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Soybean meal (SBM) is a staple proteinaceous feedstuff in diets for monogastric animals like poultry and swine. It is known that soybeans contain several anti-nutritional factors that, if untreated, results in decreased quality and bioavailability of amino acids (AA). Thermal processing via heat treatment of soybeans and SBM is essential for inactivation of these anti-nutritional factors; however, over-processing may result in extensive AA damage, particularly lysine. Feeding heat damaged SBM has been proven to be an inefficient source of AA for monogastrics as they cannot be used for any metabolic function. In typical corn-soybean meal diets for pigs and poultry, lysine is the first- and second- limiting AA, respectively. Currently, laboratory procedures are unable to accurately determine digestible lysine in SBM. The objective of this thesis was to compare SBM AA digestibility obtained from 28-day old broilers to values obtained from an in vitro digestion procedure. The correlation between AA concentration in the SBM and its in vivo standardized ileal digestibility (SID) was also analyzed. Twenty-four SBM samples (21 from U.S.A., 2 from Canada, and 1 from Mexico) were analyzed. In vivo lysine SID ranged from 69-93%. Results indicated no correlation (r = -0.16 to 0.21; P = 0.33 to 0.98) between analyzed AA content in SBM and in vivo SID. An increase in lysine SID was associated with an increase in the SID of phenylalanine, leucine, isoleucine, valine, tyrosine, alanine, threonine, glutamate, aspartate, methionine, histidine, and glycine (r² = 0.63 to 0.93; P < 0.001). Poor association was determined between lysine proline, arginine, and serine (r² = 0.14 to 0.43; P = 0.001 to 0.003). Lastly, results indicated no association (r² = 0.00 to 0.08; P = 0.17 to 0.99) between in vivo and in vitro SID for any of the AA tested. In summary, it appears that lysine may be a good indicator for SID estimations for most essential AA; however, SBM content of a particular AA is not a good indicator of its digestibility. Additionally, current in vitro digestibility techniques seemed inadequate in identifying in vivo SID differences and further analytical improvements are needed. / Master of Science

Protein tyrosine kinase and phosphatase domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue on their substrates. Although the preferred recognition motifs of many kinase and phosphatase domains have been characterized, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically-encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences and predict phosphorylation efficiencies of substrates. We then screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. Finally, we have made progress towards extending this platform to the analysis of tyrosine phosphatase domains, by optimizing methods to produce tyrosine-phosphorylated bacterial display libraries and implementing methods to detect peptide dephosphorylation on the cell surface. Collectively, these experiments demonstrate the utility of our platform for rapid profiling of sequence specificity by tyrosine kinases and will shed new light on phosphotyrosine signaling.

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. / Doctor of Philosophy / 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.

Broomrapes are achlorophyllous holoparasites of many economically important dicotyledonous crops. As weeds, they cause reductions in crop yield, adversely affect crop quality, and result in loss of cultivated land due to reduced crop alternatives. Few effective control measures exist for broomrapes. One of the most promising approaches is the use of low rates of glyphosate in hosts with tolerance to the herbicide. Recently, availability of glyphosate-resistant crops has provided an alternative in broomrape infested areas. Knowledge about the nitrogen status of broomrapes is essential for developing new control strategies. Broomrapes have two potential sources of amino acids. First, the haustorium aids in the translocation of amino acids from the host plant to the parasites. Second, broomrapes may be able to synthesize some amino acids themselves and obtain the rest from the host. However, the relative importance of these two modes of acquiring amino acids by broomrapes is not clear. Osmotic stress has been implicated as a possible reason for inhibition of broomrape germination by nitrogen. To date, there has been no attempt to correlate osmotic potential with nitrogen induced inhibition of broomrape germination. Optimum temperatures for conditioning and germination are different among broomrape species. Although temperature is known to influence germination in broomrape, its effect on subsequent development of the parasitic seedling has not been studied. Studies were conducted to determine the use of glyphosate in controlling broomrape in common vetch that is tolerant to low rates of glyphosate, and to compare this response with broomrape control in oilseed rape that has been genetically engineered for glyphosate resistance. Glyphosate dose response studies using a commercial formulation and patterns of absorption, translocation, and metabolism, using ¹⁴C-glyphosate, were determined for both host crops. Glyphosate significantly reduced the growth of broomrape at 0.18 and 0.36 kg ae ha⁻¹> in common vetch and 0.25 to 0.75 kg ha⁻¹ in oilseed rape. More than 25% of translocated ¹⁴C-glyphosate in both host crops accumulated in broomrape tubercles. Broomrape parasitism caused a redistribution of translocated ¹⁴C-glyphosate in the roots of both host crops. Glyphosate was metabolized up to 25% in common vetch, but remained intact in oilseed rape. Studies were conducted to analyze amino acid composition of both nonparasitized and broomrape-parasitized hosts and associated broomrape after hydrolysis and phenylisothiocyanate derivatization of amino acids. Results indicated that amino acid concentrations of leaves of parasitized carrot plants were lower than those of the leaves of nonparasitized carrot plants. Broomrape tubercles had equal or higher amino acid concentrations compared to those of the leaves of nonparasitized carrot plants. Levels of free alanine and arginine concentrations of broomrape callus were higher than those of any other tissue of either carrot or broomrape. The effect of glyphosate on the host-broomrape interaction regarding amino acid metabolism was examined. Glyphosate generally increased the amino acid concentrations in common vetch and oilseed rape plants, and broomrape attachments. The aromatic amino acids, phenylalanine and tyrosine, did not differ from this pattern. Concentrations of certain amino acids in broomrape were similar to those of parasitized common vetch and parasitized oilseed rape, whereas levels of several others, were higher in broomrape attachments compared to the host plants. <I>In vitro</I> studies were conducted to determine the influence of osmotic potential and temperature on broomrape germination. Osmotic potential significantly affected germination and radicle elongation of broomrapes. No correlation was found between osmotic potential and ammonium-induced inhibition of germination of broomrapes. Temperature significantly influenced germination and radicle elongation of all broomrape species tested. / Ph. D.

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. / Ph. D.

Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes. The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1. To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated. / Ph. D.

Lysine absorption by ruminal and omasal epithelia was studied using parabiotic chambers that were sampled for 60-min. Lysine appearance in serosal buffers and the accumulation of lysine in tissues increased linearly (P < .001) with time. Lysine appearance in serosal buffers of ruminal tissue increased proportionally as the concentration of lysine increased in mucosal buffers. However, lysine appearance in serosal buffers of omasal tissue increased proportionally to a substrate concentration of 1.5 mM, then plateaued. Total absorption (tissue accumulation plus serosal appearance) increased linearly for ruminal tissue; however, for omasal tissue, total absorption increased linearly to 1.5 mM (P < .001), then plateaued. Using omasal epithelium, glycyl-L-sarcosine (Gly-Sar; .1 mM) absorption was studied during co-incubation with glycine and peptide substrates (each at 5 mM). Accumulation of Gly-Sar in omasal epithelium was greatest (P < .05) when Gly-Sar was present alone. Glycine inhibited (P < .05) Gly-Sar accumulation by 20%, whereas peptide substrates inhibited (P < .05) Gly-Sar accumulation by 60 to 85%. The absorption of Gly-Sar (.1 mM) alone or during co-incubation with either 10 mM butyric acid, or a mixture of VFA was also studied. Accumulation of Gly-Sar in tissue was greatest (P < .05) when Gly-Sar was present alone; butyric acid and VFA inhibited (P < .05) Gly-Sar accumulation by 50 to 84%. These results suggest absorption of amino acids and peptides by the omasum, and also suggest the mechanism involves mediated as well as possibly paracellular transport. / Master of Science

Amino acids are essential molecules in plant metabolism. Amino acids carry reduced nitrogen while serving as precursors for protein synthesis and secondary metabolites. Translocation of amino acids in the cell is mediated by amino acid transporters. While about 100 transporters have been identified, only a dozen have been fully characterized. The regulation of amino acid transporters is not fully understood and stands as the basis of this study. Previous toxicity-based screenings of Arabidopsis thaliana mutants led to the isolation of a loss-of-function line and the phenylalanine insensitive growth (pig1) mutant capable of growth on toxic concentrations of phenylalanine (1). The pig1-1 mutants also displayed a deregulated metabolism (1). We followed this work with a similar forward genetic screening of Arabidopsis thaliana that led to the identification of 18 mutants capable of growth in the presence of amino acids at toxic concentrations. From this screen, seven mutations were confirmed to affect the amino acid transporter AAP1. Here I demonstrate that, when expressed in yeast deficient for endogenous amino acid transporters, three variant aap1 proteins restored growth similar to yeast complemented by wild type AAP1. Transport of radiolabeled Pro was abolished by variant aap1 proteins while deletion of an intracellular loop spanning the 8th and 9th transmembrane domains reduced Pro transport in yeast. Site directed mutagenesis of this loop conferred a variant aap1 protein which augmented Pro transport in yeast. Amino acid transport in loss-of-function aap1 plants display decreased uptake and increased efflux. In addition, aap1 mutant plants accumulated between 2 and 8 times more free amino acids in the leaves than the wild type. These observations are not fully compatible with the accepted role of AAP1 in transport by the root. The present work describes how the amino acid transporter AAP1 could play a role in regulating amino acid metabolism. We hypothesize that the amino acid transporter AAP1 functions as a senor that is involved in amino acid homeostasis in addition to its established role as a transporter. Is true, this would make AAP1 the first identified amino acid sensor in plants. Knowledge of the mechanism of amino acid sensing would enable us to engineer crops for improved nutrition in a more efficient way than affecting metabolic enzymes. / MSLFS / Amino acids play essential role in crop metabolism. Amino acids are nitrogen containing molecules that are used to make protein and many other molecules. They are located through-out the plant and move from organ to organ by amino acid transporters. A dozen of approximately 100 known amino acid transporters have been studied in depth and are well understood. Interestingly, not much is known about these transporters and what controls their activity. A mutant weed, Arabidopsis thaliana mutant phenylalanine insensitive growth (pig1), was identified by its ability to survive in toxic environments with high amounts of the amino acid phenylalanine and also showed an irregular metabolism of amino acids (1). The Pilot Lab and I were able to identify 18 more mutants with similar abilities to survive in toxic amino acid conditions by performing similar experiments. Seven of the new mutants were found to have mutations that effected the amino acid transporters AAP1. Using yeast incapable of growing in nitrogen restricted conditions where amino acids are the only source of nitrogen, I found that three of the variants app1 proteins we identified were able to restore growth like wild type AAP1 yeast. These variant aap1 yeast did not show the ability to transport the acid proline, while other alter versions of the aap1 protein made to alter its structure and proposed significant parts were able to increase proline transport. Plants with no or mutant aap1 proteins showed a decreased ability to uptake amino acids in addition to increased efflux of amino acids. These plants also had a higher level of amino acids in their leaves than normal wild type plants. These results obtained in both plants and yeast with altered amino acid transporter aap1 do not agree with what we understand to be the accepted function of AAP1 transporting amino acids in plant roots. The work presented in this thesis discusses how AAP1 could be involved with controlling plant amino acid metabolism. It is my hypothesis that the transporter is serving two functions by both transporting and sensing amino acids. As a sensor, AAP1 serves to maintain a proper balance of amino acids for plant metabolism. If AAP1 does this, it would be the first of its kind to be identified in plants and help enhance crop engineering for better nutrition to better feed growing populations.

The purpose of this research was to further test a stable isotope based approach as a more reliable in vivo method to determine amino acid bioavailability from a variety of ingredients. The method was used to assess feather meal (FM), blood meal (BM), soybean meal (SBM), and a rumen protected amino acid (RPAA). An abomasal infusion of raw EAAs (isoleucine, leucine and methionine) and an abomasal infusion of sodium caseinate were used as control treatments to test the accuracy of the technique. The isotope-based results were then compared to in situ, in vitro and in vivo test results. The isotope-based technique provided AA bioavailability values for five non-essential AA and seven essential AA. The raw EAA infusion had the greatest AA recovery in plasma with an estimated absorbed RUP value of 93.4± 7.35% followed by the casein infusion (86.7 ± 4.81%), SBM (54.8 ± 5.19%), FM (52.7 ± 4.81%) and BM (47.5 ± 4.81%). The RPAA treatment had the lowest bioavailability at 9.9 ± 12.73%. Numerically, SBM supplied the most absorbable EAA of the 4 feed ingredients, but was not significantly different from that of BM and FM. Simply based on the control treatments in this research (raw EAA and casein), this isotope method was a more accurate method in determining AA bioavailability values with relatively low standard errors. Ingredients are exposed to all aspects of natural digestive processes and the method is able to determine AA appearance in the blood with no use of in situ or in vitro measurements. / Master of Science / Balancing rations for essential amino acids has beneficial effects on milk production and milk protein synthesis. However, to have predictable results, accurate knowledge of essential amino acid supply deriving from ingredient rumen undegradable protein and microbial crude protein flows is required. Methods used to assess essential amino acid supply include in vivo, in vitro and in situ methods; however these methods often generate conflicting results and have significant deficiencies that have hampered development of robust, accurate predictions of essential amino acid supply to the animal. This research tested a non-steady state, stable isotope based approach as a more reliable in vivo method to determine amino acid bioavailability for feed ingredients. Two control treatments (abomasally infused casein and raw essential amino acid) and four ingredients (feather meal, blood meal, soybean meal, and a rumen protected amino acid) were tested. Based on the control treatments, the method provided a reliable assessment of amino acid bioavailability values with relatively low standard errors. This method has the advantage of assessing essential amino acid bioavailabilities in a natural state where the ingredients of interest are components of a relatively normal diet exposed to all of the natural digestive processes. Thus values derived from this approach can be expected to be representative of most normal industry diets. With some further refinement, this method can help to create a library of true values for a variety of feed ingredients, leading to more accurately balanced diets and increased milk production. More accurate values of amino acid digestibility and rumen undegradable protein measurements for ingredients will also help to better determine their fair market value.