People who endure horrific, life-threatening experiences are at risk for developing post-traumatic stress disorder (PTSD). However, only about 25% of all individuals who experience trauma develop PTSD. Recent research indicates that the presence of certain physiological conditions, such as reduced cortisol and parasympathetic inhibition, during trauma may increase one's susceptibility to developing PTSD. Thus, I attempted to develop a novel animal model of PTSD and test the hypothesis that reduced adrenal and parasympathetic activity during stress would exacerbate its long-term effects on behavior.In Experiment One, adult male rats were exposed to two stress sessions, each involving one hour of immobilization plus cat exposure. Before each session, rats were injected with vehicle, metyrapone, AF-DX 116, or both drugs. The second session occurred 10 days after the first and served to model a traumatic flashback. Stressed rats endured unstable housing conditions throughout t he experiment to add an element of daily anxiety. Three weeks after the second session, all rats underwent a battery of tests to examine the lasting effects of stress on physiology and behavior. The results indicated that stressed rats exhibited heightened anxiety on the elevated plus maze, an exaggerated startle response, and greater blood pressure, relative to controls. Moreover, metyrapone, when combined with stress, led to significant short- and long-term spatial memory impairments. Experiment Two assessed the effects of the same stress paradigm on rats' sensitivity to yohimbine, an alpha-2 adrenergic receptor antagonist. Yohimbine induces flashbacks and panic attacks in patients with PTSD; thus, I hypothesized that stressed rats would react abnormally to this agent. Stressed and unstressed rats were administered vehicle or yohimbine (1 mg/kg) 30 min prior to behavioral testing. The results indicated that stressed rats were hyperresponsive to yohimbine, as evidenced by a greater su ppression of rearing, greater avoidance of the center of the open field, and a greater suppression of activity on the elevated plus maze, relative to controls. Collectively, the findings of these studies indicate that uncontrollable and unpredictable psychological stress produces lasting changes in the physiology and behavior of rats that resemble symptoms commonly observed in people with PTSD.

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Background: An important role has been suggested for tissue-specific glucocorticoid metabolism in the development of obesity and its complications. 11ß hydroxysteroid dehydrogenase 1 (11ßHSD1) is an enzyme that catalyzes the interconversion of biologically inactive cortisone to active cortisol, thereby regulating its access to glucocorticoid receptors in target tissues. Indeed, an unfavorable metabolic outcome has been associated with increased 11ßHSD1 gene expression and activity in adipose tissue and liver in humans and rodents. Cortisol is an important regulator of phosphoenolpyruvate carboxykinase (PEPCK) a key enzyme in gluconeogenesis and lipid metabolism. In rodents, overexpression of PEPCK in adipose tissue leads to adiposity and increased fatty acid re-esterification. In human obesity, PEPCK has been positively associated with body fat, total cholesterol levels, and plasma triglycerides. However, few studies have addressed the putative reversibility of peripheral cortisol levels and disturbed fatty acid homeostasis that may accompany weight loss. The aim of this thesis was to investigate alterations in peripheral glucocorticoid metabolism in the context of obesity, and putative modulations of glucocorticoid metabolism in the context of weight changes in humans and rodents. Materials & Methods: 11ßHSD1 expression/activity in different adipose tissue depots and liver, the expression of genes involved in adipogenesis and fatty acid homeostasis, and serum levels of adipose tissue-derived adipokines were investigated in severely obese women before and after surgically induced weight loss. The same parameters were measured in female Sprague-Dawley rats fed on high-fat and control diets. Results: In severely obese women, 11ßHSD1 expression was higher in subcutaneous adipose tissue (SAT), while 11ßHSD1 activity and PEPCK expression were higher in the omental depot. In a multivariate analysis, SAT 11ßHSD1 activity was an independent predictor for central fat accumulation. Hepatic 11ßHSD1 activity and levels of intra-abdominal fat storage correlated negatively, while 11ßHSD1 correlated positively with PEPCK in adipose tissue and liver. Weight loss after gastric bypass surgery was followed by significant and metabolically beneficial reductions in subcutaneous 11ßHSD1 and leptin gene expression, as well as reduced circulating leptin and increased adiponectin levels. In contrast, PEPCK gene expression did not change with weight loss. In rats, a high-fat diet did not affect body weight, but was associated with increased serum leptin and decreased adiponectin levels. Short-term, high-fat diet feeding resulted in the up-regulation of SAT 11ßHSD1 expression, while chronic feeding led to its significant down-regulation (compared with the control diet and short-term, high-fat feeding). Interestingly, hepatic 11ßHSD1 expression was constantly downregulated in rats that were fed a high-fat diet. Conclusions: Severe obesity in women was accompanied by a metabolically adverse increase of 11ßHSD1 in adipose tissue, with a concomitant decrease in the liver. Subcutaneous 11ßHSD1 was an independent predictor for central fat accumulation. As weight loss was followed by significant down-regulation of subcutaneous 11ßHSD1, we suggest that up-regulation of this enzyme was a consequence, rather than a cause of obesity. In rodents, a high-fat diet induced dynamic changes in 11ßHSD1 in SAT and liver, both being down-regulated after chronic high-fat feeding without altered weight. In summary, weight changes and alterations in fat and liver glucocorticoid metabolism are closely linked. Moreover, a high-fat diet significantly influences 11ßHSD1 expression/activity in adipose tissue and liver without affecting body weight.

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Mountain caribou (Rangifer tarandus caribou) are endangered in British Columbia and motorized backcountry recreation has been identified as a potential threat to their persistence. My objective was to test if fecal glucocorticoids (GCs), indicative of physiological effects of ecological stress in wildlife, could be used as a non-invasive tool to quantify stress response in free-ranging caribou exposed to motorized recreation. I validated an enzyme-linked immunosorbent assay (ELISA) to measure concentration of fecal GCs for R. tarandus using an adrenocorticotropic hormone (ACTH) challenge experiment on captive reindeer exposed to extreme variation in winter weather. Female reindeer expressed elevated fecal GCs 9-11 hrs after ACTH injection. Males showed no detectable increase, perhaps due to underdosing. Fecal GCs varied markedly in both sexes in response to natural variation in weather. Overall, my results indicated fecal assays can be used to track biologically meaningful changes in adrenal activity in R.tarandus. I investigated the effects of motorized recreation on stress hormone production by measuring GCs in feces of mountain caribou exposed to snowmobile and heli-ski activity. Concentrations of fecal GCs in snowmobile and heli-ski areas were higher than those measured from caribou in areas where motorized recreation was not allowed. Caribou sampled up to 4km. 8km and I0 km distant from snowmobile activity showed elevated fecal GCs when compared to those sampled further from snowmobile activity areas. Other variables with a significant effect on fecal GCs included reproductive state, snow, aspect. minimum ambient temperature, and daily temperature range. My study indicates that measurement of fecal GCs provides a useful, noninvasive approach in the evaluation of physiological effects of environment, reproductive state, and human-induced stressors on free-ranging mountain caribou. Although research on many species indicates that chronically elevated GCs carry a variety of physiological costs, more study is needed to know whether GCs can be used as an index of human impact on population health or trend.

Glucocorticoids are commonly used to treat cows with clinical ketosis and fatty liver disease. This study investigated the effects of 10 mg/mL of FlucortRTM and PredefRTM 2X on the day of calving on blood metabolites, immune function and milk composition on 30 transitional Holstein cows. Sample of blood and milk were analyzed for energy metabolites (glucose, NEFA, BHB and insulin), mineral metabolites (Ca, P, Na, K, Cl and Mg), energy function parameters (antibody, lymphocyte), milk compositional parameters (protein, fat, lactose, SCC). There were no differences in glucose, Na, Cl, Mg, antibody, lymphocyte and milk fat, were observed among treatments. FlucortRTM treated cows had significantly lower NEFA on D1, higher BHB on D21 and D28, lower insulin on D14, higher Ca on D1 and lower P on D1. PredefRTM 2X treated cows had significantly higher BHB on D21, higher insulin on D7, lower Ca on D1, higher SCC on D1 and higher milk protein on D1. With respect to the significant data in this study, the use of glucocorticoids FlucortRTM and Predef RTM 2X in a single intramuscular injection on d1 for the treatment of ketosis is not warranted.

Lung cancer is the most common cancer worldwide and is the fourth leading cause of death in South Africa. Lung cancer is categorised into two types; non-small cell lung cancer and small cell lung cancer (SCLC). SCLC constitutes 20% of all lung cancers and is considered to be an aggressive tumour as it gains chemo-resistance and exhibits early metastasis in diagnosed patients. SCLC cells originate from the neuroendocrine cells of the bronchoepithelium and are known to secrete the neuropeptide, proopiomelanocortin (POMC). POMC undergoes proteolytic cleavage to produce the adrenocorticotropin hormone (ACTH). ACTH stimulates the production of the steroid hormone, glucocorticoid hormone (GC), through the hypothalamus-pituitary-adrenal (HPA) axis. The produced GCs mediate a negative feedback system of the HPA axis to sequester ACTH production. SCLC cells are insensitive to this negative feedback stimulus. GCs elicit their actions through the glucocorticoid receptor (GR). Studies have shown that SCLC cells have a reduced expression of GR which perpetuates the GC-insensitivity. Importantly, over-expression of exogenous GR in SCLC cells leads to cell death by apoptosis. It was postulated that SCLC cells select against GR expression for longevity. Cancer cells are known to alter/silence the expression of tumour suppressor genes by a mechanism known as methylation. Methylation occurs when the enzyme, DNA methyltransferase 1, adds a methyl group to a cytosine present in a guanine-cytosine rich region of the gene (CpG island). The GR gene has a 5’-untranslated exon 1 region that consists of eight promoter regions (1A-1J), in these promoter regions are many CpG islands that have the potential to be methylated. The first aim of this study was to determine the promoter/s utilised by SCLC cells to express the GR protein. Conventional PCR revealed that all three cell lines predominantly utilise promoters 1B and 1C for GR expression. Bioinformatic analysis revealed that these promoters contain putative CpG islands and new data suggests that the GR is silenced by methylation and that treatment with a de-methylating agent results in GR re-expression. To determine which promoter is responsible for GR re-expression after de-methylation, the SCLC cell line, DMS79, as well as two control cell lines, A549 and HEK cells, were treated with the de-methylating agent, 5-aza-2’-deoxycytidine, for 72 hours. qPCR analyses revealed that all three cell lines expressed promoters 1B and 1C with A549 cells showing no evidence of methylation. The HEK cells showed methylation in promoter 1C and not promoter 1B. The SCLC cells showed methylation in both promoter 1B and 1C, however, only promoter 1B showed a significantincrease in transcript levels. SCLC cells are induced to undergo GC-mediated apoptosis when GR expression is restored however the mechanism utilised by the GR to induce the apoptotic cascade is unknown. The GR structure is divided into three domains; ligand binding domain (LBD), DNA binding domain (DBD) and amino terminal domain (NTD). The second aim of this study was to determine the component of the GR that induces apoptosis of SCLC cells. HEK and SCLC cells were infected with empty virus and various GR construct viruses; containing either a wild-type GR, ligand binding mutant, DNA binding mutant or a transactivation mutant (NTD); for 72 hours. Both cell lines were quantified for apoptosis and cell death using microscopic analyses. In HEK cells, it was shown that apoptosis occurred in cells expressing the wild-type GR, the DNA binding mutant and transactivation mutant constructs but apoptosis was reduced in cells expressing the ligand binding viruses. This indicates that the LBD may be necessary for inducing apoptosis in HEK cells. In DMS79 cells, apoptosis occurred in cells expressing the wild-type GR, ligand binding mutant and the DNA binding mutant constructs. There was less apoptotic activity exhibited in the transactivation constructs which indicates the NTD may be necessary for apoptosis induction in these cells. The NTD of the GR is responsible for interaction with other transcription factors to mediate GR transcriptional activity and this study has shown that the transactivation domain plays a necessary role in apoptosis induction. An analysis of the various pathways the GR interacts with through the NTD domain could lead to the identification of the pathway which triggers apoptosis in SCLC cells. This discovery, together with knowledge of promoter methylation and expression may contribute to the development of new, more effective therapies for SCLC. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.