The ultimate design of a microencapsulated AI dose is to continuously release sperm over a period of time in the female reproductive tract, thus alleviating the need for estrus detection. The objective of Trial 1 was to determine in vitro sperm release times for three microcapsule membranes. Semen was collected from four bulls, pooled, extended in 20% egg yolk TEST to a concentration of 80 = 10⁶ cells/ml, and encapsulated. Microcapsule membranes were constructed from isomers of polylysine: .1% poly-L-lysine (PLL), .1% poly-D-lysine (PDL), and a 50:50 mixture of the isomers (PLPD). Microcapsules were incubated at 37°C in a buffer containing .5% heparin or .5% trypsin and evaluated at 0.5, 1, 2, 4, 8, and 16 h post-encapsulation. For sperm encapsulated there were no significant differences in sperm motility. However, peak time of maximum sperm release differed between PLL and PDL membranes at 2 and 4 h of incubation. In Trial 2, sperm viability and microcapsule membrane stability were assessed post-thaw using PLL or PDL, two encapsulating temperatures (5°C or 23°C) and two times of glycerol addition (prior or post encapsulation at 5°C). Semen was extended to 80 = 10⁶ cells/ml and encapsulated. Capsules from all treatment combinations were incubated in .5% trypsin and evaluated as in Trial 1. In addition, motility was estimated at 1, 3, 6, and 9 h post-thaw. Motility from the unencapsulated control and capsules with glycerol addition prior to encapsulation, was superior (P < .05). Additionally, sperm release from capsules prepared at 5°C with glycerol addition post encapsulation was greater than all other treatments (P < .05). Time of peak sperm release for capsules was similar to the previous trial. There was a positive correlation between average capsule diameter and sperm release for both trials (P < .05). These data suggest that a combination of PLL and PDL capsules may complement each other in timing of sperm release and may be utilized in an inseminate mixture for extending the effective release in the female / Master of Science

Master of Science / Department of Animal Sciences and Industry / Jim L. Nelssen / Seven experiments were conducted to estimate the optimal standardized ileal digestible (SID) lysine:ME ratio for growing and finishing pigs. Experiment 1 determined an optimal SID lysine:ME ratio of 3.16 g/Mcal for 38 to 65 kg gilts. Experiment 2 reported an optimal level of 2.58 g SID lysine/Mcal ME for 55 to 80 kg gilts. Experiment 3 determined an optimal SID lysine:ME ratio of 2.55 g/Mcal for 85 to 110 kg gilts. In Exp. 4 and 5, growth rates were improved with porcine circovirus type 2 vaccine, with the optimal SID lysine:ME ratio for 38 to 65 kg gilts and barrows being 2.99 and 3.36 g SID lysine/Mcal ME, respectively. In Exp. 6 and 7, the optimal SID lysine:ME ratio was 1.86 and 2.61 g/Mcal for 102 to 125 kg gilts and 98 to 118 kg barrows, respectively. These trials indicate the optimal SID lysine:ME ratio for commercial growing and finishing pigs has increased compared with earlier estimates. Four experiments were also performed to determine the effect of copper (Cu) and zinc (Zn) supplementation on growth performance of weanling pigs. In Exp. 1, both 3,000 ppm Zn from zinc oxide (ZnO) and 150 ppm Cu from tri-basic copper chloride (TBCC) independently improved growth performance in weanling pigs due to increased feed intake. Similar results were observed in Exp. 2, where 3,000 ppm Zn from ZnO increased ADG and ADFI. Also, 125 ppm Cu from copper sulfate (CuSO4) increased growth rate due to enhanced feed intake, with Cu supplementation from TBCC offering intermediate results to CuSO4 and no Cu supplementation. For the first 28-d of Exp. 3, similar additive responses were observed to adding Cu and Zn to the diets of weanling pigs. However, from d 28 to 42 the combined use of Cu and Zn produced decreased performance compared each used singularly. Similarly in Exp.4, CuSO4 and ZnO improved growth performance, however the benefit was not additive. These trials showed growth promoting advantages to adding Cu and Zn to weanling diets, but additive responses were inconsistent.

L'adaptation est un phénomène capital pour la croissance optimale et la survie des bactéries dans un environnement qui est constamment soumis à des changements physico-chimiques. Pour y parvenir, les bactéries doivent contrôler l'expression génétique de façon efficace, c'est-à-dire en ayant le moins de perte énergétique possible et ce, dans un laps de temps très court suite à la détection du stress. Chez les procaryotes, on dénombre plusieurs mécanismes différents pour réguler l'expression des gènes. Par exemple, la transcription de certains gènes peut être inhibée ou activée par des facteurs protéiques. Dans certains cas, c'est plutôt la stabilité de l'ARNm ou encore le niveau traduction du gène qui est affecté, on parle alors de régulation post-transcriptionnelle. Chez les bactéries, les petits ARN régulateurs, exprimés selon différentes conditions de stress, contrôlent majoritairement l'expression de leurs gènes cibles de manière post-transcriptionelle. En plus de ces régulations en trans , il a récemment été découvert que certaines structures conservées de l'ARN pouvaient également contrôler l'expression de gènes en cis lors de la liaison spécifique d'un ligand. Ces structures, aujourd'hui connues sous le nom de riborégulateur, sont divisées en plusieurs classes dépendamment du type de ligand qui est lié. Chez Escherichia coli , il y a six riborégulateurs dont trois riborégulateurs TPP (thiMD, thiCEFSGH et thiBPQ ), un riborégulateur lysine (lysC ), un riborégulateur FMN (ribB ) et un riborégulateur AdoCbl (btuB ). Les résultats, présentés dans ce mémoire, portent sur la caractérisation du mode de régulation du riborégulateur lysine chez E. coli . Ainsi, pour la première fois dans le domaine des riborégulateurs, nous avons démontré que le riborégulateur lysine contrôle l'expression du gène lysC par deux mécanismes distincts, soit au niveau de la traduction du gène et de la stabilité de l'ARNm. Également, nous avons mis en évidence que par un changement de structure, le riborégulateur lysine peut contrôler l'accessibilité du site de clivage à la RNase E et par le fait même, la stabilité de l'ARNm. Ce nouveau mode de régulation ne semble pas être unique au riborégulateur lysine puisqu'il semble, selon les résultats préliminaires, que le riborégulateur thiC régulerait l'expression de l'opéron thiCEFSGH par les mêmes mécanismes de régulation.

Sarcopenia is a condition that is most common in aged animals, and is characterized by the loss of skeletal muscle mass and integrity, and can lead to physical disability and poor quality of life. Since skeletal muscle protein synthesis can be limited by the availability of amino acids, supplementation of limiting amino acids to ameliorate the progression of sarcopenia has become a topic of interest in companion animal research. Although there is some data to support the idea that amino acid supplementation improves maintenance of muscle mass in aged horses, the cellular mechanisms behind that improvement have yet to be elucidated. Therefore, the objective of this study was to examine the effect of amino acid supplementation in aged horses on markers of whole body and muscle protein metabolism. In a cross-over design, six old horses were studied while receiving each of three treatments in a replicated Latin square design. For all three treatments, horses received 1.8% BW/d of timothy hay cubes and 0.5% BW/d of experimental concentrate. The three treatments included a control (CON) treatment concentrate that was designed to meet all requirements of mature horses when fed in combination with the timothy hay cubes, and two supplemented concentrates, LYS/THR with additional lysine and threonine (40 mg/kg BW/d and 31 mg/kg BW/d, respectively), and LYS/THR/MET with additional lysine, threonine, and methionine (40 mg/kg BW/d, 31 mg/kg BW/d and 11mg/kg BW/d respectively). In each 15 d period, following a 9-day adaptation, horses were fitted with a collection harness, and total urine and feces were collected for 72 hours for assessment of nitrogen balance and creatinine output. Blood samples were taken directly before feeding and 30, 60, 90, 120, 150, 180, 210, and 240 minutes post-feeding for analysis of plasma urea nitrogen (PUN), glucose, insulin, and plasma amino acid concentrations. Muscle biopsy samples were taken for analysis of proteins in the mTOR pathway. Additionally, horses underwent stable isotope infusion procedures, and comparisons of phenylalanine kinetics were used to determine whole-body rates of protein synthesis and degradation. There was no significant effect of treatment on creatinine output (P=0.58), relative abundance of proteins in the mTOR pathway (P>0.05), nitrogen retention (P=0.70), or phenylalanine kinetics (P>0.05). PUN concentrations were significantly (P=0.0058) higher for LYS/THR and LYS/THR/MET than for CON. Atrogin-1 activation was significantly higher for the pre-feeding CON sample compared to the post-feeding CON sample. Lack of significant difference in creatinine output suggests that there were not significant differences in muscle mass between treatments. Lack of significant differences in mTOR protein activation suggests that amino acid supplementation did not result in improvements in protein synthesis. Lack of significant differences in nitrogen retention and phenylalanine kinetics suggests that whole-body protein metabolism was not improved. Additionally, higher PUN concentrations in the supplemented diets suggests that the supplemented amino acids being provided were catabolized. However, increased activation of Atrogin-1 in the pre-feeding CON samples, but not the pre-feeding samples of supplemented treatments, suggests amino acid supplementation may have reduced protein degradation in the post-absorptive state. Data from the present study suggests that amino acid availability may not have been limiting protein synthesis in the sedentary aged horses in the present study.

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Histone acetylation is an epigenetic post-translational modification recognised by the bromodomain, a protein module that forms part of multi-component complexes affecting transcription. This interaction plays fundamental cellular roles, and shows association with particular diseases including inflammation and cancer. The biological roles of bromodomains and the progress of ligands developed so far has been summarised in introductory Chapter 1. Work within the group has led to the development of a nanomolar ligand for BRD4, a BET bromodomain implicated in cancer and numerous diseases. Evaluation in an NCI-60 cancer cell screen indicated antiproliferative activity in a variety of cancer types. However, metabolic predictions indicate that this compound is unoptimised for use in vivo. Chapter 2 describes synthesis of a collection of analogues to improve the physical and pharmacokinetic properties of this series of compounds. This work identified compounds with equivalent affinity but greater predicted metabolic stability, as well as more potent derivatives. This research will direct the design of potent and metabolically stable derivatives that can be used in animal models. Chapter 3 describes work carried out towards the development of small molecules to target bromodomains for which there are no known ligands, using the FALZ bromodomain as an initial target. A fragment-based approach has identified a number of compounds that bind to different non-BET bromodomains. These fragments will be a useful starting point for the development of more potent and selective non-BET bromodomain ligands. As well as acetylated lysines, a number of other acylation post-translational modifications occur on lysine residues. Chapter 4 describes work carried out to investigate the interaction of other acylated lysine residues with bromodomains. This work highlighted that other acylated lysines can interact with bromodomains, and selectivity for particular bromodomains can also be achieved. These modified lysines could be incorporated into cognate peptides to improve in vitro peptide displacement assays, aiding the development of small molecular bromodomain probes.

Master of Science / Department of Animal Sciences and Industry / Joel DeRouchey / Jason Woodworth / Two experiments using a total of 646 nursery pigs were used to determine the effects of increasing phytase on nursery pig growth performance and bone ash characteristics. Two experiments using a total of 821 sows were used to determine the impact of increasing standardized ileal digestible (SID) lysine (Lys) in lactating sows. Experiment 1 determined the available phosphorus (aP) release of Natuphos E 5,000 G phytase in nursery pigs. Increasing phytase from 0 to 1,000 FTU/kg in phosphorus deficient diets improved nursery pig performance and bone ash characteristics. Using percentage bone ash and formulated phytase concentrations, an equation was developed to predict aP release up to 1,000 FTU/kg of Natuphos E phytase. Experiment 2 was conducted to determine the effect of Superdosing Natuphos E 5,000 G phytase on nursery pig performance and bone ash characteristics. Increasing phytase in diets marginal in P improved pig performance and bone ash values. Increasing phytase in P sufficient diets improved bone ash percent and tended to improve feed efficiency. Experiments 3 and 4 determined the impacts of increasing SID Lys in primiparous and multiparous lactating sows and their litters. In Exp. 3, increasing SID Lys above 0.80% in primiparous sows decreased backfat loss, but had no effect on sow BW loss, ADFI or litter gain. Conception rate at d 30 and percentage born alive tended to improve at 0.95% SID Lys. In Exp. 4 with mixed parity sows, increasing SID Lys to 1.05% increased sow weaning BW, litter gain, and reduced weight loss in lactation. Sow backfat loss increased as SID Lys increased from 0.75 to 1.20%, however loin eye depth loss was reduced as SID Lys increased. Percentage of females bred by d 7 after weaning was improved in primiparous females with increasing SID Lys, however no difference was observed in multiparous sows.

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.

Dissertation (MSD) --Boston University, Goldman School of Dental Medicine, 2012 (Department of Oral Biology). / Includes bibliographic references: leaves 71-77. / Lysyl oxidases constitute a family of enzymes responsible for the formation of crosslinks in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. [TRUNCATED]