Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007. / Committee Chair: Sheldon W. May ; Committee Members: Donald F. Doyle, Leslie T. Gelbaum, Stanley H. Pollock, and James Powers. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 136-141). Also available via the Internet.

Production of poly(3-hydroxybutyrate) (PHB) was done in a cyclone bioreactor using various culture methods; including batch (lab and pilot scale) fed-batch and self-cycling fermentation with and without starvation periods. Alcaligenes eutrophus DSM 545 was used to accumulate about 87% (wt/wt) PHB to a total of 6.2 g L$ sp{-1}$ PHB at the end of a 48 hour batch. Similar pilot scale experiments contained a maximum of 96% (wt/wt) PHB with 4.9 g L$ sp{-1}$ accumulated. Fed-batch culture of A. eutrophus produced 96% (wt/wt) PHB with a final PHB concentration of 22.2 g L$ sp{-1}$ after 54 h. Self-cycling fermentation (SCF) production of PHB resulted in an average of 35% (wt/wt) PHB without starvation periods with production rates reaching 0.24 g L$ rm sp{-1} hr sp{-1}.$ With starvation periods of 4, 6 and 8 h extended on the cycle times, production of PHB decreased except in the 8 hour starvation period which was 59% (wt/wt). However, the rates of production all decreased to below 0.13 g $ rm L sp{-1} h sp{-1}$ as the lengths of the starvation periods were increased.

The vitamin B3 precursor nicotinamide riboside (NR) and the ketone DL-body beta-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. NR increases nicotinamide adenine dinucleotide (NAD) levels to activate sirtuin protein deacetylases and BHB is an anti-aging calorie restriction (CR) mimetic. Caenorhabditis elegans is a 1 mm long nematode worm used as a model system to study aging with a mean lifespan of roughly 2-3 weeks depending upon the exact temperature of culture. NR and BHB have previously been shown to increase lifespan when administered to C. elegans by roughly 20%. However, the effect on lifespan when both compounds are added together has not yet been studied. It is hypothesized that when added together the effect on lifespan will be slightly larger than when either compound is given alone, due to the activation of complementary signaling pathways. The results will help determine if humans could benefit from taking both compounds simultaneously as most signaling pathways that regulate lifespan are conserved from nematodes to humans. For these experiments cultures of mixed age C. elegans nematodes were first treated with alkaline-bleach to kill adult worms leaving only eggs that are protected by their thick eggshell. Using this protocol isolated eggs are age-synchronized to within 9 hours of each other. The eggs were then transferred into E. coli-permeable, but nematode impermeable 8 micron cell culture inserts placed in twelve well microplates with roughly 25-40 eggs per insert. 1.35 mL of liquid S-media containing 9 x109 E. coli cells/mL as food was added to each well. Microplates were shaken to provide culture aeration. After three days, the worms reached adulthood and 0.4 mM fluorodeoxyuridine (FUdR), a DNA synthesis inhibitor, was added to prevent C. elegans egg-laying to maintain age-synchrony. Every Monday, Wednesday, and Friday for the approximate 4 weeks of the lifespan experiments the worms were counted under a microscope and the culture media and bacteria replaced. Results were analyzed using Kaplan-Meier survival curves and Log-rank analysis. Results indicated that individual treatment with BHB or NR or both combined increased lifespan in the two trials performed thus far. Another trial is currently underway, and results will be analyzed after it is completed to determine if the combined treatment has a greater benefit on lifespan then either individual treatment. Future studies could also be performed to determine if either NR or BHB can further extend the lifespan of animals given rapamycin, a TOR kinase inhibitor, another promising anti-aging therapeutic.

Elevated ketone production and utilization results in a host of health benefits. The aim of this study was to assess the rate of ketone production during a prolonged fast and to evaluate how an initial bout of exercise influences this production. Mood and hunger, along with plasma insulin and glucagon, were also assessed. In this crossover study, 20 adult subjects (11 Male, 9 Female) completed two 36-hour fasts, with one protocol requiring the subject to complete an intense treadmill exercise session at the beginning of the fast. Ketone levels were assessed via blood ketone meter and recorded every two hours. Subjective mood and hunger ratings were also recorded every two hours. Venipuncture was performed every 12 hours to assess plasma insulin and glucagon. The mean area under the ketone production curve for the nonexercise intervention was 19.19 ± 2.59 mmol/L and 27.49 ± 2.59 mmol/L for the exercise intervention, resulting in a significant 8.30 mmol/L difference between conditions (95% probability interval was 1.94 to 14.82 mmol/L). The mean time to nutritional ketosis was 21.07 ± 2.95 hours with fasting alone, and 17.5 ± 1.69 hours when combined with exercise (posterior probability = 0.89). There was a significant decrease in insulin over time (F(3,133) = 61.75, p < 0.0001). There was also a significant increase in glucagon over time (F(3,133) = 21.10, p < 0.0001). Hunger and stomach discomfort did not differ between conditions. Anger (F(10,394) = 2.74, p = 0.0028), depression (F(10,394) = 2.91, p = 0.0016), tension (F(10,394) = 2.29, p = 0.0128), vigor (F(10,394) = 11.65, p < 0.0001), and fatigue (F(10,394) = 10.60, p = 0.0001) increased over the course of the fast, but did not differ between conditions. Completing aerobic exercise at the beginning of a 36-hour fast results in significantly more ketone production. The impact of exercise on ketone production comes at little or no impact on hunger, stomach discomfort and negative moods. A difference in time to achieving nutritional ketosis between conditions may exist, but this was not observed in this study.

No description available.

The importance of Poly(3-hydroxybutyrate) (PHB) as a biodegradable material is well known. Due to ever increasing environmental awareness, significant efforts have been made to utilize PHB or its derivatives in producing disposable products. However, brittle mechanical properties of PHB hinder the direct application of this material in useful commodity items. In order to achieve toughened PHB, blending with other polymers which possess high relaxation behavior at room temperature seems attractive. Prior to such development, the fundamental characterization of the relaxation behavior of PHB itself is extremely important in order to assess the effect of any attempt to improve the situation in a quantitative manner. Dielectric thermal analysis was used in the study of the relaxation behavior of melt processed PHB. The approach was largely phenomenological, that was, based on the macroscopic theory of dielectric relaxation. The mean relaxation time of melt processed PHB was evaluated while PHB was undergoing crystallization at room temperature. The experimental conditions were kept as close as possible to actual shelf-life conditions. Dynamic temperature sweep experiments revealed multiple relaxation peaks at the glass transition region. Temperature plane curve resolution revealed, in the early stage of crystallization, two dynamically changing peaks whose behaviors, as the extent of crystallization progressed, were quite opposite in terms of the magnitude of the loss property. By analyzing the temperature dependence of loss property and mean relaxation time, it was concluded that the peak located at the lower temperature is related to pure amorphous chain movement, and the peak located at the higher temperature is related to the movement of amorphous chains which are confined in-between crystalline phases, such as lamellae and spherulites. For the evaluation of the mean relaxation time of binary blends or multiple relaxations arising from homopolymers and copolymers, an empirical model has been developed which is grounded in the theory of linear viscoelasticity with the aim of quantitatively assessing the effect of attempts to improve the toughness of PHB. In the course of data reduction and model development, the majority of empirical dielectric relaxation functions has been reviewed including the Havriliak-Negami model and the Kohlrausch-Williams-Watts stretched exponential function. It was found that the center of relaxation time in the Havriliak-Negami model was skewed toward short time scale of relaxation, while mean relaxation time reflected the relaxation behavior of PHB chains on average, including movement of chains which relax with difficulty as the extent of crystallization progresses. / Ph. D.

Em animais, dietas cetogênicas induzem o Browning de tecidos adiposos brancos, fenômeno caracterizado pelo de aumento de adipócitos capazes de expressar a proteína desacopladora 1 (UCP1) e outros marcadores de gordura marrom em meio a gordura branca. Estudo anterior demonstrou que o &beta;-hidroxibutirato (&beta;HB), principal corpo cetônico, aumenta marcadores do processo de browning em adipócitos brancos (in vitro) após 24 horas de incubação. No entanto, as doses testadas foram suprafisiológicas (50 mM) ou apenas encontradas durante a cetoacidose (25 mM). As dietas cetogênicas aumentam a cetonemia em torno de 1-3 mM. O jejum prolongado pode aumentá-lo para 4-7 mM. Uma vez que poderia ser provocada in vivo através de intervenções dietéticas, estudamos o impacto de concentrações fisiológicas de &beta;HB no metabolismo e marcadores de Browning em adipócitos brancos / tecido adiposo em diferentes modelos: adipócitos isolados de ratos Wistar, células 3T3 -L1 e in vivo, através da suplementação de sais de &beta;HB em ratos Wistar. Demostramos que o &beta;HB: não induz o aparecimento diferentes marcadores de Browning (tais como o incremento: da capacidade oxidativa, da atividade de citrato sintase e de genes relacionados ao Browning) em adipócitos isolados após 24 ou 48 horas de tratamento; não exerce efeito permissivo no browning induzido por agonismo &beta;-adrenérgico. Além disso, os adipócitos 3T3-L1 diferenciados com &beta;HB (4mM) tiveram diminuição de 52% na expressão de Ucp1, resultado que foi reproduzido no tecido adiposo inguinal subcutâneo de ratos Wistar após a ingestão de sais DL- &beta;HB, onde a expressão gênica de Ucp1 foi indetectável. Em conclusão, embora as causas de browning do tecido adiposo branco induzido por dietas cetogênicas permaneçam inconclusivas, nosso estudo demonstra a incapacidade de, em concentrações fisiológicas, os corpos cetônicos serem, por si, responsáveis por esse fenômeno. Pelo contrário, em algumas situações, o &beta;HB pode prejudicar a expressão da Ucp1. / In animals, ketogenic diets induce browning of white adipose tissue, a phenomenon characterized by the increase of adipocytes capable of expressing the uncoupling protein 1 (UCP1) and other markers of brown fat in among white fat. A previous study demonstrated that &beta;-hydroxybutyrate (&beta;HB), the major ketone body, increases markers of the browning process in white adipocytes (in vitro) after 24 hours of incubation. However, the doses tested were supraphysiological (50 mM) or only found during ketoacidosis (25 mM). Ketogenic diets increase ketonemia by about 1-3 mM. Prolonged fasting can increase it to 4-7 mM. Since it could be elicited in vivo through dietary interventions, we studied the impact of physiological concentrations of &beta;HB on metabolism and browning markers on white adipocytes/adipose tissue in different models: adipocytes isolated from Wistar rats, 3T3-L1 cells and in vivo, through the supplementation of &beta;HB salts in Wistar rats. We demonstrate that &beta;HB does not increase any browning markers (such as: oxidative capacity, citrate synthase activity, and browning related genes expression) in isolated adipocytes after 24 or 48 hours of treatment; does not exert a permissive effect on browning induced by &beta;-adrenergic agonism. In addition, 3T3-L1 adipocytes differentiated with &beta;HB (4mM) had a 52% decrease in Ucp1 expression, a result that was reproduced in the subcutaneous inguinal adipose tissue of Wistar rats after ingestion of DL-&beta;HB salts, where Ucp1 expression was undetectable. In conclusion, although the causes of browning of white adipose tissue during ketogenic diets remain inconclusive, our study demonstrates the inability, in physiological concentrations, of ketone bodies themselves to be responsible for this phenomenon. In contrast, in some situations, &beta;HB may impair the expression of Ucp1.

This dissertation evaluated nutritional approaches such as the addition of betaine, prebiotics, probiotics, transitional metabolic substrates, and β-mercaptoacetate (MAA; a compound which inhibits β-oxidation) to the diet of lactating dairy cows to determine their impact on physiological, metabolic, hormonal and production responses during thermal stress. The first objective was to evaluate the use of an organic osmolyte, betaine to reduce the impact of heat stress (HS). Cows were fed either 0 (control; CON), 57 mg/kg BW (mid) or 114 mg/kg (high; HI) body weight (BW) betaine and subjected to thermoneutral (TN) and HS conditions. There was an increase in milk yield during TN with HI betaine over controls (P< 0.01), but the advantage was lost during HS. Plasma glucose increased during HS in HI dose cows compared to control (P < 0.01) as did plasma insulin (P = 0.01). Betaine increased milk production during TN and plasma glucose in HS, but did not improve the HS response. Objective two evaluated the use of a probiotic or direct fed microbial (DFM), Calsporin (Bacillus subtilus C-3102) to decrease the effects of HS in dairy cows. We hypothesized that feeding Calsporin prior to and during HS would reduce pathogenic strains of bacteria, maintain commensal microbes, and improve ruminal anaerobic fermentation resulting in improved milk yield (MY). Milk yield was numerically increased (1.26 kg, P = 0.11) in cows fed Calsporin during TN but was reduced under HS (-2.67 kg, P < 0.01) and milk protein content was decreased (P = 0.05). The DFM tended to decrease somatic cell count (SCC) across periods (P = 0.07). Calsporin addition to the diet did not affect respiration rates and was associated with higher rectal temperature at 1800 in HS (P = 0.02). The expression of heat shock protein 27 (HSP27) was decreased with Calsporin treatment (P = 0.03) and in both HS and TN. The fecal microbial count did not change with the exception of the Calsporin strain in treated animals (P < 0.01). The third objective was to feed OmniGen-AF (OG) to dairy cows before and during thermal stress. We hypothesized that feeding OG to HS dairy cows will improve the immune response, and decrease production losses associated with HS. Cows fed OG maintained lower SCC compared to control (P < 0.01) during the recovery period. We did not detect differences between groups in serum calcium while serum non-esterified fatty acid (NEFA) concentrations (P = 0.10) tended to be greater in OG fed cows across the Agricultural Research Center (ARC) portion including HS. Serum Adrenocorticotropic hormone (ACTH) levels were greater in OG cows (P<0.0001) across all sample days. Feeding OG reduced the HS response including serum Cortisol. The final study measured the effects of the metabolic substrate β-hydroxybutyrate (BHB) during HS on feed intake and metabolites. Under TN conditions the cows received a bolus dose of BHB and dry matter intake (DMI) and metabolites were measured. The second part of this study used a bolus of MAA to limit the up-stream production of acetyl-CoA available for ketogenesis by inhibiting ß-oxidation. We proposed that dosing lactating dairy cows with BHB would decrease DMI, increase plasma insulin, decrease NEFAs and increase skin temperature by vasodilatation. The same cows were then subjected to HS and dosed with saline and MAA on different test days. The infusion of BHB increased skin temperature (time 0.5, 1, 2, 3 and 4°C r² =0.98 with serum BHB) and decreased serum NEFA levels (P < 0.01). There was no change in mean DMI, glucose or insulin. The bolus of MAA decreased feed intake, vaginal temperature, and insulin. There was an increase in serum BHB with the initial dose of MAA and an initial decrease in serum glucose (P < 0.0001) with MAA. Serum glucose increased as insulin decreased with MAA. The infusion of BHB did not alter feed intake in this study despite high plasma levels of BHB.

This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.