Horses with high energy requirements are generally fed two large concentrate meals per day, either in the form of grain or pellets. The postprandial elevation of blood glucose resulting from this type of feeding has the potential to alter production of hormones such as leptin. Leptin is an adipose-derived protein that promotes satiety in normal animals. The objective of this study was to determine if feeding large amounts of concentrate twice each day would alter serum leptin concentration. Nine horses were placed into three groups (A, B, and C) and each group was rotated through three feeding schedules (2x, 3x, and 4x) in a 3 x 3 Latin square design. Horses were fed twice per day on the 2x schedule, three times per day on the 3x schedule, and four times per day on the 4x schedule. Horses were fed the same total amount of concentrate per day throughout the study, although meal size varied with the number of times the horse was fed per day. Horses were weighed and scored for body condition on the first day of each period. Each treatment period lasted for 11 days. Blood was drawn on days one, four, and seven of each period and leptin concentration was determined by radioimmunoassay. On the afternoon of the tenth day of each period, horses were fitted with jugular catheters and blood was drawn every two hours for 24-hours to determine the circadian rhythm of leptin secretion. Additionally, blood was taken 30 minutes prior to and every 30 minutes after the morning meal to determine postprandial plasma glucose concentrations. Mean and peak glucose values were higher on the 2x schedule than the 3x or 4x schedules (P < 0.05). Leptin concentration was highest in horses on the 3x schedule, although when these data were normalized to baseline (day one) values, leptin was highest on the 2x schedule (P < 0.05). Serum leptin concentration was highly correlated with body condition score (P < 0.01), but not gender (P = 0.82), and leptin increased throughout the study (P < 0.05). Data from the 24-hour collection showed that serum leptin concentration varied with time in horses on the 2x but not the 4x schedule (P < 0.05). Linear regression of data from the 2x schedule indicates that the pattern of change may be modeled by a quadratic equation (P < 0.05). This study demonstrates that feeding horses large carbohydrate meals twice per day disrupts the normal pattern of leptin in the horse, possibly affecting appetite and other physiological processes.

Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.

Background: Fibroblast growth factor 23 (FGF23) is the earliest marker of disturbed mineral metabolism as renal function decreases. Its serum levels are associated with mortality in dialysis patients, persons with chronic kidney disease (CKD) and prevalent cardiovascular disease (CVD), and it is associated with atherosclerosis, endothelial dysfunction and left ventricular hypertrophy in the general population. The primary aim of this study is to examine the association between FGF23 and mortality, in relation to renal function in the community. A secondary aim is to examine the association between FGF23 and CVD related death. Methods: The population-based cohort of MrOS Sweden included 3014 men (age 69-81 years). At inclusion intact FGF23, intact parathyroid hormone (PTH), 25 hydroxyl vitamin D (25D), calcium and phosphate were measured. Mortality data were collected after an average of 4.5 years follow-up. 352 deaths occurred, 132 of CVD. Association between FGF23 and mortality was analyzed in quartiles of FGF23. Kaplan-Meier curves and Log-rank test were used to examine time to events. Cox proportional hazards regression was used to examine the association between FGF23, in quartiles and as a continuous variable, with mortality. The associations were also analyzed in the sub-cohort with estimated glomerular filtration rate (eGFR) above 60 ml/min/1.73 m(2). Results: There was no association between FGF23 and all-cause mortality, Hazard ratio (HR) 95% confidence interval (CI): 1.02 (0.89-1.17). For CVD death the HR (95% CI) was 1.26 (0.99 - 1.59)/(1-SD) increase in log(10) FGF23 after adjustment for eGFR, and other confounders. In the sub-cohort with eGFR &gt; 60 ml/min/1.73 m(2) the HR (95% CI) for CVD death was 55% (13-111)/(1-SD) increase in log(10) FGF23. Conclusions: FGF23 is not associated with mortality of all-cause in elderly community living men, but there is a weak association with CVD death, even after adjustment for eGFR and the other confounders. The association with CVD death is noticeable only in the sub-cohort with preserved renal function.

Over the past decade, the constraint-based approach to metabolic modeling has become an important tool for understanding and controlling biology. Unfortunately, the application of this novel approach to systems biology in biotechnology has been limited by three significant technical issues: existing metabolic modeling methods cannot completely model the overflow metabolism, cannot model the metabolism of microbial communities, and cannot design strains optimized for productivity and titer. Three computational methods – the Flux Balance Analysis with Membrane Economics (FBAME) method, the Dynamic Multi-species Metabolic Modeling (DyMMM) framework, and the Dynamic Strain Scanning Optimization (DySScO) strategy – have been developed to resolve these issues respectively. First, the FBAME method, which adopts the membrane occupancy limitation hypothesis, was used to explain and predict the phenomenon of overflow metabolism, an important metabolic phenomenon in industrial fermentation, in Escherichia coli. Then, the DyMMM framework was used to investigate the community metabolism during uranium bioremediation, and demonstrated that the simultaneous addition of acetate and Fe(III) may be a theoretically viable uranium bioremediation strategy. Lastly, the DySScO strategy, which combines the DyMMM framework with existing strain design algorithms, was used to design commodity-chemical producing E. coli optimized for a balanced product yield, titer, and volumetric productivity. These novel computational methods allow for broader applications of constraint-based metabolic models in biotechnology settings.

3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

3-Methylcholanthrene (MC) is a model polycyclic aromatic hydrocarbon that induces cytochrome P450 1A1 (CYP1A1) expression. This laboratory has shown previously that aromatic hydrocarbons, which are important environmental toxicants, down-regulate the expression of rat liver CYP2C11. Recent observations also suggested that CYP2C8, a human enzyme that metabolizes antineoplastic and antidiabetic drugs, among others, is down-regulated in response to aromatic hydrocarbon exposure in primary human hepatocytes. I examined the regulation of CYP2C8 at the mRNA level by MC in two human hepatocellular carcinoma cell lines, HepG2 and HepaRG. MC down-regulated CYP2C8 mRNA levels in HepG2 cells at 24 hours and in HepaRG cells at 48 hours. CYP1A1 mRNA was induced by MC in both cell lines and HepaRG cells appeared to be more sensitive than HepG2 cells to MC-induced cytotoxicity. Further studies are warranted to define the mechanisms and functional impacts of the modulation of this important human CYP by environmental toxicants.

Glycerophosphodiesters are products of phospholipase B-mediated hydrolysis of phospholipids. Their transport and metabolism is elaborated in the fungal pathogen, Candida albicans, as compared to the non-pathogenic Saccharomyces cerevisiae. C. albicans contains four ORFs (CaGIT1-4) predicted to encode transporters for glycerophosphodiesters, compared to one (ScGIT1) in S. cerevisiae. Here I have identified the gene products responsible for glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) transport. C. albicans strain lacking ORF 19.34, which codes for CaGit1, is unable to transport intact GroPIns. Transport activity can be rescued by reintegration of one copy of CaGIT1 back into the genome. Similarly, a strain lacking CaGIT3 (ORF 19.1979) and CaGIT4 (ORF 19.1980) is unable to transport intact GroPCho into the cell. Reintegrating one copy of either CaGIT3 or CaGIT4 can rescue GroPCho transport activity. Initial transport assays and kinetic analyses indicate that CaGit3 is responsible for the majority of GroPCho transport activity. In addition, I present evidence that CaGDE1 (ORF 19.3936) codes for an enzyme with glycerophosphodiesterase activity against GroPCho. Homozygous deletion of CaGDE1 results in a buildup of internal GroPCho, which is restored to wild type accumulation by reintegration of one copy of CaGDE1 into the genome. The transcriptional regulator, CaPho4, was shown to positively regulate the expression of CaGIT1, CaGIT3, CaGIT4, and CaGDE1. Finally, glycerophosphodiester transport and metabolism was active under physiological relevant conditions that C. albicans may experience in the human host. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

Monounsaturated fatty acids (MUFA) have been viewed as either beneficial or neutral with respect to health; however, recent evidence suggests that MUFA may be associated with obesity and cardiovascular disease. Sex differences in MUFA composition have been reported in both rats and humans that may be mediated by sex hormones. Therefore, differences in fatty acid and enzyme expression were examined in male and female rats. HepG2 cells treated with 17β-estradiol, progesterone and testosterone (0, 10, 30 and 100 nM), and ovariectomized female rats with hormone implants (sham, no treatment, estradiol, progesterone, and estradiol plus progesterone) examined the role of sex hormones in MUFA metabolism. MUFA concentrations were determined by gas chromatography. The mRNA and protein expression of stearoyl-CoA desaturase and elongase 6, key enzymes involved in MUFA biosynthesis, were measured by real-time PCR and immunoblotting, respectively. Elongase 6 protein expression was higher in females as compared with males, increased with estradiol and progesterone treatment of HepG2 cells, and was higher in ovariectomized rats treated with estradiol. Elongase 6 expression was also decreased in HepG2 cells treated with testosterone. In contrast, the expression of stearoyl-CoA desaturase did not appear to be associated with sex or sex hormones in HepG2 cells, although ovariectomized rats treated with estradiol plus progesterone had increased stearoyl-CoA desaturase protein levels compared with sham controls. Sex differences and differences after hormonal treatments were observed in the fatty acid concentrations of MUFA and immediate MUFA substrates including 16:0, 16:1n-7, 18:0, 18:1n-7 and 18:1n-9. These differences in MUFA are consistent with the changes in elongase 6 expression. The effect of sex and hormone status on elongase 6 has not been previously examined, but these results suggest elongase 6 is an important factor in determining MUFA composition. These sex and hormonal differences in MUFA composition may contribute to sex differences in obesity and cardiovascular disease. Further work examining other factors involved in MUFA composition including oxidation, synthesis of complex lipids and the effect of diet is required.

Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.

This study was conducted to assess the regulation of glycolysis by 6phosphofructokinase (PFK) during the postmortem metabolism of beef muscle. In the first experiment, M. sternocephalicus pars mandibularis samples were excised from six randomly-selected steers. Two samples were obtained from each steer immediately postmortem; one sample was quickly immersed in liquid nitrogen and the other was stored at 4oC for 4 d. Glycogen concentrations decreased 45% from d 0 to d 4, and 39.6 ?mol/g of glycogen was still present in the tissue at d 4. Concentrations of free glucose increased (P < 0.001) from 0.84 ?mol/g at d 0 to 6.54 ?mol/g at d 4. Fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P) increased (P < 0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33 ?mol/g) and was elevated to 45.9 ?mol/g by d 4. Glycolytic potential was 34.4 ?mol/g higher (P < 0.05) when measured at d 0 than at d 4. The greatest activity of PFK was measured in fresh muscle extracts, between pH 7.4-7.8; by reducing the pH to 7.0, PFK activity was depressed by nearly 50% at 1 mM F6P. In a second experiment, M. longissimus lumborum samples were excised at the 13th thoracic rib location from six randomly-selected steers. Samples were obtained at intervals ranging from 40 min to 24 h postmortem. Glycogen concentrations decreased 45% between 40 and 100 min, and tended (P &#8804; 0.10) to decrease between 100 min and 24 h (from 47 to 32 ?mol/g). Concentrations of free glucose increased (P &#8804; 0.009) from 1.0 ?mol/g at 40 min to 5.0 ?mol/g at 24 h. Concentrations of F6P and G6P increased dramatically after 100 min (muscle pH &#8804; 6.5), whereas glycogen depletion appeared to halt by 100 min. Lactate began accumulating almost immediately and tripled in concentration by 24 h. The elevation of G6P and F6P, coupled with the pH sensitivity of PFK, indicate that the postmortem decline in pH ultimately inactivates PFK prior to glycogen depletion.