Dissertations / Theses on the topic 'Essential and non-essential amino acids'

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva- derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

Thesis (PhD(Agric) (Animal Sciences))--University of Stellenbosch, 2006.<br>This project consisted of five studies. The objectives were to determine the essential amino acid (AA) requirements of growing woolled lambs (Merino and Dohne Merino) and the essential AA profile of duodenal digesta pre-dominantly derived from microbial protein. The limiting essential AA`s in high rumen degradable protein (RDP) diets to growing lambs, where microbial protein is the primary source of AA`s, were also identified. The first study determined the essential AA profile of duodenal protein on a high rumen degradable diet and evaluated the impact of dietary RDP concentration and source [true RDP vs. non-protein nitrogen (NPN)] on the AA composition of supplied in the duodenum. The first trial in this study evaluated the effects of increasing true RDP levels on the essential AA composition of duodenal protein primarily derived from rumen microbes. The lambs had free access to wheat straw and fresh water. The daily RDP supplements were administered in two equal doses into the rumens through rumen cannulas at 07:00 and 19:00. Duodenal digesta was extracted with 6h intervals through T-type cannulas, inserted proximally to the common bile duct. Sampling time was advanced 2h every day to obtain duodenal samples on every even hour of a 24h period after three days. As expected, deficient RDP limited the supply of essential AA`s in the iv duodenum. When the true RDP supplements increased, the duodenal flow of essential AA`s also increased concomitantly, but appeared to level off at the higher RDP levels. Despite the positive quantitative effects of true RDP supplementation on AA supply to the duodenum, the AA profile in the duodenum was unaltered. Consequently, the essential AA profile of duodenal protein of sheep receiving high RDP diets, where microbial protein is the primary source of AA`s in the duodenum, is relatively constant and insensitive to dietary RDP concentration. In the second trial the effects of RDP source (true RDP vs. NPN) on the essential AA profile of duodenal protein on high RDP diets were evaluated by substituting increasing amounts of urea for true RDP in isonitrogenous teatments. Higher NPN increments reduced the daily supply of essential AA`s in the duodenum. In corroboration of the first trial, the AA profile of the duodenal protein was very constant, irrespective of the RDP source. Since microbial protein is the major source of duodenal AA`s on high RDP diets, this study supports the view that microbial protein has a relatively constant AA profile, but microbial protein yield varies according to several rate limiting factors in the rumen. A constant microbial AA profile allows accurate estimates of microbial essential AA supply in the small intestine if microbial protein production and fluid and particulate outflow rates from the rumen can be accurately predicted. This allows the development of more accurate undegradable protein (UDP) supplementation strategies, based on the essential AA requirements of animals. In the second study growing male Merino and Dohne Merino lambs were slaughtered at different weights and body condition scores. The digesta was removed from the stomachs and intestines and every organ or body part were weighed to determine the whole empty body (WEB) composition. The WEB was partitioned into the carcass, internall offal (stomachs, intestines, organs and blood) and external offal (head, feet, skin and wool). No differences were apparent in the proportional weight distribution of similar body components of the same breed at different ages. In a comparison between breeds, the proportional weight contributions of the carcasses from both breeds to the WEB weight were remarkably similar at both slaughtering stages. The Dohne Merino lambs had proportionally larger internal offals and smaller external offals than the Merino lambs at both slaughters. Unless the essential amino acid compositions of the internal and external offals were identical to the carcass, the dissimilarities in weight and protein allocation to these two components within the WEB`s of Merino and Dohne Merino lambs imply a distinct WEB essential AA composition for each breed. The apparent digestibilities of dry matter (DM), crude protein (CP), energy, acid detergent fibre (ADF), neutral detergent fibre (NDF), fat and ash did not differ between Merino and Dohne Merino lambs. Energy retention was also similar for the two breeds, but the Merino lambs retained considerably more N than the Dohne Merino lambs. This may also impact on the respective amino acid requirements of the lambs. Since the Merino lambs utilise N more efficiently, they may have potentially lower essential amino acid requirements to achieve a similar growth rate. The WEB essential AA compositions of growing Merino and Dohne Merino lambs were determined in the third study. Based on the ideal protein concept, the WEB essential AA profile was accepted as representative of the AA requirements for growth. The use of a single body part as a representation of the WEB AA profile was also evaluated. Differences in the proportional weight and protein contribution of the three body components (carcass, internal offal and external offal) of the two breeds strongly suggested that the WEB AA composition of the breeds would differ, because of likely differences in the AA profiles of these components. The essential AA profiles of the carcasses from the two breeds were surprisingly similar. However, the essential AA compositions of the internal offal and external offal differed substantially from each other, as well as from the carcass. In addition, the internal offal and external offals of each breed had characteristic essential AA profiles. Inevitably, the WEB essential AA profiles of Merino and Dohne Merino lambs differed considerably. Only the leucine and phenylalanine concentrations in the WEB`s of Merino and Dohne Merino lambs did not differ. Significant differences in the concentrations of eight essential AA`s implied that the two breeds have different AA requirements for growth. The different AA compositions of the internal and external offal within each breed also illustrated that the use of a single body component, like the carcass, as a predictor of WEB essential AA composition contains considerable inaccuracies. The essential AA index indicated that the duodenal protein, primarily derived from rumen microbes, provided approximately 81 % of the qualitative AA requirements of growing lambs. During periods of sufficient availability of very low-quality forage, as the diet in this study simulated, microbial protein is not able to support maximum growth. The first two limiting AA`s (histidine and methionine) could not even support daily growth rates of 100 g/d. This is very low and stresses the need for effective undegradable AA supplementation under these conditions. Chemical scores identified histidine as the first limiting AA in high RDP diets (predominantly microbial protein), followed by methionine, leucine, arginine and phenylalanine. However, the requirements for histidine and arginine are frequently over estimated and these AA`s should actually be considered semi-essential, which could render methionine, leucine and phenylalanine the first three limiting AA`s to growing lambs receiving high RDP diets. Because of the limitations of static measurement systems for the determination of AA requirements, a more comprehensive evaluation method was introduced for determination of the limiting AA`s in duodenal protein of lambs on high RDP diets, in the fourth study. The fourth study focused on the identification of limiting AA`s to growing lambs being limit-fed a high RDP diet. The diet consisted primarily of soybean hulls, for its’ low rumen UDP content. Microbial protein production was calculated as 13 % of total digestible nutrient intake and complementary AA supplements prepared to simulate the WEB AA profile, determined in the previous study, in the small intestine. To eliminate the influence of the rumen on the AA supplements, the latter were infused into the abomasums via flexible tubing. Each essential AA was in turn removed from the control treatment (simulating the WEB composition) and the effect on N retention measured. When methionine or the branched-chain amino acids (BCAA`s) were removed from the infusate, N retention of the lambs was reduced. Consequently, methionine and at least one of the BCAA`s limited growth performance of young lambs when microbial protein was the predominant source of AA`s. The concomitant increased plasma concentrations of total AA`s when methionine or the BCAA`s were removed from the infusate corroborates the effects on N retention, since it indicates that AA utilisation was reduced when these AA imbalances were introduced. Amino acid imbalances had no effect on apparent DM, organic matter (OM) or NDF digestion, but N digestibility was reduced. The final study verified whether the BCAA’s were co-limiting the growth of lambs, or if any single BCAA was responsible for the limitation. Again the WEB AA profile of growing lambs was simulated in the small intestine via abomasal infusions to lambs receiving a soybean hull-based diet. Leucine, isoleucine and valine were individually or simultaneously removed from the infusate and the impact on N retention measured. On an individual basis valine had the largest negative impact on the efficiency of N utilisation. However, the simultaneous removal of the BCAA`s resulted in the lowest N retention, suggesting that valine might be limiting, but the three BCAA`s are more likely to be co-limiting in diets to growing lambs where microbial protein is the primary source of AA`s. Once again, neither DM, OM or NDF digestibility were affected by the AA imbalances. Nitrogen digestibility was, however, negatively affected by AA imbalances. This project succeeded in establishing the essential AA profile of duodenal protein in sheep receiving high RDP diets. The WEB essential AA compositions of growing lambs from two prominent sheep breeds in South Afica were then determined and the duodenal essential AA profile evaluated against the calculated AA requirements. Finally, the AA`s that limit growth in diets where microbial protein is the predominant source thereof were identified. These results contribute to the current knowledge of AA requirements in growing lambs, and highlight areas for future research, as discussed in the General Conclusion.

Thesis (MScAgric)--University of Stellenbosch, 2002.<br>ENGLISH ABSTRACT: The Dorper is numerically the second most abundent of all the sheep breeds in South Africa. It is the most numerous sheep breed in the dry arid areas of South Africa which make out a very big portion of the natural grazing in South Africa. In this study the feedlot performance was specifically investigated since so little research has been done in this regard. In the first study (Chapter 1), the production efficiency, body components and commercial yield of 13 Dorper and 12 Merino lambs were investigated. A pelleted diet was fed ad lib and the water intake per day was measured. Lambs were slaughtered at 30kg and at 40kg live weight and cut up according to the South African commercial cuts. The weights of the organs, head, feet, skin and carcass were also recorded. Results from the first study showed that Dorpers had a lower (P<0.05) water efficiency (285.97 mVkgO75/day) than the Merinos (240.07 mVkg075/day), but had a higher (P<0.05) nitrogen retention. The head, feet, skin, heart, lungs, kidneys and diaphragm of Merino lambs were heavier (P<0.05). Merinos had heavier (P<0.05) breast and shank and thick rib (only at 40kg live weight) cuts. But the Dorper had a significantly heavier hindquarter (buttock and loin) which is the high-priced cut of any carcass. It seems that the Dorper will do well under feedlot conditions, and would possibly make a better commercial meat producer than the Merino under all circumstances. In the second study (Chapter 2), the free-ranging Dorper on natural grazing were compared with 7 Dorper lambs in the feedlot study. The essential amino acids in the duodenal contents were compared. The free-ranging Dorpers were randomly slaughtered at 40 kg live weight and a duodenal sample was collected. The feedlot Dorpers were also slaughtered at 40 kg live weight and a duodenal sample was taken. The second study indicated no significant differences between the EAA composition of the duodenal samples, whether protein quantity was taken into account (gAA/100g crude protein (CP)) or not (expressed as % of lysine). The chemical scores indicated that the two most limiting amino acids in the duodenal digesta for whole empty body growth were: arginine and histidine. Excess levels of amino acids tend to be present in all three duodenal digesta samples (isoleucine, leucine, lusine, methionine, phenylalaline, threonine and valine). The whole empty body EAA composition (g AA/100 g crude protein) was as follows 7.10 arginine; 2.40 histidine; 3.31 isoleucine; 7.22 leucine; 6.61 lysine; 1.62 methionine; 3.91 phenylalanine; 3.77 threonine and 4.85 valine. The composition can serve as an example of the ideal EAA requirements for whole empty body growth between 30 and 40 kg live weight of Dorper ram lambs. The duodenal amino acid concentrations of the free-ranging Dorper compared well with those of the feedlot Dorpers which suggests that the microbial organisms have a big influence on the amino acid concentration which reaches the intestines.<br>AFRIKAANSE OPSOMMING: Die voerkraal prestasie en essentiele aminosuur behoeftes van groeiende Dorper lammers: The Dorper is getalgewys die tweede mees talryke skaapras in Suid-Afrika. Dit is die mees talryke skaapras wat in die drce woestynagtige gebiede van Suid-Afrika aangetref word. Die gebiede beslaan die grootste oppervlakte van Suid-Afrika se natuurlike weiveld. In die studie is voerkraal prestasie van die Dorper nagevors, spesifiek omdat daar so min navorsing op hierdie gebied gedoen is. In die eerste studie (Hoofstuk 1) is die produksie doeltreffendheid, liggaams komponente en kornrnersiele produksie van 13 Dorper- en 12 Merino lammers ondersoek. 'n Verpilde rantsoen is ad lib gevoer en die water inname per dag is ook gekontroleer. Die lammers is geslag op 30 en 40 kg lewende gewig en die karkasse is daarna opgesny volgens die Suid-Afrikaanse kornmersiele karkassnitte. Die resultate van die eerste studie het getoon dat Dorpers 'n laer water potentiaal (P<0.05) (285 97 mflkgO 75/dag) as die Merinos (240.07 mVkg075/dag) het. Dorper lammers het ook 'n hoer (P<0.05) stikstof retensie. Die kop, vel, pote, hart, longe, niere en diafragma van Merino lammers was swaarder (P<0.05). Merinos se brisket en dikrib snitte weeg ook swaarder (P<0.05). Die Dorper het 'n beduidende swaarder agterkwart (len de en boud) gehad, wat die duurste snit is van enige karkas. Die Dorper het baie goed gedoen in die voerkraal toestande en sal waarskynlik onder aile omstandighede 'n hcer vleisproduksie op die Merino lewer. In die tweede studie (Hoofstuk 2), is die vryweiende Dopers op natuurlike weiding vergelyk met die Dorpers in 'n voerkraal. Die twee groepe Dorpers se essensiele aminosuur samestelling in hul dunderms is bepaal en vergelyk om te bepaal of daar enige tekorte teenwoordig is. Die Dorpers in die veld en in die voerkraal is op 40 kg lewende gewig geslag en monsters van die dunderm inhoud is gekollekteer. Die monsters is ontleed om te bepaal of daar enige betekenisvolle verskille in die essensiele aminosure van die dunderm inhoud tussen die veld Dorpers en voerkraal Dorpers was. Die tweede studie het geen betekenisvolle verskille getoon tussen die dunderm monsters se essensiele aminosure, of die hoeveelheid proteien (gAA/100g) in aanmerking geneem is of nie (uitgedruk as % lisien). Die chemiese telling dui aan dat die twee mees beperkende aminosure in die dunderm inhoud vir lee liggaam groei arginien en histidien was. Oormaat vlakke van aminosure kom voor in al drie dunderm inhoud monsters (isoleusien, leusien, lisien, metionien, femielalanien, treonien en valien). Die totale lee liggaam essensiele aminosuursamestelling (g aminosuur/100g ruprotein) was soos volg: 7.10 arginien; 2.40 histidien; 3.31 isoleusien; 7.22 leusien; 6.61 lisien; 1.62 metionien; 3.91 fenielalanien; 3.77 treonien en 4.85 valien. Hierdie aminosuursamestelling kan as 'n aanduiding dien vir die ideale aminosuurbehoeftes van Dorper ram lammers vir totale lee liggaamsgroei vanaf 30 tot 40 kg lewende massa. Die dunderm aminosuur konsentrasie van die Dorper in die veld stem goed ooreen met die van die voerkraal Dorper. Dit toon waarskynlik dat die mikrobe organismes in die rumen In groot invloed het op die aminosuur samestelling wat die dunderm bereik.

Dairy cows inefficiently convert dietary protein to milk protein causing economic and environmental costs. Amino acids (AA), insulin, and glucose significantly enhance muscle protein synthesis efficiencies. The objectives of this research project were 1) to investigate the regulatory effects of essential AA (EAA) and their interactions with insulin, glucose and acetate on mammary protein synthesis rates, 2) to investigate whether branched chain amino acids (BCAA): leucine , isoleucine , and valine , become limiting for milk protein synthesis when Met and Lys supply were not limiting, and 3) to develop a mathematical representation for the EAA and insulin effects on cellular signals for protein synthesis. MAC-T cells were treated with EAA, insulin, glucose, and acetate to observe their individual and interactive effects on phosphorylation of mTOR, rpS6, S6K1, 4EBP1, eEF2, eIF2α, Akt, and AMPK. These signaling effects on protein synthesis rates were examined with mammary tissue slices. A mathematical representation of the insulin and EAA effects was developed. The effects of supplementing BCAA on milk protein synthesis were investigated using nine Holstein cows, assigned to 7 d continuous jugular infusions of saline, Met and Lys, and Met and Lys plus BCAA. Multiple essential amino acids, Leu, Ile, Met, and Thr were able to substantially regulate protein synthesis rates in bovine mammary cells by increasing (P < 0.05) phosphorylation of mTOR, S6k1, 4EBP1, and decreasing (P < 0.10) eEF2 phosphorylation. Insulin considerably (P < 0.10) exerted similar signaling effects in MAC-T cells, independent of EAA. Supplementation of only acetate increased (P = 0.09) mammary cell energy status as indicated by reduced AMPK phosphorylation in MAC-T cells. Neither acetate nor glucose had substantial regulatory effects on mammary protein synthesis rates. Although Met and Lys supplementation increased (P < 0.01) milk protein yields and protein efficiencies, there were no apparent benefits of BCAA supplementation under the feeding circumstances of our study. The developed mathematical model adequately represented the regulatory effects of EAA and insulin. Such mathematical representations of regulatory effects of EAA and their interaction with other nutrients may improve our current AA requirement models to predict AA requirements of dairy cows with increased accuracy.<br>Ph. D.

Thesis (MScAgric)--University of Stellenbosch, 2001.<br>ENGLISH ABSTRACT: Regardless of the extensive game eradication during most of the previous century, the game farming industry in South Africa has shown dynamic growth over the past few decades. Currently the approximately 8 000 game farms have a total income of more than R850 million per annum. Although there are various game species included in these farming enterprises, three of the most important species for commercial farming and meat marketing are springbok iAntidorcas marsupialisï, blesbok (Damaliscus dorcas phillipsiï and impala (Aepyceros melampus). Since nutrition is a key factor in the productivity of animals, any information on this subject is essential for the successful management of a game farm. However, apart from the general lack in applied research on game animals (due to the different approaches by biologists and agricultural researchers), there is also only limited information available on the nutritional requirements of game species or any of the aspects affected by nutrition. In order to address the above shortcomings in game animal nutrition, two separate studies were conducted. The purpose of the first study was to determine the physical body component and nitrogen (N) distribution in the springbok, blesbok and impala. The chemical composition of the three-rib cut was also compared with that of the carcass. Amino acid requirements for growth of the three game species were investigated in the second study according to the ideal protein concept. The essential amino acid (EAA) profile of the whole empty body of the three game species was compared with the EAA profile of the duodenal digesta in order to detect any imbalances for optimal growth. The same animals were used in both studies, namely eight of each of the three game species. Characterization of the physical body composition revealed that the dressing percentages (% of body weight; % of empty body weight) for the three game species were: springbok (57.1 ± 2.4 %; 64.0 ± 2.5 %), blesbok (50.2 ± 2.1 %; 62.8 ± 1.4 %) and impala (57.4 ± 2.2 %; 65.6 ± 2.0 %). The proportional distribution ofN between the carcass, external offal and internal offal was also determined. The mean carcass N concentration of the impala was higher (P<0.05) than that of the springbok and blesbok on a dry matter basis. According to the carcass chemical composition, the three game species seem to have a better potential for lean meat production (fat content of carcass 4.7-5.2 %) than domestic sheep (17-25 % fat). Male animals had a lower (P<0.05) fat and higher (P<0.05) moisture percentage in the carcass and threerib cut than the females. The fat correlation between the three-rib cut and the carcass on both an as-is and dry matter basis was significant across species and sexes. The protein content of the same cuts did, however, not yield a significant correlation on an as-is basis, and only the protein content of the springbok three-rib cut related (P<0.05) to that in the carcass on a dry matter basis. The second study concentrated on the nutritional requirements of springbok, blesbok and impala, and more specifically the EAA requirements. Differences (P<0.05) were found between the whole empty body EAA composition of the three game species. These differences were present whether protein quantity was included in the calculations (g AA/100 g crude protein) or not (expressed as % of lysine). The chemical scores indicated that the four most limiting amino acids (three for impala) in the duodenal digesta for whole empty body growth were: arginine, histidine, threonine and methionine (springbok); methionine, arginine, histidine and lysine (blesbok); and methionine, histidine and arginine (impala). Further disproportion of the amino acids in the duodenal digesta was present in excess levels of isoleucine (blesbok) and lysine, valine, leucine, threonine, isoleucine and phenylalanine (impala). The EAA:Lysine ratios of the whole empty body indicated a similar trend for the three game species.<br>AFRIKAANSE OPSOMMING: Die essensiële aminosuurbenodighede van springbok, blesbok en rooibok vir optimale groei: Ten spyte van die feit dat wild grootskaals uitgedun is gedurende die vorige eeu, het die wildboerdery-bedryf in Suid-Afrika oor die laaste paar dekades dinamiese groei getoon. Die totale inkomste van die ongeveer 8000 wildsplase beloop tans meer as R850 miljoen per jaar. Alhoewel daar verskeie wildspesies is wat by hierdie tipe boerdery ingesluit word, is die springbok (Antidorcas marsupialis), blesbok (Damaliscus dorcas phillipsi) en rooibok (Aepyceros melampus) van die belangrikste spesies vir kommersiële boerdery en vleisbemarking. Voeding IS 'n sleutelfaktor in die produktiwitieit van diere en daarom is emge inligting oor hierdie onderwerp noodsaaklik vir die suksesvolle bestuur van 'n wildsplaas. Afgesien van die algemene tekort aan toegepaste navorsing op wilde diere (as gevolg van die verskillende benaderings van bioloë en landboukundige navorsers), is daar ook slegs beperkte inligting beskikbaar oor die voedingsbehoeftes van wilde dierspesies of enige van die faktore wat deur voeding beïnvloed word. In 'n poging om bogenoemde tekortkominge in die voeding van wilde diere aan te spreek, is daar twee aparte studies uitgevoer. Die doel van die eerste studie was om die fisiese liggaamsamestelling en die stikstof (N) verspreiding in die springbok, blesbok en rooibok te bepaal. Die chemiese samestelling van die drie-ribsnit is ook met die van die karkas vergelyk. In die tweede studie is ondersoek ingestel na die aminosuurbehoeftes van die drie wildspesies deur van die ideale proteïen konsep gebruik te maak. Die bepaling van die essensiële aminosuurprofiel van die totale leë liggaam van die onderskeie drie wildspesies is bepaal en met die essensiële aminosuurprofiel van die duodenale-inhoud vergelyk, ten einde enige wanbalans vir optimale groei vas te stel. Dieselfde diere is in beide studies gebruik, naamlik ag springbokke, blesbokke en rooibokke. 'n Beskrywing van die fisiese liggaamsamestelling het aangetoon dat die uitslagpersentasies (% van liggaamsmassa; % van leë liggaamsmassa) van die drie wildspesies as volg was: springbok (57.1 ± 2.4 %; 64.0 ± 2.5 %), blesbok (50.2 ± 2.1 %; 62.8 ± 1.4 %) en rooibok (57.4 ± 2.2 %; 65.6 ± 2.0 %). Die proporsionele verspreiding van N in die karkas, uitwendige afval en inwendige afval is ook bepaal. Die gemiddelde N konsentrasie in die karkas van die rooibok was hoër (P<0.05) as die van die springbok en blesbok op 'n droë materiaal basis. Dit blyk vanaf die chemiese samestelling van die karkas dat die drie wildspesies 'n groter potensiaal het vir die produksie van maer vleis (vet-inhoud van karkas 4.7-5.2 %) as skape (17-25 % vet). Manlike diere het 'n laer (P<0.05) vetpersentasie en 'n hoër (P<0.05) vogpersentasie in beide die karkas en die drie-ribsnit teenoor die vroulike diere gehad. 'n Betekenisvolle korrelasie is tussen die persentasie vet in die drie-ribsnit en die karkas van al drie wildspesies en vir beide geslagte gevind, op beide 'n nat en droë materiaal basis. Die proteïeninhoud van dieselfde snitte was egter nie op 'n nat basis betekenisvol gekorreleerd nie, en slegs die proteïen-inhoud van die springbok se drieribsnit het verband gehou (P<0.05) met die in die karkas op 'n droë materiaal basis. Die klem van die tweede studie was op die voedingsbehoeftes van springbok, blesbok en rooibok, en meer spesifiek die essensiële aminosuurbenodighede. Daar is verskille (P<0.05) tussen die totale leë liggaam essensiële aminosuursamestelling van die drie wildspesies gevind. Hierdie verskille was aanwesig afgesien daarvan of die hoeveelheid proteïen in berekening gebring is (g aminosure /100 g ruproteïen) of nie (uitgedruk as % van lisien). Volgens die chemiese tellings was die vier mees beperkende aminosure (drie vir rooibok) in die duodenale-inhoud vir totale leë liggaamsgroei as volg: arginien, histidien, treonien en metionien (springbok); metionien, arginien, histidien en lisien (blesbok); en metionien, histidien en arginien (rooibok). Verdere wanbalanse in die armnosure van die duodenale-inhoud was aanwesig in die oormaat isoleusien (blesbok) en lisien, valien, leusien, treonien, isoleusien en fenielalanien (rooibok). Die verhouding van die essensiële aminosure tot lisien van die totale leë liggaam het by al drie wildspesies dieselfde tendens getoon.

Thesis (MScAgric)--University of Stellenbosch, 2001.<br>ENGLISH ABSTRACT: The purpose of this study was to determine the essential amino acid requirements of beef cattle under feedlot conditions through evaluation of the duodenal and whole empty body essential amino acid compositions. To define the ideal protein required for growth, the whole empty body essential amino acid compositions of 8 beef steers (Simmental and Hereford crosses) was investigated. The amino acid composition of the components (carcass, metabolic organs and residual fraction), was pooled relative to their respective mass and protein contribution, resulting in the calculated whole empty body amino acid composition: arginine 6.81; histidine 2.69; isoleucine 4.02; leucine 6.96; lysine 7.43; methionine 2.01; phenylalanine 4.03; threonine 4.01; valine 5.30; tryptophan 0.82. Variations in profiles were found between scientific reports, either indicating that ratios change with growth and implants or possibly through genotype. The present study's body amino acid ratios were used to estimate dietary amino acid requirements through evaluation of the duodenal essential amino acid compositions from three different maize based feedlot diets. Although there was a general increase in the biological value of protein after rumen fermentation, the duodenal essential amino acids in comparison with the whole empty body recorded deficient/unbalanced profiles of essential amino acids for growth. The chemical scores suggested that the first-to-thirdlimiting amino acids in the duodenal digesta of beef cattle, that received three different commercially available feedlot diets, were: histidine, lysine, methionine/arginine (Diet 1), histidine, arginine, lysine (Diet 2) and arginine, methionine, histidine (Diet 3). Thedisproportionate duodenal amino acid concentrations obtained from the three diets, emphasise the necessity to enhance the intestinal delivery of amino acid profiles through different undegradable protein sources, with the objective to maximise protein utilisation and obtain the genetic potential for optimal growth in feedlot cattle. When amino acid requirements and flows to the duodenum were simulated using the Cornell Net Carbohydrate and Protein system (CNCPS), predictions indicated that lysine amino acid flow was limiting the metabolizable allowable average daily gain in Diet 1 and 3. Predicted profiles indicated that the order of limitation was: lysine, arginine, histidine (Diet 1), lysine, arginine, histidine, methionine (Diet 2) and lysine, arginine, histidine (Diet 3). The predicted profiles were in accordance with observed duodenal values, except for methionine that was observed limiting in Diet 1 and 3; however, the sequence and extent of limitation varied. Results indicate that prediction models have potential in predicting requirements; however there are still limitations for use to accurately define requirements for particular EAA's. From the present study, it is clear that the protein accretion was constrained by quantity and/or disproportionality of amino acids available for absorption. Further research should therefore be directed towards obtaining a more desirable array of amino acids to the lower digestive tract that is digestible, absorbable and an economically viable option for the feedlot operator.<br>AFRIKAANSE OPSOMMING: Aminosuurbehoeftes van Voerkraalbeeste Volgens die Duodenale en Totale Lee Liggaam Essensiele Aminosuur Profiel: Die doel van hierdie studie was om die essensiele aminosuur vereistes van vleisbeeste onder voerkraal toestande te bepaal deur die evaluering van die duodenale en totale lee liggaam essensiele aminosuursamesteilings. Om die idea Ie proteiene wat vir groei benodig word te definieer, is 'n ondersoek ingestel na die totale lee liggaam essensiele aminosuursamesteilings van 8 vleisbees osse (Simmetailer en Hereford kruise). Die aminosuursamesteiling van die komponente (karkas, metaboliese organe en residuele fraksie), se massa en proteien bydrae is gebruik om die totale lee liggaam aminosuursamestelling 5005 volg te bereken: arginien 6.81; histidien 2.69; isoleusien 4.02; leusien 6.96; lisien 7.43; metionien 2.01; fenielalanien 4.03; treonien 4.01; valien 5.3; triptofaan 0.82. Variasies in profiele tussen wetenskaplike verslae is gevind. Dit dui daarop dat groei en inplantings of moontlik genotipe 'n invloed op profiele kan he. Die huidige studie se liggaam aminosuurverhoudings is gebruik om die dieet aminosuurbehoeftes te skat deur evaluering van die duodenale essensiele aminosuursamesteilings van drie verskiilende mielie-gebaseerde voerkraal diete. Alhoewel daar 'n algemene toe name in die biologiese waarde van die proteien na rumen fermentasie was, het die duodenale essensiele aminosure in vergelyking met die totale lee liggaam, ongebalanseerde profiele van essensiele aminosure vir groei getoon. Die chemiese teilings toon aan dat die eerste-tot-derde-beperkende aminosure in die duodenale inhoud van vleisbeeste wat drie verskillende kommersieel beskikbare voerkraal diete ontvang het, soos volg is: histidien, lisien, metionieniarginien (Dieet 1), histidien, arginien, lisien (Dieet 2) en arginien, metionien, histidien (Dieet 3). Die ongebalanseerde aminosuur konsentrasies wat in die duodenum van die drie dlete verkry is, beklemtoon die noodsaaklikheid om die intestinale lewering van aminosuurprofiele te verbeter deur verskillende nie-degradeerbare proteien bronne te voer. Die doelwit moet wees om proteien verbruik te optimaliseer en daardeur die genetiese potensiaal vir optimale groei in voerkraalbeeste te bereik. Met die simulering van aminosuurbehoeftes en vloei na die duodenum van die drie standaard voerkraal diete met behulp van die "Cornell Net Carbohydrate and Protein System" (CNCPS), het voorspellings gewys dat lisien aminosuurvloei die metaboliseerbare toelaatbare gemiddelde daaglikse toename in Dieet 1 en 3 beperk het. Voorspelde profiele wys dat die volgorde van beperking soos volg sou wees: lisien, arginien, histidien (Dieet 1), lisien, arginien, histidien, metionien (Dieet 2)' en lisien arginien, histidien (Dieet 3). Die voorspelde profiele was in ooreenstemming met die waargeneemde duodenale waardes, behalwe vir metionien wat beperkend was in Dieet 1 en 3; die volgorde en mate van beperking was egter verskillend. Resultate wys dat voorspellingsmodelle die potensiaal het om behoeftes te voorspel. Vir die akkurate definisie van behoeftes vir spesifieke essensiele aminosuure is daar egter nog beperkinge. Uit die huidige studie, is dit duidelik dat protetenneerleqqinq deur die hoeveelheid en/of oneweredigheid van geabsorbeerde aminosure beperk is. Verdere navorsing moet dus fokus op die verkryging van In meer geskikte profiel van aminosure wat verteerbaar, opneembaar en 'n ekonomiese lewensvatbare opsie vir die voerkraalbestuurder is.

Determining the AA availability and metabolism in ruminant is a big challenge due to the rumen fermentation and complicated post absorption utilization. Current techniques used for direct determination of AA absorption and metabolism are laborious and expensive with large variation. The objectives of this project were to investigate AA availability of rumen undegradable protein, develop a stable isotope technique for determination of microbial protein and to evaluate the metabolism of amino acids in mammary glands of dairy cattle using stable isotope-based approaches. In the first experiment, seven heifers (258 ± 28 kg BW) were randomly chosen and assigned to 8 treatment sequences in a 7 x 8, incomplete, Latin square design. Treatments were a basal diet (BD), and 10% (DM basis) of BD replaced by corn silage (CS), grass hay (GH), alfalfa hay (AH), dried distillers grain (DDGS), soybean hulls (SH), wet brewers grain (BG), or corn grain (CG). Individual essential AA availabilities for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain and corn grain were 33.4, 29.9, 34.1, 40.6, 28.8, 41.2, and 36.5% of the essential AA in each of the respective ingredients when a loss of 8.27% to splanchnic utilization during first pass was assumed; however, availability varied across individual essential AA. In the second experiment, twelve cows were blocked into 3 groups according to days in milk and randomly assigned to 4 treatments in a repeated 4 x 4 Latin square design with 2 factors to evaluate the essential AA availability from microbial protein and rumen undegradable protein under different rumen fermentation conditions. The 4 treatments were high rumen undegradable protein and high starch (HPHS), low rumen undegradable protein and high starch (LPHS), high rumen undegradable protein and low starch (HPLS) and low rumen undegradable protein and low starch (LPLS). Microbial protein synthesis calculated from purine derivatives was positively associated with rumen degradable protein, which was consistent with total microbial AA entry derived from the isotope dilution model indicating that the isotope based approach was representative. The individual essential AA availability from microbial protein was determined by isotope technique, whereas the PD method was just total PD absorption reflecting CP absorption. The metabolizable AA estimates from NDS nutritional model was similar to results from isotope dilution models, but with smaller difference among treatments. The microbial protein estimated from White's model showed the same trend among treatments compared to isotope dilution model, which may imply it represents the rumen fermentation better. The average essential AA digestibility for microbial AA was 82%, which varied across individual AA and treatments. In the third experiment, four cows (78 ± 10 DIM) were used to study the effects of jugular infusion of 2 groups of AA on essential AA uptake and metabolism by mammary glands in a 4 x 4 Latin square design. Treatments were jugular infusion of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL (MKH+IL). The MKH increased milk protein yield in high producing dairy cows. The IL infusion increased milk and milk lactose yields. The production response was associated with a change in mammary plasma flow together with changes in AA uptake and metabolism in mammary gland. Mammary uptake of essential AA was 135 % of milk protein output. Efflux of EAA from the mammary to blood was 13-61% of influx, which was high for BCAA but low for Met and Lys. Changes in influx and efflux resulted in net uptake difference of infused essential AA that were responsive to varying supplies resulting in maintenance of homeostasis. The proportion of AA catabolized and used for milk protein was affected by EAA infusion, which demonstrated plasticity of mammary gland in AA metabolism. Overall, results suggested essential AA availability from rumen undegraded protein and microbial protein varied across individual AA and diets and can be affected by rumen fermentation. After absorption, EAA transport into mammary tissue was bi-directional and their metabolism was affected by AA supply and energy. Using a single coefficient to represent all AA digestibility in MCP or feed ingredient and an integrated efficiency of MP-AA converted into milk protein is inaccurate.<br>Doctor of Philosophy<br>Studies in monogastric animals have showed that balancing AA supply with animal requirements can improve the efficiency of N utilization. In order to build a model for AA balanced diet formulation, the composition of feed ingredients, the profile and digestibility of EAA for the rumen undegradable protein and microbial protein, the partition and efficiency of EAA utilization in mammary glands must be determined accurately. However, current AA degradation, digestibility and metabolism data used in nutritional models are from in vitro and in situ studies, which have not been fully validated against in vivo observations. This research used an in vivo stable isotope-based approach to determine amino acid availability for commonly used feed ingredients in dairy industry. The microbial protein AA and rumen undegradable protein AA availability was determined by adapting this isotope technique and introducing another isotope into rumen to label microbes. In addition, by coupling stable isotope tracers with arterio-venous difference technique and compartmental modelling, essential AA metabolism in mammary glands of dairy cows were qualified. Total essential AA availabilities for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain and corn grain were similar to values from meta-analysis of mobile bag results, but the availabilities of individual AA were more variable compared to in vitro and in situ results. The model derived microbial AA availability was consistent with the microbial protein calculated from NDS and Felming's model. However, our model predicted a lower proportion of metabolizable AA from microbial protein under diets including low rumen degradable protein, which might imply the NDS nutritional model overestimates microbial protein under low protein diets. The microbial protein estimated from White's model showed the same trend among treatments compared to isotope dilution model, which may imply it represents the rumen fermentation better. The averaged essential AA digestibility form microbial protein was 82%, which varied across individual AA and treatments. After absorption, mammary uptake of essential AA was 135 % of milk protein output. Cellular efflux represented 13 to 61% of essential AA uptake. The proportion of AA catabolized and used for milk protein was affected by essential AA infusion, which demonstrated the plasticity of mammary glands in AA metabolism. In conclusion, the results from isotope technique quantified the essential AA availability from rumen undegradable protein for various feed ingredients and from microbial protein under different feeding conditions. The essential AA transport and metabolism in mammary glands were regulated by multi factors and essential AA supply.

Two approaches were proposed and developed using artificial cells containing leucine dehydrogenase multienzyme systems with dextran-NAD$ sp{+}$ recycling. These approaches were intended to specifically convert waste metabolites (ammonia or urea) into essential amino acids (L-leucine, L-valine, and L-isoleucine) required by the body. Known technology of enzyme immobilization was used to develop these new approaches. Characterization, optimization, and feasibility studies were carried out to test the therapeutic potentials. The maximum conversion ratios were higher than 90% in both batch and packed-bed column bioreactors. Microencapsulated multienzyme systems showed a greater conversion activity than the systems free in solution. An orthogonal test showed that dextran-NAD$ sp{+}$ was the primary factor affecting the reaction rate within artificial cells. The D-glucose and glucose dehydrogenase system was the most promising system for dextran-NADH regeneration. The optimum pH and temperature were 8.5-9.0 and 35-40$ sp circ$C. Kinetic analysis suggested that the substrates of leucine dehydrogenase, $ alpha$-ketoisocaproate, $ alpha$-ketoisovalerate, and DL-$ alpha$-keto-$ beta$-methyl-n-valerate acted as competitive inhibitors with respect to the others. Their inhibitor constants were their corresponding Km values.

Hindbrain patterning requires many factors involved in early segmentation and later segment identity of the specific domains of the hindbrain. Hox proteins and their cofactors are of great importance during segmentation of the hindbrain, because segmentation and/or segment identity are lost when any of them are lost. Previously, we have reported that Meis proteins synergize with Pbx, another Hox cofactor, and Hox proteins expressed in the hindbrain. To further investigate Meis function during hindbrain development, we utilized a Meis dominant-negative molecule, ΔCPbx4, and expressed it in zebrafish embryos. We find that ΔCPbx4 affects gene expression and neuronal differentiation especially in r3 through r5. Further, we combined ΔCPbx4 with another Meis dominant-negative molecule (ΔHDCMeis) to disrupt Meis function more extensively. Under these conditions, we find that the entire hindbrain loses gene expression as well as its complement of neuronal differentiation. This phenotype is strikingly similar to that of loss of Pbx function, suggesting that Meis proteins act in the same pathway as Pbx. Therefore, Meis family proteins are indispensable for the entire hindbrain segmentation. In addition to the milder effect on hindbrain patterning, we also found upon expressing ΔCPbx4 that the caudal hindbrain transforms to r4-like fates, supported by expression of r4-specific marker gene (hoxbla) and specification of r4-specifc Mauthner neurons in the domain. This phenotype is not reported upon loss of Pbx function, suggesting that Meis proteins may play a more modulatory role, while Pbx is absolutely required during hindbrain development. Through several in vivo assays, we find that this r4 transformation is induced by Hox PG1 proteins and that vhnf1 represses r4 fates in the caudal hindbrain to further specify caudal fates in this region. Based on these results, we propose a model by which hindbrain patterning is achieved. Initially, un-segmented hindbrain is segmented into two domains wherein the caudal domain displays an r4 fate. This caudal r4 fate is then repressed by vhnf1 function which restricts the r4 fate to the presumptive r4 domain and specifies r5 and r6 by inducing its downstream genes such as valentino and hox PG3. Taken together, we conclude that Meis family proteins are essentially involved in function of Hox complexes to specify distinct rhombomeres during segmentation of the zebrafish hindbrain.

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are post-translationally removed from L27 by a site-specific protease so that conserved residues important for tRNA stabilization at the peptidyl transferase center are exposed. We have identified a novel cysteine protease in S. aureus that performs this cleavage. This protease, which we have named Prp, is conserved in all bacteria containing the L27 N-terminal extension. L27 cleavage was shown to be essential in S. aureus; un-cleavable L27 did not complement an L27 deletion. Cleavage appears to play an essential regulatory role, as a variant of L27 lacking the cleavage motif could not complement. Ribosomal biology in eubacteria has largely been studied in E. coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of E. coli. This research lays the foundation for the development of new therapeutic approaches that target this novel, essential pathway.

Objetivo: avaliar a ingestão de nutrientes no período pré-concepcional e níveis séricos de ácidos graxos, durante a gestação, em gestantes portadoras de fetos com gastrosquise e gestantes portadoras de fetos normais. Métodos: estudo prospectivo caso-controle realizado no período de Julho de 2013 a Julho de 2015 no setor de Medicina Fetal do Hospital das Clínicas. O grupo gastrosquise (GG) foi constituído de 57 gestantes com gestações únicas, idade gestacional inferior a 34 semanas e feto com gastrosquise isolada. O grupo controle (GC) foi constituído de 114 gestantes portadoras de fetos normais pareadas de acordo com idade materna (± 2 anos), idade gestacional (± 2 semanas) e mesma classificação de índice de massa corpórea (IMC) no período pré-concepcional. Os dados referentes ao consumo dietético das gestantes foram obtidos a partir do questionário de frequência e consumo alimentar (QFCA) e o cálculo da ingestão dos nutrientes (macronutrientes; micronutrientes, ácidos graxos e aminoácidos) foi obtido a partir de programas específicos: Dietwin Profissional 2.0® and Virtuanutri®. Para a avaliação de níveis séricos de ácidos graxos (AG), as gestantes foram submetidas à coleta de sangue na entrada no estudo e no momento do parto. A comparação de AG foi realizada durante a gestação e no momento do parto. Com o objetivo de avaliar se as diferenças entre os grupos eram mais frequentes na primeira ou na segunda metade da gestação, uma nova análise foi realizada subdividindo o período gestacional 25 semanas e < 34 semanas. Resultados: no período pré-concepcional, a media diária de calorias ingerida foi maior (2382,43 vs. 2198,81; p = 0,041) no GG em comparação com GC. O consumo médio de metionina (763,89 vs 906,34; p = 0,036), treonina (1248,34 vs. 1437,01; p = 0,018) e crômio (54,66 vs. 59,49 p = 0,014) foi menor no GG em comparação ao GC. Na análise de ácidos graxos, observa-se que o total AG (p = 0,008), AG insaturados (p = 0,002) e a razão C18:1n9/C18:00 (p = 0,021) foi menor no GG em comparação ao GC durante a gestação; entretanto, a razão C16:00 / C18:2n6 (p = 0,018) foi maior no GG em comparação ao GC no mesmo período. Total AG (p = 0,044) e AG insaturados (p = 0,024) foi menor no GG em comparação ao GC no período <= 25 . AG insaturados (p = 0,025) e a razão C18:1n9/C18:00 (p = 0,013) foi menor no GG em comparação ao GC no período > 25 semanas e < 34 semanas. Conclusão: gestantes portadoras de fetos com gastrosquise apresentam dieta de baixa qualidade nutricional, com alto valor calórico e pobre em aminoácidos essenciais, no período pré-concepcional, e baixos níveis séricos de ácidos graxos durante a gestação<br>Objective: To evaluate the nutrients intake during the preconceptional period and the serum fatty acid levels during the gestation period of pregnant women with fetuses with gastroschisis and pregnant women with normal fetuses. Methods: A prospective case-control study was conducted at the Fetal Medicine Unit at Hospital das Clínicas from July 2013 to July 2015. The gastroschisis group (GG) comprised 57 pregnant women with singleton pregnancies of less than 34 weeks with fetuses with isolated gastroschisis, and the control group (CG) comprised 114 pregnant women with normal fetuses matched for maternal age (± 2 years), gestational age (± 2 weeks), and the same preconceptional body mass index (BMI). Nutritional assessments related to the preconceptional period were obtained using the Food Consumption Frequency Questionnaire and nutrient intakes (macronutrient, micronutrient, fatty acid and amino acid) were calculated using nutrition programs: Dietwin Profissional 20 ® and Virtuanutri ®. For the evaluation of serum fatty acid levels (FA), a blood sample was collected from each subject at the time they entered the study and at the time of delivery. The FA comparison was performed during gestation and at the time of delivery. In order to evaluate whether the differences between both groups were more frequent in the first or second half of gestation, a new analysis was performed, subdividing gesta 25 weeks and < 34 weeks. Results: during the preconceptional period, the median daily calorie intake was higher (2382.43 versus 2198.81; p = 0.041) in the GG than in the CG. The median intakes of methionine (763.89 versus 906.34; p = 0.036), threonine (1248.34 versus 1437.01; p = 0.018) and chromium (54.66 versus 59.49 p = 0.014) were lower in the GG than in the CG. By analyzing the serum fatty acid levels, total FA (p = 0.008), unsaturated FA (p = 0.002) and the C18:1n9/C18:00 ratio (p = 0.021) were lower in the GG than in the CG during gestation; however, the C16:00 / C18:2n6 ratio (p = 0.018) was higher in the GG than in the CG during the indicated period. Total FA (p = 0.044) and unsaturated FA (p = 0.024) were lower in the GG than in the CG at period <= 25 w k , and unsaturated FA (p = 0.025) and the C18:1n9/C18:00 ratio (p = 0.013) were lower in the GG than in the CG at period > 25 weeks and < 34 weeks. Conclusion: Pregnant women with fetuses with gastroschisis have low-nutritional-quality diet, which is both high in calories and poor in essential amino acids during the preconceptional period, and have low serum FA levels during pregnancy

Esta pesquisa bibliográfica teve o propósito de elucidar a via do acido aspártico, apontando como fonte deste estudo os cereais. O objetivo principal desta pesquisa consistiu em estudar a via do acido aspártico, visando descrever uma variedade de técnicas de engenharia genética e bioquímicas que podem ser empregadas para aumentar a qualidade nutricional de cereais, podendo, assim, compreender o que acarreta o aumento do acumulo de lisina, metionina e treonina nos grãos para suprir essa necessidade na formação de uma proteína balanceada nutricionalmente. Foi realizada uma busca exaustiva em bases de dados Google Scholar, Portal Capes, ISI web of Science, no período de publicação de 1970 a 2012. Foram adotados textos de referencia internacional e nacional. Esta pesquisa foi dividida em três etapas: via do acido aspártico e seus aminoácidos derivados em plantas superiores de 1970 a 1997, via metabólica do acido aspártico no período de 1997 a 2006 e estratégias interessantes para aumentar o nível dos aminoácidos essenciais da via do acido aspártico em plantas no período de 2006 ate o momento. A primeira etapa foi desenvolvida relatando o acido aspártico como precursor dos aminoácidos essenciais: metionina, lisina, treonina e isoleucina. Entre os essenciais, a lisina e um dos mais estudados devido a escassez em muitos cereais, o que contribuiu para o estudo extensivo da via do acido aspártico, revelando, assim, a importância da aspartato quinase (AK), homoserina desidrogenase (HSDH) e dihidrodipicolinato sintase (DHDPS) como enzimas chaves para a regulação da síntese de lisina. A aspartato quinase (AK) exerce um controle sobre via do acido aspártico. A enzima dihidrodipicolinato sintase (DHDPS) regula a síntese de lisina. Na segunda etapa foi apresentada a importância dos aminoácidos sintetizados nas plantas através de complexas vias metabólicas que são controladas por enzimas, intermediários, substratos e aminoácidos. Este estudo também relata os aspectos importantes para uma melhor compreensão da síntese e o acumulo de aminoácidos solúveis e incorporados em proteínas. A terceira etapa foi apresentar estratégias interessantes para utilização em estudos, visando aumentar o nível de aminoácidos essenciais através da manipulação de genes já existentes, como também a introdução de genes estranhos nas plantas. Devido a importância nutricional, essa via tem sido extensivamente estudada, utilizando técnicas de engenharia genética e bioquímica. Pesquisadores tem apresentado esforços considerados no estudo desta via a fim de contribuir para futuras manipulações genéticas, cujo objetivo e produzir plantas com alto conteúdo de lisina, metionina e treonina.<br>The aim of this research was to elucidate the aspartate metabolic pathway using grains of cereal as a source of study. Therefore, it was necessary to understand the aspartate metabolic pathway in order to depict various biochemical and genetic techniques which can be used to enhance the nutritional value in cereals. After studying theses issues, it was possible to understand the results of having cereals with a high lysine, methionine, and threonine content, so that grains can have balanced protein content. For that reason, an exhaustive research was done by using international and national scientific data published in 1970 to 2012. These data were found in Google Scholar, Portal Capes, and ISI Web of Science. This research was divided in three parts: studies of aspartate metabolic pathway and their essential amino acids derived from plants published in 1970 to 1997, studies of aspartate metabolic pathway published in the period of 1997 to 2006, and interesting strategies to enhance the level of essential amino acids of the aspartate metabolic pathway in plants from 2006 to this moment. Firstly, this investigation reported about the aspartic acid as a precursor of essential amino acids such as methionine, lysine, threonine, and isoleucyne. Among the essential amino acids, lysine has been the most researched due to its lack in many kinds of grains. Needless to say, it has contributed to the intensive study showing the relevancy of aspartate kinase (AK), homoserine dehydrogenase (HSDH), dihydrodipicolinate synthase (DHDPS), as key enzymes for lysine regulation. The aspartate kinase (AK) has an important role on the aspartate metabolic pathway, meanwhile the dihydrodipicolinate synthase (DHDPS) is intrinsically involved on lysine synthesis regulation. Secondly, this investigation presented the importance of the amino acids which are synthesized by plants through metabolic pathways that are controlled by enzymes, intermediates, substrates, and amino acids. In addition, this research reported relevant aspects whereby scientists can improve their understanding about the synthesis and accumulation of soluble amino acids which are incorporated in proteins. Finally, the third part showed interesting strategies which can be used in future researches in order to increase not only the level of essential amino acids by manipulating genes, but also the introduction of odd genes in plants. Given the nutritional relevancy, this pathway has been extensively investigated by using techniques used by biochemical and genetic engineering. Hence, researchers have demonstrated a considerable effort on this matter contributing for future genetic manipulations, so that plants with high lysine, methionine, and threonine content can be produced.

Thesis (M. Sc. (Botany)) -- University of Limpopo, 2015<br>The aim of this study was to investigate the chemical composition of indigenous watermelon landraces (Citrullus lanatus) seeds grown in two districts in the Limpopo Province. Watermelon seeds are the most undermined oilseeds. The seeds have nutritional values that compare favourably with those of soybean, sunflower and ground nuts. Many cucurbit seeds such as watermelon seeds are rich in protein and oil, although none of these products have been used on an industrial scale. Nine indigenous watermelon landraces seeds (four from the Sekhukhune district and five from the Capricorn district) were examined. The landrace 06CDGM was found to have the highest mass per one hundred seeds (10.95 g per 100 seeds) with 07CDGM having the lowest mass (8.05 g per 100 seeds). The landrace 10CDGM was found to have the highest oil (41.5%), protein (20.39%) and fibre content (23.98%) with 01SDPW having the lowest oil yield (30.00%), 02SDPW was found to have the highest saponification value (184.57 mg KOH/g oil) and 09CDGM had the highest iodine value (138.575 g I2/100 g oil). The landrace 01SDPW had the highest total sugar content. All landraces were found to have the essential amino acid leucine. Antioxidant activity (66.95%) and total flavonoids (0.295 mg/g as catechin equivalents) were found to be highest in the 09CDGM landrace, and total phenolic content (0.91 mg/g gallic acid equivalent) was found to be highest in 05SDPW. The landrace 06CDGM was found to have the highest copper content (0.088 mg/g); 02SDPW was found to have the highest iron content (0.194 mg/g); 10CDGM had the highest zinc (0.312 mg/g) and sulphate content (0.129 mg/g); while both 10CDGM and 03SDPW had the highest content of calcium (9.13 mg/g). The landraces were found to differ slightly in the content and quality of components tested. Each of the landraces was found to have some qualities that render them superior to the other landraces. The qualities and content of the landraces compare favourably with those of commercial oil seeds such as the sunflower and soybean. Overall, the landraces from the Capricorn district were observed to have superior qualities than those from the Sekhukhune district. The findings indicated that the landraces from Capricorn district had more mean oil, mean protein, crude fibre content, total phenolics and total flavonoid content than those from the Sekhukhune district. The landraces from Sekhukhune district had high carbohydrate, ash content and high content of the minerals iron and calcium. The landrace 10CDGM was found to have overall high vi values with regard to most analyses, thus making the landrace superior to the others. The results indicate that landraces from both districts have complementary qualities to each other; however the landraces from the Capricorn are more superior in most aspects. The watermelon seeds have the potential to be used as a cost-effective and easily accessible source of nutrients and oil to meet nutritional challenges in developing countries. KEY CONCEPTS Citrullus lanatus; Landrace; Sekhukhune district; Capricorn district; Indigenous watermelon; Oil seeds; Essential amino acids; Antioxidant activity; Total phenolics; Total flavonoids; Mineral content; Total sugars; Crude fibre; Oil content; Saponification value; Iodine value.

Objective: to counteract muscle mass, muscle strength and power loss during aging, and to study age-related change of neuromuscular manifestation of fatigue in relation to nutritional supplementation. Design: randomized controlled double-blind study. Setting: twice-daily consumption for 12 weeks of an Essential Amino Acids (EAA)-based multi-ingredient nutritional supplement containing EAA, creatine, vitamin D and Muscle Restore Complex®. Participants: 38 healthy elderly subjects (8 male, 30 female; age: 68.91±4.60 years; body weight: 69.40±15.58 kg; height: 1.60±0.09 m) were randomized and allocated in supplement (SUPP) or placebo (PLA) group. Mean Measurements: vitamin D blood level; Appendicular Lean Mass (ALM); Visceral Adipose Tissue (VAT); Maximal Voluntary Contraction (MVC) and Peak Power (PP); myoelectric descriptors of fatigue: Fractal Dimension and Conduction Velocity initial values (FD iv, CV iv), their rates of change (FD slopes, CV slopes) and the Time to perform the Task (TtT). Mean Results: significant changes were found in SUPP compared to baseline: vitamin D (+8.73 ng/ml; p<0.001); ALM (+0.34 kg; p<0.001); VAT (-76.25 g; p<0.001); MVC (+0.52 kg; p<0.001); PP (+4.82 W; p<0.001). Between group analysis (SUPP Vs. PLA) showed improvements: vitamin D blood levels (+11,72 ng/ml; p<0.001); Legs FFM (+443.7 g; p<0.05); ALM (+0.53 kg; p<0.05); MVC (+1.38 kg; p<0.05); PP (+9.87 W; p<0.05). No statistical changes were found for FD iv, CV iv, FD and CV slopes and TtT, either compared to baseline or between groups. Significant correlations between mean differences in SUPP group were also found. Conclusion: the study demonstrates that in healthy elderly subjects an EAA-based multi-ingredient nutritional supplementation of 12 weeks is not effective to change myoelectric manifestation of fatigue and TtT failure but can positively affect muscle mass, muscle strength, muscle power and VAT, counterbalancing more than one year of age-related loss of muscle mass and strength. Keywords: essential amino acids, creatine, vitamin D, antioxidants, muscle aging, muscle strength, muscle power, muscle fatigue.<br>Objective: to counteract muscle mass, muscle strength and power loss during aging, and to study age-related change of neuromuscular manifestation of fatigue in relation to nutritional supplementation. Design: randomized controlled double-blind study. Setting: twice-daily consumption for 12 weeks of an Essential Amino Acids (EAA)-based multi-ingredient nutritional supplement containing EAA, creatine, vitamin D and Muscle Restore Complex®. Participants: 38 healthy elderly subjects (8 male, 30 female; age: 68.91±4.60 years; body weight: 69.40±15.58 kg; height: 1.60±0.09 m) were randomized and allocated in supplement (SUPP) or placebo (PLA) group. Mean Measurements: vitamin D blood level; Appendicular Lean Mass (ALM); Visceral Adipose Tissue (VAT); Maximal Voluntary Contraction (MVC) and Peak Power (PP); myoelectric descriptors of fatigue: Fractal Dimension and Conduction Velocity initial values (FD iv, CV iv), their rates of change (FD slopes, CV slopes) and the Time to perform the Task (TtT). Mean Results: significant changes were found in SUPP compared to baseline: vitamin D (+8.73 ng/ml; p<0.001); ALM (+0.34 kg; p<0.001); VAT (-76.25 g; p<0.001); MVC (+0.52 kg; p<0.001); PP (+4.82 W; p<0.001). Between group analysis (SUPP Vs. PLA) showed improvements: vitamin D blood levels (+11,72 ng/ml; p<0.001); Legs FFM (+443.7 g; p<0.05); ALM (+0.53 kg; p<0.05); MVC (+1.38 kg; p<0.05); PP (+9.87 W; p<0.05). No statistical changes were found for FD iv, CV iv, FD and CV slopes and TtT, either compared to baseline or between groups. Significant correlations between mean differences in SUPP group were also found. Conclusion: the study demonstrates that in healthy elderly subjects an EAA-based multi-ingredient nutritional supplementation of 12 weeks is not effective to change myoelectric manifestation of fatigue and TtT failure but can positively affect muscle mass, muscle strength, muscle power and VAT, counterbalancing more than one year of age-related loss of muscle mass and strength. Keywords: essential amino acids, creatine, vitamin D, antioxidants, muscle aging, muscle strength, muscle power, muscle fatigue.

Made available in DSpace on 2015-03-26T14:02:14Z (GMT). No. of bitstreams: 1 texto completo.pdf: 282982 bytes, checksum: 02ab6e3afc4b760df02b71b3ecefbab5 (MD5) Previous issue date: 2009-05-29<br>Pasture is the main source of cattle feed, but it is not capable of supplying the animal nutritional requirements completely. Therefore, an adequate management is necessary to improve productivity and reduce costs related to long term production. The search for technology to increase animal performance may be a feasible alternative to reduce costs with cattle nutrition. Fiber digesting ruminal bacteria utilize ammonia as their main source of nitrogen. Also, they have specific requirements for amino acids and an optimum growth when ruminal pH is around 6.2 to 6.8. For that, maximization of fiber fermentation is essential to increase ruminant production on pasture. Results of several experiments have shown that the supplementation of branched-chain amino acids (BCAA) increases feed intake, celluloses digestion, ruminal microbial growth, and daily gain for growing beef cattle. In this revision, studies show raise in milk production and daily gain in ruminants fed with forage based diets with the combination of some or all BCAA. In addition, this work makes possible to conclude that the supplementation with BCAA in association with rumen undegradable protein sources can increase animal production through the maximization of metabolizable protein.<br>O pasto constitui a principal fonte de alimentos dos bovinos, mas não é capaz de suprir todas as exigências nutricionais dos animais. Portanto, um manejo adequado é necessário para melhorar a produtividade e reduzir os custos associados à produção em longo prazo. A busca por tecnologias para o aumento do desempenho destes animais pode ser uma alternativa viável para reduzir os custos com alimentação do rebanho. Bactérias que fermentam carboidratos fibrosos (CF) utilizam a amônia como principal fonte de nitrogênio, possuem requerimentos específicos para aminoácidos e possuem um crescimento ótimo quando o pH ruminal está ao redor de 6,2 a 6,8. A maximização da fermentação de fibra é essencial para se aumentar a produção de ruminantes a pasto. Os resultados de vários experimentos têm mostrado que a adição de aminoácidos essenciais de cadeia ramificada aumenta a ingestão alimentar, a digestão da celulose, o crescimento dos microrganismos ruminais e o ganho de peso para animais em crescimento. Nesta revisão bibliográfica, os estudos mostram o aumento na produção de leite e no ganho de peso com a combinação de alguns, ou de todos os ácidos graxos voláteis de cadeia ramificada (AGVR) na dieta de ruminantes, compostas basicamente por volumosos. Além disso, através deste trabalho, conclui-se que é possível proporcionar um incremento na produção animal através da maximização da proteína metabolizável, associando AGVR a fontes de proteína não degradáveis no rúmen.

Submitted by PAULO SÉRGIO MARINELLI (professor.marinelli@gmail.com) on 2016-07-22T17:54:29Z No. of bitstreams: 1 TesePAULOSERGIOMARINELLIFINAL2016.pdf: 927674 bytes, checksum: beb1c758b76a3f632022e11378f2bcab (MD5)<br>Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-22T19:40:11Z (GMT) No. of bitstreams: 1 marinelli_ps_dr_bauru.pdf: 927674 bytes, checksum: beb1c758b76a3f632022e11378f2bcab (MD5)<br>Made available in DSpace on 2016-07-22T19:40:11Z (GMT). No. of bitstreams: 1 marinelli_ps_dr_bauru.pdf: 927674 bytes, checksum: beb1c758b76a3f632022e11378f2bcab (MD5) Previous issue date: 2016-07-11<br>As hortaliças não-convencionais como ora-pro-nóbis (Pereskia aculeata Mill.) e moringa (Moringa oleifera Lam.) são uma alternativa alimentar com excelente valor nutricional, de fácil cultivo e baixo custo. Têm sido descritas como importantes fontes de proteínas de composição equilibrada de aminoácidos, alto teor de fibras e de lipídios, principalmente ácidos graxos insaturados. Assim, este trabalho teve como objetivo a elaboração e caracterização das farinhas das folhas de Pereskia aculeata Mill. e Moringa oleifera Lam., além da avaliação sobre ratos Wistar de parâmetros bioquímicos e motilidade intestinal dos efeitos da administração destas farinhas. A caracterização química das farinhas foi feita por meio da determinação de umidade, cinzas, lipídios, composição de ácidos graxos, proteínas, aminograma, carboidratos, fibras, sódio, cálcio, e potássio. Os dados obtidos foram submetidos à análise de variância (ANOVA) complementada com o teste de Tukey. As farinhas estudadas apresentaram valores de umidade considerados seguros pela legislação brasileira. O alto teor de carboidratos das farinhas é indicativo de uma planta potencialmente energética. Verificou-se ainda que as farinhas apresentaram altos teores de proteína, fibras, cálcio e na composição de ácidos graxos, destacou-se o α-linolênico. Com relação aos estudos bioquímicos e antropométricos, observou-se que a utilização das farinhas melhorou o perfil lipídico, evitou ganho de peso e não apresentou efeitos adversos significativos. O uso das farinhas também influenciou positivamente a motilidade intestinal. Pelos resultados obtidos nas análises, pode-se explorar o uso dessas farinhas em produtos alimentícios com a finalidade de agregar valor nutricional e produzir alimentos que contribuam para o bem-estar e a saúde do consumidor.<br>Unconventional vegetables like ora-pro-nobis (Pereskia aculeata Mill.) and moringa (Moringa oleifera Lam.) are an alternative food with excellent nutritional value, easy to grow and low cost. They have been described as important sources of protein balanced amino acid composition, high fiber content and lipids, especially unsaturated fatty acids. Thus, this study aimed to the preparation and characterization of flour leaves of Pereskia aculeata Mill. Moringa oleifera Lam, apart from review of biochemical parameters of Wistar rats and intestinal motility effects of administration of these flour. The chemical characterization of flour was made by the moisture determination, ash, lipids, fatty acid composition, protein, aminogram, carbohydrates, fiber, sodium, calcium, and potassium. The data were submitted to analysis of variance (ANOVA) complemented by the Tukey test. Flours studied showed humidity values considered safe by Brazilian law. The high carbohydrate content of the flours is indicative of a potentially energy plant. It was also found that the meal had high levels of protein, fiber, calcium and fatty acid composition, the highlight was the α-linolenic. With regard to the anthropometric and biochemical studies it was observed that the use of flour has improved lipid profile avoided weight gain and has no significant adverse effects. The use of flours also positively influenced the intestinal motility. The results obtained in the analysis, one can explore the use of these flours in food products in order to add nutritional value and produce foods that contribute to the welfare and consumer health.

Made available in DSpace on 2015-03-26T13:54:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 108398 bytes, checksum: 5b60b137e550825f68202b0f7d2f561d (MD5) Previous issue date: 2010-02-09<br>Fundação de Amparo a Pesquisa do Estado de Minas Gerais<br>A 21-d experiment with 126 pigs was conducted to evaluate growth performance, weight of liver, pancreas and spleen, morph-physiological conditions of the small intestine, and, and diarrhea occurrence of nursery pigs fed different levels of crude protein. Pigs (6.05 ± 0.35 kg of initial weight) were allotted in pens with three pigs each on day 21 after weaning, with six treatments, and seven blocks on a randomized blocks experimental design. The treatments were six corn-soybean meal based diets with decreased levels of crude protein (24%; 23%; 22%; 21%; 20%; and 19%). Increased levels of synthetic amino acid were added to the diets in order to keep the ratio between lysine and each one of the following amino acids constant (methionine + cystine, threonine, tryptophan, valine, and isoleucine). Lactose levels were the same in all diets. Decreasing levels of crude protein from 24% to 19% did not influence daily gain (P>0.05), average daily feed intake (P>0.05), feed:gain (P>0.05) and carcass weight (P>0.05).. At the end of experimental period, one animal of each treatment was slaughtered for pH measurement of the content in the stomach and duodenum, relative weight of the liver, pancreas and spleen, villi height and crypt depth of the duodenum, jejunum and ileum. Effects (P<0.05) on duodenum pH were observed, but not in the stomach pH. The treatments did not influence the villi height and crypt depth (P>0.05), the weight of liver and spleen (P>0.05) but (P>0.05). No diarrhea occurrence was observed. In conclusion, decreasing levels of crude protein in nursery diets from 24% to 19% does not affect the performance of the piglets, the weight of organs, and the morph- physiological parameters of the gastrointestinal tract of piglets weaned at 21 days of age through 42 days of age.<br>Objetivando-se determinar o nível de redução de proteína (RP) a ser usado em substituição ao farelo de soja em dietas para leitões desmamados aos 21 dias de idade foram usados 126 leitões com peso inicial de 6,00± 0,95 Kg, distribuídos em experimento com delineamento em blocos casualizados, com seis tratamentos, sete repetições e três animais por unidade experimental. Os tratamentos consistiram de seqüências de níveis de RP de 24,0; 23,0; 22,0; 21,0; 20,0; 19,0% nas dietas dos leitões. As rações foram suplementadas com níveis crescentes de aminoácidos industriais, resultando em rações com relações constantes entre metionina + cistina, treonina, triptofano, valina e isoleucina, com base na lisina. Ao final do experimento, um animal (unidade experimental) de cada tratamento foi sacrificado para mensuração do pH do conteúdo estomacal e duodenal, peso relativo do fígado, pâncreas e baço e altura das vilosidades e profundidade das criptas do duodeno, jejuno e íleo. Considerando o período experimental total (22 a 42 dias), constatou-se que não houve diferença significativa no ganho de peso médio diário, no consumo de ração diário e na conversão alimentar dos animais que receberam menor quantidade de proteína na dieta. Constatou-se que não houve diferença significativa no pH do estomago entretanto no pH do duodeno foi encontrado diferença significativa. Quanto ao peso relativo dos órgãos o baço e o figado não apresentaram efeito significativo enquanto o pâncreas apresentou diferença significativa. Quanto aos resultados para altura de vilosidades e profundidade de criptas do duodeno, jejuno e íleo não foi encontrado diferenças significativa. Conclui-se que as dietas de creche para leitões desmamados aos 21 dias de idade devem conter 19,0% de proteína bruta desde que suplementadas com metionina, treonina, triptofano, valina e isoleucina industriais.

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.18.06 – технологія жирів, ефірних масел і парфумерно-косметичних продуктів. – Національний технічний університет «Харківський політехнічний інститут» Міністерства освіти і науки України, Харків, 2019 р. Дисертація присвячена науковому обґрунтуванню та розробці технології білково-жирових сумішей підвищеної харчової цінності спеціального призначення. Сформульовано робочу гіпотезу щодо використання в складі білково-жирової суміші насіння олійних культур соняшника, льону та кунжуту як джерел незамінних амінокислот, ессенціальних жирних кислот, вітамінів, мікроелементів, рослинних антиоксидантів. Науково обґрунтовано склад білково-жирової суміші підвищеної харчової цінності спеціального призначення (насіння льону; насіння кунжуту; насіння соняшника; рафінована кукурудзяна олія). Визначено органолептичні, фізико-хімічні, мікробіологічні та технологічні показники якості білково-жирової суміші. Розраховано залежність періоду індукції окиснення ліпідів білково-жирової суміші в закритій тарі від температури зберігання. Розроблено технологічне рішення щодо зниження активності інгібіторів протеолітичних ферментів білково-жирової суміші для максимально можливого збільшення її харчової цінності, яке полягає в її зволоженні та обробці надвисокочастотним випромінюванням. Запропоновано технологічну схему виробництва білково-жирової суміші підвищеної харчової цінності на основі олійної сировини, розроблено та узгоджено проект технічних умов на білково-жирові суміші з олійного насіння. Науково обґрунтовано рецептури жировмісних кондитерських виробів – кремової цукеркової маси для виробництва цукерок типу «трюфель» і шоколадної пасти з додаванням білково-жирової суміші підвищеної харчової цінності. Оцінено фізико-хімічні показники розроблених кондитерських виробів, вони відповідають нормам.<br>The dissertation for obtaining the scientific Degree of Candidate of Technical Sciences in the specialty 05.18.06 – technology of fats, essential oils and perfumery and cosmetic products. – National Technical University «Kharkov Polytechnic Institute», Ministry of Education and Science of Ukraine, Kharkiv, 2019. The thesis is devoted to the scientific substantiation and development of technology of protein-fat mixtures with increased nutritional value for nutrition of athletes. A working hypothesis on the use of sunflower, flax and sesame oilseeds as sources of essential amino acids, essential fatty acids, vitamins, microelements, and plant antioxidants in the composition of the protein-fat mixture has been formulated. The composition of protein-fat mixture of increased nutritional value of a special purpose (flax seeds; sesame seeds; sunflower seeds; refined corn oil) has been scientifically substantiated. Organoleptic, physico-chemical and technological quality control parameters of the protein-fat mixture were determined. The dependence of the induction period of lipid oxidation of the protein-fat mixture in a closed container on the storage temperature was calculated. A technological solution to reduce the proteolytic enzymes inhibitors activity of the protein-fat mixture for maximize its nutritional value, which consists in its wetting and processing by microwave radiation, has been developed. The technological scheme for the production of a protein-fat mixture of increased nutritional value based on oilseeds was proposed, a draft technical specification for protein-oil mixtures from oilseeds was developed and agreed upon. Recipes of fat-containing confectionery products – cream candy mass for the production of "truffle" cream candies and chocolate paste with the addition of protein-fat mixture of high nutritional value were scientifically based. Physical and chemical quality control parameters of the developed confectionery were evaluated, they corresponded to the norms.

Дисертація на здобуття наукового ступеня кандидата технічних наук (доктора філософії) за спеціальністю 05.18.06 «Технологія жирів, ефірних масел і парфумерно-косметичних продуктів» (18 – Виробництво та технології). – Національний технічний університет «Харківський політехнічний інститут», Міністерства освіти і науки України, Харків, 2019. Дисертацію присвячено науковому обґрунтуванню та розробці технології білково-жирових сумішей підвищеної харчової цінності. Актуальність теми дослідження пов’язана з тим, що дотепер практично немає вітчизняних спеціалізованих продуктів, зокрема для харчування спортсменів. Відомо, що найбільш важливими нутрієнтами для відновлення організму спортсмена є білки з певним співвідношенням амінокислот, поліненасичені жирні кислоти (ПНЖК) -3 групи, вітаміни та мінеральні речовини, які в недостатній кількості присутні в традиційних харчових продуктах. Тому часто спортсмени змушені вживати різноманітні біологічно активні добавки, що позиціонуються як спортивне харчування, нормативна база яких в Україні відсутня. Створення новітніх білково-жирових продуктів на науково обґрунтованій основі збагатить раціон харчування спортсменів, що, в свою чергу, позитивно вплине на повноцінне тренування та покращення спортивних результатів. Перспективною сировинною базою для означених продуктів, які є концентрованим джерелом поживних речовин, зокрема, незамінних нутрієнтів, є олійне насіння. Розробка науково обґрунтованої технології білково-жирових сумішей підвищеної харчової цінності для харчування спортсменів є актуальним науковим завданням, яке вирішується в дисертаційній роботі. Актуальність теми та вагомість результатів дисертаційної роботи підтверджується тим, що вона виконувалась згідно плану проведення науково-дослідних робіт Українського науково-дослідного інституту олій та жирів (м. Харків) у рамках держбюджетних науково-дослідних робіт НААН України: «Розробка білково-жирової основи для раціонального харчування спортсменів» (ДР № 0117U002374) та «Розробка способу інактивації інгібіторів протеїназ в білково-жировій основі для раціонального харчування» (ДР № 0116U000178), де здобувач був відповідальним виконавцем окремих етапів роботи. Практичне значення роботи для олійножирової промисловості полягає в обґрунтуванні раціонального співвідношення компонентів білково-жирової суміші (насіння льону, кунжуту, соняшника та кукурудзяної олії) для харчування спортсменів, яке є ефективним за вмістом незамінних амінокислот з розгалуженим ланцюгом (лейцином, ізолейцином, валіном) та триптофаном, максимально збагачене ПНЖК ω-3 групи та рослинними антиоксидантами; визначенні технологічних умов її обробки надвисокочастотним випромінюванням для збільшення харчової цінності; обґрунтуванні рецептур кремових цукерок типу «трюфель» і шоколадної пасти, збагачених білково-жировою сумішшю. Розроблено та узгоджено проект технічних умов: ТУ У 10.86.1 – 02071180.001:2018 «Білково-жирові суміші з олійного насіння». Проведено апробацію виробництва білково-жирової суміші з насіння льону, кунжуту, соняшника та рафінованої дезодорованої кукурудзяної олії, а також шоколадної пасти, збагаченої білково-жировою сумішшю, в промислових умовах ТОВ «Вегетус» (м. Харків). Результати дисертаційних досліджень впроваджено в навчальний процес кафедри органічного синтезу і нанотехнологій НТУ «ХПІ» під час викладання дисциплін «Хімічні технології харчових добавок та косметичних засобів», «Розробка комплексних харчових добавок» та «Застосування харчових добавок», в курсовому та дипломному проектуванні, а також у науково-дослідній роботі студентів. У дисертаційній роботі вперше одержано нові наукові дані щодо особливостей складу жирних кислот, стеринової фракції, комплексу антиоксидантів, амінокислот білків та сортових особливостей до накопичення ксенобіотиків насіння досліджених олійних культур, адаптованих до вирощування в Україні; науково обґрунтовано склад білково-жирової суміші (насіння льону, кунжуту, соняшника та кукурудзяної олії) спеціального призначення, яка є ефективною за вмістом ессенціальних жирних кислот, незамінних амінокислот та рослинних антиоксидантів; експериментально визначено у вигляді апроксимаційних моделей залежність періоду індукції окиснення ліпідів білково-жирової суміші від вмісту сезамолу, сезамоліну, α-ліноленової жирної кислоти та масової частки вологи. У дисертаційній роботі набуло подальшого розвитку експериментальне обґрунтування високої харчової цінності насіння олійних культур – соняшника, льону та кунжуту не тільки як джерела есенціальних жирних кислот, але й незамінних амінокислот – лейцину, ізолейцину і валіну; застосування надвисокочастотного випромінювання та зволоження олійної сировини для зниження активності інгібіторів протеолітичних ферментів і збільшення засвоюваності компонентів білково-жирової суміші. Компонентами білково-жирової суміші обрано підсушене насіння льону, кунжуту та соняшника. Вибір обґрунтовано тим, що насіння цих олійних культур поки ще не містить модифікованих генів і може стати джерелом білків рослинного походження, ессенціальних ПНЖК, мікроелементів, а також низки цінних сполук, які потрібні для здоров’я спортсменів. Досліджено загальний хімічний склад, жирнокислотний склад, склад стеринової і токоферольної фракцій, амінокислотний, вітамінний та мінеральний склад насіння льону (сорти Південна ніч, Ківіка, Симпатік), кунжуту (сорти Ілона, Кадет, Боярин) та соняшника (кондитерські сорти Лакомка, Алмаз, Запорізький кондитерський), адаптованих до вирощування в Україні. Насіння льону характеризується найбільшим вмістом α-ліноленової кислоти. Кунжутне насіння містить антиоксиданти сезамол і токофероли. Сезамол здатен посилювати антиокиснювальну дію токоферолів в оліях, а тому такий комплекс можна пропонувати для використання як антиоксиданту в жирових продуктах для спортсменів. В свою чергу, соняшникове насіння містить значну кількість токоферолів. Обране олійне насіння також містить фітостерини, які мають низку лікувально-профілактичних властивостей, зокрема знижують рівень загального холестерину в організмі. Сировина багата на амінокислоти лейцин, ізолейцин, валін, які є незамінним матеріалом для побудови м’язової тканини та приймають участь у процесах її анаболізму й відновлення. Саме ці амінокислоти в плазмі крові людини конкурують з амінокислотою триптофан, що визначає швидкість проникнення триптофану в мозок і зниження моторної активності та працездатності, тобто виникнення втоми. Насіння олійних культур обраних сортів містить значну кількість вітамінів групи В та вітаміну Е. Проаналізовано здатність олійного насіння обраних сортів щодо накопичення токсичних речовин, а саме важких металів та нітратів. Результати досліджень свідчать про те, що кількість солей важких металів – міді, цинку, свинцю та кадмію, а також і нітратів в усіх сортах значно менша за ГДК. В результаті аналізу отриманих даних як сировину для виробництва білково-жирової суміші обрано наступні сорти олійного насіння: Південна ніч (льон), Ілона (кунжут), Лакомка (соняшник). Науково обґрунтовано склад білково-жирової суміші підвищеної харчової цінності спеціального призначення: насіння льону – 51,00 ± 2,55 %; насіння кунжуту – 21,25 ± 1,06 %; насіння соняшника – 12,75 ± 0,63 %, рафінована дезодорована кукурудзяна олія – 15,00 ± 0,75 %. Вміст жиру в суміші становить 55,92 ± 2,80 %, вміст білку – 17,50 ± 0,88 %. Співвідношення лінолевої та -ліноленової жирних кислот у суміші становить близько 1,2 : 1,0; а незамінні амінокислоти з розгалуженим ланцюгом знаходяться в співвідношенні лейцин : ізолейцин : валін, яке дорівнює близько 2,0 : 1,2 : 1,4. Визначено органолептичні, фізико-хімічні та технологічні показники якості білково-жирової суміші. Розраховано залежність періоду індукції окиснення ліпідів білково-жирової суміші в закритій тарі від температури зберігання. Встановлено, що період індукції окиснення ліпідів білково-жирової суміші за 85±2 °С при запропонованому співвідношенні компонентів знаходиться в інтервалі 175 – 195 хв., що перевищує приблизно в 2,7 – 3,0 рази – період індукції окиснення лляного насіння як найбільш термолабільного компоненту та приблизно в 1,3–1,4 рази період індукції соняшникового насіння. Окисну стабільність білково-жирової суміші гарантує те, що при виготовленні її використано компоненти, що не піддавалися тривалій температурній обробці. Але присутність в складі олійного насіння інгібіторів протеолітичних ферментів знижують засвоєння білка організмом та істотно обмежують його застосування в харчовій промисловості. У зв’язку з цим розроблено технологічне рішення щодо зниження активності інгібіторів протеолітичних ферментів олійної сировини білково-жирової суміші для максимально можливого збільшення її харчової цінності, яке полягає в її зволоженні та обробці надвисокочастотним випромінюванням ( = 2450 МГц). Експериментально встановлено раціональні умови обробки олійної сировини: зволоження до вмісту вологи 11 – 13 % і час обробки надвисокочастотним випромінюванням 210 – 260 с, що дозволить підвищити біологічну цінність суміші, а саме збільшити ступінь розщеплення і засвоюваності білків у шлунково-кишковому тракті. Запропоновано технологічну схему виробництва білково-жирової суміші підвищеної харчової цінності на основі олійної сировини, розроблено та узгоджено проект технічних умов ТУ. У. 10.86.1 – 02071180.001:2018 на білково-жирові суміші з олійного насіння. Науково обґрунтовано рецептури жировмісних кондитерських виробів – кремової цукеркової маси для виробництва цукерок типу «трюфель» і шоколадної пасти з додаванням 15 % білково-жирової суміші підвищеної харчової цінності. Експериментально доведено, що періоди індукції цукеркової маси і шоколадної пасти запропонованого складу перевищують контрольні показники на 44 і 55 % відповідно. Для визначення впливу білково-жирової суміші на споживчі властивості кондитерських виробів проведено органолептичну оцінку дослідних зразків, на базі якої виявлено, що використання білково-жирової суміші в складі кремових цукеркових мас і шоколадної пасти впливає на такі органолептичні показники як смак і аромат, які у дослідних зразків вище, ніж у контрольних. Зокрема, в дослідних зразках кондитерських виробів проявляється характерний пікантний горіховий аромат і смак; довше зберігається відчуття «наповненості» у роті («mouth-feeling»). Подальше збільшення вмісту суміші – понад 15 % – приводить до появи борошнистого присмаку і характерного аромату лляного насіння. Виходячи з результатів органолептичної оцінки, обрано ефективну концентрацію білково-жирової суміші в кондитерських виробах на рівні 15 %. Результати визначення фізико-хімічних показників якості кондитерських виробів свідчать, що при додаванні в кремову конфетну масу і шоколадну пасту 15 % білково-жирової суміші зміна масової частки вологи та масової частки жиру відбувається в рамках нормованих величин. Зниження вмісту загального цукру не впливає на органолептичні властивості кондитерських виробів, але знижує собівартість продукції. Розроблені продукти порівняно з контролем мають вищій вміст незамінних амінокислот, змінюється їх ліпідний склад – значною мірою зростає вміст ПНЖК ω-3 групи. У складі розроблених кондитерських виробів також присутня значна кількість харчових волокон – нутрієнту, який прискорює відчуття насичення, а також є фактором формування нормальної кишкової мікрофлори і попередження ожиріння.<br>The dissertation for obtaining the scientific degree of candidate of technical sciences (PhD.) in the specialty 05.18.06 – technology of fats, essential oils and perfumery and cosmetic products. – National Technical University «Kharkov Polytechnic Institute», Ministry of Education and Science of Ukraine, Kharkiv, 2019. The dissertation is devoted to scientific substantiation and development of the technology of protein-fat mixtures of increased nutritional value. The relevance of the research topic is related to the fact that up to now there are practically no domestic specialized products in particular for nutrition of sportsmen. It is known that the most important nutrients for the recovery of the athlete's organism are proteins with a certain ratio of amino acids, polyunsaturated fatty acids (PUFA) -3 groups, vitamins and minerals that are present in insufficient amounts in traditional foods. Therefore, athletes are often forced to take various dietary supplements, which are positioned as sports nutrition, and there is no regulatory framework for these products in Ukraine. Creating the newest protein-fat products on a scientifically based basis will enrich the diet of athletes, which, in turn, will positively affect the full-fledged training and improved athletic performance. Oilseeds are a promising raw material base for these products, which are a concentrated source of nutrients, in particular, essential nutrients. The development of scientifically based technology of protein-fat mixtures of increased nutritional value for sportsmen is a relevant scientific task, which is solved in the dissertation. Relevancy of a subject and significance of dissertation is confirmed by the fact that it has been carried out according to the plan of researches of the Ukrainian research institute of oils and fats (Kharkiv) within the state budgetary research of NAAS of Ukraine: «Development of a protein-fat base for rational nutrition of athletes» (RW № 0117U002374) and «Development of a method for inactivating proteinase inhibitors in a protein-fat base for a balanced nutrition» (RW № 0116U000178), where the applicant was an executive of separate stages of work.. Practical value of work for the oil and fat industry consists in justify the rational ratio of the components of the protein-fat mixture (flax, sesame, sunflower and corn oil) for nutrition of athletes, which is effective for essential amino acids with branched chain (leucine, isoleucine, valine) and tryptophan, the most enriched with PUFA ω-3 groups and plant antioxidants; in determining the technological conditions of its processing by microwave radiation to increase the nutritional value; in substantiation of the recipes of «truffle» cream candies and chocolate paste enriched with protein-fat mixture. The draft technical specification TU U 10.86.1 – 02071180.001:2018 «Protein-fat mixtures from oilseeds» was developed and agreed. Approbation of the production of protein-fat mixture from flax seeds, sesame, sunflower and refined deodorized corn oil, as well as chocolate paste, enriched with protein-fat mixture, was conducted in industrial conditions of Vegetus LLC (Kharkiv). Results of dissertation researches were introduced in educational process of organic synthesis and nanotechnologies department of NTU «КPI» when teaching disciplines «Chemical technologies of food additives and cosmetics», «Development of complex food additives» and «Use of food additives», in course and degree design, as well as in a students research work. For the first time, new scientific data on the composition of fatty acids, sterol fraction, antioxidant complex, amino acid composition and varietal characteristics to the accumulation xenobiotics in oilseeds adapted for cultivation in Ukraine has been obtained; scientifically based composition of protein-fat mixture for special purposes (flax seeds, sesame, sunflower and corn oil), which is effective relative to the content of essential fatty acids, essential amino acids and plant antioxidants; the dependence of the induction period of lipid oxidation of the protein-fat mixture on the content of sesamol, sesamolin, α-linolenic fatty acid and the mass fraction of moisture in the form of approximation models was experimentally determined. The experimental substantiation of the high nutritional value of oilseeds – sunflower, flax, and sesame, – not only as sources of essential fatty acids, but also essential amino acids – leucine, isoleucine, and valine, – received further development; the use of microwave radiation and wetting oilseeds to reduce the activity of inhibitors of proteolytic enzymes and increase the digestibility of the components of the protein-fat mixture were received further development in the thesis. The dried flax, sesame and sunflower seeds were selected as components of the protein-fat mixture. The choice is justified by the fact that the seeds of these oilseeds still do not contain modified genes and can become a source of plant-derived proteins, essential PUFAs, trace elements, as well as a number of valuable compounds necessary for the health of athletes. The overall chemical composition, fatty acid composition, composition of sterol and tocopherol fractions, amino acid, vitamin and mineral composition of flax seeds (Southern Night, Civica, Sympatik), sesame (Ilona, Cadet, Boyarin) and sunflower varieties (confectionery varieties Lakomka, Almaz, Zaporizkyi kondyterskyi), adapted to growing in Ukraine, were investigated Flax seeds are characterized by the highest content of α-linolenic acid. Sesame seed contains antioxidants sesamol and tocopherols. Sesamol is able to enhance the antioxidant effect of tocopherols in oils, so this complex can be offered for use as an antioxidant in fatty products for athletes. In turn, sunflower seeds contain significant amounts of tocopherols. Selected oilseeds also contain phytosterols, which have a number of therapeutic and prophylactic properties, in particular, reduce the level of total cholesterol in the body. Raw materials are rich in amino acids (leucine, isoleucine, valine), which are indispensable materials for building muscle tissue and are involved in the processes of its anabolism and recovery. It is these amino acids in human blood plasma that compete with tryptophan amino acid, which determines the rate of tryptophan penetration into the brain and the decrease in physical activity and efficiency, that is, the occurrence of fatigue. Seeds of oilseeds of selected varieties contain significant amounts of B vitamins and E vitamin. The ability of oilseeds of selected varieties of accumulation of toxic substances, namely heavy metals and nitrates was analyzed. Research results indicate that the amount of salts of heavy metals – copper, zinc, lead and cadmium, – as well as nitrates in all grades is much less than the MPC. As a result of the analysis of the obtained data the following varieties of oilseeds were selected as raw materials for the production of protein-fat mixture: Southern night (flax), Ilona (sesame), Lakomka (sunflower). The composition of protein-fat mixture of increased nutritional value of special purpose was scientifically substantiated: flax seeds – 51.00 ± 2.55 %; sesame seeds – 21.25 ± 1.06 %; sunflower seeds – 12.75 ± 0.63 %, refined deodorized corn oil – 15.00 ± 0.75 %. The fat content in the mixture is 55.92 ± 2.80 %, the protein content was 17.50 ± 0.88 %. The ratio of linoleic and -linolenic fatty acids in the mixture was about 1.2: 1.0; and the essential branched-chain amino acids (leucine: isoleucine: valine) were in the 2.0 : 1.2 : 1.4 ratio. Organoleptic, physico-chemical and technological quality control parameters of the protein-fat mixture were determined. The dependence of the induction period of lipid oxidation of protein-fat mixture in a closed container from storage temperature was calculated. It was established that the induction period of lipid oxidation of protein-fat mixture with the proposed ratio of components was in the range of 175–195 minutes at 85 ± 2 °C, which was about 2.7–3.0 times longer than the induction period of flaxseed component and about 1.3–1.4 times – the induction period of sunflower seeds. The oxidative stability of the protein-fat mixture was ensured by the fact that components, which were used in its manufacture, had not been subjected to prolonged heat treatment. But the presence of proteolytic enzymes inhibitors in the composition of oil seeds reduces the digestion of protein by the body and significantly limits its use in the food industry. In this regard, a technological solution to reduce the activity of proteolytic enzymes inhibitors of oilseeds of protein-fat mixture to maximize its nutritional value has been developed. This solution consists in its wetting and processing by microwave radiation ( = 2450 MHz). Rational conditions of oilseeds processing (hydration to 11–13 % moisture content and 210–260 s microwave radiation processing time) were experimentally established. These conditions will increase the biological value of the mixture, namely, increase the degree of cleavage and digestibility of proteins in the gastrointestinal tract. The technological scheme for the production of a protein-fat mixture of increased nutritional value based on oilseeds was proposed, a draft technical specification TU U 10.86.1 – 02071180.001:2018 for protein-oil mixtures from oilseeds was developed and agreed upon. Recipes of fat-containing confectionery – cream candy mass for the production of truffle-type sweets and chocolate paste with the addition of 15 % protein-fat mixture of high nutritional value were scientifically substantiated. It was experimentally proved that the induction periods of the candy mass and chocolate paste of the proposed composition exceed the control values by 44 and 55 %, respectively. The organoleptic evaluation of test samples was carried out to determine the effect of protein-fat mixture on consumer properties of confectionery products. Based on its results it was determined that the use of protein-fat mixture in cream candy masses and chocolate paste influenced on such organoleptic indicators like taste and aroma that were higher than in control samples. In particular, there were a characteristic savory nutty aroma and taste, longer remains «mouth-feeling»in the prototypes of confectionery products. A further increase in the content of the mixture – more than 15 % – leaded to the appearance of a mealy taste and characteristic aroma of flaxseed. The effective concentration of the protein-fat mixture in the confectionery products was chosen at the level of 15 % based on the results of the organoleptic assessment The results of determining the physico-chemical quality control parameters of confectionery indicated that when 15% of the protein-fat mixture was added to a creamy candy mass and chocolate paste, the change in the mass fraction of moisture and the mass fraction of fat occurred within the framework of normalized values. Reducing the total sugar content did not affect the organoleptic properties of confectionery, but reduced the cost of production. The developed products have a higher content of essential amino acids compared to the control, their lipid composition changes – the content of PUFAs of the ω-3 group increases significantly. As part of the developed confectionery products, there is also a significant amount of dietary fiber – a nutrient that accelerates the feeling of fullness, as well as a factor in the formation of normal intestinal microflora and the obesity prevention.

The purpose of this research was to evaluate four objectives: 1) update and evaluate predictions of essential amino acid (EAA) outflows from the rumen, 2) predict EAA use and release by the portal drained viscera (PDV) and liver (LIV) of dairy cows, 3) predict EAA use by the mammary (MAM) and non-splanchnic, non-mammary (OTH) tissues, and 4) predict milk protein production from MAM use. To evaluate the first objective, a model was constructed using previously derived equations for ruminally undegraded (RUP), microbial (MiP) and endogenous protein (EndP) flow from the rumen and refit to literature data. Corrections were included in the model to address recovery of EAA during 24-h acid hydrolysis. Upon initial evaluation, all EAA, except Leu, were over predicted and slope bias (P < 0.01) was present for all except Met and Leu. Because of the bias, residuals were regressed on the EAA from each protein flow and adjustments were made to the protein flows. The added adjustments removed all mean bias for the EAA; however, a small slope bias was introduced for Lys and Thr. To evaluate the second objective, equations of Hanigan et al. (2004b) were tested and modifications were made to determine which equation form best represented EAA use by the tissue. Upon initial evaluation of the PDV model of Hanigan et al. (2004b), significant slope bias was present and addressed by deriving alternative forms of the equation. Initial predicted EAA use displayed a mean bias ranging from 0.15 to 45 % and a slope bias ranging from 0.02 to 76% mean square error. The alternative equation forms derived reduced the overall mean and slope bias and improved other fit statistics (RMSE, CCC). To evaluate the third objective, previously derived equations from Hanigan et al. (1998b) were tested using literature data and modifications were made to address deficiencies for each EAA. Upon initial evaluation of the MAM model, significant mean and slope bias was present and was further addressed by derivation of alternative equation forms. Initial evaluation of the OTH model displayed significant mean and slope bias for majority of the EAA ranging from 0.3 to 26 % for mean and 46 to 61 % for slope. For the last objective, several models, both linear and non-linear were evaluated to determine which EAA have a significant impact on milk protein. All models derived has prediction errors below 18-20 % which is comparable or a s light improvement as compared to previous literature data (Moraes et al., 2018). Overall, the equations evaluated show promise in accurately predicting dietary EAA from the time of absorption to their use within the tissues (PDV, LIV, MAM, and OTH) and further impact on milk protein production.

Specific AA have been demonstrated to activate signaling pathways that regulate<br />translation initiation and to stimulate protein synthesis in mammary tissue. The<br />objectives of this research were to determine the response to Ile, Leu, Met, and Thr in<br />cellular signaling and "-S1 casein fractional synthesis rates (CFSR). An experiment was<br />developed as a composite design. The experiment was replicated in tissue corresponding<br />to 5 cows. Mammary tissue slices (0.12 ± 0.02 g) from lactating dairy cows were<br />incubated 4 h in treatment media enriched with 2H5 Phe. Following incubation, slices<br />were homogenized in lysis buffer and caseins were precipitated by acidification to pH<br />4.6. An aliquot of the pellet was trypsinized and 2H5 Phe enrichment in the 34-<br />NLLRFFVAPFPE-45 peptide of "-S1 casein was measured by MALDI TOF-MS and<br />used to determine CFSR (%/h). Western immunoblotting was performed to identify total<br />and site-specific phosphorylated mammalian target of rapamycin (mTOR, Ser2448),<br />eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein (rp) S6 (Ser235/236),<br />and eukaryotic initiation factor (eIF) 2" (Ser51). Addition of Ile, Leu, Met, or Thr had<br />no effect on eIF2" phosphorylation. Isoleucine positively affected mTOR, and rpS6, and<br />negatively affected eEF2 phosphorylation. Leu had a similar effect on eEF2, but not on<br />mTOR or rpS6, and these two AA inhibited each other. Thr negatively interacted with<br />Ile on mTOR and rpS6, and with Leu on eEF2. Increasing concentrations of Ile, Leu,<br />Met, and Thr caused curvilinear increases in CFSR. The maximum response to Ile, Leu,<br />iii<br />Met, and Thr was at 71, 49, 60, and 65% of DMEM concentrations, respectively. All<br />maximums were above plasma AA concentrations observed in lactating cows fed to meet<br />NRC requirements. The CFSR estimated at those maximums were similar between AA<br />(3.6 ± 0.6 %/h). Individual AA effects on CFSR did not correlate with mTOR signaling.<br />Independent CFSR responses to individual essential AA observed in this study contradict<br />the single-limiting AA theory assumed in current requirement systems. The saturable<br />responses of CFSR to these 4 AA also demonstrate the deficiencies of a fixed postabsorptive<br />AA efficiency approach for determining AA requirements for milk protein<br />synthesis.<br>Ph. D.

Mouse embryonic stem cells (ESCs) are capable of differentiating into any somatic cell type and are known to display fragile immune privilege in vivo and in vitro. The extent to which the depletion of essential amino acids (EAAs) by ESCs contributes to this phenomenon was investigated. ESCs were found to express various enzymes capable of catabolising EAAs within the culture medium. In particular, depletion of threonine, valine and lysine was found to have significant impact on T cell proliferation and differentiation, biasing their polarisation towards a FoxP3⁺ T regulatory (T_reg) phenotype. Supplementing ESC conditioned medium with these three EAAs alone rescued normal T cell proliferation, whereas artificially limiting their availability was sufficient to induce T_reg cell differentiation to a level equivalent to general EAA depletion. The pattern of EAA catabolism by mouse ESC was shared by induced pluripotent stem cells, while mouse melanoma cell lines and human ESCs displayed distinct patterns of EAA depletion. The cytosolic branched chain aminotransferase enzyme, Bcat1, catalyses the first step of branched chain amino acid catabolism (isoleucine, leucine and valine), and is highly expressed by both mouse and human ESCs. The contribution of this enzyme to the establishment of acquired immune privilege by ESC-derived tissues was, therefore, investigated. ESC lines were derived from mice lacking Bcat1 activity and were characterised. Bcat1^−/− ESC lines displayed no difference to their wildtype counterparts (Bcat1^LoxP) in terms of in vitro proliferation and their capacity to form teratomas in vivo. Furthermore, the loss of Bcat1 function had little impact on the inhibition of T cell proliferation in culture, ability to induce T_reg cell commitment or their ability to prevent rejection by T cell receptor transgenic recipients, suggesting the minimal contribution of Bcat1 to the depletion of EAAs by ESCs. In conclusion, EAA depletion by mouse ESC may provide a mechanistic explanation for the previously described immune-suppressive capacity of ESC.

En condicions d’estrès pot existir un dèficit relatiu de glutamina (Gln). Aquesta actua com a font energètica i en la protecció cel·lular, activant l’expressió de les “Heat Shock Proteins” (HSP). Disminucions en la seva concentració mitja provoquen una alteració en la regulació inflamatòria. La seva suplementació estimula mecanismes de mort bacteriana i disminueix l’explosió citotòxica. OBJECTIUS: Determinar la resposta inflamatòria en els pacients pediàtrics suplementats amb Gln parenteral respecte els que reben nutrició estàndard. Valorar la morbi-mortalitat. METODOLOGIA: Estudi prospectiu, a doble cec i aleatoritzat amb blocs balancejats. Es van recollir mostres analítiques i dades de la història clínica dels pacients ingressats en la Unitat de Cures Intensives Pediàtriques (UCIP) de l’Hospital Sant Joan de Déu de Barcelona. Criteris d’inclusió: pacients (1 mes – 14 anys), que van requerir nutrició parenteral (NPT), afectes d’infeccions sistèmiques o focals greus, i post operats cirurgia major abdominal. Criteris d’exclusió: pacients amb patologia prèvia de base i derivats d’altres centres amb evolució clínica de més de 48 h. Aleatorització en dos grups: Grup 1 o grup experimental al que es va administrar una solució d’aminoàcids (Aminopaed® o Vamin®) i es va suplementar amb Gln (Dipeptiven®), anomenat NPS + Gln. Grup 2 o grup control al que es va administrar una solució d’aminoàcids(Aminopaed® o Vamin®) no suplementada, anomenat NPS. Es van recollir 73 pacients en cada grup. Les variables a estudi van ser: edat; sexe; Pediatric Risk Score of Mortality II (PRISM-II) a l’ingrés; malaltia actual; nivells de limfòcits CD4, CD8 i CD4/CD8; valors d’interleuquines (IL) IL-6, IL-10 i de HSP-70 a les 0 hores (h) a les 48h al cinquè dia; i presència de sobre infecció, fallida multi orgànica i mortalitat. RESULTATS: Els nivells de Gln no van mostrar diferències entre els grups. El dia 5, els pacients del grup experimental presentaren nivells d’HSP-70 significativament superiors que el grup control (68,6 vs 5,4, p = 0,014). En tots dos grups, els nivells d’IL-6 van tenir un descens significatiu des de el dia 0 al dia (NPS: 42,24 vs 9,39, p < 0,001; NPS + Gln: 35,20 vs 13,80, p<0,001), però tan sols en el grup experimental va existir un descens significatiu entre el dia 2 i el dia 5 (13,80 vs 10,55, p = 0,013). Els nivells d’IL-10 no van variar durant les visites excepte en el grup control entre el dia 0 i el dia 2 (9,55 vs 5,356, p < 0,001). Al final de l’estudi no s’observaren diferències significatives entre els grups referent a l’estància en UCIP o a l’estància hospitalària, tot i que va haver una menor estada a intensius en els pacients suplementats amb Gln. No es van detectar efectes adversos en cap grup. CONCLUSIONS: La suplementació amb Gln en pacients crítics contribueix al manteniment de majors nivells d’HSP-70, durant més temps. Aquesta suplementació no influeix en els nivells d’IL-10 i no mostra disminució significativa dels nivells d’IL-6. La Gln podria disminuir la estada en la UCIP.<br>In a stress situation, there is a Glutamine (Gln) deficit owing to an increase in its consumption as much as a decrease of its availability. This amino acid acts as a source of energy and also intervenes in the cellular and tissue protection activating the Heat Shock Proteins (HSP) expression. The Gln average concentration value is associated with a fall in immune response. It has been also proved that, if a supplement of Gln is added, it stimulates the mechanisms of bacteria death and diminishes the cytotoxic response. OBJECTIVES: To determine if there exist differences in the inflammatory response in seriously ill patients who received Gln supplemented nutrition with regard to those who received standard nutrition. To evaluate the patient clinical response and mortality. METHODOLOGY: It was a prospective, interventional, double blind, randomized and stratified clinical trial. Collection of samples and data for patients admitted at the Pediatric Intensive Care Unit (PICU) of the “Sant Joan de Déu” Hospital in Barcelona. Inclusion criteria: Patients (1 month to 14 years) who require parenteral nutrition, with one of the following diagnosis: Systemic or local infection and Major abdominal surgery. Exclusion criteria: patient with prior illnesses and patients coming from other centers with more than 48 hours clinical evolution. Sample selection: Group 1: Standard total parenteral nutrition (NPS) (Aminopaed® o Vamin®) and Gln perfusion (Dipeptiven®), denominated NPS + Gln. Group 2: Standard total parenteral nutrition (Aminopaed® o Vamin®) without Gln, denominated NPS. It will be necessary to include 73 experimental units in the reference group and 73 units in the experimental group. Variables: Age; sex; Pediatric Risk Score of Mortality (PRISM-II) score at admission; illness cause for the admission; CD4, CD8 i CD4/CD8 lymphocyte levels; determination at 0 hours (h), 48 h and 5th day of Interleukins(IL), IL-6, IL-10 and HSP-70; and infection complication, multiorganic failure and death. RESULTS: Gln levels failed to show statistical differences between groups. At day 5, patients in the experimental group had significantly higher levels of HSP-70 as compared with the control group (68.6 vs 5.4, p = 0.014). In both groups, IL-6 levels showed a remarkable descent from baseline and day 2 (SPN: 42.24 vs 9.39, p < 0.001; SPN + Gln: 35.20 vs 13.80, p<0.001) but only the treatment group showed a statistically significant decrease between day 2 and day 5 (13.80 vs 10.55, p = 0.013). Levels of IL-10 did not vary among visits except in the SPN between baseline and day 2 (9.55 vs 5.356, p < 0.001). At the end of the study, no significant differences between groups for PICU and hospital stay were observed. Although there was a shorter stay in the supplemented patients. No adverse events were detected in any group. CONCLUSIONS: Gln supplementation in critically-ill children contributed to maintain high HSP-70 levels for longer. Glutamine supplementation had no influence on IL-10 and failed to show a significant reduction of IL-6 levels. The Gln could reduce PICU stay.

An increase in myofibrillar protein accretion can occur in the very early post-exercise period and can be potentiated by dietary protein intakes. The positive effect of nutrition on net muscle protein balance is credited to essential amino acids (EAAs), and particularly to the branched-chain amino acid leucine. Thus, leucine plays a key role in feeding-induced muscle protein synthesis by its unique ability to activate signalling pathways modulating translational initiation (Dreyer et al. 2008). Furthermore, strength exercise induces important disturbances in protein turnover, especially in novice athletes, and unaccustomed physical exercise, particularly repeated eccentric muscle contractions, induces muscle soreness and alterations in muscle cellular structure. Still, muscle strength gains induced by resistance training are mainly attributed to neural adaptations in the initial part of training, and subsequently to changes in the force capacity of muscle fibres due to hypertrophy (Duchateau et al. 2006; Sale, 1988). Since acute post-exercise muscle protein accretion does not inform on chronic muscle adaptations, the main purpose of this work is to test the effectiveness of an EAA supplementation on muscle hypertrophy and strength gains in response to resistance training, and on repair-oriented remodelling of disrupted muscle fibres in the early recovery period of an unaccustomed exercise. The first investigations aim at evaluating the effects of an EAA supplementation on muscle mass, architecture, and strength in the early stages of a heavy-load training programme, and at establishing if the enrichment of the supplement with leucine induces greater improvements in these muscle functional adaptations. In a second part, central and peripheral adaptations are examined following training to compare whether EAA supplementation modifies the relative contribution of neural and muscular factors to the increase in muscle torque, and promotes adaptations in muscle mechanical and contractile properties. The purpose of the third investigation is to study the potential effects of an EAA supplementation on the reduction in the efflux of indirect markers of muscle damage and the delayed onset muscle soreness in the week following a heavy-load eccentric training session.In the first and in the second investigation, young males trained for 12 weeks. They were divided into a placebo (PLA) group (n = 14), an EAA group (n = 15) and a leucine (LEU) group (n = 14). At baseline, daily food intakes and nitrogenous balance were assessed with a food questionnaire over 7 days and two 24h urine collections. The effect of training on muscle mass was assessed by anthropometric techniques. Muscle thickness and pennation angle were recorded by ultrasonography of the medial gastrocnemius (MG). Maximal strength during squat and bench press exercises was tested on an isokinetic ergometer. The torque produced by the plantar flexors and the surface electromyogram (EMG) from the soleus (Sol) and MG were recorded during maximal voluntary isometric contractions (MVC). Central activation was tested by the superimposed electrical stimulation method during MVC and by computing the ratio between voluntary average EMG and compound muscle action potential (M wave) induced by electrical stimulation (average EMG/M-wave). Contractile properties of the plantar flexor muscles were investigated by recording the mechanical responses to single and paired maximal stimuli. In the third investigation, young males performed a bench press exercise in eccentric condition. They were subdivided into a PLA group (n=11) and an EAA group (n=12). The effect of the training session was assessed by analysing two indirect markers of muscle damage, namely creatine kinase (CK) and myoglobin (Mb). Plasma concentrations were measured before, immediately after, and on post-workout days 1, 2, 3, 4 and 7. Muscle soreness was evaluated by a visual analogic scale (VAS) at the same time points as the markers of muscle damage.Resistance training resulted in significant increases in muscle mass and strength in all PLA, EAA and LEU groups with no statistical differences between groups. A positive linear regression was found between nitrogen balance and the increase in muscle mass in the PLA group only. When strength was normalised to skeletal muscle mass, negative linear regressions were observed between the normalised initial strength and the increase in muscle strength in both EAA and LEU groups. EAA and LEU ingestion induced changes in MG muscle architecture in response to training. After training, plantar flexor torque recorded during MVC was increased in PLA, EAA and LEU groups. Central activation level was enhanced with no effect of EAA supplementation. Twitch torque evoked by single and paired supramaximal stimuli, maximal rate of torque development to single stimulus and rate of torque relaxation to single and paired stimulus were significantly improved in the LEU group in response to training. The eccentric training session induced a significant increase in muscle soreness in both PLA and EAA groups. Plasma CK release increased significantly at D+3 and D+4 and Mb efflux rose at D+3 in PLA group only. No statistical differences were observed between groups for the two indirect markers of muscle damage. Gaussian distribution was found to be the best fit model for plasma Mb and CK concentration curves. F-test showed that individual curves were statistically distinguishable when comparing the best-fit values of the three parameters (area, SD and mean) between PLA and EAA group data sets (P < 0.01).In summary, the data indicates that EAA supplementation results in similar muscle mass and strength adaptations in response to resistance training with no additional effect by the enrichment of the supplement with LEU. However, the nutritional interventions appear to be more effective in subject having a lower initial nitrogen balance and/or a lower initial strength, potentially by promoting changes in muscle architecture. EAA ingestion does not potentiate neural adaptations, but leucine-enriched EAA supplementation may induce earlier peripheral adaptations, resulting in enhanced torque production and transmission. Finally, EAA supplementation could have minor effects on the overall plasmatic release of indirect markers of muscle damage during the recovery period without any impact on delayed onset muscle soreness.<br>Doctorat en Sciences de la motricité<br>info:eu-repo/semantics/nonPublished

Important to the evaluation of potential feed ingredients for inclusion in fish diets should be their digestibility and amino acid requirement so that feeds can be formulated on a digestible basis rather than a gross nutrient basis. This thesis established techniques for faecal collection, the validity of digestibility markers, it determined apparent digestibility coefficients for various novel feed ingredients, and it established the optimal lysine requirement for Argyrosomus japonicus, which was used to estimate each of the essential amino acid requirements by using the ideal protein concept. There were no significant differences in dry matter apparent digestibility coefficients when faeces were collected by stripping (77.0 %), dissection (80.1 %) or settlement (83.5 %). Faecal collection by the settlement method produced the most reliable digestibility data. Apparent digestibility coefficients for dry matter, crude protein and energy calculated using acid-insoluble ash were higher (84.0 %, 93.2 % and 93.0 %, respectively) than those using chromic oxide (55.7 %, 79.1 % and 78.2 %) and titanium dioxide (58.6 %, 79.7 % and 80.5 %). The magnitude of variation in digestibility coefficients obtained using acid-insoluble ash was always lower than that obtained with the other markers. Therefore, acid-insoluble ash was preferred as a dietary marker. Using the above protocol, protein and amino acid digestibility of some animal and plant protein ingredients were evaluated in a series of experiments. The first trial compared the apparent digestibility coefficients of some animal products included as single protein source in the test diets. Apparent protein digestibility values were 84.5 %, 83.8 %, 85.8 % and 83.1 % for fishmeal-prime, fishmeal-standard, poultry meal and pork meal, respectively. Apparent digestibility coefficients for poultry meal were comparable to those of fishmeal, which indicate its potential as a substitute for fishmeal in the diets of A. japonicus. The second trial determined the apparent coefficients of plant and animal protein sources included at 30 % into a practical reference diet (70 %). Apparent protein digestibility ranged from 92.4 % in sunflower meal to 85.5 % in corn gluten meal. Soybean meal is a promising feed ingredient in A. japonicus due to the high apparent digestibility of its protein (92.0 %) and essential amino acid digestibility (mean average 91.4 %). A fundamental assumption in fish feed formulation is that the digestibility of nutrients is additive, i.e., digestibility of a nutrient in one ingredient does not interact with the digestibility of the same nutrient in another ingredient. In the third trial, additivity of feed ingredients was tested using pork meal and poultry meal. The results indicate that the apparent digestibility coefficient of animal protein ingredients could be calculated from compound diets to accurately determine protein and amino acid digestibility in A. japonicus, and possibly other carnivorous fish species. Dietary essential amino acid requirements were determined for juvenile A. japonicus in two trials. A dose-response study was conducted using crystalline lysine to determine the optimal requirement of dietary lysine for A. japonicus. Optimal dietary lysine was estimated at 31.7 g kg-¹ dry diet, corresponding to 73.5 g kg-¹ of dietary protein, based on specific growth rate and broken-line segmented regression analyses. Dietary requirements for other essential amino acids ranged from 22 g kg-¹ (histidine) to 71 g kg-¹ (leucine) crude protein. The results of the present study provided a research tool that could be used to assess and verify the conclusions of earlier dietary work on A. japonicus and in further studies to develop least cost diet formulations for this species. The study also adds to the knowledge of the nutritional requirements of A. japonicus by providing information on the digestibility of plants and animal protein ingredients. It also contributes to future dietary research for this species because this study determines, for the first time, the most suitable methods for investigating the digestibility of raw materials for A. japonicus.

<p>In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are involved in these defence systems (Korsloot et al. 2004). Since an organism’s resources may be considered to be limited, the ability to restore homeostasis depends on the severity of the different forms of stress it experiences. It has been proposed that pollutants present in the environment may alter the ability to respond to climatic stressors like e.g. low temperature, desiccation (Holmstrup 2002).</p><p>This work deals with the possible consequences of combined stress from metal exposure and low temperature in cold hardy insects. Many of these insects produce so called <i>antifreeze proteins</i> that protect them from lethal freezing. Metallothioneins are metal binding proteins that are considered to be important in detoxification when animals are exposed to metals. Metallothioneins and most forms of antifreeze proteins from insects are known to contain unusually high amounts cysteine. Cysteine is considered to be semi-essential, since it must be derived from the essential amino acid methionine (Choen 2004). Induction of one of these two types of proteins may potentially deplete the cysteine pool and thus reduce the capacity to produce the other type. Alternatively, the animals might have evolved other structures to avoid a potential competition for cysteine. The purpose of the present work was to explore these possible scenarios.</p><br>Paper I and II reprinted with kind permission of Elsevier, Sciencedirect.com

Sarcopenia, loss of skeletal muscle in older adults, can lead to serious health consequences. Several non-pharmacological strategies have been suggested to prevent sarcopenia, one of which is increased protein consumption above the current RDA and/or reaching a certain threshold (approximately equal to 10 grams of essential amino acids) of quality protein at each meal. This study examined the association between the amount of skeletal muscle mass in older active adults with regard to their total protein intake and essential amino acid threshold frequency (EAATF) (number of meals per day that provided at least 10 grams of essential amino acids). Fifty-one healthy male (n=22) and female (n=29) active older adults participated in this cross-sectional study. Average daily total protein (grams) and EAATF were determined from a seven-day dietary record. Dietary records and subsequent amino acid profiling were analyzed using a computer software program. Daily food records were averaged across seven-days to give an average representation of total protein intake and EAATF. Total and appendicular lean mass (aLM) were determined by Dual Energy X-Ray Absorptiometry (DXA). Relative lean mass (RLM, total lean mass/height2) and appendicular lean mass index (aLM/height2) were also calculated. Data were analyzed using Pearson Partial Correlation Coefficients controlling for body weight and physical activity level with an alpha level of 0.05. EAATF (1.1 + 0.6 times/day) was positively associated with RLM (r = 0.621, p < 0.001) as well as aLM index (r = 0.583, p < 0.001). Total protein intake (91.1 + 29.1) was also positively associated with RLM (r = 0.582, p < 0.001) and aLM index (r = 0.598, p < 0.001). Our data suggest that, not only total protein intake, but also consuming a certain threshold of quality protein with each meal throughout a day may be important for maintaining muscle mass during aging.<br>Thesis (M.Ed.)--Wichita State University, College of Education, Dept. of Human Performance Studies

Doctor of Philosophy<br>Department of Human Nutrition<br>Tonatiuh Melgarejo<br>Background and Objective: Crohn’s disease (CD) patients express low levels of host defense peptides (HDPs) especially β-defensins, which may compromise intestinal barrier function. Antibiotic treatment for bacterial infections in CD is limited and rarely curative, making it necessary to find alternative therapeutic approaches. We therefore investigated to what extent an essential amino acid; L-isoleucine (L-ILE) might induce the expression of human β-defensins (HBDs) in colonic epithelial cells as an alternative approach to help patients with CD. Antimicrobial activity of HBD2 was also assessed against four bacterial isolates which can cause secondary infections in CD. Methods: HTB-37 Caco-2 cells were stimulated with L-ILE at a concentration of 0 - 500µg/ml for 6 hours. Total RNA was extracted using RNeasy Micro Kit (QIAGEN). Reverse transcription was carried out with Superscript ®III First-Strand Synthesis System. The cDNA was amplified using specific primers for HBD1-3. Antimicrobial activity of HBD2 was determined using the broth dilution assay. Results: HBD1 was constitutively expressed under all conditions. HBD2 was expressed in HTB-37 cells after stimulation with L-ILE. Below 25µg/ml L- ILE stimulation, no expression of HBD2 was observed. HBD2 exhibited antimicrobial activity against bacterial isolates tested, with a MIC of 32, 64 and 128 µg/ml for both strains of E. coli, S. aureus and P. aeruginosa respectively. Conclusions: Our results indicate that L-ILE stimulation of HTB-37 Caco-2 cells can induce HBD2 expression. Data collected from our in vitro studies might have major implications for modifying the intestinal microbiota towards a healthier state in CD patients. Promoting the expression of HBD2 by colonic cells may lead to a lower rate of infection in these patients. Future in vivo studies are warranted to determine the potential clinical use of intra colonic administration of L-ILE in CD patients. The observed antimicrobial activity of HBD2 against bacterial isolates provides evidence that it is a crucial component of mucosal epithelial defense against infections which can complicate disease symptoms in CD.

Made available in DSpace on 2017-07-10T18:13:19Z (GMT). No. of bitstreams: 1 Romulo Batista Rodrigues.pdf: 1330851 bytes, checksum: 4991e2d69fa6f7d58830a99378844e76 (MD5) Previous issue date: 2016-02-26<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>The valine is part of the branched chain amino group together with leucine and isoleucine, and has an important structural role and anabolic. Two experiments were conducted in order to determine the requirement of valine for fingerlings and juveniles of tilapia, by assessing the growth performance, and analysis of chemical composition of housing, hematological and biochemical blood, and white muscle morphometric. In the first experiment were used 270 tilapia fingerlings whit initial average weight of 1.57 ± 0.05 g total initial length of 4.16 ± 0.46 cm, distributed in completely randomized design with six treatments and three replications, using 18 boxes with 250 liter of capacity during the experimental period of 79 days. In the second experiment were used 216 tilapia juveniles with initial average weight of 21.40 ± 0.42g and initial total length of 10.07 ± 1.00 cm, distributed in 18 boxes whit 500 liters of capacity during the experimental period of 77 days. The treatments consisted of diets with increasing concentrations of valine, being for fingerlings (0.82, 0.86, 0.98, 1.04, 1.10 and 1.26% valine) and juvenile (0.54, 0.63, 0.72, 0.81, 0.90 and 0.99% valine). Feed has been held four times a day to apparent satiation. Evaluated the growth performance (weight gain, feed conversion, daily weight gain, protein efficiency ratio, condition factor, survival, protein retention efficiency, visceral fat, liver somatic index, specific growth rate and uniformity of the lot) for fingerlings and juveniles; fish proximate composition (moisture, crude protein, lipids and ash) for fingerlings and juveniles; hematological and biochemical indices of the blood (hemoglobin, erythrocytes count, hematocrit percent, total leukocytes, total thrombocytes, plasma protein, glucose, triglyceride and cholesterol) for juvenile and distribution of muscle fibers to fingerlings and juveniles. There was no statistical difference (P>0.05) for the performance variables, chemical composition and histology between the different treatments evaluated to fingerlings. For juvenile tilapia, on the performance were observed significant differences (P<0.05) between treatments for weight gain, daily weight gain and feed conversion. For hematological and biochemical parameters of blood, there were significant differences (P<0.05) between treatments for hemoglobin, total triglycerides and total cholesterol. Significant differences in the chemical composition of fish and frequency of muscle fibers were observed. It is concluded that the nutritional requirements are met with valine 0.82% valine in the diet (2.65% valine crude protein) for minnows and 0.81% of valine in the diet (2.90% valine crude protein) for juvenile Nile tilapia.<br>A valina faz parte do grupo de aminoácidos de cadeia ramificada, juntamente com a leucina e isoleucina, e possui importante papel estrutural e anabólico. Foram realizados dois experimentos com o objetivo de determinar a exigência nutricional de valina para alevinos e juvenis de tilápias do Nilo, por meio da avaliação do desempenho zootécnico, e das análises de composição química da carcaça, perfil hematológico e bioquímico do sangue, e da morfometria do músculo esquelético. No primeiro experimento, foram utilizados 270 alevinos de tilápia com peso médio inicial de 1,57 ± 0,05 g e comprimento total inicial de 4,16 ± 0,46 cm, distribuídos em um delineamento inteiramente ao acaso com seis tratamentos e três repetições, utilizando 18 caixas com 250 litros de capacidade, durante período experimental de 79 dias. No segundo experimento, foram utilizados 216 juvenis de tilápia com peso médio inicial de 21,40 ± 0,42 g e comprimento total inicial de 10,07 ± 1,00 cm, distribuídos em um delineamento inteiramente ao acaso com seis tratamentos e três repetições, utilizando 18 caixas com 500 litros de capacidade, durante período experimental de 77 dias. Os tratamentos consistiam em dietas com concentrações crescentes de valina, sendo para alevinos (0,82; 0,86; 0,98; 1,04; 1,10 e 1,26 % de valina na dieta) e para juvenis (0,54; 0,63; 0,72; 0,81; 0,90 e 0,99 % de valina na dieta). A alimentação foi realizada quatro vezes ao dia até a saciedade aparente. Avaliou-se o desempenho zootécnico (ganho em peso, conversão alimentar aparente, ganho em peso diário, taxa de eficiência proteica, fator de condição, sobrevivência, eficiência de retenção proteica, gordura visceral, índice hepatossomático, taxa de crescimento específico e uniformidade do lote) para alevinos e juvenis; a composição centesimal dos peixes (umidade, proteína bruta, extrato etéreo e cinzas) para alevinos e juvenis; os índices hematológicos e bioquímicos do sangue (taxa de hemoglobina, contagem de eritrócitos, percentual de hematócritos, leucócitos totais, trombócitos totais, proteína plasmática, glicose, triglicerídeos e colesterol) para juvenis e a distribuição das fibras musculares para alevinos e juvenis. Não houve diferença estatística (P>0,05) para as variáveis de desempenho, composição centesimal e histologia entre os diferentes tratamentos avaliados para alevinos. Para os juvenis de tilápia, no desempenho zootécnico observaram-se diferenças significativas (P<0,05) entre os tratamentos para o ganho em peso, ganho em peso diário e conversão alimentar aparente. Para os parâmetros hematológicos e bioquímicos do sangue, observaram-se diferenças significativas (P<0,05) entre os tratamentos para taxa de hemoglobina, triglicerídeos totais e colesterol total. Não foram observadas diferenças significativas para a composição centesimal dos peixes e frequência das fibras musculares. Conclui-se que as exigências nutricionais de valina são atendidas com 0,82% de valina na dieta (2,65% de valina da proteína bruta) para alevinos e 0,81% de valina na dieta (2,90% de valina) para juvenis de tilápias do Nilo.

The gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) require n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for optimal growth and health. Due to the rapid growth of global aquaculture the quantity of marine oils used in aquafeeds has been limited, yet the overall quantity of oil in an aquafeed has increased by the addition of vegetable oil (VO) to supply dietary energy. For aquaculture to continue to grow more fish must be produced with less marine ingredients, yet EPA and DHA must be maintained at levels above fish requirements. This project set out to re-evaluate the requirement for EPA and DHA in gilthead seabream and European seabass. Two dose-response studies were designed and executed where juvenile seabream and seabass were fed one of six levels of EPA+DHA (0.2 – 3.2 % as fed). Biometric data were collected and analysed to determine new requirement estimates for EPA+DHA for fish of two weight ranges (24 – 80 g and 80 – 200 g). The effects of the dietary LC-PUFA gradient on lipid composition and metabolism were also considered.

碩士<br>臺灣大學<br>微生物與生化學研究所<br>98<br>Phenylalanine ammonia lyase (PAL; EC 4.3.1.5), the entry enzyme for phenylpropanoid biosynthesis, is often encoded by mutigene families in plant. It catalyzes the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia. In monocotyledon plants, PAL may exhibit the similar substrate specificities for tyrosine and phenylalanine and the protein-catalyst serves double duty. BoPAL1 showed 96％ sequence identity with BoPAL2, but had very different situation in substrate specificity. By exchanging the different amino acid nearby active site to figure out which amino acid was important for substrate specificity, the fusion proteins BoPAL1 F133H and BoPAL2 I198V, F134H, A149P, G389N showed the poor substrate affinity to phenylalanine than those of wild-type PALs. To tyrosine substrate,fusion protein BoPAL1 F133H and BoPAL2 A149P, G389N increase enzyme specific activities. BoPAL4 is one of the Bamboo PAL gene families. Expression constructs pTrcHisA/BoPAL4 was transformed into E.coli Top10. Fusion protein BoPAL4 contains (histidine)6-tag in N-terminus and can be purified by Ni2+-column chromatography. By using gel filtration chromatography, the molecular mass of expressed BoPAL4 was estimated to be 290 kD, and monomer mass was determined to be 70 kD by SDS-PAGE. As the result, BoPAL4 was estimated to be homotetramer. The optimum temperature and pH for PAL activity were 50℃ and 9.0, respectively. The activation energy was 24.7 kcal/mol for BoPAL4. The Km value for phenylalanine was 2072 μM, and kcat/Km was 7.87. For tyrosine substrate, Km value was 97 μM, and kcat/Km was 5.56. Expressed BoPAL4 has highly tyrosine ammonia lyase activity than that of BoPAL2.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Nikola Holotíková Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Can essential amino acids be considered as copper chelators? Copper is a trace element playing an important role in the human organism. Copper levels in the body have to be carefully controlled because disruption of copper homeostasis can cause oxidative damage which may lead to various pathologies. Chelation of copper can potentially be used as a therapeutic tool. Medical chelators should ideally mimic chelators, especially peptides and proteins which are important for regulation of copper homeostasis. Their structure is formed by amino acids linked by peptide bonds. For this reason, the study is focused on selected amino acids. Proteinogenic amino acids are classified into essential and nonessential. Essential amino acids must be obtained from the diet. The aim of this study was to compare the copper chelation and reduction activity of essential amino acids L-histidine and L-methionine, and nonessential amino acids L-cysteine, its dimer L-cystine, L-aspartic acid and L-glutamic acid. Also a L-cysteine-derived compound, N-acetylcysteine, was tested. All of them were tested using spectrophotometric methods at pHs which...

碩士<br>國立臺灣大學<br>漁業科學研究所<br>88<br>Among the genus of Lutjanus, the Red striped snapper (Lutjanus erythropterus) with red-colored body, is suitable for the taste of Chinese people. It is most likely developing as the main kind of cultured fish using the seawater net cage culture in Taiwan. The aim of this study is to evaluate the effect of that using fermented soybean meal instead of fish meal for feed of the Red striped snapper and to explore its possible application. This study is divided into two parts. One is that to add separately 30%, 20% and 10% of fermented soybean meal and essential amino acid into the feed to discuss the most suitable amount of fermented soybean meal in the feed of the Red striped snapper. The other is that in the case of fixed amount (20%) of fermented soybean meal , 3.5%, 8.5% and 13.5% of fish oil are added, respectively, into the feed to explore the utilization of lipid for the Red striped snapper. After 50 days with the adding amount of fermented soybean meal up to 30 %, the weight gain (%) of the Red striped snapper is distinctly lower than controled. In the cases that the adding amount of fermented soybean meal are 20% and 10%, respectively, the weight gains are similar in between. According to the Broken-line analysis, the most suitable amount of fermented soybean meal added in the feed is 14.9%. If the extra amount of essential amino acid is added, the adding amount of fermented soybean meal in the feed is increasing up to 16.7% based on the Broken-line analysis. After 50 days of feeding, the weight gain of the Red striped snapper increases with increasing lipid amount added in their feed. When the adding amount of lipid in their feed is 17.8%, their weight gain and feed conversion rate are 227% and 0.75, which are the best and 70% and 0.22 higher than controled, respectively.

The Cu$\rm\sb{A}$ center, located in subunit II of cytochrome c oxidase, is the primary site for electron entry from cytochrome c. The recent crystal structure confirms this site to be a binuclear copper center with a dithiolate, dihistidyl coordination. A methionine and the carbonyl oxygen of Glu$\sp{198}$ are also ligands. To further characterize the structure and function of the Cu$\sb{A}$ center, site directed mutagenesis has been implemented in subunit II of S. cerevisiae. Substitutions of direct ligands to the Cu$\rm\sb{A}$ site result in loss of cellular respiration. Cytochrome a from cytochrome c oxidase is not detected in the visible absorption spectrum. Furthermore, subunit II does not accumulate to wild-type levels as observed by immunodetection. These results suggest that the enzyme is not assembling, most likely due to the disruption of copper binding, and that subunit II is being degraded. In contrast, substitutions to Glu$\sp{198},$ whose carbonyl oxygen is a ligand to one of the coppers, merely results in cells with reduced respiratory function. In an attempt to perturb the Cu$\rm\sb{A}$ site without disrupting protein stability, nearby amino acids were substituted. Asp$\sp{158}$ is known to hydrogen bond to the Cu$\rm\sb{A}$ ligand His$\sp{161}$, and Ser$\sp{197}$ is adjacent to the Cu$\rm\sb{A}$ ligand Cys$\sp{196}$. Substitutions at these positions result in reduced cellular respiration and may perturb the Cu$\rm\sb{A}$ site. Substitutions to conserved residues Gly$\sp{194}$ and Gly$\sp{201}$ result in a loss of cellular respiration and the underaccumulation of subunit II, while substitutions at other conserved positions have no observable effect on respiration. These results are discussed in relation to the homologous quinol oxidases and nitrous oxide reductases. To begin to characterize residues which may be involved in binding cytochrome c, substitutions were made to various conserved carboxylates which had been implicated in binding cytochrome c. The recent crystal structure demonstrates that Glu$\sp{198}$ and Asp$\sp{158}$ are not available for direct interaction with cytochrome c. However, the crystal structure does point to other conserved carboxylates. Asp$\sp{112}$ may be at the periphery of a cytochrome c binding site since replacement of this residue by Arg results in only a small decrease in cellular respiration.

碩士<br>國立臺灣大學<br>生化科學研究所<br>92<br>Isoprenoids are widely distributed natural polymers containing isopentenyl pyrophosphate (IPP) as building block. The biosyntheses of isoprenoids are carried out by a group of enzymes called prenyltransferases which catalyze the head to tail condensation between IPP and farnesyl pyrophosphate (FPP) to make linear long-chain products. These FPP-utilizing enzymes play essential biological role, e.g. the C55 undecaprenyl pyrophosphate (UPP) generated by UPP synthase (UPPs) serves as a lipid carrier to transport lipid II across the bacterial cell membrane for peptidoglycan synthesis of the cell wall; Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate (FPP) to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Previously, we have solved the complete kinetic pathways and 3-D structures of E. coli UPPs and OPPs. Based on the crystal structure, some amino acids in neighborhood of the active site are proposed to play important roles in substrate binding and the reaction. However, the amino acids which act as general acid and base in the catalysis have not been identified. In this study, we utilized pH profile in conjunction with site-directed mutagenesis to examine the important residues. Some novel method of study has also been developed.