Carbohydrate-binding proteins play vital roles in many vital biological processes and study of these interactions, at residue level, are useful in treating many critical diseases. Analyzing the local sequential environments of the binding and non-binding regions to predict the protein-carbohydrate binding sites is one of the challenging problems in molecular and computational biology. Prediction of such binding sites, directly from sequences, using computational methods, can be useful to fast annotate the binding sites and guide the experimental process. Because the number of carbohydrate-binding residues is significantly lower than non-carbohydrate-binding residues, most of the methods developed are biased towards over predicting the non-carbohydrate-binding residues. Here, we propose a balanced predictor, called StackCBPred, which utilizes features, extracted from evolution-driven sequence profile, called the position-specific scoring matrix (PSSM) and several predicted structural properties of amino acids to effectively train a stacking-based machine learning method for the accurate prediction of protein-carbohydrate binding sites.

acase@tulane.edu

Lactococci are widely used in the cheese industry as a starter culture. Starter cultures face carbohydrate starvation due to the absence of a fermentable carbohydrate in the cheese curd after pressing. Starvation leads to a decreased ability to synthesize ATP, generate a proton motive force, and accumulate nutrients necessary to maintain viability. The aim of this work was to investigate the culturability of lactococci grown with and without lactose in a chemically defined medium, and to define the metabolic changes that occur during carbohydrate starvation. Lactose metabolism provided energy for logarithmic phase growth and greater cell density in L. lactis ssp. lactis ML3 and L. lactis ssp. cremoris S2. However, the rate of lactose metabolism was strain dependent in that L. lactis ssp. lactis 11454 did not metabolize lactose as rapidly as did ML3 and S2. In the absence of lactose the cells became nonculturable on agar. In addition to becoming nonculturable, the aminopeptidase and lipase/ esterase activity became nonmeasurable after 21 d, and cellular metabolism was altered because of carbohydrate starvation. Nevertheless, the cells remained viable for up to 42 d in spent media as measured by fluorescent viability stains and intracellular ATP content. Fluorescent viability staining demonstrated that the cells maintained an intact cell membrane to contain their DNA, as well as to contain enzymes and ATP necessary to maintain viability and metabolic activity. With the addition of arginine to the basal medium, the survival time, cell number, and ATP concentration increased. Amino acids, including arginine, provided energy after carbohydrate exhaustion. At the onset of lactose exhaustion, the extracellular concentrations of arginine, glycine/valine, glutamate, and glutamine decreased in the media when energy was present for their transport. There was a significant increase in serine and methionine concentrations in the spent media over the same time period. These data indicated lactococci remained viable and metabolically active, but were nonculturable in response to carbohydrate starvation. Additionally, amino acids are in a dynamic state during carbohydrate starvation, and utilization of amino acids, such as arginine and serine, could facilitate lactococcal cells in maintaining viability in harsh environments such as ripening cheese.

The atmospheric CO2 concentration is rising. These higher CO2 concentrations will certainly affect growth but may also affect nutrient responses of crop plants. The overall aim of this thesis was to investigate the effect of elevated CO2 on the nitrogen and phosphorus nutrition of two different crop species: wheat and cotton. Once establishing the new nutrient requirements of these crop species at elevated CO2 the study focussed on the interactive effects of elevated CO2 and N supply on growth and leaf N concentrations in cotton and wheat, carbohydrate metabolism, source:sink interactions and photosynthetic enzyme levels in the two species. Having established these effects, the role of N supply in controlling source:sink effects on Rubisco concentration were studied at the molecular level in wheat. Finally, some implications of the effect of elevated CO2 on wheat were examined, specifically on the quality of flour produced from wheat grain grown at elevated CO2. The foliar nitrogen concentrations, required to produce maximum shoot growth (critical concentrations) were lower at elevated CO2 while the critical phosphorus concentrations were higher. / Doctor of Philosophy (PhD)

This thesis presents studies relating to effects of different macronutrients, predominantly fat and carbohydrate, on gastrointestinal motility, hormone release/suppression, appetite and energy intake in healthy subjects, and on symptom generation in patients with functional dyspepsia. The three broad areas that have been investigated in these studies are: (i) the effect of load, and duration, of small intestinal nutrient exposure on gastric motility, gastrointestinal hormone release/suppression, appetite and energy intake in healthy subjects, (ii) the dietary factors that may contribute to symptom generation in patients with functional dyspepsia, through analysis of diet diaries and acute nutrient challenges, and (iii) the effects of the herbal medication, Iberogast®, on gastric motility in healthy subjects. The ingestion of nutrients, triggers a number of gastrointestinal responses, including the modulation of antropyloroduodenal motility, gastrointestinal hormone release/suppression, and the suppression of appetite and energy intake, resulting in a slowing of gastric emptying to an average rate of 1 - 3 kcal/min, which is required for efficient nutrient digestion and absorption. Additionally, the rate at which glucose enters the small intestine influences postprandial glycaemia and incretin responses. These responses have been demonstrated in animals to be dependent on the length, and region, of the small intestine exposed to fat and glucose, however, this has not been directly investigated in humans. Functional dyspepsia is a clinical condition, characterised by chronic upper abdominal symptoms, such as nausea, bloating and early fullness, without a known cause, which affects approximately 11 - 29 % of the population. Many studies have reported that disturbed gastric motor activity may be the cause of these symptoms, but patients frequently experience symptoms following ingestion of food, and some patients report to eat smaller meals more frequently and avoid fatty and spicy foods. In addition, laboratory-based studies have indicated that functional dyspepsia patients may be hypersensitive to fat, but not carbohydrate. To date, the treatments used to reduce symptoms are frequently directed at the normalisation of gastroduodenal motility, using prokinetics. However, the beneficial effect of these drugs is relatively small and variable, and their adverse effects can be substantial. Herbal drug preparations have recently received considerable interest as an alternative treatment option in functional dyspepsia. A commercially available herbal preparation, Iberogast® which contains nine plant extracts, has been reported to improve upper abdominal symptoms in functional dyspepsia and to decrease fundic tone, increase antral contractility and decrease afferent nerve sensitivity in experimental animals. The effects of Iberogast® in the human gastrointestinal tract have not been investigated. The first three studies presented in this thesis have focused on the effects of delivering fat and glucose into the small intestine at different loads (Chapter 5, 6 and 7), lower, comparable to, and higher than gastric emptying normally occurs, and at different durations of infusion (but still at similar caloric loads - Chapter 5, fat only), on gastrointestinal motility, plasma hormone release/suppression, glycaemia, and energy intake in healthy male subjects. The study in Chapter 5 demonstrated that antral pressure waves and pressure wave sequences were suppressed, and basal pyloric pressure, isolated pyloric pressure waves, and plasma cholecystokinin and peptide YY stimulated, during both the low (1.33 kcal/min for 50 min: 67 kcal/min), and high (4 kcal/min for 50 min: 200 kcal), loads of lipid. The effect of the 4 kcal/min load was sustained so that the suppression of antral pressure waves and pressure wave sequences and increase in peptide YY remained evident after cessation of the infusion. The prolonged lipid infusion (1.33 kcal/min for 150 min: 200 kcal) suppressed antral pressure waves, stimulated cholecystokinin and peptide YY and basal pyloric pressure and tended to stimulate isolated pyloric pressure waves when compared with saline throughout the entire infusion period. These results indicate that both the load, and duration, of small intestinal lipid have an influence on antropyloroduodenal motility and patterns of cholecystokinin and peptide YY release. Chapter 6 demonstrated that lipid loads lower than gastric emptying normally occurs (0.25 kcal/min for 50 min: 12.5 kcal) transiently stimulated isolated pyloric pressure waves and cholecystokinin release and suppressed pressure wave sequences and hunger scores. Loads comparable to (1.5 kcal/min for 50 min: 75 kcal) and higher (4 kcal/min for 50 min: 200 kcal), than the normal rate of gastric emptying, were required to stimulate basal pyloric tone and peptide YY release and suppress antral and duodenal pressure waves. Only the 4 kcal/min load suppressed energy intake. The effects of lipid on all parameters, with the exception of hunger, were load-dependent. In addition, there were relationships between antropyloroduodenal motility and cholecystokinin and peptide YY concentrations with energy/food intake. The study in Chapter 7 demonstrated that loads of glucose lower than (1 kcal/min for 120 min: 120 kcal), comparable to (2 kcal/min for 120 min: 240 kcal) and higher than (4 kcal/min for 120 min: 480 kcal) the rate gastric emptying normally occurs, stimulated blood glucose, plasma insulin, glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide and cholecystokinin concentrations and suppressed the number of antral pressure waves, 2 and 4 kcal/min loads were required for the suppression of duodenal pressure waves and pressure wave sequences and the stimulation of basal pyloric pressure and suppression of energy intake only after the 4 kcal/min loads. There were also relationships between glucagon-like peptide-1 and glucose-dependent insulinotropic peptide with basal pyloric tone, and food/energy intake with pyloric pressures. The studies presented in the subsequent three chapters investigated the contribution of dietary factors on the generation of symptoms in patients with functional dyspepsia when compared with healthy subjects (Chapter 8 and 9) and the effect of Iberogast® on motility in the healthy gastrointestinal tract (Chapter 10). The effects of equi-caloric high-carbohydrate vs. high-fat yoghurt preloads on symptom generation, plasma hormone concentrations, antral area and energy intake were compared between functional dyspepsia patients and healthy subjects (Chapter 8). Nausea and pain were greater in patients after the high-fat, when compared with high-carbohydrate and control, preloads and with healthy subjects. Discomfort was greater after all preloads in patients when compared with healthy subjects. Fasting cholecystokinin and stimulation of cholecystokinin by the high-fat preload were greater in patients, while fasting and postprandial peptide YY were lower in patients than in healthy subjects, with no differences in fasting, or postprandial, plasma ghrelin between patients and healthy subjects. Fasting antral area was greater in patients, with no differences postprandially between patients and healthy subjects. There were no differences in energy intake between the two groups. The relationship between the effect of dietary intake and eating behaviour over a 7-day period on the occurrence and severity of abdominal symptoms was compared between patients and healthy subjects (Chapter 9). The symptoms experienced by the patients included nausea, fullness discomfort, bloating and upper abdominal, and epigastric, pain, of a modest severity, which occurred within 30 min of eating. The number of “meals” ingested was significantly less in functional dyspepsia patients and there was a trend for total energy and fat intake to be less. The occurrence of these symptoms was also statistically related to the ingestion of fat and energy intake. The results of these studies indicate that diet, particularly the ingestion of fat, influences the development of symptoms in a subgroup of patients with functional dyspepsia. The study in Chapter 10 evaluated the effect of the herbal drug Iberogast® on gastric motility in the gastrointestinal tract. Iberogast® increased proximal gastric volume, increased antral pressure waves without affecting pyloric or duodenal pressures, and slightly increased the retention of liquid in the total stomach, but had no effect on gastric emptying of solids or intragastric distribution. These results demonstrate that Iberogast® affects gastric motility in humans, and the stimulation of gastric relaxation and antral motility may contribute to the reported therapeutic efficacy of Iberogast® in functional dyspepsia. The studies reported in this thesis provide new information about the regulation of gastric motility, hormone release/suppression, appetite and energy intake, by varying the loads of lipid and glucose infused into the small intestine in healthy subjects, which may have implications in patients with altered gastric motor functions, such as obese, type-2 diabetes and functional dyspepsia patients. In addition, studies in functional dyspepsia patients revealed that diet, in particular the ingestion of fat, contribute to the cause of their symptoms, and these findings may have important implications for the development of diet-based therapies for the treatment of functional dyspepsia. Furthermore, functional dyspepsia patients with impaired gastric relaxation and antral dysmotility may benefit from the effects of Iberogast® as demonstrated in the healthy gastrointestinal tract. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1331606 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

Under terminal water deficit, the impact of stem carbohydrate remobilization has greater significance because post-anthesis assimilation is limited, and grain growth depends on translocation of carbohydrate reserves. The working hypothesis of this thesis is that increases in stem carbohydrates facilitate tolerance to terminal drought in wheat. The goals of this thesis are to examine this hypothesis using physiological and genetic tools; identify genes that are related to QTL for stem carbohydrate; work with wheat and barley breeders to integrate findings into the breeding program of the Department of Agricultural and Food Western Australia. The physiological data of three drought experiments (two years in a glasshouse and one year in the field) suggested the maximum level of stem water soluble carbohydrate (WSC) is not consistently related to grain weight, especially, under water deficit. The patterns of WSC accumulation after anthesis differed depending on variety and suggested that WSC degradation and translocation have different genetic determinants. Most of the carbohydrates in stem WSC in wheat are fructans. Because 1-FEH gene was an important gene in fructan degradation, the three copies of this gene (1-FEH w1, 1-FEH w2 and 1-FEH w3) were isolated from the respective genomes of bread wheat. In addition, the genes were mapped to chromosome locations and coincided with QTL for grain weight. The results of gene expression studies show that 1-FEH w3 had significantly higher levels in the stem and sheath which negatively corresponded to the level of stem WSC in two wheat varieties in both water-deficit and well-watered treatments. Strikingly, the 1-FEH w3 appeared to be activated by water deficit in Westonia but not in Kauz. The results suggest that stem WSC level is not, on its own, a reliable criterion to identify potential grain yield in wheat exposed to water deficit during grain filling. The expression of 1-FEH w3 may provide a better indicator when linked to instantaneous water use efficiency, osmotic potential and green leaf retention, and this requires validation in field grown plants. In view of the location of the contribution to grain filling of stem WSC, this is a potential candidate gene contributing to grain filling. The numerous differences of intron sequences of 1-FEH genes would provide more opportunities to find markers associated with the QTL. A new FEH gene was partially isolated from Chinese Spring and the sequence was closely related to 1-FEH genes. This gene, FEH w4, was mapped to 6AS using Chinese Spring deletion bin lines. The polymorphism of this gene was found between different bread varieties using PCRs and RFLPs, and this allowed the gene to be mapped to two populations of Hanxuan 10 × Lumai 14 and Cranbrook × Halberd. In the population of Hanxuan 10 × Lumai 14, it was close to SSR marker xgwm334 and wmc297 where the QTL of thousand grain weight and grain filling efficiency were located. This result indicated this gene might be another possible candidate gene for grain weight and grain filling in wheat.

Thesis (Ph. D.)--Pennsylvania State University, 1968. / Includes bibliographical references.

Diorhabda elongata elongata leaf beetles were released at two field locations in the upper Colorado River watershed of Texas in 2003 and 2004 for the biological control of invasive Tamarix, exotic trees deteriorating riparian ecosystems of western North America. Establishment and biological success were monitored using trees on transects from the release points. D. elongata elongata released at the Lake Thomas site in August 2003 successfully overwintered and were recovered in the spring 2004; however, beetles were not present after June 2004. The April 2004 release at Beals Creek led to establishment and survival during 2005 and 2006. Mean abundance increased from less than five insects per tree per 2 minute count in August 2004 to more than 40 insects per tree per 2 minute count in August 2006. By then the population was dispersed throughout an area of approximately 12 hectares and beetles were present on 100% of the 47 trees surveyed, 57% of which were at least 90% defoliated. To measure the impact of beetle defoliation on Tamarix, nonstructural carbohydrates (NCHOs) were measured in manipulative field cage experiments in Texas and natural experiments in Nevada. There was no significant difference in NCHOs between trees with versus trees without beetle herbivory in the cage experiment, although spring foliage regrowth was reduced by 35% in trees defoliated the previous fall. In Nevada, root crown tissue was sampled in 2005 and 2006 from trees that had experienced 0-4 years of defoliation. In 2005, NCHO concentrations differed between tree stands and ranged from 9.0 ± 0.8% (Mean ± SE) in non-defoliated trees to 3.2 ± 0.4%, 2.1 ± 0.4% and 2.3 ± 0.4% in trees defoliated for 1, 2 and 3 successive years, respectively. NCHO concentrations in 2006 were similar, ranging from 13.6 ± 0.9% in non-defoliated trees to 7.6 ± 0.8%, 2.3 ± 0.4%, 1.5 ± 0.3% and 1.7 ± 0.4% in trees defoliated for 1, 2, 3 and 4 years, respectively. The establishment, biological success and host impact of D. elongata leaf beetles suggest there is potential for biological control of Tamarix in the United States.

Nanoporous carbon materials are widely used in industry as adsorbents or catalyst supports, whilst becoming increasingly critical to the developing fields of energy storage / generation or separation technologies. In this thesis, the combined use of carbohydrate hydrothermal carbonisation (HTC) and templating strategies is demonstrated as an efficient route to nanostructured carbonaceous materials. HTC is an aqueous-phase, low-temperature (e.g. 130 – 200 °C) carbonisation, which proceeds via dehydration / poly-condensation of carbon precursors (e.g. carbohydrates and their derivatives), allowing facile access to highly functional carbonaceous materials. Whilst possessing utile, modifiable surface functional groups (e.g. -OH and -C=O-containing moieties), materials synthesised via HTC typically present limited accessible surface area or pore volume. Therefore, this thesis focuses on the development of fabrication routes to HTC materials which present enhanced textural properties and well-defined porosity. In the first discussed synthesis, a combined hard templating / HTC route was investigated using a range of sacrificial inorganic templates (e.g. mesoporous silica beads and macroporous alumina membranes (AAO)). Via pore impregnation of mesoporous silica beads with a biomass-derived carbon source (e.g. 2-furaldehyde) and subsequent HTC at 180 oC, an inorganic / carbonaceous hybrid material was produced. Removal of the template component by acid etching revealed the replication of the silica into mesoporous carbonaceous spheres (particle size ~ 5 μm), representing the inverse morphological structure of the original inorganic body. Surface analysis (e.g. FTIR) indicated a material decorated with hydrophilic (oxygenated) functional groups. Further thermal treatment at increasingly elevated temperatures (e.g. at 350, 550, 750 oC) under inert atmosphere allowed manipulation of functionalities from polar hydrophilic to increasingly non-polar / hydrophobic structural motifs (e.g. extension of the aromatic / pseudo-graphitic nature), thus demonstrating a process capable of simultaneous control of nanostructure and surface / bulk chemistry. As an extension of this approach, carbonaceous tubular nanostructures with controlled surface functionality were synthesised by the nanocasting of uniform, linear macropores of an AAO template (~ 200 nm). In this example, material porosity could be controlled, showing increasingly microporous tube wall features as post carbonisation temperature increased. Additionally, by taking advantage of modifiable surface groups, the introduction of useful polymeric moieties (i.e. grafting of thermoresponsive poly(N-isopropylacrylamide)) was also demonstrated, potentially enabling application of these interesting tubular structures in the fields of biotechnology (e.g. enzyme immobilization) and medicine (e.g. as drug micro-containers). Complimentary to these hard templating routes, a combined HTC / soft templating route for the direct synthesis of ordered porous carbonaceous materials was also developed. After selection of structural directing agents and optimisation of synthesis composition, the F127 triblock copolymer (i.e. ethylene oxide (EO)106 propylene oxide (PO)70 ethylene oxide (EO)106) / D-Fructose system was extensively studied. D-Fructose was found to be a useful carbon precursor as the HTC process could be performed at 130 oC, thus allowing access to stable micellular phase. Thermolytic template removal from the synthesised ordered copolymer / carbon composite yielded functional cuboctahedron single crystalline-like particles (~ 5 μm) with well ordered pore structure of a near perfect cubic Im3m symmetry. N2 sorption analysis revealed a predominantly microporous carbonaceous material (i.e. Type I isotherm, SBET = 257 m2g-1, 79 % microporosity) possessing a pore size of ca. 0.9 nm. The addition of a simple pore swelling additive (e.g. trimethylbenzene (TMB)) to this system was found to direct pore size into the mesopore size domain (i.e. Type IV isotherm, SBET = 116 m2g-1, 60 % mesoporosity) generating pore size of ca. 4 nm. It is proposed that in both cases as HTC proceeds to generate a polyfuran-like network, the organised block copolymer micellular phase is essentially “templated”, either via hydrogen bonding between hydrophilic poly(EO) moiety and the carbohydrate or via hydrophobic interaction between hydrophobic poly(PO) moiety and forming polyfuran-like network, whilst the additive TMB presumably interact with poly(PO) moieties, thus swelling the hydrophobic region expanding the micelle template size further into the mesopore range. / Nanoporöse kohlenstoffbasierte Materialien sind in der Industrie als Adsorbentien und Katalysatorträger weit verbreitet und gewinnen im aufstrebenden Bereich der Energiespeicherung/erzeugung und für Trennverfahren an wachsender Bedeutung. In der vorliegenden Arbeit wird gezeigt, dass die Kombination aus hydrothermaler Karbonisierung von Zuckern (HTC) mit Templatierungsstrategien einen effizienten Weg zu nanostrukturierten kohlenstoffbasierten Materialien darstellt. HTC ist ein in Wasser und bei niedrigen Temperaturen (130 - 200 °C) durchgeführter Karbonisierungsprozess, bei dem Zucker und deren Derivate einen einfachen Zugang zu hochfunktionalisierten Materialien erlauben. Obwohl diese sauerstoffhaltige Funktionalitäten auf der Oberfläche besitzen, an welche andere chemische Gruppen gebunden werden könnten, was die Verwendung für Trennverfahren und in der verzögerten Wirkstofffreisetzung ermöglichen sollte, ist die mittels HTC hergestellte Kohle für solche Anwendungen nicht porös genug. Das Ziel dieser Arbeit ist es daher, Methoden zu entwickeln, um wohldefinierte Poren in solchen Materialien zu erzeugen. Hierbei führte unter anderem der Einsatz von anorganischen formgebenden mesoporösen Silikapartikeln und makroporösen Aluminiumoxid-Membranen zum Erfolg. Durch Zugabe einer Kohlenstoffquelle (z. B. 2-Furfural), HTC und anschließender Entfernung des Templats konnten poröse kohlenstoffbasierte Partikel und röhrenförmige Nanostrukturen hergestellt werden. Gleichzeitig konnte durch eine zusätzliche Nachbehandlung bei hoher Temperatur (350-750 °C) auch noch die Oberflächenfunktionalität hin zu aromatischen Systemen verschoben werden. Analog zur Formgebung durch anorganische Template konnte mit sog. Soft-Templaten, z. B. PEO-PPO-PEO Blockcopolymeren, eine funktionelle poröse Struktur induziert werden. Hierbei machte man sich die Ausbildung geordneter Mizellen mit der Kohlenstoffquelle D-Fructose zu Nutze. Das erhaltene Material wies hochgeordnete Mikroporen mit einem Durchmesser von ca. 0,9 nm auf. Dieser konnte desweiteren durch Zugabe von Quell-Additiven (z. B. Trimethylbenzol) auf 4 nm in den mesoporösen Bereich vergrößert werden. Zusammenfassend lässt sich sagen, dass beide untersuchten Synthesewege nanostrukturierte kohlenstoffbasierte Materialien mit vielfältiger Oberflächenchemie liefern, und das mittels einer bei relativ niedriger Temperatur in Wasser ablaufenden Reaktion und einer billigen, nachhaltigen Kohlenstoffquelle. Die so hergestellten Produkte eröffnen vielseitige Anwendungsmöglichkeiten, z. B. zur Molekültrennung in der Flüssigchromatographie, in der Energiespeicherung als Anodenmaterial in Li-Ionen Akkus oder Superkondensatoren, oder als Trägermaterial für die gezielte Pharmakotherapie.

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar-cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono- and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high-performance liquid chromatography (HPLC) and the products were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The main products are mono- and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D-glucose and D-fructose, followed by D-xylose and D-maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross-linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented.