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Recent recommendations are that older adults increase their dietary protein intake to intakes higher than are currently recommended to mitigate sarcopenia-induced muscle loss caused in part by anabolic resistance. Protein supplementation may serve as an effective strategy to meet protein intake goals; however, protein supplements vary in their quality, which may impact muscle protein turnover. Protein quality is determined by the digestibility and content of essential amino acids in a protein source and may play an important role in mitigating the loss of muscle mass and muscle protein synthesis (MPS) during energy restriction (ER), acute reductions in physical activity, which we modeled using enforced step reduction (SR), and during recovery from SR. We aimed to determine whether the quality of a protein supplement – whey protein (higher quality) versus collagen peptides (lower quality) – would impact the reduction in fat-free bone-free mass (FBFM) and MPS (Study 1), and also to compare differences in functional variables: strength loss in men and women, and single fibre function with SR in men (Study 2). In Studies 1 and 2 we compared supplementation with whey protein (WP) and collagen peptides (CP), higher and lower quality proteins respectively, as part of a higher protein diet provided to older adults during one week of ER (-500 kcal/d), two weeks of step reduction (< 750 steps/d) (ER+SR) and one week of recovery (RC). Two weeks of ER+SR significantly reduced FBFM in both the WP and CP groups with greater FBFM recovery with WP. MPS was significantly reduced following ER in both groups and did not decrease further during ER+SR. MPS was increased above ER+SR following 1 week of RC in the WP group only. ER+SR significantly reduced maximum voluntary contraction (MVC) in both men and women; however, following RC men fully recovered their strength and women did not. In Study 3, we aimed to determine the impact of WP and CP supplementation combined with unilateral resistance exercise (RE) to augment the acute and longer term MPS response in healthy older women. Acutely, rates of MPS were elevated following WP+RE and with WP alone while MPS was elevated only in CP+RE. Six days of supplementation increased MPS in WP and WP+RE with no increase in MPS with CP or CP+RE. Collectively, these studies demonstrate that protein quality is an important variable to consider in selecting a protein supplement for older adults and for recovering from inactivity. / Thesis / Doctor of Science (PhD) / At the end of the 5th decade of life, adults will have lost an appreciable amount of muscle mass and strength versus what they had in their 3rd decade of life. This age-related loss of muscle mass and strength is known as sarcopenia. Additionally, as they age, adults will experience brief periods of reduced physical activity due to illness, injury, or recovery from surgery. Such periods are associated with a rapid loss of muscle and strength creating a brief period of ‘accelerated sarcopenia’. Strategies to combat the loss of muscle and strength in these periods include increasing protein intake and even periodic exercise which may help to reduce the negative impact of physical inactivity. In particular, higher quality protein sources (protein derived from animal sources or soy) and weightlifting may better help muscles recover from inactivity. Our main findings were that consuming high quality protein (whey protein) stimulated the process of muscle building that is normally reduced with inactivity. Importantly, when combined with resistance exercise, we were able to increase the rate at which healthy older women built muscle with whey protein in comparison to a lower quality protein source (collagen peptides). These findings provide novel and insightful information for the recommendations of protein supplement types to older adults to increase daily protein intake to preserve muscle mass with age.

In many situations in the world, the amount of resources available for use is limited. This statement is especially true in the cells of living organisms. During the translation process in protein synthesis, ribosomes move along the mRNA strand constructing proteins based on the sequence of codons that form a gene. The totally asymmetric simple exclusion process (TASEP) models well the translation process. However, these genes are constantly competing for ribosomes and other resources in the cell. To see how finite resources and competition affects such a system, we must construct a simple model to account for the limited resources. We consider coupling multiple TASEPs to a finite reservoir of particles where the entry rate of particles into the TASEPs depends on the number of particles left in the reservoir. Starting with a single TASEP connected to the reservoir, we study the system using both Monte Carlo simulations and theoretical approaches. We explore how the average overall density, density profile, and current change as a function of the number of particles initially in the reservoir for various parameters. New features arise not seen in the ordinary TASEP model, even for a single TASEP connected to the pool of particles. These features include a localized shock in the density profile. To explain what is seen in the simulations, we use an appropriately generalized version of a domain wall theory. The dynamics of the TASEPs with finite resources are also studied through the power spectra associated with the total particle occupancy of each TASEP and the reservoir. Again, we find new phenomena not seen in the power spectrum of the ordinary TASEP. For a single constrained TASEP, we find a suppression at low frequencies when compared to the power spectrum of the ordinary TASEP. The severity of this suppression is found to depend on how the entry rate changes with respect to the number of particles in the pool. For two TASEPs of different lengths, we find an enhancement of the power spectrum of the smaller TASEP when compared to the ordinary TASEP's power spectrum. We explain these findings using a linearized Langevin equation. Finally, we model competition between ten genes found in Escherichia coli using a modified version of the TASEP. This modified version includes extended objects and inhomogeneous internal hopping rates. We use the insight gained from the previous studies of finite resources and competition as well as other studies to gain some insight into how competition affects protein production. / Ph. D.

Dairy cows are extremely inefficient at converting dietary nitrogen (N) to productive N. Approximately 25-30% of dietary N is used for milk protein while the remaining N is lost to the environment. According to National Research Council (NRC, 2001) recommendations, dairy cow rations are formulated in terms of metabolizable protein (MP) which often causes many amino acids (AA) to be fed in excess. A better understanding of protein and AA requirements could help to improve the nitrogen efficiency of dairy cows. The objective of this work was to examine the effects of feeding a low protein diet supplemented with rumen protected (RP) AA on production and N efficiency of dairy cows. Twenty-four Holstein and 24 Holstein x Jersey crossbred cows were used in a Youden square design consisting of 3 periods. Cows were randomly assigned to one of 8 treatments: 1) a standard diet containing 17% crude protein (+Con), 2) a 15% crude protein diet (-Con), 3) â Con plus RP methionine (+M, 16g/d), 4) â Con plus RP lysine (+K, 47g/d), 5) â Con plus RP leucine (+L, 181g/d), 6) â Con plus RP methionine and lysine (+MK), 7) â Con plus RP methionine and leucine (+ML), and 8) â Con plus RP methionine, lysine, and leucine (+MKL). Cows fed the â Con as well as the +MKL diet experienced a reduction in milk production and milk protein yield (P < 0.05). Dry matter intake decreased only for those animals on the +ML diet (P < 0.05). Milk urea N (MUN) decreased for all diets when compared to the +Con treatment (P < 0.05). In accordance with the decrease in MUN, N efficiency was numerically increased in the diets supplemented with RP AA, but this improvement was not significant. Phosphorylation of signaling proteins important for protein synthesis were also examined. Animals fed the +MK treatment increased phosphorylated and total forms of eukaryotic elongation factor 2 (eEF2) when compared to the +Con and â Con (P < 0.05), but this increase in abundance did not affect the ratio of phosphorylated to total abundance. Feeding dairy cows a low protein diet supplemented with RP AA has the ability to alleviate the loss in milk production associated with feeding a low protein diet as well as to increase nitrogen efficiency. / Master of Science

Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States. / Ph. D.

Postnatal skeletal muscle growth occurs through myonuclear accretion and high protein turnover rate. While fiber type composition of the muscle could affect protein turnover rate, less is known about how fiber type influences the regulation of protein synthesis and degradation signaling pathways. Thus, the hypothesis of this work was that variation in fiber type composition will differentially affect the regulation of signaling pathways related to protein turnover in skeletal muscle hypertrophy in growing pigs. Downregulated protein synthesis signaling and reduced expression of type II MyHC isoforms have been reported in skeletal muscles of low birth weight (LBWT) neonatal pigs. Therefore, we sought to determine whether these changes are sustained until weaning and would explain the reduction in LBWT pig growth compared to their normal birth weight (NBWT) sibling at weaning. Another objective was to determine whether the regulation of protein turnover signaling pathways are correlated to fiber type differences in skeletal muscles. Our data suggest that the longissimus dorsi (LD, glycolytic) muscle of LBWT pigs experienced compensatory growth while the soleus (oxidative) remained proportionally smaller. Growth of the LD was accompanied by upregulation of translation initiation. Additionally, there was no difference in expression of MyHC isoforms between NBWT and LBWT pigs. These data suggest the rapid growth of the LD of LBWT pigs may be attributed to an upregulation of protein synthesis signaling and occurred only in glycolytic muscles. A caveat in LBWT pig model is that the reduction in type II MyHC at birth is not the only factor that could influence muscle growth, and that other factors may have confounded our results. This is why we aimed to use β-adrenergic agonist as a means to induce a shift fiber type in muscles to a more glycolytic phenotype. Our objective was to determine the influence of the β-adrenergic agonist Ractopamine (RAC) induced slow-to-fast fiber type transformation on the regulation of protein synthesis and degradation pathways. Although supplementation improved translational capacity, enhanced S6K1 phosphorylation, and reduced the abundance of calcium-dependent proteases, RAC feeding had no effect on body or muscle weights. These results suggest that a fiber type transformation without other physiological influences does not alter protein turnover signaling in favor of hypertrophy in growing pigs. / Master of Science / Skeletal muscles grow by increasing the amount of protein contained within them. The amount of protein deposited is determined by the net balance between the rates at which proteins are synthesized and degraded. However, not all skeletal muscles grow at the same rate. One factor that is thought to influence protein synthesis and degradation rates is the types of muscle fibers that are present within a muscle. These fibers can display a range of contractile and metabolic characteristics, from slow-twitch oxidative fibers to fast-twitch glycolytic fibers. In the presented studies, we sought to determine whether changes in fiber type composition result in difference to the signaling pathways the regulate protein synthesis and degradation, ultimately leading to differences in the muscle growth of young pigs. We have previously shown reduced activation of the protein synthesis pathway in the skeletal muscle of low birth weight (LBWT) newborn pigs. These pigs also had lower expression of glycolytic fibers. In experiment 1, we aimed to compare the signaling pathways regulating protein synthesis and degradation in LBWT and normal birth weight (NBWT) pigs at weaning. We also sought to determine if the regulation of these signaling pathways changed between muscles with differing fiber type compositions. The glycolytic longissimus dorsi (LD) muscle of LBWT pigs grew rapidly between birth and weaning whereas the highly oxidative soleus did not. In addition, the LD of LBWT pigs had greater protein synthesis signaling and similar expression of muscle fibers compared with NBWT pigs, suggesting the improvement in protein synthesis signaling of LBWT pigs between birth and weaning may be related to a shift in fiber type. In experiment 2, we used a compound called ractopamine hydrochloride (RAC) to promote a slow-to-fast fiber type switch in the muscle of young pigs. With this study, we sought to determine the effect of this fiber type transformation, without the influence of birth weight, on the regulation of protein synthesis and degradation pathways. Although RAC-fed pigs showed some minor changes that could improve protein synthesis and decrease protein degradation, RAC feeding had no observable effect on body weight or muscle growth. These results suggest that a fiber type transformation alone is not enough to promote muscle growth in growing pigs.

Skeletal muscle adapts differently to specific modes of exercise, where resistance training results in muscle growth and endurance training induces mitochondrial biogenesis. These are results of molecular events that occur after each exercise session, increasing the expression of specific genes and the rate of both synthesis and breakdown of protein. The rate of protein synthesis is controlled by the mTORC1 signaling pathway, which is potently stimulated by resistance exercise and amino acid, and their combined effect is needed for muscle growth. The essential amino acids (EAA) are responsible for the stimulation of protein synthesis and here leucine has been attributed specific attention, but its particular role among the EAA, and the involvement of the other branched-chain amino acids (BCAA) is unclear. Endurance exercise activates the protein AMPK which, in animal models, has been shown to inhibit mTORC1 signaling and protein synthesis.  Suggesting that concurrent endurance and resistance exercise could restrain muscle growth, but it is unknown if this mechanism is relevant in exercising human muscle. Little is known about the regulation of protein breakdown and although much attention has been given the proteins MuRF-1 and MAFbx which target proteins for degradation, their role requires further investigation. The aim of thesis was to address the mentioned uncertainties by examining how different modes of exercise and amino acids affect mTORC1 signaling, protein synthesis and markers of protein breakdown in human muscle. In study I, the influence of high intensity endurance exercise on subsequent resistance exercised induced mTORC1 signaling was examined. Despite robust activation of AMPK by the endurance exercise there was no inhibition of mTORC1 signaling or protein synthesis during recovery from resistance exercise. Study II utilized a similar set up, but with the difference that resistance exercise was performed with the triceps. The cycling exercise reduced the resistance exercise stimulated mTORC1 signaling immediately after the exercise, but during the recovery period mTORC1 signaling and protein synthesis was similar between trials. Concurrent exercise induced the mRNA expression of MuRF-1 and that of PGC-1α, the master regulator of mitochondrial biogenesis, in both studies, despite that the exercise modes in study II were separated between legs and arms. In study III, the effect of an EAA supplement with or without leucine, in the stimulation of mTORC1 signaling in connection with resistance exercise was examined. Intake of EAA robustly stimulated mTORC1 signaling after exercise, but this was only minor when leucine was excluded from the supplement. In study IV, subjects were supplied with leucine, BCAA, EAA or placebo in a randomized fashion during four sessions of resistance exercise. Leucine alone stimulated mTORC1 signaling after the exercise, but both the amplitude and extent of stimulation was substantially greater with EAA, an effect that was largely mediated by the BCAA as a group. In conclusion, endurance exercise prior to resistance exercise using the leg or arm muscles does not affect mTORC1 signaling or protein synthesis during the three hour recovery period from exercise, supporting compatibility between resistance- and endurance exercise induced signaling. Concurrent exercise increases the expression of the proteolytic marker MuRF-1 compared to resistance exercise only, which could indicate both and increased demand of cellular adaptive remodeling or a more direct detrimental proteolytic effect. Leucine is crucial among the EAA in the stimulation of mTORC1 signaling after exercise, its effect is however potentiated by intake of the remaining EAA. As a supplement a mixture of EAA must be regarded preferable, although the effect is largely mediated by the BCAA as a group.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Members of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase (2OG oxygenase) superfamily catalyse a wide range of oxidative reactions in biology. 2OG oxygenases require Fe(II) and atmospheric oxygen for their activity, and couple substrate oxidation with the decarboxylation of 2OG into succinate and carbon dioxide. There are more than sixty known 2OG oxygenases in the human genome; they modify small molecules, nucleic acids and proteins implicated in diverse biological processes. Importantly, the seemingly disparate functions of 2OG oxygenases often converge to regulate gene expression. 2OG oxygenases have been shown to affect epigenetic reprogramming, chromatin remodelling, transcription factor activity and mRNA splicing. Emerging evidence indicates that 2OG oxygenases are also involved in the translational control of gene expression. Oxygenases TYW5, ALKBH8, ALKBH5 and FTO were found to catalyse modifications of tRNA and mRNA. The work in this thesis extends these observations by demonstrating that 2OG oxygenase-catalysed protein hydroxylations also play an important role in protein synthesis. The catalytic activities of two oxygenases belonging to the JmjC-only family, NO66 and JMJD4, are described. NO66 catalyses the histidinyl hydroxylation of 60S ribosomal subunit protein L8. NO66 is part of a conserved group of ribosomal protein hydroxylases that can be traced back to prokaryotes. JMJD4 is a lysyl hydroxylase of eRF1, the eukaryotic release factor responsible for translation termination. The hydroxylation of eRF1 takes place on a conserved NIKS motif important for release factor activity, and promotes effcient translational termination. JMJD4 is further implicated in cell growth and cancer, though the link between its activity and tumourigenesis remains to be determined. These results highlight the potential of 2OG oxygenases as regulators of protein synthesis, and further extend the scope of 2OG oxygenase function. The small molecule inhibition of 2OG oxygenases presents a novel therapeutic possibility targeting translational control in cancer and other diseases.