Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although the effect of inhaled corticosteroids (ICS) on the hypothalamic- pituitary-adrenal axis (HPA) has been regarded as a “benign physiological response”, a survey published in 2002 suggested that adrenal crisis is more common in asthmatic children on ICS than previously thought. Relying on clinical features to detect chronic adrenal insufficiency secondary to corticosteroids may not be wise, as these are non-specific and can therefore easily be missed. Accurate biochemical assessment of the whole axis to detect subclinical HPA suppression (HPAS) is thus desirable. A review of the literature indicates that basal adrenal function tests, including plasma cortisol profiles, do not identify which children can appropriately respond to stress. There is no evidence to suggest that the degree of the physiological adjustment of the HPA to ICS and/or nasal steroids (by reducing basal cortisol production), predicts HPAS. Cortisol profiles should therefore only be used to demonstrate differences in systemic activity of various ICS and delivery devices. Only two tests, considered as gold standard adrenal function tests [the insulin tolerance test (ITT) and the metyrapone test] can assess the integrity of the whole axis. / AFRIKAANSE OPSOMMING: Die outeurs van ´n opname wat in 2002 gepubliseer is stel voor dat ´n bynierkrisis meer algemeen by asmatiese kinders, wat inhalasie kortikosteroïede ontvang, voorkom as wat voorheen gedink is. Dit is strydig met die gevestigde opvatting dat die effek van IKS op die hipotalamiese-hipofise-bynier-as (HHB) ’n “goedaardige fisiologiese reaksie” is. Die kliniese kenmerke van kroniese bynierontoereikendheid sekondêr tot die gebruik van kortikosteroïede (KS) is nie-spesifiek en gevolglik onbetroubaar. ´n Akkurate biochemiese toets van subkliniese HBB onderdrukking (HHBO) sou gevolglik waardevol wees. ´n Literatuur oorsig toon dat toetse van basale bynierfunksie, insluitend plasma kortisol (K) profiele, nie kinders uitken wat toepaslik op stres sal reageer nie. Daar is geen bewyse dat die graad van fisiologiese aanpassing van die HHB, soos aangedui deur laer K-vlakke, na die gebruik van IKS en/of nasale steroïede (NS), HHBO voorspel nie. Serum K profiele is dus slegs van waarde om die sistemiese aktiwiteit van verskillende IKS en toedieningsstelsels te ondersoek. Slegs twee toetse, naamlik die insulien toleransie toets (ITT) en die metyrapone -(MTP)-toets (wat beide as die goue standaard van bynier funksie beskou word), kan die integriteit van die hele as meet.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 44-48).

Pineal-mediated depressions in prolactin cell activity after light deprivation were studied in the male and female Golden Syrian hamster. Prolactin cell activity was determined by measuring radioimmunoassayable prolactin, newly synthesized prolactin and prolactin mRNA levels in the pituitary. Serum prolactin was also measured by radioimmunoassay. Use of the recombinant DNA plasmid, pPRL-1, which contains the rat prolactin complimentary DNA sequence, was validated in this dissertation for measuring prolactin mRNA in the hamster. Male hamsters blinded for 11, 21, or 42 days showed significant and progressively greater declines in prolactin mRNA levels which were completely prevented by pinealectomy. The decline seen after 11 days is the earliest depression in prolactin cell activity reported after light deprivation in the hamster. Female hamsters blinded for 28 days, however, showed no such decreases in prolactin cell activity if they continued to display estrous cyclicity. This supports the hypothesis that, unlike the male, there is not a gradual decline in prolactin cell activity after blinding in the female hamster and that loss of estrous cyclicity may precede or possibly accompany declines in prolactin cell activity. After 12 weeks of blinding, females were acyclic and had dramatically depressed levels of prolactin cell activity. However, pinealectomy did not completely prevent this decline due to blinding unless the females continue to display estrous cyclicity. Thus, when pinealectomy was ineffective in preventing the loss of estrous cyclicity due to blinding, it was also ineffective in preventing declines in prolactin cell activity. In ovariectomized females, blinding caused a decline in prolactin cell activity. Pinealectomy was not consistently effective in preventing this decline after 12 weeks of treatment, although, in females blinded for 4 weeks (at which time all animals were cycling) and then ovariectomized for an additional 4 weeks, pinealectomy completely prevented this decline in prolactin cell activity. In a separate study, significant changes in prolactin cell activity during the estrous cycle were seen in untreated normally cycling female hamsters. These changes in prolactin mRNA, prolactin synthesis, and radioimmunoassayable prolactin in the pituitary were measured in the morning, when, consistent with other reports, no differences in serum prolactin were observed.

To test the hypothesis that the hypothalamic tuberoinfundibular dopaminergic neuronal system plays a role in the dynamic regulation of pituitary prolactin secretion, its activity was correlated with experimentally-induced prolactin secretory episodes in the male rat. Direct estimates of tuberoinfundibular neuronal activity were made by measuring its rates of dopamine and norepinephrine synthesis or release. Prolactin secretion was assessed in vivo by measuring radioimmunoassayable prolactin levels in peripheral blood and the pituitary and in vitro by measuring prolactin concentrations released into incubation media. The anesthetic urethane and a substance isolated from the pineal gland were both demonstrated to inhibit prolactin secretion. Significant elevations of newly synthesized tuberoinfundibular dopamine were observed concomitant with this decreased prolactin secretion suggesting that acute increases in tuberoinfundibular dopaminergic neuronal activity were perhaps causally related to acute decreases in prolactin secretion since these substances were without a direct effect on the pituitary in vitro. Conversely, acute decreases in tuberoinfundibular neuronal activity induced by dopamine biosynthesis inhibition or mimicked by pituitary receptor blockade induced acute increases in prolactin secretion. As another prerequisite for its involvement in the dynamic regulation of prolactin secretion, the tuberoinfundibular neuronal system was demonstrated to be involved in the negative feedback control of prolactin over its own secretion. Elevated circulating prolactin levels produced by pituitary homografts transplanted beneath the kidney capsule accelerated tuberoinfundibular dopaminergic neuronal activity. In two unrelated experimental conditions, rats rendered blind and anosmic or hyperprolactinemic, the chronic inhibition of prolactin secretion was not associated with the maintenance of an increased tuberoinfundibular neuronal activity, but rather with a supersensitivity of the anterior pituitary to the prolactin-release-inhibitory action of dopamine. Long-lasting alterations in tuberoinfundibular dopaminergic neuronal activity appeared to induce this pituitary supersensitivity to dopamine. The tuberoinfundibular neuronal system appears to have the capacity to modulate prolactin secretory episodes via the alteration of its dopaminergic activity. Long-lasting alterations in this activity may induce changes in anterior pituitary sensitivity to dopamine essential for the chronic inhibition of pituitary prolactin secretion.

Call number: LD2668 .T4 1950 S666 / Master of Science

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). "June 2006." Includes bibliographical references (leaves 67-75). Also available via the World Wide Web.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (p. 143-152). Also available via the World Wide Web.

The growth and development of an organism requires the coordinated actions of many factors. During development individual cells undergo proliferation, migration and differentiation to form the adult organism. Two structurally related members of the glucagon superfamily, growth hormone releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), are thought to modulate vertebrate development. In mammals, GRF modulates the development of pituitary somatotrophs and the release of fetal growth hormone. In contrast, PACAP appears to have a more general role during development. PACAP may be involved in the patterning of the embryonic axis and in the development of the neural tube. The objectives of my study were to isolate GRF, PACAP and their receptors from the zebrafish, characterize their expression in the developing embryo and adult embryo and examine the role of PACAP during brain development. To study the role of GRF and PACAP, I isolated a genomic clone encoding the GRF and PACAP peptides from the zebrafish genomic library and characterized its gene copy number and adult tissue expression pattern. The GRF-PACAP gene isolated from the zebrafish was comprised of five exons with the GRF peptide encoded on the fourth exon and the PACAP peptide encoded on the fifth exon. This gene structure is similar to that found in other non-mammalian vertebrates and supports the hypothesis that the gene duplication leading to the encoding of the GRF and PACAP peptides on separate genes occurred later in evolution. In addition, the zebrafish genome was found to contain only one copy of the GRF-PACAP gene. The GRF-PACAP gene was widely expressed in the adult zebrafish in tissues developmentally derived from all three germ layers, suggesting that the gene may be widely expressed in the embryo as well. To examine the functional significance of the co-expression of GRF and PACAP in zebrafish, I isolated the GRF and PACAP receptors and characterized their expression pattern. I isolated three distinct cDNAs from zebrafish encoding the GRF receptor, the PACAP specific PAC1 receptor and the shared vasoactive intestinal peptide/PACAP receptor VPAC1. In addition, four isoforms of the PAC1 receptor were isolated from zebrafish including a novel isoform found in the gill. All three receptors were widely expressed in adult zebrafish and receptors for both GRF and PACAP were found in most tissues. This indicates that GRF and PACAP may modulate each other’s function. To determine the developmental role of GRF and PACAP, I characterized the expression pattern of the GRF-PACAP gene and the GRF, PAC1 and VPAC1 receptors in the zebrafish embryo. The GRF and PAC1 receptors are the earliest to be expressed in development starting at the cleavage stage. Later, the GRF-PACAP gene and the VPAC1 receptor are first expressed at the late blastula/early gastrula stage in the zebrafish and are expressed throughout the developmental period. Strong expression of the GRF, PACAP and their receptors during mid gastrulation indicates that these peptides may be involved in modulating the formation of the embryonic axis. During the segmentation period the GRF-PACAP gene is widely expressed in the zebrafish embryo and the PAC1 receptor short and hop isoforms are differentially expressed. Therefore, PACAP may regulate cell cycle exit or cell proliferation through activation of different PAC1 receptor isoforms during the segmentation stage. In the subsequent pharyngula period, the GRF-PACAP transcript is localized mainly to the hatching gland. However, expression is seen also in tissues that undergo differentiation during this stage. Therefore, the timing of the expression of the GRF-PACAP gene indicates that it may be involved in early patteming events and promoting cell cycle exit prior to differentiation. To investigate the role of GRF and PACAP in the developing brain, I localized the expression of GRF, PACAP and the PAC1 receptor in neuroblasts derived from an embryonic day 3.5 chick. PACAP was found to stimulate the cAMP pathway in these cells, indicating that PACAP may modulate brain development. This work indicates that GRF and PACAP play an important role in vertebrate development. / Graduate

Growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of the PACAP/Glucagon superfamily. The family is proposed to have developed from an ancestral PACAP-like molecule in invertebrates. Through successive exon, gene and genome duplications the family has grown to include seven other members. In mammals GRF and PACAP are located on different genes, but in fish, amphibians and birds they are located on the same gene. The main function of GRF is the release of growth hormone (GH) from the pituitary. Also, during development GRF influences the fetal pituitary and stimulates GH release during late gestation. In contrast, the functions of PACAP are extremely varied. PACAP is the newest member of the superfamily and there is still much work to be done before its actions are well understood. Like GRF, PACAP is a releasing hormone acting on the pituitary and in addition, the adrenal gland, pancreas and heart, as well as other organs. Also, PACAP regulates smooth muscle in the vascular system, gut, respiratory tract and reproductive tract During development PACAP affects proliferation, differentiation and apoptosis. GRF and PACAP are expressed throughout development in fish, beginning during the blastula period in rainbow trout and at the end of gastrulation in zebrafish (earliest stage examined). In rainbow trout the grf/pacap gene is expressed as two transcripts, a short and a long transcript. The short transcript is produced by alternative splicing of the gene and does not include the fourth exon which codes for GRF. The long transcript includes the coding regions for both GRF and PACAP. By this means PACAP can be regulated separately from GRF. With the extensive role PACAP appears to play in development, separate regulation of the hormone may be necessary. Expression of the grf/pacap gene in zebrafish is widespread early in development and gradually becomes localized. Of particular interest is the expression of the grf/pacap transcript in regions associated with the prechordal plate, an important organizing center in development. Although it is not yet confirmed, there is evidence to suggest GRF and PACAP are expressed in the prechordal plate and its derivatives in the gut and hatching gland. In addition, expression of the grf/pacap transcript is observed in the neuroectoderm (eye, brain and spinal cord) and the developing heart. Considering the expression pattern of GRF and PACAP, I propose that one of both of these hormones may be involved in patterning during vertebrate embryogenesis. The evolution of gene families is thought to occur through successive exon, gene and genome duplications. Duplicate exons or genes become differentiated and eventually gain new functions or become functionless. During evolution of the grf/pacap lineage, several duplication events have occurred. Analysis of rainbow trout leads me to think that this fish and other salmonids possess two copies of the grf/pacap gene. This is not unexpected considering the tetraploid nature of salmonids. Present day mammals encode GRF and PACAP on separate genes. At some point during the evolution of this lineage a duplication event has occurred, possibly in early mammals or prior to the divergence of birds. The study of multigene families is a useful way to understand evolutionary processes. To this end I examined three members of multigene families from sockeye salmon. Therefore, in addition to the evolutionary mechanisms and pathways that directed grf/pacap gene evolution, I examined the ferritin-H subunit, the alpha-tubulin subunit and the beta-globin subunit. These cDNA sequences are similar to their counterparts in other teleost. The evolution of the ferritin gene family is particularly interesting because it involves the addition or deletion of DNA sequences that affect regulation and cytosolic location. / Graduate