Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

The effect of diet and parasite load on female phonotaxis in the Texas field cricket, Gryllus integer

Female crickets respond selectively to variations in species-specific male calling songs. This selectivity has been shown to be age-dependent; older females are less choosy. However, female quality should also affect female selectivity. The effect of female quality on mate choice was examined in Gryllus integer by comparing the phonotactic responses of females on different diets and with different parasite loads to various synthetic models of conspecific calling song. Test females were virgin, 11-14 days old, and had been maintained on one of five diets varying in protein and fat content. Phonotaxis was quantified using a non-compensating Kugel treadmill which generates vector scores incorporating the speed and direction of movement of each female. Test females were presented with four calling song models which differed in pulse rate, but were still within the natural range of the species for the experimental temperature. After testing, females were dissected and the number of gregarine parasites within the digestive tract counted. There were no significant effects of either diet or parasitism on female motivation to mate although the combined effects of these variables seem to have an effect with no apparent trend. Control females did not discriminate among song types, but there was a trend of female preferences for lower pulse rates which are closest to the mean pulse rate for the species. Heavily parasitized females did not discriminate among pulse rates altho~gh there was a similar trend of high vector scores for low pulse rates. Diet, however, affected selectivity with poorly-fed females showing significantly high vector scores for pulse rates near the species mean. Such findings raise interesting questions about energy allocation and costs and risks of phonotaxis and mate choice in acoustic Orthoptera. These results are discussed in terms of sexual selection and female mate choice.

Iridium(I) complexes of phenanthroline-derived benzimidazolylidenes : synthetic, structural and catalytic studies

N-heterocyclic carbenes (NHCs) have undergone rapid development in recent years. Due to their strong a-electron donation and structural variability properties, NHCs are becoming a major class of ligands in organometallic chemistry. Compared with the other two types of NHCs (imidazolylidenes and imidazolinylidenes), benzimidazolylidenes have not been well represented. Limited synthetic approaches may impede the development ofbenzimidazolylidenes. This thesis is focused on the synthesis of phenanthroline-derived benzimidazolylidene ligands and their metal complexes. A series of benzimidazolylidene-iridium complexes were synthesized and characterized spectroscopically and crystallographic ally. All of the new complexes showed varying degrees of catalytic activity and enantioselectivity toward transfer hydrogenation and asymmetric hydrogenation. The best results were achieved in hydrogenation of methyl-2-acetamidoacrylate, which afforded (-)-(R)-methyl-2-acetamidopropanoate in 97% yield and 81 % ee.

Interactions between the cholinergic and dopaminergic systems during the production of ultrasonic vocalizations in rats

Rats produce ultrasonic vocalizations that can be categorized into two types of ultrasonic calls based on their sonographic structure. One group contains 22-kHz ultrasonic vocalization (USVs), characterized by relatively constant (flat) frequency with peak frequency ranging from 19 to 28-kHz, and a call duration ranging between 100 – 3000 ms. These vocalization can be induced by cholinomimetic agents injected into the ascending mesolimbic cholinergic system that terminates in the anterior hypothalamic-preoptic area (AH-MPO) and lateral septum (LS). The other group of USVs contains 50-kHz USVs, characterized by high peak frequency, ranging from 39 to 90-kHz, short duration ranging from 10-90 ms, and varying frequency and complex sonographic morphology. These vocalizations can be induced by dopaminergic agents injected into the nucleus accumbens, the target area for the mesolimbic dopaminergic system. 22-kHz USVs are emitted in situations that are highly aversive, such as proximity of a predator or anticipation of a foot shock, while 50 kHz USVs are emitted in rewarding and appetitive situations, such as juvenile play behaviour or anticipation of rewarding electrical brain stimulation. The activities of these two mesolimbic systems were postulated to be antagonistic to each other. The current thesis is focused on the interaction of these systems indexed by emission of relevant USVs. It was hypothesized that emission of 22 kHz USVs will be antagonized by prior activation of the dopaminergic system while emission of 50 kHz will be antagonized by prior activation of the cholinergic system. It was found that injection of apomorphine into the shell of the nucleus accumbens significantly decreased the number of carbachol-induced 22 kHz USVs from both AH-MPO and LS. Injection of carbachol into the LS significantly decreased the number of apomorphine-induced 50 kHz USVs from the shell of the nucleus accumbens. The results of the study supported the main hypotheses that the mesolimbic dopaminergic and cholinergic systems function in antagonism to each other.

Interactions between the cholinergic and dopaminergic system during the production of ultrasonic vocalizations in rats

Rats produce ultrasonic vocalizations that can be categorized into two types of ultrasonic calls based on their sonographic structure. One group contains 22-kHz ultrasonic vocalization (USVs), characterized by relatively constant (flat) frequency with peak frequency ranging from 19 to 28-kHz, and a call duration ranging between 100 – 3000 ms. These vocalization can be induced by cholinomimetic agents injected into the ascending mesolimbic cholinergic system that terminates in the anterior hypothalamic-preoptic area (AH-MPO) and lateral septum (LS). The other group of USVs contains 50-kHz USVs, characterized by high peak frequency, ranging from 39 to 90-kHz, short duration ranging from 10-90 ms, and varying frequency and complex sonographic morphology. These vocalizations can be induced by dopaminergic agents injected into the nucleus accumbens, the target area for the mesolimbic dopaminergic system. 22-kHz USVs are emitted in situations that are highly aversive, such as proximity of a predator or anticipation of a foot shock, while 50 kHz USVs are emitted in rewarding and appetitive situations, such as juvenile play behaviour or anticipation of rewarding electrical brain stimulation. The activities of these two mesolimbic systems were postulated to be antagonistic to each other. The current thesis is focused on the interaction of these systems indexed by emission of relevant USVs. It was hypothesized that emission of 22 kHz USVs will be antagonized by prior activation of the dopaminergic system while emission of 50 kHz will be antagonized by prior activation of the cholinergic system. It was found that injection of apomorphine into the shell of the nucleus accumbens significantly decreased the number of carbachol-induced 22 kHz USVs from both AH-MPO and LS. Injection of carbachol into the LS significantly decreased the number of apomorphine-induced 50 kHz USVs from the shell of the nucleus accumbens. The results of the study supported the main hypotheses that the mesolimbic dopaminergic and cholinergic systems function in antagonism to each other.

Two Levels of Motivation and Two Types of Well-Being: Relations Between General and Goal-Specific Motivations, and Eudaimonic and Hedonic Well-Being

In this thesis I assess the individual and joint predictive associations and effects between multiple motivation and well-being concepts. In particular, three pairs of motivation concepts (intrinsic/extrinsic, approach/avoidance, and eudaimonic/hedonic) are assessed simultaneously at two levels of analysis (disposition and goal) and examined in relation to two types of well-being (eudaimonic and hedonic) in two studies, one correlational and the other experimental. Study 1: Using a correlational design, participants (N = 325, M age = 19.10, 87% female) completed self-report measures assessing six motivation and two well-being concepts. Exploratory factor analyses were used to assess patterns of associations among the motivational constructs. Results indicated that constructs displaying conceptual and empirical similarities co-occur, particularly, intrinsic, approach and eudaimonic motivation. Regression models were used to assess predictive relations between the motivational constructs and well-being. Both types of well-being were predicted by approach and avoidance dispositions, and hedonic goals. Additionally, eudaimonic well-being was uniquely predicted by eudaimonic dispositions and goals, and intrinsic dispositions; and hedonic well-being was uniquely predicted by extrinsic dispositions and approach goals. The patterns of associations among motivational constructs, and similarities and differences in the ways they predict each type of well-being, are discussed. Study 2: Using an experimental design, participants (N = 447, M age = 19.30, 88% female) were randomly assigned to one of eight experimental conditions, each involving a manipulation aimed at priming combinations of the three pairs of motivational constructs at the goal level. Participants then completed measures of both types of well-being. ANOVAs were used to assess the main effects and interactions of experimental condition for each of the three pairs of motivational constructs on well-being. Main effects of experimental conditions were non-significant. However, results indicated that focus on each of the three pairs of motivational constructs predicted well-being and that the manipulation impacted well-being indirectly, through experimentally-shifted motivational focus. Few interactions emerged. Implications for future experimental research and the conceptual integration of motivation and well-being constructs are discussed. In conclusion, Studies 1 and 2 inform the motivation and well-being fields in novel ways and provide preliminary steps towards studying these fields from an integrated and comprehensive motivational framework.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Reservoirs for irrigation, water-power and domestic water- supply ; with an account of various types of dams and the...

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