The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

Eight cyclic analogues of Substance P were made in order to investigate the conformation of the C-terminal end of the peptide. These analogues were designed to test three literature models describing the active conformation of substance P. Although the potencies of the analogues were low (in the micromolar range), our results support Cotrait's and Hospital's model (1986). Several substance P antagonists were synthesized. These compounds did not demonstrate agonistic activity nor anatagonistic activity. The tryptophan side chain is contributing to the antagonistic activity of these analogues, and not just the chirality of the α-carbon. Highly potent and selective photoaffinity ligands of H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP) were synthesized. These compounds will be useful in the isolation of δ and μ opioid receptors. Several new amino acids designed and synthesized to contain both the natural amino acid side chain and a thiol group which can be used to make disulfide constraints. The racemic amino acids made were as follows: (1) 2-amino-4-methyl-2- [(p-methylbenzyl)thiomethyl] pentanoic acid; (2) 2-amino-2- [(p-methylbenzyl)thiomethyl] -3-phenylpropanoic acid; (3) 2-amino-e- [(p-methylbenzyl)thio] pentanoic acid; and (4) 2-amino-3- [(p-methylbenzyl)-thio] -3-phenyl-pentanoic acid. These amino acids will be useful in the conformational restriction of peptides. To investigate the δ-opioid receptor conformation proposed for DPDPE by Hruby et al. (1988) and the μ-opioid receptor conformation proposed for Tyr-c [Abu₂,Gly,Phe,Leu] by Mierke et al. (1988), constrained phenylalanine amino acids were incorporated into H-Try-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) in the four position. Our results indicate that these models are correct. And in an investigation into the physical-chemical properties of the delta opioid receptor, our results suggest that the δ receptor topochemical site for the Phe⁴ residue contains a partial positive charge on its surface and has specific steric requirements.

The first half of this dissertation describes the synthesis and biological activities of a series of amino aldehydes; which were derivatives of the basic amino acids, arginine, lysine and ornithine. The synthesis of the amino aldehydes was complicated by the difficulty of producing an intermediate oxidation state (the aldehyde) in the presence of two other functional groups (the α-amino, and the side chain functionality). The amino aldehydes were of biological interest due to the fact that they were inhibitors of the proteolytic enzymes called kallikreins. The kallikreins are known to be involved with the renin-angiotensin system, arginine vasopressin, and the prostaglandins, in the regulation of blood pressure. The aldehydes were assayed for their ability to inhibit the kallikrein-mediated production of kinins, and by the inhibition of the cleavage of Nᵅ-tosyl arginine methyl ester (TAME) to the carboxylic acid. Two of the amino aldehydes (Nᵅ-t-Boc-Nᴳ-nitro-L-argininal and Nᵅ-t-Boc-Nᴳ-tosyl-L-argininal) were effective inhibitors in both bioassays at micromolar concentrations. The second part of the dissertation details the development of two syntheses of β-substituted analogues of cysteine. The first method was based on sulfenylation of Nᵅ-formyl-α, β-dehydro amino acid esters, followed by protection of the sulfhydryl group as the benzyl or para-methylbenzyl thioether. The Nᵅ-formyl and ester groups were cleaved by acidic hydrolysis, and the amino group was then blocked as the t-butyloxycarbonyl derivative. This procedure gave cysteine analogues which were suitable for direct use in solid phase peptide synthesis. A second, more efficient preparation of the cysteine analogues was based on the conjugate addition of lithium benzylthiolate (or lithium para-methylbenzylthiolate) to the Nᵅ-formyl-α, β-dehydroamino acid esters. This synthesis was more efficient since the cysteine analogues were generated directly in S-protected form. The fully protected intermediates were deprotected at the amino and carboxyl groups, followed by treatment with di-tert-butyl dicarbonate. The Nᵅ- t-Boc-β-S-benzyl cysteine analogues (or Nᵅ -t-Boc-β-S-para-methylbenzyl) also were suitable for direct use in solid phase peptide synthesis.

Biomolecular systems, in particular those involving proteins and their constituents, have been the focus of much research in the last century. The relationship between experiment, development of models and simulation has enabled vast improvements in our knowledge of subjects such as protein folding and the processes by which key biomolecules affect the human body. In particular, vital information can be obtained from understanding the building blocks of polypeptides and proteins involved in these processes. This work focuses on simulating two such building blocks; glutamate, the salt of the proteinogenic amino acid, glutamic acid, and glycine-proline-glutamate, or GPE, a related tripeptide. Both are important in neurotransmission processes in the brain. Glutamate is the most abundant neurotransmitter in the central nervous system and GPE is an important neuroprotective agent. This work aims to elucidate the key structural properties of aqueous solutions of glutamate and GPE, focusing on the solute-solute as well as the solute-solvent interactions. Both systems were considered with classical empirical potentials using the CHARMM22 force-fi eld. The glutamate system has also been studied using Car-Parrinello Molecular Dynamics and classical parallel tempering. In both the aqueous glutamate and GPE systems the molecules formed a large proportion of bifurcated bonding motifs with both carboxyl groups, but not with the amin (N-terminal) of the molecules. Bifurcated bonds form between solute molecules as well as in the solute-solvent interactions. The structure of the glutamate solution was found to be dependent on the initial con figuration and thus the parallel tempering simulations enabled better sampling of the conformational landscape. In addition, in the glutamate system single water molecules form a stable structure by bonding to both the amine (N-terminal) and C -carboxyl within the same glutamate molecule.

Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a global issue affecting about 24 million individuals. Because AD is a systemic pathology, dementia is not the only leading factor contributing to loss of independence in AD patients. AD may also impair skeletal muscle metabolism and function. Creatine (CR) supplementation may enhance skeletal muscle hypertrophy/mass and function in sarcopenia and muscular dystrophies, but has yet to be studied in AD. This study examined the effect of oral CR on muscle metabolism in a triple-transgenic (3xTg) AD mouse model. Twenty-four, 3×Tg AD mice (~8 month-old) were randomly assigned to control (CON) or CR (3% w/w) diet. Bodyweights and feed intakes were measured throughout the 8-week study. Lower limb (quadriceps muscle; QM and gastrocnemius; GM) and upper limb muscles (triceps; TM) were collected to analyze levels of CR, total protein, DNA, RNA, amino acids (AA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), total and phosphorylated p70 ribosomal S6 kinase (p70S6K). Data (mean ± SEM) were assessed by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) post hoc test. In comparison to the CON group, CR supplementation increased CR content in both GM (p=0.002) and QM (p=0.037), with higher (p=0.032) ATP/ADP ratio in CR in comparison with CON in QM. A higher protein concentration (p<0.0001) was notable in GM of CR supplemented group vs. CON. Total branched-chain AA levels in QM increased 2-fold (p< 0.0001) in CR groups. Additionally, CR resulted in a higher (p<0.05) protein/DNA ratio; an index of muscle cell size, in both QM and GM for CR groups. The index of cell capacity for protein synthesis (RNA/DNA ratio) in GM was also higher (p=0.001) in CR groups. However, phosphorylation (activation) level of p70S6K, an integral component in protein synthesis signalling pathway, did not show any significant differences in female (p=0.161) and male (p=0.292) CR supplemented groups compared with CON. To conclude, CR supplementation is capable of inducing muscle hypertrophy/growth parameters in the 3×Tg AD mouse model, thereby enhancing protein synthesis capacity in skeletal muscles, thus possibly promoting muscle function in AD. / May 2016

Transition metal complexes derived from Schiff bases have rendered an important contribution to the development of modern coordination chemistry. Various stable compounds have been prepared having synthetic, biological, and physicochemical interest. In particular, complexes of salicylaldimines, B-ketoamines, and closely related ligand systems have been investigated.

The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

The sense of taste is one of the most important factors in regulating ingestive decisions. This is central to a number of disease conditions including but not limited to obesity, diabetes, anorexia, hypertension, coronary artery diseases and malnutrition. The detection of the molecules eliciting taste qualities in food is mediated by the coordinated actions of distinct types of taste sensory cells (TSCs) housed in taste buds within specialized papillae throughout the oral cavity. Taste receptors in the taste sensory cells that detect food molecules are the key players in selecting dietary nutrients. One such example is L-amino acids, a critical part of one's diet. L-glutamate is the prototypical umami compound and is known to increase palatability of food. A unique characteristic of umami taste is the response potentiation of glutamate by 5' ribonucleotide monophosphates, such as inosine 5' monophosphate (IMP), which is also capable of eliciting an umami taste. Candidate receptors for umami taste include a heterodimer T1r1+T1r3, brain variants of mGluR1 and mGluR4, and the truncated variants of mGluR1 and mGluR4. Studies using heterogeneous expression of T1r1+T1r3 suggest it is an umami and a broadly tuned L-amino acid receptor. While much attention is devoted to understanding glutamate transduction, the detection mechanisms for other L-amino acids by TSCs are less well understood. Here calcium imaging of isolated TSCs and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice was performed to determine if other receptors are involved in the detection of L-amino acids and IMP. Ratiometric imaging with Fura-2 was used to study calcium responses to IMP and four L-amino acids (monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine) with and without IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across TSCs. Only a small subset of cells responded to all stimuli. Interestingly, L-amino acids other than glutamate elicited synergistic responses in a subset of TSCs. Additionally IMP alone elicited a response in a large number of TSCs. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex. Next the roles of mGluR1 and mGluR4 in the detection of the IMP and L-amino acids were investigated. Selective agonists for mGluR1, (RS)-3, 5-dihydroxyphenylglycine (DHPG; a group I mGluR agonist), and mGluR4, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; a group III mGluR4 agonist) elicited responses in TSCs. In addition, TSCs responsive to these agonists were also responsive to L-amino acids and IMP. More importantly, selective antagonists against different mGluRs such as (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; a group I mGluR antagonist), and (RS)-α-methylserine-O-phosphate (MSOP; a group III mGluR antagonist) significantly suppressed L-amino acid- and IMP-mediated responses in TSCs of T1r3 knockout mice. Collectively, these data provide evidence for the involvement of taste and the brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the hypothesis that multiple receptors contribute to the IMP and L-amino acid tastes.

Master of Science / Department of Animal Sciences and Industry / Joel DeRouchey / Michael Tokach / Six experiments using a total of 2,974 nursery pigs were used to determine the effects of monosodium glutamate (MSG) and amino acids (AA) on nursery pig growth performance. Experiments 1 and 2 evaluated increasing dietary MSG for nursery pigs. Increasing dietary MSG up to 2% without balancing for sodium and chloride content decreased nursery pig performance, and feeding sodium levels equivalent to 1% MSG also decreased performance. When sodium and chloride were balanced, there were marginal effects of increasing dietary MSG on pig performance. Experiment 3 was conducted to determine the standardized ileal digestible (SID) lysine (Lys) requirement for pigs weighing 7- to 11- kg. The SID Lys requirement was estimated to be 1.45% and greater than 1.60% depending on the statistical model applied for both ADG and G:F. This experiment served to validate the SID Lys requirement for use in formulating diets for the subsequent experiments. Experiment 4 evaluated increasing SID valine (Val) to Lys ratio for nursery pigs weighing 7- to 10- kg. A SID Val:Lys ratio of 62.9% optimized ADG. Maximum feed efficiency (G:F) was captured using 71.7% SID Val:Lys ratio, however, 99% of maximum was achieved with SID Val at 64.4% of Lys. For ADFI, maximum performance was at 74% SID Val:Lys ratio, with 99% of maximum intake achieved at 68%. Experiments 5 and 6 investigated increasing SID isoleucine (Ile) to Lys ratio for 6- to 11- kg pigs. When ADG and ADFI were modeled, broken-line models reported maxima of 52.0% Ile:Lys ratio while quadratic models were as high as 64% of Lys.

This thesis is concerned with the development of methodology for the regioselective ring-opening of aziridines and aziridinium intermediates and its subsequent application to the asymmetric synthesis of &beta;-substituted-&alpha;-amino acids and 1,2,3,4-tetrahydroisoquinolines. Chapter 1 introduces methods for the formation of aziridines and aziridinium ions and focusses on their utility as intermediates in synthesis. Chapter 2 describes studies into the synthesis of aziridines from enantiopure &alpha;-hydroxy-&beta;-amino esters and their subsequent conversion to the corresponding &beta;-hydroxy-&alpha;-amino acids via either a regioselective ring-opening with Cl₃CCO₂H, or a rearrangement promoted by Cl3CCO2H. Application of this procedure to both syn- and anti-configured substrates enabled the syntheses of (S,S)-allo-threonine, (2R,3S)-threonine, (R,R)-3-hydroxyphenylalanine and (2S,3R)-3-hydroxyphenylalanine. Chapter 3 details attempts to truncate the synthesis described in Chapter 2 by investigating the synthesis of enantiopure anti-&beta;-hydroxy-&alpha;-amino acids via the intermediacy of aziridinium ions. These studies culminated in the development of a regioselective and stereospecific one-pot aziridinium formation and ring-opening protocol, leading to the synthesis of a range of C(3)-aryl and C(3)-alkyl substituted anti-&beta;-hydroxy-&alpha;-amino acids. Chapter 4 discusses the conversion of enantiopure anti-&alpha;-hydroxy-&beta;-amino esters to anti-&beta;-fluoro-&alpha;-amino esters via the regioselective and stereospecific ring-opening of an aziridinium intermediates in situ. The subsequent development of a one-pot deprotection strategy leads to a concise and expedient synthesis of anti-&beta;-fluorophenylalanines. The extension of this methodology to access a representative anti-&alpha;,&beta;-diamino acid is also demonstrated. Chapter 5 describes the development of a one-pot diastereoselective rearrangement of enantiopure &alpha;-hydroxy-&beta;-amino esters to 1,2,3,4-tetrahydroisoquinolines. The substrate scope of this reaction manifold is examined and application to the asymmetric synthesis of enantiopure 1,2,3,4-tetrahydroisoquinolines also discussed. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.