The objectives of the research were to examine the effects of high dietary levels of fibrous feeds, and of supplementation with Roxazyme® G2 (RX), on the digestive metabolic and physiological responses of growing pigs fed maize-soybean diets. The nutrient and dietary fibre (DF) composition, the swelling and water-binding capacities of maize (MM), its hominy chop (HC) and cobs (MC), dehulled soybean (dSBM) and the hulls (SH), brewer’s grains (BG), lucerne hay (LH) and wheat bran (WB) were evaluated using standard procedures. Feed fibre fractions were isolated by simulating upper tract digestion in an Ankom® DaisyII Incubator, whereby each feed was digested in pepsin (porcine, 200 FIP-U/g, Merck No, 7190), followed by pancreatin (porcine, grade IV, Sigma No P-1750), with recovery of the fibrous residues. In a third step to complete the simulated pig gastro-intestinal digestion, the pepsin-pancreatin fibre extracts were digested by RX or Viscozyme L ® V2010 (VZ). Enzyme activity was measured as the coefficients of partial degradability (solubilisation) of the washed fibre extracts. The kinetics and products of fermentation of the DF were evaluated in an AnkomRF gas production system, using buffered faecal inoculum. Among the feed ingredients, dissimilar, fibre source-dependent activities between RX (0.02 to 0.12) and VZ (0.04-0.33) were observed. The lowest RX activities were observed on the maize and soybean derived fibres, with similarly low VZ activity on MC fibre. Variation in the activity of faecal microbial enzymes was similarly indicated by the variable production of fermentation gas (51.8-299.4 mL g-1 DM) and short chain fatty acids (SCFA) (2.3-6.0 mMol g-1 DM). Soy hull, dSBH, MM and HC fibres were highly fermentable, with low fermentability of BG, MC and WB fibres. The fibres differed in the composition of fermentation SCFA, whereby SH, LH and MC shifted fermentation to Ace, and BG, dSBM, WB, MM, HC favoured Pro, while MM and HC favoured But production. The same nutritional properties were similarly evaluated in complete diets which were formulated from the ingredients for growth, and metabolic trials. For the growth trial, a standard (STD) (control), 141 g total dietary fibre (TDF) kg-1 dry matter (DM) maize-soybean growing pig diet, and five iso-nutritive, 246 g TDF kg-1 DM nutritionally balanced diets were formulated. The high DF was achieved by partial replacement of the MM and dSBM in the STD diet with MC, SH, BG, LH or WB. The differences in RX and VZ activities and in the fermentation characteristics which were observed on the fibre extracts from the high fibre ingredients were reflected in the DF from the respective complete diets in which they were included. However, the fibre from the basal dietary ingredients reduced the absolute values and the variation in the activities of RX (0.03-0.06) and VZ (0.16-0.22), and similarly reduced the variation in gas (126.6-187.6 mL g-1 DM) and SCFA (4.1-5.4 mMol g-1 DM) production of the DF from the fibrous diets. Enzyme activities on the STD DF were low for RX (0.03) and high for VZ (0.25). The STD DF produced 205.3 mL gas g-1 DM, which was similar to SH DF, and higher than all the other diets. The STD DF produced 5.0-mMol SCFA g-1 DM, which was quantitatively, and not statistically higher than the other fibres. The composition of SCFA was similar across all diets, except for the high percent Ace, with low Pro by the SH DF. Compared to the STD, the high DF diets increased percent Ace, with reduced Pro and But. The STD, MC, SH, BG, LH and WB diets were each prepared in duplicate mixes, one of which was fortified with 200 mg RX kg-1 feed (as fed). Seventy-two intact Large White X Landrace, male, 32.0 ± 5.6 kg live weight (LW) pigs were allocated to the diets in two completely randomised weight blocks in a 2 (fibre source) X 2 (enzyme) factorial arrangement. The pigs were fed ad libitum for 10 weeks. Cumulative LW gain and feed intake were measured at different stages of growth, and at slaughter. Apparent total tract digestibility (ATTD) of nutrients was estimated at 65-70 kg LW, using 0.2% (as fed) chromium oxide as the indigestible marker. Ileal tissue was sampled 50 cm above the ileo-caecal valve, on which villi height and area, and crypt depth were evaluated by computerised image analysis. Blood was sampled at slaughter from the severed vena jugularis, 16 hours after feeding. Serum urea, creatinine, triglycerides, glucose, and total cholesterol were analysed chemically. The serum metabolome was further explored using Proton Nuclear Magnetic Resonance Spectroscopy (1H -NMRS). There was fibre X RX interaction for villi height, whereby the enzyme reduced the villi height in pigs on the SH, STD and WB diets, with an opposite effect on pigs on the MC, BG, LH diets. The soluble fibre content was negatively correlated with crypt depth. Chemical analysis did not detect differences in metabolite concentration between the STD and the high fibre diets. However, more serum cholesterol was observed in pigs fed the WB compared to the LH and MC diets. 1H-NMRS indicated that feeding pigs the WB diet increased serum Cys and His, while supplementation of RX increased serum formate, glucose, and urea. There was diet X enzyme interaction for fructose, glucose, Arg, Cys, Ser, and Trp, whereby RX increased the levels in pigs on MC and WB, with an opposite effect in pigs on the other diets. There was large DF source-dependent variation among diets in ATTD of DM (0.80-0.85), organic matter (OM) (0.81-0.87), gross energy (GE) (079-0.85) and CP (0.81-0.85), whereby, relative to the STD diet, high DF reduced the ATTD of DM (all diets except SH), organic matter (OM) and energy and CP (all diets except the MC). Positive correlation was observed between fermentability and the ATTD digestibility of DM, OM, energy, ADF, NDF, and fat. Negative correlation was observed between the swelling capacity and the ATTD of DM, OM, energy and protein, between DF solubility and DM, OM, protein, ADF and NDF, and between water binding capacity and ATTD of DM and OM, energy and NDF. At slaughter, there was similarly large, and DF source-dependent variation among the high fibre diets in feed intake (2.31-2.71 kg as fed day-1), live weight gain (0.75-0.86 kg day-1), and feed: gain ratio (2.73-3.00). Corresponding values for the STD diet were 2.44 kg day-1, 0.83 kg day-1and 2.86 kg day-1, respectively. Relative to the STD, LH reduced feed intake and live weight gain, and MC increased the feed: gain ratio. Predictions based on the in vitro fermentability of DF and feed intake suggested that due to poor fermentability, and or restriction of feed intake, relative to a standard fibre diet, high dietary levels of MC, WB and BG may reduce fermentation in the lower gut, while similar dietary levels of SH and LH may result in substantial increases in fermentation. At 50 kg LW, the fermentability of DF was positively correlated with feed intake and with weight gain, while water binding capacity and solubility of DF were negatively correlated with feed intake. At slaughter, the solubility of DF was negatively correlated with feed intake and feed: gain ratio. Large variation among the high fibre diets was also observed in the slaughter weight (89.2-96.8 kg), dressing % (68.6-76.4), meat colour (80.4-82.3), lean % (69.5-71.2), and fat % (10.1-12.6). In comparison, pigs on the STD diet scored 94.7 kg slaughter weight, 75.1% dressing, 81.6 cm carcass length, 82.5 meat colour, 68.4% lean, and 15.0% fat. Relative to the STD, LH reduced dressing and fat %. Lucerne hay and WB increased the lean%. For the metabolic trial, two iso-nutritive, mixed high fibre (319 g TDF kg-1 DM), nutritionally balanced diets were formulated to contain DF of high (HF) versus low (LF) fermentability. The diets had similar content of soluble DF and similar swelling and water binding capacities. Viscozyme was more active than RX on both the HF (0.20 versus 0.04) and the LF (0.17 versus 0.07) DF. The combination of RX and VZ statistically increased the enzyme activity on the HF (0.25) and quantitatively increased enzyme activity on the LF (0.18) DF, suggesting additive or synergistic effects. More gas was produced by the HF (159.5 mL g-1 DM) compared to the LF DF (96.6 mL g-1 DM). More SCFA were produced by HF (5.0 mMol g-1 DM), compared to the LF DF (3.6 mMol g-1 DM). Compared to the STD, HF DF increased percent Ace, with reduced Pro and But. The LF DF increased percent Ace, with quantitative, and not statistical reduction of Pro and But. In a metabolic trial, the HF and LF diets, and their duplicates containing 0.270 g RX kg-1 DM of feed (as fed) were fed ad libitum to eight ileum T-cannulised, intact Large White X Landrace male pigs weighing 65.0 ± 5.1 kg. The diets were allocated to the pigs in a duplicate 4 x 4 Latin Square design, in a 2 (enzyme) x 2 (fermentability) factorial arrangement. Each period consisted of two weeks of adaptation followed by five days of sampling. The ileal digesta was collected in each period and was similarly subjected to the fermentation test. Apparent ileal digestibility (AID) and ATTD were determined using 0.2% (as fed) chromium oxide as the indigestible marker. N excretion in faeces and urine were measured, and N retention was calculated. Blood was sampled by vena jugularis puncture on the last day of each period. Two blood samples were collected, the first 15 hours after removal from feed (15-hour serum), and the second 3 hours after re-introduction to feed (3-hour serum). Serum metabolites were evaluated by both chemical analyses and by 1H-NMRS, as described for the growth trial. Roxazyme did not affect the fermentation characteristics of the ileal digesta. In similar proportion to the fermentability of the PP digesta, the HF ileal digesta was more fermentable (65.4 mL gas g-1 DM and 6.1 mMol SCFA g-1 DM) than the LF ileal digesta (46.7 mL gas g-1 DM and 4.4 mMol SCFA g-1 DM SCFA). Prediction based on the in vitro fermentability of DF and feed intake suggested the HF diet could support one half times more fermentation in the lower gut compared to the LF diet. The HF diet had higher AID of DM (62.5 vs. 58.6), OM (65.6 vs. 62.1), energy (64.4 vs. 61.0), fat (85.8 vs. 81.7) and ash (41.8 vs. 32.7). The AID of HO-Pro, Met and Val were higher for the LF diet. There was diet X enzyme interaction on the AID of Met, whereby the RX reduced the AID of met in the LF diet, and not that of the HF diet. The ATTD was higher for the HF diet for DM (74.2 vs. 68.4), NDF (64.7 vs. 57.4), and ADF (35.1 vs. 21.0). There was positive correlation between the fermentability of DF and the AID DM, OM, ash, ash, fat and energy. The solubility of DF was negatively correlated with the AID of DM, OM, ash, fat, ADF and energy, and with the ATTD of DM, OM, ash, fat, energy, NDF, and ADF. Negative correlation was also observed between the swelling capacity of DF and the AID of protein, Trp and Lys. The solubility of DF was positively correlated with Ser, Ala, Val, Iso-Leu and His. There was diet X enzyme interaction for urea in the 15- hour serum, whereby RX tended to reduce the urea in the LF diet, while it increased that of the HF diet. Fermentability negatively correlated with urea in the 15- hour serum, and positively correlated with serum glucose in the 3-hour serum. In the 3-hour sample, 1H-NMRS indicated higher fucose, Pro and cholesterol in the LF diet. 1H-NMRS also indicated fermentability x RX interaction for Ser, Tyr, Lys, creatine, and possibly, glucose or fructose, glycerol or Gly and His or Arg, whereby RX increased the levels in the LF diets, with opposite effect in the HF diet. In conclusion, enzyme activities and fermentability were higly variable among different DF sources, and the effects were evident in the fibrous complete diets. The results of the in vitro studies supported the application of the methods to formulate fermentable insoluble fibre-rich, maize-soybean-mixed co-product diets. Correlation analyses suggested that DF fermentability, and solubility, swelling and water binding capacities explained significant proportions of the variances of the metabolic and physiological responses of the pigs to different feeds. Predictions based on the in vitro fermentability of DF and feed intake suggested that a strategy whereby pig diets are enriched in DF after the feedstuffs are screened on DF fermentability could substantially increase fermentation in the lower gut. Overall, the results suggested that productivity can be maintained in growing pigs fed diets containing up to twice the standard levels of DF, provided producers target co-product feeds that contain highly fermentable DF. The use of RX to improve nutrient digestion and to stimulate gut fermentation was not justified. / Environmental Sciences / Ph.D. (Environmental Sciences)

El presente trabajo analiza la influencia de los regímenes cambiarios sobre la dolarización de los pasivos empresariales, focalizándose en la diferencia entre regímenes fijos y flexibles. Para hacerlo se utiliza una muestra de 237 empresas de Argentina, Brasil, Colombia y México para el período 1992-2000, la metodología de estimación GMM-system para modelos de panel dinámicos y dos clasificaciones de regímenes cambiarios. Los resultados sugieren que los regímenes cambiarios fijos, así como su duración y volatilidad, influyen positivamente sobre la proporción de deuda en moneda extranjera mantenida por las firmas. Asimismo, se exploran otros determinantes del grado de dolarización de los pasivos corporativos, introduciéndose variables sugeridas por la literatura pero no analizadas hasta el momento. Se observa que la inestabilidad de la economía afecta las decisiones de cartera de las firmas. A su vez, las expectativas de salvataje por parte del Estado y las regulaciones generan problemas de información asimétrica, incentivando a las firmas a tomar un mayor riesgo cambiario. Por último se explora la relación entre la dolarización de los pasivos corporativos y el original sin interno, concluyendo que la reducción de éste último es, en parte, alcanzada vía dolarización de la deuda de largo plazo.

The objectives of the research were to examine the effects of high dietary levels of fibrous feeds, and of supplementation with Roxazyme® G2 (RX), on the digestive metabolic and physiological responses of growing pigs fed maize-soybean diets. The nutrient and dietary fibre (DF) composition, the swelling and water-binding capacities of maize (MM), its hominy chop (HC) and cobs (MC), dehulled soybean (dSBM) and the hulls (SH), brewer’s grains (BG), lucerne hay (LH) and wheat bran (WB) were evaluated using standard procedures. Feed fibre fractions were isolated by simulating upper tract digestion in an Ankom® DaisyII Incubator, whereby each feed was digested in pepsin (porcine, 200 FIP-U/g, Merck No, 7190), followed by pancreatin (porcine, grade IV, Sigma No P-1750), with recovery of the fibrous residues. In a third step to complete the simulated pig gastro-intestinal digestion, the pepsin-pancreatin fibre extracts were digested by RX or Viscozyme L ® V2010 (VZ). Enzyme activity was measured as the coefficients of partial degradability (solubilisation) of the washed fibre extracts. The kinetics and products of fermentation of the DF were evaluated in an AnkomRF gas production system, using buffered faecal inoculum. Among the feed ingredients, dissimilar, fibre source-dependent activities between RX (0.02 to 0.12) and VZ (0.04-0.33) were observed. The lowest RX activities were observed on the maize and soybean derived fibres, with similarly low VZ activity on MC fibre. Variation in the activity of faecal microbial enzymes was similarly indicated by the variable production of fermentation gas (51.8-299.4 mL g-1 DM) and short chain fatty acids (SCFA) (2.3-6.0 mMol g-1 DM). Soy hull, dSBH, MM and HC fibres were highly fermentable, with low fermentability of BG, MC and WB fibres. The fibres differed in the composition of fermentation SCFA, whereby SH, LH and MC shifted fermentation to Ace, and BG, dSBM, WB, MM, HC favoured Pro, while MM and HC favoured But production. The same nutritional properties were similarly evaluated in complete diets which were formulated from the ingredients for growth, and metabolic trials. For the growth trial, a standard (STD) (control), 141 g total dietary fibre (TDF) kg-1 dry matter (DM) maize-soybean growing pig diet, and five iso-nutritive, 246 g TDF kg-1 DM nutritionally balanced diets were formulated. The high DF was achieved by partial replacement of the MM and dSBM in the STD diet with MC, SH, BG, LH or WB. The differences in RX and VZ activities and in the fermentation characteristics which were observed on the fibre extracts from the high fibre ingredients were reflected in the DF from the respective complete diets in which they were included. However, the fibre from the basal dietary ingredients reduced the absolute values and the variation in the activities of RX (0.03-0.06) and VZ (0.16-0.22), and similarly reduced the variation in gas (126.6-187.6 mL g-1 DM) and SCFA (4.1-5.4 mMol g-1 DM) production of the DF from the fibrous diets. Enzyme activities on the STD DF were low for RX (0.03) and high for VZ (0.25). The STD DF produced 205.3 mL gas g-1 DM, which was similar to SH DF, and higher than all the other diets. The STD DF produced 5.0-mMol SCFA g-1 DM, which was quantitatively, and not statistically higher than the other fibres. The composition of SCFA was similar across all diets, except for the high percent Ace, with low Pro by the SH DF. Compared to the STD, the high DF diets increased percent Ace, with reduced Pro and But. The STD, MC, SH, BG, LH and WB diets were each prepared in duplicate mixes, one of which was fortified with 200 mg RX kg-1 feed (as fed). Seventy-two intact Large White X Landrace, male, 32.0 ± 5.6 kg live weight (LW) pigs were allocated to the diets in two completely randomised weight blocks in a 2 (fibre source) X 2 (enzyme) factorial arrangement. The pigs were fed ad libitum for 10 weeks. Cumulative LW gain and feed intake were measured at different stages of growth, and at slaughter. Apparent total tract digestibility (ATTD) of nutrients was estimated at 65-70 kg LW, using 0.2% (as fed) chromium oxide as the indigestible marker. Ileal tissue was sampled 50 cm above the ileo-caecal valve, on which villi height and area, and crypt depth were evaluated by computerised image analysis. Blood was sampled at slaughter from the severed vena jugularis, 16 hours after feeding. Serum urea, creatinine, triglycerides, glucose, and total cholesterol were analysed chemically. The serum metabolome was further explored using Proton Nuclear Magnetic Resonance Spectroscopy (1H -NMRS). There was fibre X RX interaction for villi height, whereby the enzyme reduced the villi height in pigs on the SH, STD and WB diets, with an opposite effect on pigs on the MC, BG, LH diets. The soluble fibre content was negatively correlated with crypt depth. Chemical analysis did not detect differences in metabolite concentration between the STD and the high fibre diets. However, more serum cholesterol was observed in pigs fed the WB compared to the LH and MC diets. 1H-NMRS indicated that feeding pigs the WB diet increased serum Cys and His, while supplementation of RX increased serum formate, glucose, and urea. There was diet X enzyme interaction for fructose, glucose, Arg, Cys, Ser, and Trp, whereby RX increased the levels in pigs on MC and WB, with an opposite effect in pigs on the other diets. There was large DF source-dependent variation among diets in ATTD of DM (0.80-0.85), organic matter (OM) (0.81-0.87), gross energy (GE) (079-0.85) and CP (0.81-0.85), whereby, relative to the STD diet, high DF reduced the ATTD of DM (all diets except SH), organic matter (OM) and energy and CP (all diets except the MC). Positive correlation was observed between fermentability and the ATTD digestibility of DM, OM, energy, ADF, NDF, and fat. Negative correlation was observed between the swelling capacity and the ATTD of DM, OM, energy and protein, between DF solubility and DM, OM, protein, ADF and NDF, and between water binding capacity and ATTD of DM and OM, energy and NDF. At slaughter, there was similarly large, and DF source-dependent variation among the high fibre diets in feed intake (2.31-2.71 kg as fed day-1), live weight gain (0.75-0.86 kg day-1), and feed: gain ratio (2.73-3.00). Corresponding values for the STD diet were 2.44 kg day-1, 0.83 kg day-1and 2.86 kg day-1, respectively. Relative to the STD, LH reduced feed intake and live weight gain, and MC increased the feed: gain ratio. Predictions based on the in vitro fermentability of DF and feed intake suggested that due to poor fermentability, and or restriction of feed intake, relative to a standard fibre diet, high dietary levels of MC, WB and BG may reduce fermentation in the lower gut, while similar dietary levels of SH and LH may result in substantial increases in fermentation. At 50 kg LW, the fermentability of DF was positively correlated with feed intake and with weight gain, while water binding capacity and solubility of DF were negatively correlated with feed intake. At slaughter, the solubility of DF was negatively correlated with feed intake and feed: gain ratio. Large variation among the high fibre diets was also observed in the slaughter weight (89.2-96.8 kg), dressing % (68.6-76.4), meat colour (80.4-82.3), lean % (69.5-71.2), and fat % (10.1-12.6). In comparison, pigs on the STD diet scored 94.7 kg slaughter weight, 75.1% dressing, 81.6 cm carcass length, 82.5 meat colour, 68.4% lean, and 15.0% fat. Relative to the STD, LH reduced dressing and fat %. Lucerne hay and WB increased the lean%. For the metabolic trial, two iso-nutritive, mixed high fibre (319 g TDF kg-1 DM), nutritionally balanced diets were formulated to contain DF of high (HF) versus low (LF) fermentability. The diets had similar content of soluble DF and similar swelling and water binding capacities. Viscozyme was more active than RX on both the HF (0.20 versus 0.04) and the LF (0.17 versus 0.07) DF. The combination of RX and VZ statistically increased the enzyme activity on the HF (0.25) and quantitatively increased enzyme activity on the LF (0.18) DF, suggesting additive or synergistic effects. More gas was produced by the HF (159.5 mL g-1 DM) compared to the LF DF (96.6 mL g-1 DM). More SCFA were produced by HF (5.0 mMol g-1 DM), compared to the LF DF (3.6 mMol g-1 DM). Compared to the STD, HF DF increased percent Ace, with reduced Pro and But. The LF DF increased percent Ace, with quantitative, and not statistical reduction of Pro and But. In a metabolic trial, the HF and LF diets, and their duplicates containing 0.270 g RX kg-1 DM of feed (as fed) were fed ad libitum to eight ileum T-cannulised, intact Large White X Landrace male pigs weighing 65.0 ± 5.1 kg. The diets were allocated to the pigs in a duplicate 4 x 4 Latin Square design, in a 2 (enzyme) x 2 (fermentability) factorial arrangement. Each period consisted of two weeks of adaptation followed by five days of sampling. The ileal digesta was collected in each period and was similarly subjected to the fermentation test. Apparent ileal digestibility (AID) and ATTD were determined using 0.2% (as fed) chromium oxide as the indigestible marker. N excretion in faeces and urine were measured, and N retention was calculated. Blood was sampled by vena jugularis puncture on the last day of each period. Two blood samples were collected, the first 15 hours after removal from feed (15-hour serum), and the second 3 hours after re-introduction to feed (3-hour serum). Serum metabolites were evaluated by both chemical analyses and by 1H-NMRS, as described for the growth trial. Roxazyme did not affect the fermentation characteristics of the ileal digesta. In similar proportion to the fermentability of the PP digesta, the HF ileal digesta was more fermentable (65.4 mL gas g-1 DM and 6.1 mMol SCFA g-1 DM) than the LF ileal digesta (46.7 mL gas g-1 DM and 4.4 mMol SCFA g-1 DM SCFA). Prediction based on the in vitro fermentability of DF and feed intake suggested the HF diet could support one half times more fermentation in the lower gut compared to the LF diet. The HF diet had higher AID of DM (62.5 vs. 58.6), OM (65.6 vs. 62.1), energy (64.4 vs. 61.0), fat (85.8 vs. 81.7) and ash (41.8 vs. 32.7). The AID of HO-Pro, Met and Val were higher for the LF diet. There was diet X enzyme interaction on the AID of Met, whereby the RX reduced the AID of met in the LF diet, and not that of the HF diet. The ATTD was higher for the HF diet for DM (74.2 vs. 68.4), NDF (64.7 vs. 57.4), and ADF (35.1 vs. 21.0). There was positive correlation between the fermentability of DF and the AID DM, OM, ash, ash, fat and energy. The solubility of DF was negatively correlated with the AID of DM, OM, ash, fat, ADF and energy, and with the ATTD of DM, OM, ash, fat, energy, NDF, and ADF. Negative correlation was also observed between the swelling capacity of DF and the AID of protein, Trp and Lys. The solubility of DF was positively correlated with Ser, Ala, Val, Iso-Leu and His. There was diet X enzyme interaction for urea in the 15- hour serum, whereby RX tended to reduce the urea in the LF diet, while it increased that of the HF diet. Fermentability negatively correlated with urea in the 15- hour serum, and positively correlated with serum glucose in the 3-hour serum. In the 3-hour sample, 1H-NMRS indicated higher fucose, Pro and cholesterol in the LF diet. 1H-NMRS also indicated fermentability x RX interaction for Ser, Tyr, Lys, creatine, and possibly, glucose or fructose, glycerol or Gly and His or Arg, whereby RX increased the levels in the LF diets, with opposite effect in the HF diet. In conclusion, enzyme activities and fermentability were higly variable among different DF sources, and the effects were evident in the fibrous complete diets. The results of the in vitro studies supported the application of the methods to formulate fermentable insoluble fibre-rich, maize-soybean-mixed co-product diets. Correlation analyses suggested that DF fermentability, and solubility, swelling and water binding capacities explained significant proportions of the variances of the metabolic and physiological responses of the pigs to different feeds. Predictions based on the in vitro fermentability of DF and feed intake suggested that a strategy whereby pig diets are enriched in DF after the feedstuffs are screened on DF fermentability could substantially increase fermentation in the lower gut. Overall, the results suggested that productivity can be maintained in growing pigs fed diets containing up to twice the standard levels of DF, provided producers target co-product feeds that contain highly fermentable DF. The use of RX to improve nutrient digestion and to stimulate gut fermentation was not justified. / Environmental Sciences / Ph.D. (Environmental Sciences)

The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium. Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex. / QC 20101125

Scintillation of Global Positioning Systems (GPS) signals have been extensively studied at low and high latitude regions of the Earth. It has been shown in past studies that amplitude scintillation is severe at low latitudes and phase scintillation is severe at high latitudes. Unlike low and high latitude regions, mid-latitude scintillation has not been extensively studied. Further, it has been suggested that mid-latitude scintillation is negligible. The purpose of this research is to challenge this belief. A multi-constellation and multi-frequency receiver, that tracks American, Russian, and European satellites, was used to monitor scintillation activity at the Virginia Tech Space Center. Analysis was performed on collected data from various days and compared to past research done at high, mid, and low latitudes. The results are discussed in this thesis. / Master of Science

Das humane Zytomegalievirus, ist ein ubiquitäres Pathogen, welches akute und persistierende Infektionen verursacht. Bei immunsupprimierten Patienten kann das Virus zu schweren Erkrankungen, wie Hepatitis, Pneumonie und bei kongenitaler Infektion außerdem zu Schädigungen des ZNS führen. HCMV blockiert die Zellproliferation durch einen Arrest am G1/S-Übergang des Zellzyklus, andererseits wird aber gleichzeitig die Expression S-Phase spezifischer Gene aktiviert. Teilweise lässt sich dies durch eine Virus vermittelte spezifische Inhibition der zellulären DNA-Repliaktion sowie durch eine massive Deregulation Zyklin-assozzierter Kinasen erklären. Zellkulturexperimente deuten darauf hin, dass die Zellzyklusalterationen wichtige Voraussetzungen für eine erfolgreiche Virusreplikation darstellen. Es ist hingegen nicht bekannt, welche Relevanz sie für die Virusvermehrung in vivo und das pathologische Erscheinungsbild im erkrankten Organismus besitzen. Diese Frage kann nur in einem Tiermodell sinnvoll angegangen werden. Aufgrund der Wirtsspezifität der Zytomegalieviren, ist man dabei auf die Verwendung der jeweiligen artspezifischen CMV angewiesen. Murines CMV (MCMV) und Ratten-CMV (RCMV) sind dabei die bislang bestuntersuchtesten Systeme. Das Anliegen dieser Arbeit war es zu prüfen, inwieweit die für HCMV beschriebenen Zellzyklusregulationen in MCMV und RCMV auf Zellkulturbasis konserviert sind. Es konnte gezeigt werden, dass sowohl RCMV als auch MCMV einen antiproliferativen Effekt auf infizierte Zellen besitzen und ebenso wie HCMV zu einem Zellzyklusarrest führen. Nager-Zytomegalieviren können Zellen auch in der G2-Phase arretieren und in dieser Zellzyklusphase auch effizient replizieren können. Die Infektion mit Nager-CMV führt außerdem auf breiter Basis zur Veränderung Zyklin-assoziierter Kinaseaktivitäten. Allen Zytomegalieviren ist die Hemmung der zellulären DNA-Synthese am G1/S-Übergang durch die Inhibition des replication licensing, dem Beginn der DNA-Synthese gemein. Durch diese vergleichende Studie wird einerseits deutlich, dass wesentliche funktionelle Schritte der Zellzyklusregulation zwischen den Zytomegalieviren konserviert sind, aber andererseits die zu Grunde liegenden molekularen Mechanismen zum Teil deutlich variieren. / Human Cytomegalovirus (HCMV) is an ubiquitous, species-specific beta-herpesvirus that, like other herpesviruses, can establish lifelong latency following primary infection. HCMV infection becomes virulent only in immunocompromised patients such as premature infants, transplant recipients and AIDS patients where the virus causes severe disease like hepatitis, pneumonitis and retinitis. Congenital infection produces birth defects, most commonly hearing loss. To develop rational-based strategies for prevention and treatment of HCMV infection, it is crucial to understand the interactions between the virus and its host cell that support the establishment and progression of the virus replicative cycle. In general, herpesviruses are known to replicate most efficiently in the absence of cellular DNA synthesis. What is more, they have evolved mechanisms to avoid the cell´s DNA replication phase by blocking cell cycle progression outside S phase. HCMV has been shown to specifically inhibit the onset of cellular DNA synthesis resulting in cells arrested with a G1 DNA content. Towards a better understanding of CMV-mediated cell cycle alterations in vivo, we tested murine and rat CMV (MCMV/RCMV), being common animal models for CMV infection, for their influence on the host cell cycle. It was found that both MCMV and RCMV exhibit a strong anti-proliferative capacity on immortalised and primary embryonic fibroblasts after lytic infection. This results from specific cell cycle blocks in G1 and G2 as demonstrated by flow cytometry analysis. The G1 arrest is at least in part caused by a specific inhibition of cellular DNA synthesis and involves both the formation and activation of the cells’ DNA replication machinery. Interestingly, and in contrast to HCMV, the replicative cycle of rodent CMVs started from G2 as efficiently as from G1. Whilst the cell cycle arrest is accompanied by a broad induction of cyclin-cdk2 and cyclin-cdk1 activity, cyclin D1-cdk4/6 activity is selectively suppressed in MCMV and RCMV infected cells. Thus, given that both rodent and human CMVs are anti-proliferative and arrest cell cycle progression we found a surprising divergence of some of the underlying mechanisms. Therefore, any question put forward to a rodent CMV model involving cell cycle regulation has to be well defined in order to extrapolate meaningful information for the human system.

No description available.

<p>У оквиру ове докторске дисертације испитана је биолошка активност екстраката плодних тела и потопљених култура (мицелије и филтрата) аутохтоних врста гљива <em>Coprinus comatus</em> и<em> Coprinellus truncorum</em>. Такође, испитан је&nbsp; метаболизам фосфата мицелија обе врсте употребом нуклеарно магнетне резонантне спректроскопије (³¹Р NMR), утицај ванадијума на метаболизам фосфата као и идентификација облика ванадата присутних у ћелији мицелије (⁵¹V NMR). Утврђена је антирадикалска и антиоксидативна активност&nbsp; етанолних,метанолних и водених екстраката гљива при чему су се екстракти потопљених култура издвојили по антирадикалској, а екстракти плодних тела по антиоксидативној активности. Екстракти потопљених култура истакли су се и у погледу антибактеријске активности, где се као најпотентнији показао&nbsp; хлороформски екстракт филтрата потопљене културе<em> C. comatus</em>. Такође, етанолни екстракт филтрата потопљене културе <em>C. comatus</em> показао се као најпотентнији у анти-ацетилхолинестеразној активности у односу на&nbsp; конвенционални лек донепезил. Испитан је и утицај екстраката на вијабилност ћелијских линија HepG2 (хумане хепатома ћелије) и Rin-5F (&szlig; ћелије панкреаса пацова).</p><p>Спектрофотометријским методама одређен је укупан садржај фенола и флавоноида у већини анализираних екстраката.</p><p>LC/MS идентификацијом и квантификацијом фенолних киселина уочена је разлика између фенолних једињења присутних у плодном телу, мицелији и филтрату потопљене културе. Екстракти потопљених култура бележе већи број и већи садржај једињења. Укупан садржај протеина одређен само у воденим екстрактима, а укупан садржај угљених хидрата у полисахаридним екстрактима.Употребом Фуријеве инфрацрвене спектроскопске методе (FTIR) детектоване су везе између угљених хидрата&nbsp; присутних у полисахаридним екстрактима, а планарном&nbsp; хроматографијом показано је да екстракти плодног тела и филтрата врсте <em>С. truncorum</em>, као и екстракт плодног тела врсте <em>C</em>. <em>comatus</em>, садрже велику&nbsp; количину D-глукозе, док екстракт мицелије <em>C. truncorum</em>, баш као и екстракти филтрата и мицелије <em>C. comatus</em>, садрже највише галактозе. Квалитативном и квантитативном елементарном анализом (ААS) утврђен је виши садржај&nbsp; калијума и гвожђа у анализираним узорцима. GC-МS идентификацијом и квантификацијом масних киселина указано је на значајно присуство линолне киселине код обе врсте.&nbsp;<br />Како за аутохтону врсту&nbsp; <em>C.truncorum </em>постоји мало података у литератури, подаци о њеном хемијском саставу могу се сматрати иновативним.<br />Компаративним прегледом биолошке активности и хемијског састава екстраката плодног тела и мицелије и филтрата (потопљених култура) указано је да су анализирани екстракти извори биоактивних супстанци са медицинским потенцијалом, а потопљене културе датих гљива представљају атрактивне кандидате за даља биотехнолошка истраживања.</p> / <p>U okviru ove doktorske disertacije ispitana je biološka aktivnost ekstrakata plodnih tela i potopljenih kultura (micelije i filtrata) autohtonih vrsta gljiva <em>Coprinus comatus</em> i<em> Coprinellus truncorum</em>. Takođe, ispitan je&nbsp; metabolizam fosfata micelija obe vrste upotrebom nuklearno magnetne rezonantne sprektroskopije (³¹R NMR), uticaj vanadijuma na metabolizam fosfata kao i identifikacija oblika vanadata prisutnih u ćeliji micelije (⁵¹V NMR). Utvrđena je antiradikalska i antioksidativna aktivnost&nbsp; etanolnih,metanolnih i vodenih ekstrakata gljiva pri čemu su se ekstrakti potopljenih kultura izdvojili po antiradikalskoj, a ekstrakti plodnih tela po antioksidativnoj aktivnosti. Ekstrakti potopljenih kultura istakli su se i u pogledu antibakterijske aktivnosti, gde se kao najpotentniji pokazao&nbsp; hloroformski ekstrakt filtrata potopljene kulture<em> C. comatus</em>. Takođe, etanolni ekstrakt filtrata potopljene kulture <em>C. comatus</em> pokazao se kao najpotentniji u anti-acetilholinesteraznoj aktivnosti u odnosu na&nbsp; konvencionalni lek donepezil. Ispitan je i uticaj ekstrakata na vijabilnost ćelijskih linija HepG2 (humane hepatoma ćelije) i Rin-5F (&szlig; ćelije pankreasa pacova).</p><p>Spektrofotometrijskim metodama određen je ukupan sadržaj fenola i flavonoida u većini analiziranih ekstrakata.</p><p>LC/MS identifikacijom i kvantifikacijom fenolnih kiselina uočena je razlika između fenolnih jedinjenja prisutnih u plodnom telu, miceliji i filtratu potopljene kulture. Ekstrakti potopljenih kultura beleže veći broj i veći sadržaj jedinjenja. Ukupan sadržaj proteina određen samo u vodenim ekstraktima, a ukupan sadržaj ugljenih hidrata u polisaharidnim ekstraktima.Upotrebom Furijeve infracrvene spektroskopske metode (FTIR) detektovane su veze između ugljenih hidrata&nbsp; prisutnih u polisaharidnim ekstraktima, a planarnom&nbsp; hromatografijom pokazano je da ekstrakti plodnog tela i filtrata vrste <em>S. truncorum</em>, kao i ekstrakt plodnog tela vrste <em>C</em>. <em>comatus</em>, sadrže veliku&nbsp; količinu D-glukoze, dok ekstrakt micelije <em>C. truncorum</em>, baš kao i ekstrakti filtrata i micelije <em>C. comatus</em>, sadrže najviše galaktoze. Kvalitativnom i kvantitativnom elementarnom analizom (AAS) utvrđen je viši sadržaj&nbsp; kalijuma i gvožđa u analiziranim uzorcima. GC-MS identifikacijom i kvantifikacijom masnih kiselina ukazano je na značajno prisustvo linolne kiseline kod obe vrste.&nbsp;<br />Kako za autohtonu vrstu&nbsp; <em>C.truncorum </em>postoji malo podataka u literaturi, podaci o njenom hemijskom sastavu mogu se smatrati inovativnim.<br />Komparativnim pregledom biološke aktivnosti i hemijskog sastava ekstrakata plodnog tela i micelije i filtrata (potopljenih kultura) ukazano je da su analizirani ekstrakti izvori bioaktivnih supstanci sa medicinskim potencijalom, a potopljene kulture datih gljiva predstavljaju atraktivne kandidate za dalja biotehnološka istraživanja.</p> / <p>The biological activity of extracts of basidiocarps (fruiting bodies)&nbsp; and submerged cultures (mycelium and filtrate) of autochthonous mushroom species&nbsp; <em>Coprinus comatus</em> and&nbsp; <em>Coprinellus truncorum&nbsp;</em> was examined. Furthermore, the metabolism of phosphate&nbsp; of mycelia&nbsp; of both types was studied using nuclear magnetic&nbsp; resonance spectros-copy ( ³¹ R NMR), the influence of vanadium on phosphate metabolism and the identification of vanadate oxidation states present in the mycelia cell ( ⁵¹ V NMR). The antiradical and antioxidant activity of methanolic, ethanolic and water fungal extracts was determined. Extracts of submerged cultures achieved the best anti- radical activity while fruit body extracts showed the best antioxidant activity. Extracts of submerged cultures also highlighted in terms of antibacterial activity, where the chloroform extract of the submerged culture&nbsp; <em>C. comatus</em>&nbsp; showed as the most potent. Also, the ethanolic extract of the submerged culture of<em>&nbsp; C. comatus</em>&nbsp; was found to be most relevant in anti-acetylcholinesterase activity&nbsp; compared with&nbsp; the conventional donepezil drug. The influence of extracts on the viability of cell lines HepG2 (human hepatocytes cells) and Rin-5F (&szlig; pancreatic cells of the rat) was also examined.</p><p>Spectrophotometric methods determined the total con-tent of phenol and flavonoids in most of the analyzed extracts.</p><p>The LC/MS identification and quantification of phenolic acids revealed the difference between the phenolic compounds present in the fruiting body, mycelium, and the submerged culture filtrate. Extracts of submerged cultures record a greater number and higher content of compounds.</p><p>The total content of proteins determined only in water extracts&nbsp; and the total content of&nbsp; carbohydrates in poly-saccharide extracts. Using the Fourier infrared spectro-scopic method (FTIR), the links between the sugar pre-sent in the&nbsp; polysaccharide extracts were detected, and planar chromatography showed that the extracts&nbsp; of the fruiting body and the filtrate of type<em>&nbsp; C. truncorum</em>, as well as the extract of the fruiting body of the species&nbsp; <em>C. comatus</em>, contain a large amount of D-glucose, while the extract of the&nbsp;<em> C</em>. <em>truncorum</em>&nbsp; mycelia&nbsp; and&nbsp; mycelia&nbsp; of&nbsp; <em>C. comatus</em>, contain the most galactose. GC-MS identification and quantification of fatty acids indicated a significant presence of linoleic acid in both species, while qualitative and quantitative elemental analysis (AAS) has determined a higher content of potas-sium and iron in the analyzed samples. Since there is no data in the literature for the autochtho-nous species&nbsp;<em> C</em>. <em>truncorum</em>, the studies on its chemical composition can be considered advanced аs innovative. A comparative review of the biological activity and the chemical composition of the extracts of the fruiting body and&nbsp; mycelia&nbsp; and filtrates&nbsp; of&nbsp; medium of&nbsp; submerged cultures&nbsp; indicated that the extracts were analyzed by sources of bioactive substances with medical potential, and the submerged cultures of these mushrooms are attractive candidates for biotechnological research.</p> / <p>В рамках данной работы была исследованна биологическая активность экстракта плодородных тел и погружонных видов култур (мицелии и филтрата) автотоных видов грибов <em>Coprinus comatus</em> и <em>Coprinellus truncorum</em>. Также, исследованн метаболизм фосфата обеих видов&nbsp; мицелий с помощью ядерного магнитного резонанса спектроскопии (³¹Р ЯМР), влияние на содержание ванадия в метаболизме фосфата, а также идентификация формы ванадата присущего в клеток мицеллий (⁵¹V ЯМР). Установленная антирадикальная и антиоксидантная активность метанольных, этанольных и водных экстрактов гриб, причём выделяются экстракты погружённых культур по антирадикальной активности и&nbsp; экстракты плодородных тел по антиоксидантной активности.</p><p>Экстракты погружённых культур выделялись и в плане антибактериальной активности, причем,&nbsp; наиболее мощным из филтратов показался экстракт хлороформа погруженной культуры <em>C. comatus.</em> А также этанольный экстракт филтрата погружённой культуры<em> C. comatus</em> оказался найболее мощным в анти-ацетихолинестеразной активностипо сравнению с традиционным лекарством донепезилом. Было исследовано и влияние экстрактов на виябильность клеток линий&nbsp;&nbsp; HepG2 (гуманые хепатома клетки) и Rin-5F (&szlig; клетки поджелудочной железы крыс).<br />Методом спектрофотометрии определена совокупность фенола и флавоноида в большинстве проанализированных экстрактах.<br />С помощью ЛС ̸МС идентификации и квантификации фенолных кислот была замечена разница между соединениями фенола, присущих в плодородном теле, и мицелии, и филтрата погружённой культуры. Экстракты погружённых культур отражают больше количество и более высокое содержание соединений.<br />Общее содержание белков выделен только в водяных экстрактах, и общее содержание углеводов в полисахаридных экстрактах. Используя инфракрасный метод спектроскопии Фурия (ИКМСФ) были обнаружены связи между сахарами, присущими в полисахаридных экстрактах, а планарной хромотографиой было показано, что экстракты плодородного тела и филтратов вида <em>С. truncorum</em>,&nbsp; а&nbsp; также и экстракты плодородного тела вида <em>C. comatus</em> содержат большое количество D-глюкозы, в то время как экстракт мицелии <em>C. truncorum,</em> именно как и экстракт фильтрата и мицелии <em>C. comatus</em>, содержат больше всего галактозы.<br />GC-МS идентификацией и квантификацией жирных кислот показано значительное наличие линолевой кислоты у обоих видах. А качественным и квантитативным элементарным анализом установленно большее содержание калиума и железа в анализированых шаблонах.<br />Из-за того, что для автохтонного вида <em>C. truncorum</em> практически не было данных в литературе, данные о её химическом составе можно считать прогрессивным и инновационным.<br />Сравнительный анализ биологической активности и химического состава экстрактов плодородного тела и мицелии и фильтрат (погружённых культур) показаывает, что проанализированные экстракты &mdash; источники биологически активных веществ с медицинским потенциалом, и погружённые культуры данных гриб являются привлекательными кандидатами для биотехнологических исследований.</p>

Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.