N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term …

The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide …

This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to …

The primary objective of this work was to determine the tissue-specific bioconcentration factors (BCFs) of the selected pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER), clozapine (CLZ) and fenofibrate (FFB) in two freshwater fishes: fathead minnow and channel catfish. BCF tests on fathead followed the standard OECD 42-day test while a 14-day abridged test design was used in catfish exposures. Additional objectives included a) comparing the measured BCFs to the US EPA's BCFWIN model predicted values, b) comparing the BCF results from the standard and reduced tests, and c) prediction of chronic risk of the pharmaceuticals in fish using the human therapeutic plasma concentrations. Each test included uptake and depuration phases to measure tissue-specific kinetic BCFs. The results indicated that all the pharmaceuticals, except IBU, have the potential for accumulation in fish. Estimated BCFs for NET, VER and FFB may not be significant in view of the current regulatory trigger level (BCF ≥ 2000); however, CLZ's BCF in the liver had approached the criterion level. Significant differences were noticed in the tissue-specific uptake levels of the pharmaceuticals with the following general trend: (liver/kidney) > (gill/brain) > (heart/muscle) > plasma. IBU uptake was highest in the plasma. When compared to the measured …

Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb …

Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active …

The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway …

This research focuses on the spatial analysis of the habitat of two vulnerable species, the one-horn rhinoceros in the grasslands of southern Nepal, and the red-cockaded woodpecker in the Piney woods of southeast Texas, in the USA. A study sites relevant for biodiversity conservation was selected in each country: Chitwan National Park in Nepal, and areas near the Big Thicket National Preserve in Texas. Land-use differs in the two study areas: the first is still undergoing agrarian development while the second is in a technological phase and undergoing urbanization processes. Satellite remote sensing images were used to derive land-cover maps by supervised classification. These maps were then processed by Geographic Information Systems methods to apply habitat models based on basic resources (food and cover) and obtain habitat suitability maps. Several landscape metrics were computed to quantify the habitat characteristics especially the composition and configuration of suitable habitat patches. Sensitivity analyses were performed as the nominal values of some of the model parameters were arbitrary. Development potential probability models were used to hypothesize changes in land-use of the second study site. Various scenarios were employed to examine the impact of development on the habitat of red-cockaded woodpecker. The method derived in …

Northern bobwhite populations have declined concurrent with global warming. The focal period of this study was the 12-d pre-incubation period, when bobwhite eggs remain in the nest without the thermal protection of the incubating parent. This study first established the storage and thermal limits of bobwhite eggs, then investigated how global warming may impact oviparous embryos and how bobwhite embryos react to acute and chronic doses of simulated drought temperatures during pre-incubation. First, the maximum storage limit of bobwhite eggs was determined by storing eggs ≤21 d and measuring hatching success and pH of egg albumen and yolk. Hatching success of stored eggs declined after 14 d, when yolk and albumen pH reached levels detrimental to embryonic development. Secondly, thermal limits were determined by exposing bobwhite eggs to hyperthermic temperatures (38-52 °C). Bobwhite embryos survived 50 °C for 1 h, 49 °C for 3 h and 46 °C for 6 h. Results indicate an adaptation to the naturally occurring temperature extremes that can occur in the bobwhite's southern range during pre-incubation. Subsequently, bobwhite eggs were exposed to either low constant (LC), low fluctuating (LF), high constant (HC), or high fluctuating (HF) temperatures during pre-incubation to determine if the nature of …

Ischemic events of even a very short duration are not tolerated Ill in humans. The human cost of ischemia, when looked at as combined cardiovascular disease, dwarfs all other causes of death in the United States. Annually, CVD kills as many people in the US as does cancer, chronic lower respiratory disease, accidents, and diabetes mellitus combined. In 2005 (the latest year for which final statistics are available), CVD was responsible for 864,480 deaths or 35.3 percent of total deaths for the year. In my study, I have used the nematode Caenorhabditis elegans to determine genetic and environmental modulators of oxygen deprivation a key component of ischemia. I have found that animals with mutations in insulin like signaling pathways, neuronal function, electron transport chain components, germline function, and animals that are preconditioned by being raised on a diet of E. coli HT115 bacteria at 25°C have an enhanced ability to survive long-term (>72 hours) anoxia (<.005 kPa O2) at 20°C. The enhanced anoxia survival phenotype partially correlates with increased levels of carbohydrate stores in the nematodes. Suppression of this enhanced anoxia survival phenotype is possible by altering expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, the FOXO transcription factor DAF-16, and …