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Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves 138-152). Also available on the Internet.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Toxicology." Includes bibliographical references (p. 135-152).

Thesis (M.S.) University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (May 18, 2007) Includes bibliographical references.

Glucocorticoids (GC) are powerful metabolic hormones with anti-inflammatory actions. Despite major side effects they remain widely prescribed therapies. GC regulates gene expression through an intracellular receptor (GR), which is a ligand activated transcription factor. Macrophages are innate immune cells and major targets of GC. Traditionally repression of pro-inflammatory genes in the context of an inflammatory stimulus has been considered the primary mode of action of GC in macrophages. The work described in this thesis has demonstrated that GC act primarily as inducers of gene expression in primary macrophages from both mouse and man, but the set of induced genes is very different between the two species. Chromatin immunoprecipitation and sequencing (ChIP-seq) in each species using anti-GR antibodies revealed candidate enhancers in the vicinity of inducible genes that were generally not shared between mouse and man. The differences in binding were correlated with DNA sequence changes at the enhancer sites between the two species, that caused gain or loss of predicted GR receptor-binding motifs. The mechanism of action of GC was investigated by imaging several different target loci using fluorescence in situ hybridisation in macrophage nuclei. Chromatin at specific GC responsive loci was found to decondense within minutes of exposure of macrophages to the ligand. The apparent decondensation was effect was maintained for at least 24 hours and was not prevented by inhibitors of transcription. The general principles of the GC response were shared between species. However the divergence found underlines the caution that must be used when translating specific findings from mouse to man. Additionally, the data support a role for GR driven changes to chromatin structure in gene regulation in macrophages.

Human plasma contains cortisol (F) and corticosterone (B) at a ratio of ~10:1. B is well studied in mice and rats, which do not produce F due to absent adrenal Cyp17, but is largely neglected in humans. Differential transmembrane export of F > B by ABCB1 may account for accumulation of B in the CNS. Conversely, ABCC1, expressed in human adipose tissue, preferentially exports B>F. Here we tested the hypotheses that: (i) negative feedback suppression of the hypothalamic-pituitary-adrenal (HPA) axis is disproportionately sensitive to B; (ii) adipose tissue is disproportionately sensitive to F; and (iii) low plasma B contributes to impaired HPA axis negative feedback and increased F action in metabolic syndrome. We validated a stable isotope tracer for B in vitro and demonstrated distinct kinetics of B and F in vivo. In a randomised crossover study, we undertook ramped steady state infusion of B or F in 10 patients with Addison’s disease. Although levels of B were marginally lower than F, ACTH was similarly suppressed, and yet glucocorticoid-responsive transcripts in adipose tissue were much higher following F than B (PER1 2.2-fold and LPL 1.3-fold; p < 0.05). We assessed associations of ACTH-stimulated plasma B and F with features of metabolic syndrome in a cross-sectional study (n=279). Glucose tolerance was impaired with higher F (β=0.146, p=0.01) but lower B (β = -0.056, p = 0.05). These data support the concept of differential tissue sensitivity to B and F, whereby B suppresses the HPA axis more effectively than it induces adverse effects in adipose tissue. Enhanced CYP17 activity, causing ‘relative B deficiency’, may contribute to HPA axis activation and enhanced F action in adipose tissue in obesity. B therapy might allow control of HPA axis activation without inducing adverse metabolic effects. The ‘neglected second glucocorticoid’, corticosterone, may optimise glucocorticoid action in the human CNS, and simultaneously limit adverse metabolic effects driven by cortisol excess.

Glucocorticoids are known to affect metabolic activities of cells. The mechanism of glucocorticoid actions in adult human dermal and mouse L-929 fibroblasts have yet to be fully ascertained. This study endeavors to examine the effects of one glucocorticoid, triamcinolone acetonide, on cellular proliferation and collagen synthesis and to compare such effects in the human and mouse cell lines. Cellular proliferation and collagen synthesis are analyzed and quantitated by cell counts and selective digestion of the protein by bacterial collagenase, respectively. Further analysis of collagen synthesis is provided by polyacrylamide gel electrophoresis. One-tenth triamcinolone acetonide per ml suppresses cellular proliferation of mouse L-929 fibroblasts. Proline incorporation into total and collagenase-sensitive protein is enhanced in the cell layer; that of medium is altered inconsistently. Polyacrylamide gel electrophoresis of proteins treated with pepsin show the abolition of total and collagenase-sensitive protein in the cell layer. Aberrations in hydroxylation and/or deformation in physical structure of protein may confer greater susceptibility to pepsin digestion. Cellular proliferation and proline incorporation into total and collagenase-sensitive protein of adult human dermal fibroblasts are affected inconsistently by the same dose of triamcinolone acetonide. Except for the consistent suppression of cellular proliferation in the murine L-929 fibroblasts by triamcinolone acetonide, all observations pertaining to human dermal fibroblasts are incompatible with those obtained by other workers. Manipulation of culture conditions and glucocorticoid treatment dictate, to a large extent, the kind of responses observed. This could account for the wide variability and frequent contradictory findings reported in the literature. / Pharmaceutical Sciences, Faculty of / Graduate

Accurately examining animal endocrine profiles pose unique challenges due to possible human interaction influencing basal values. Standard methods of gathering information about an animal’s endocrine status are often dependent upon restraint and use of invasive methodologies. However to accurately monitor the influence management practices, blood sampling sometimes requires that hormone measurements be observed from animals in a relaxed state. To this end, methods for non-invasive monitoring (NIM) are greatly needed to obtain basal endocrine measurements. Such methods include fecal collections followed by hormone extraction, and remote sampling technologies for obtaining blood samples without handling. The overall objective of this study was to use NIM techniques to effectively collect and monitor hormone profiles from domestic and non-domestic species in an effort to more completely understand stress responses and reproductive cyclicity in animals in which handling may not be possible or desired.

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Introduction Glucocorticoids (GCs) are synthesised endogenously in mammals by the hypothalamic pituitary adrenal (HPA) axis in response to stress. These hormones can elicit a number of physiological roles by binding to and activating specific receptors (glucocorticoid or mineralocorticoid receptors- GR or MR). GCs are important in tissue development and maturation and commonly used therapeutically. Mammalian animal studies have suggested that over-exposure to GCs, whether pharmacologically or through induction of maternal stress, is associated with increased cardiovascular disease risk in adult life. The underlying mechanisms underpinning this early life programming are poorly understood, however GC exposure during development may have direct and indirect effects on the structure and function of developing tissues and organs which may predispose to disease in later life. Current mammalian models of programming do not lend themselves well to studying organ development during embryogenesis. The zebrafish provides an ideal model to study this phenomenon due to the transparent nature of developing larvae and the availability of transgenic lines expressing fluorescent markers. Methods GC pathways were comprehensively characterised during zebrafish embryo development using qRT-PCR and steroid ELISAs. The physiological roles of GCs were assessed during early zebrafish development (first 120 hours post fertilisation (hpf)) assessing stress response, swim activity and global development following various genetic and pharmacological manipulations of the GC system. The impact that GC manipulation had on the cardiovascular system was also investigated. Embryos which had been exposed to GC manipulation during early development were then allowed to develop to adulthood in order to assess the long term impact. The same parameters were investigated in the adult as in the embryo. Results The key components of the GC system are present and functional in the developing embryo with de novo cortisol biosynthesis evident from 48hpf. A functioning hypothalamic pituitary inter-renal (HPI) axis is demonstrable from 72hpf. Manipulation of specific components of the GC pathway during early embryonic development influences growth-rate, head-trunk angle, chorion hatch-rate and swim behaviour. Manipulation of GCs during embryogenesis resulted in altered body weight, length and girth in adulthood, with altered stress response and swim behaviour also detected. Embryonic heart development was also affected with a reduction in ventricle cardiomyocyte number, cardiac gene abundance (vhmc) and cardiac function during embryogenesis resulting in structural abnormalities such as fewer trabeculae and increased intra-ventricular space. Embryonic GC manipulation also alters the formation and patterning of intersegmental blood vessels by 120hpf. In adulthood this manifests as a reduced angiogenic capacity. Conclusion The zebrafish embryo represents a valid and physiologically relevant model for GC research. Manipulation of GCs during early development results in altered growth, gene abundance and cardiovascular structure. These findings have significant implications for on-going research addressing GC mediated programming and suggest that the zebrafish is a highly suitable model for GC research.