-defined, soilless conditions and to examine two N sources. Shoot biomass increased with N fertilization with an observed inflection point at 210 kg N ha-1. In these pot studies, root biomass increased with N only to 115 kg N ha-1. No significant effect of P above 30 kg ha-1 was observed in shoot or root biomass. Biomass yield and tiller number were highly correlated. Biomass production was two times greater with ammonium sulfate than with urea when each was applied at equivalent N rates. Taken together, these findings suggest soils in the upper Southeast USA can supply significant amounts of the N needs of switchgrass, especially when harvested once at the end of the season. / Ph. D.

Biosolids have been used as soil conditioners and fertilizers in agriculture and mine land reclamation, but application of Exceptional Quality (EQ) biosolids to rehabilitate anthropogenic soils for urban agriculture is recent and requires greater study to ensure their appropriate use. The objectives were: 1) to quantify plant available nitrogen (PAN) of new EQ biosolids in a greenhouse bioassay; 2) to quantify PAN of EQ biosolids applied to an urban degraded subsoil via tall fescue N fertilizer equivalency, and compare field results to laboratory tests; 3) to investigate EQ biosolids and inorganic fertilizer effects on urban soil properties, vegetable yields, and potential N and phosphorus (P) loss. Biosolids evaluated were products of thermal hydrolysis plus anaerobic digestion (BLOOM), blending with woody mulch (BM) and sand/sawdust (BSS), composting (LBC), and heat-drying (OCB). Organic N mineralization of new blended biosolids products ranged between 20-25% in the greenhouse bioassay. Products BLOOM, BM, and OCB had the highest organic N mineralization as estimated by the 7-day anaerobic incubation, and this test and soil nitrate-N had the highest correlations with tall fescue N uptake (r=0.49 and r=0.505, respectively). We conducted a two-year field study with four growing seasons (fall 2016-2017 and summer 2017-2018) in an urban disturbed subsoil where EQ biosolids were applied seasonally at agronomic N rates, and yearly at reclamation rates (5x agronomic N). Cabbage yields were greater with reclamation rates (~3.0 kg m-2) and bell pepper yields were greater with BLOOM reclamation rate (~1.0 kg m-2) than with the inorganic fertilizer (1.0 kg m-2 and 0.2 kg m-2, respectively) during second year growing seasons. Soil carbon (C) accumulation (%C remaining in the soil) two years after biosolids additions ranged between 37 to 84%. Soil N availability and mineralization were limited most likely due to lack of residual soil C and N, and high clay content. Nitrogen leaching losses from reclamation rates were not greater than agronomic N rates. Leachate P was below detection during most of the experiment. Despite limiting soil conditions, biosolids amendment at reclamation rates showed greatest potential to increase vegetable yield and improve soil properties after two years of application, while not impairing water quality. / Doctor of Philosophy / Exceptional Quality (EQ) biosolids are by-products of wastewater treatment plants that have been processed to destroy pathogens, reduce attraction by disease-spreading organisms (e.g. flies, mosquitoes, rodents, etc.), and limit heavy metal concentrations. These characteristics make EQ biosolids safe for use by home gardeners for growing food crops. There is limited information on optimal recommended rates at which these products should be applied to urban gardens. The purpose of our research was to determine optimum application rates of EQ biosolids to urban gardens based on their essential plant nutrient (esp., nitrogen and phosphorus) availability. We learned that the EQ biosolids we studied are less concentrated in plant available nitrogen and phosphorus than biosolids applied to conventional agricultural fields. This is because we diluted our biosolids with sawdust, sand, and woody mulch to facilitate their storage, handling, and ease of application. We learned that high EQ biosolids application rates reduce soil compaction and increase essential plant nutrient availability and crop yields for agriculture practiced in urban soils. The high application rates of EQ biosolids accomplished such soil-improving and yield-increasing benefits without impairing local water quality.

Climate change resulting from emissions of fossil fuel combustion has sparked considerable interest in renewable energy and fuel production research, particularly energy derived from cellulosic ethanol, which is derived from biomass such as wood and grass. Cellulosic ethanol demonstrates a more promising future as a global energy source than corn-derived ethanol because it does not displace food crops, irrigation is not required, and chemical application rates are much lower than for annual crops, such as corn. Growing cellulosic biomass for energy can help reduce greenhouse gas emissions via carbon (C) sequestration and by reducing demand for fossil fuel production. The objective of this study was to investigate how land use change affects soil properties and selected soil C and nitrogen (N) dynamics among alternative cellulosic biofuel treatments at the Weyerhaeuser Alabama Cellulosic Biofuel Research site in west-central Alabama. Composite soils for characterization, along with forest floor, were collected at year 1 and year 2 after treatment establishment at 0-15cm and 15-30cm depths at six locations along three hillslope-riparian transects in five experimental watershed treatments. Decomposition of loblolly pine needles was assessed in each watershed using an in situ litter bag method. Seasonal in situ net nitrogen mineralization was measured using a sequential core method, and an anaerobic incubation for N mineralization potential of composite soils was performed in the laboratory. Results revealed high variability of soil properties and processes within these watersheds, along with no consistent treatment effects. This study provides baseline data for these watershed treatments for future studies. / Master of Science

Magmatic-hydrothermal systems typically have vertical extents of several hundred<br />meters and their geochemical characteristics (e.g. mineral assemblages) vary considerably<br />over that vertical extent. As a consequence the expression in outcrop varies depending on<br />the level of erosion. Therefore understanding the geochemical zonation of magmatic-hydrothermal<br />ore deposits opens the possibility to detect deep magmatic-hydrothermal<br />systems, and to assess qualitatively the degree of erosion that has taken place in the area<br />and at which level the mineralization may occur. This dissertation presents the<br />characterization of two shallow hydrothermal systems and their potential relations with<br />deeper magmatic-hydrothermal systems. In addition, this dissertation develops the<br />equations to directly interpret thermometric data from the fluid inclusion type dominant in<br />one of those deposits (fluid inclusions that homogenize by halite disappearance).<br />Red Mountain, AZ is a porphyry copper system with a well-preserved lithocap<br />providing an ideal candidate to characterize the shallow expression of porphyry copper<br />systems in the southwestern US. The distribution of fluid inclusions, alteration mineralogy<br />and grade indicate that the intrusive responsible for the mineralization was only partially<br />intercepted during the exploration program and that one single magmatic event was likely<br />responsible for the mineralization detected. Fluid inclusion types and clay minerals are<br />systematically distributed within the deposit. The fluid responsible for the shallow<br />hypogene mineralization was a low pH-intermediate temperature-low density fluid while a<br />high salinity fluid was responsible for deep mineralization.<br />Wutong is a Pb-Zn-Ag deposit in the Nanling belt (southeast China). The combination<br />of fluid inclusion and mineral thermometry indicates that the Wutong deposit formed at<br />relatively low pressures. The age and isotopic composition of the mineralization indicates<br />that the deposit formed during the Cretaceous from crustal derived fluids. The occurrence<br />of a shallow magmatic-hydrothermal system of Cretaceous age in this region suggests that<br />Cretaceous intrusions, despite not outcropping very commonly in this particular region may<br />occur at deeper levels. / Ph. D.

Water stress is the most critical environmental factor limiting crop production in the US Piedmont. The presence of humic substances in composted organic amendments may increase crop tolerance to water stress through their hormone-like effects on plant metabolism. The objectives of this study were to calculate N mineralization rates of composted and non-composted organic materials used in this long-term field study, and to determine differences in soil physiochemical properties, corn and soybean leaf physical and biochemical properties yield and seed quality between organically amended and inorganically fertilized treatments. Nitrogen mineralization rates were greatest in the poultry litter (21%) and Panorama yard waste compost (4.5%) amended plots. Nitrogen uptake (120 mg/pot, 133 mg/pot, respectively) in these treatments were greater than that in the control (0N) (91.3 mg/pot) treatment. Wolf Creek biosolids compost and Huck's Hen Blend yard waste compost induced N immobilization (-5.0% and 0.18%, respectively), and had N uptake values similar to the control (92.6 mg/pot and 95.7 mg/pot). Rivanna biosolids compost immobilized N (-14.8%) but N uptake (136 mg/pot) was greater than that in the control due to the relatively high inorganic N content in the amendment. The total N concentration and C:N values were less reliable variables in predicting N mineralization when a significant portion of the total N was in the inorganic form. The annual application of poultry litter, Rivanna biosolids compost, and Panorama yard waste compost at 100% agronomic nitrogen and 30 % agronomic nitrogen rates in the field study improved soil fertility and increased total organic and humified carbon contents relative to the inorganically fertilized and control treatments. The amended treatments had slightly greater plant available water contents (average 10.0 cm/15 cm) than the control (8.38 cm/15 cm). Leaf water potential measurements revealed that neither crop experienced water stress during the sampling season. Treatment differences in leaf antioxidant activity were only observed in corn. All corn plants that were fertilized with amendments supplying the crop's nitrogen needs, regardless of the source, had greater leaf nitrogen (+29%), chlorophyll (+33%), and protein contents (+37%), lower superoxide dismutase (-29%) and ascorbate peroxidase (-17%) activities, and lower malondialdehyde (-33%) contents relative to the control and low nitrogen treatments. There were no observed differences in catalase activity, which was likely due to the evolutionary advantage of C4 metabolism. Yield was strongly related to midseason leaf nitrogen contents (R2=0.87, p<0.0001) and not soil humified carbon (R2=0.02, p=0.0543). There were no observed treatment differences in soybean leaf physiology and metabolism. Differences, however, were observed over time. As the leaves senesced, leaf chlorophyll, protein, superoxide dismutase and catalase activities decreased, and the malondialdehyde content increased. Ascorbate peroxidase activity slightly increased with time. Catalase activity in soybean was primarily driven by the oxidation of glycolate, a product of photorespiration, and not the formation of reactive oxygen species in the chloroplasts. The organically amended treatments had higher yields (9-21% increase), greater protein contents (4-9% increase), and seed weights (5-14% increase) relative to the fertilizer and control treatments. It was concluded that differences in soybean yield and seed quality were due to non-nutritive benefits of the organic amendments and not available water or plant nutrition. / Master of Science

Nitrogen deficiency is characteristic of many mid-rotation loblolly pine (Pinus taeda L.) plantations in the Piedmont region of the southeastern USA. Fertilization with urea is the most common method used to correct this deficiency. Previous studies show that urea fertilization produces a rapid pulse of available nitrogen (N) with only a portion being utilized by plantation trees. Controlled release fertilizers release available N more slowly over a longer period of time and therefore may result in greater uptake efficiency. The objective of this study was to compare Nitroform®, a urea-formaldehyde controlled release N fertilizer versus urea and a control by measuring the effects of the two fertilizer treatments on N availability and loss as: total KCl extractable-N, total ion exchange membrane-N (IEM-N), N mineralization, and N volatilization, in a mid-rotation loblolly pine plantation in the Piedmont of Virginia. In addition, mid-summer and mid-winter fertilizations were compared to assess fertilizer uptake as a function of season. After the summer fertilization, Nitroform&reg; significantly increased total KCl-extractable N, IEM-N, and N mineralization for two to three months over urea and the control. Three hundred times more N volatilized from urea than from controlled release Nitroform®. Interestingly, seven months after the summer application, the controlled release Nitroform® showed marked immobilization for three months while urea demonstrated greater N mineralization. After the winter application, fertilization with urea demonstrated greater soil inorganic N concentrations for two to three months over Nitroform®, very little N was immobilized, and volatilization was only 10 times that of Nitroform®. After summer and winter fertilizations, both fertilizer treatments significantly increased soil inorganic N concentrations and N volatilization over controls, however did not significantly increase N mineralization over controls when average response was tested over the entire sampling period. In addition to the fertilizer effects measured, a thinning only treatment was also incorporated into this study with soil N-availability indices compared to a control with no thinning or fertilization. The results from the thinning only treatment demonstrated no significant increases over the control in total KCl extractable-N, IEM-N, N-mineralization, or N volatilization when average responses were tested over the entire sampling period. / Master of Science

Cette étude porte sur l’application de différents procédés d’oxydation avancée, POA (plasma d'air humide, électro-Fenton, photo-Fenton et oxydation anodique avec BDD) pour le traitement des colorants de textile. La particularité de ces procédés tient à la génération dans le milieu d’entités très réactives et très oxydantes, les radicaux hydroxyles •OH qui sont capables d’oxyder n’importe quelle molécule organique jusqu’au stade ultime d'oxydation, c'est-à-dire la minéralisation (transformation en CO2 et H2O). Le plasma d'air humide a été appliqué pour l'oxydation d'un colorant azoïque, l'OD 61. Différents catalyseurs (Fe2+, Fe3+ et TiO2) ont été ajoutés dans leurs conditions optimisées afin d'améliorer les performances du système Glidarc. La combinaison des deux catalyseurs: Fe2+ et TiO2 a permis de décolorer 91% de l'OD 61 au bout de 3 heures et d'atteindre un taux d'abattement du COT de l'ordre de 52% après 10 heures de traitement. La méthodologie de la recherche expérimentale a été appliquée dans ce mémoire afin d'étudier l'influence de: l'intensité du courant, la concentration du colorant et le temps d'électrolyse sur la vitesse de disparition de l'OD 61 et afin de déterminer les conditions optimales de sa minéralisation. Dans les conditions optimales obtenues ([colorant] = 0,53.10-3 mol.L-1, I = 250 mA), le procédé électro-Fenton (EF) permet d'atteindre des taux de minéralisation de l'ordre de 98% dans le cas de l'OD 61 et l'AO 7 et de 88% dans le cas de l'indigo carmine. L’identification des produits intermédiaires au cours de l’électrolyse a permis de proposer un mécanisme de minéralisation de l'AO7. Les constantes cinétiques apparentes et absolues ont été déterminées. La dégradation de l'indigo a été étudiée par oxydation anodique avec BDD (OA-BDD) et par procédé photo-Fenton (PF). Cette étude a montré que l'électrolyse de l'indigo suit une cinétique de pseudo premier ordre et que le taux d'abattement du COT était de l'ordre de 97% et 63% respectivement avec OA-BDD et PF. Une étude comparative pour l'oxydation de l'AO 7 a été menée par trois procédés d'oxydation avancée: PF, OA- BDD et EF-Pt et EF-BDD. Cette étude a montré que le procédé photo-Fenton permet d'atteindre des taux d'abattement supérieurs à 90% après seulement 2 heures de traitement. Toutefois, le PF s'est révélé le plus coûteux suite à l'utilisation de la lumière artificielle UV et l'ajout des réactifs. Par ailleurs, le traitement d'un effluent réel issu de l'industrie de textile par le procédé électro-Fenton avec une anode de platine a permis la minéralisation presque totale du rejet initial (94% du COT initial ont été éliminés) / This study concerns the application of various advanced oxidation processes, AOP (humid air plasma, electro-Fenton, photo-Fenton and anodic oxidation with BDD) to treatment of wastewater containing the persistent organic pollutants such as textile dyes. The characteristic of these processes is due to the generation of very reactive and very oxidizing species, hydroxyl radicals •OH which are able to oxidize any organic molecule until the ultimate oxidation stage, i.e. mineralization (transformation into CO2 and H2O). The humid air plasma was applied for DO 61degradation. Various catalysts (Fe2+, Fe3+ and TiO2) were added under their optimized conditions in order to improve the performances of Glidarc system. The combination of Fe2+and TiO2 lead to reach 91% of DO 61 degradation after 3 hours and 52% of TOC abatement after 10 H of treatment. The experimental design methodology was applied in this work in order to investigate the influence of experimental parameters (current intensity, dye concentration and electrolysis time) on the degradation rate of DO 61 dye and then for determining the optimal mineralization conditions. Under the optimal obtained conditions ([dye] = 0.53 10-3 mol.L-1, I = 250 mA), electro-Fenton process can lead to a complete mineralization of dyes: 98% of TOC abatement are obtained in the case of the DO 61 and the AO 7 and 88% in the case of indigo carmine. The identification of the intermediates during electrolysis permitted to propose a mineralization of AO 7 dye under examination. The apparent and absolute kinetic constants were determined. The degradation of indigo dye was studied by anodic oxidation with BDD (AO-BDD) and photo-Fenton process (PF). This study shows a pseudo first order reaction kinetics for indigo degradation. The mineralization efficiency is about 97% and 63% of TOC abatement, respectively with OA-BDD and PF A comparative study of the mineralization efficiency of AO 7 dye was been investigated by different advanced oxidation processes (PF, AO-BDD, EF-Pt, EF-BDD). This study shows that PF process lead to reach more than 95% of TOC abatement after only 2 hours. Nevertheless, this process is the most expensive due to the use artificial UV light and the addition of reagents. The treatment of an industrial textile effluent by electro-Fenton process lead to a complete mineralization (94% of initial TOC were eliminated)

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