Acid-sensing ion channels (ASICs) are voltage-independent proton-gated ion channels belonging to the amiloride-sensitive degenerin/epithelial Na+ channel (DEG/ENaC) family of receptor channels. Six subunits (ASICla, lb, 2a, 2b, 3 and 4) encoded for by four genes, have been identified so far. All ASIC subunits are expressed in dorsal root ganglia (DRG) and have been implicated in physiological sensory processes such as nociception associated with tissue acidosis, cutaneous and visceral mechanosensation, sour taste and cochlear function. However, some ASIC subunits also show a wide distribution throughout the brain, where they are thought to modulate synaptic communication. Supporting this hypothesis several studies demonstrated in mice a role for ASICs in learning, memory and fear behaviour. Recently ASIC la channels were also shown to make a major contribution to hippocampal neuronal damage in stroke through Ca2+ overload. ASIC4 is broadly expressed in the nervous system but is not gated by protons and has no known function. This thesis describes a genetic analysis carried out to identify interacting partners of ASICs in sensory neurons, in order to shed light on the possible role of ASIC4, and to better understand the functional roles of ASIC 1-3 and their regulatory mechanisms. A rat dorsal root ganglion cDNA library was screened in a yeast two-hybrid assay and a number of proteins interacting with the N-terminal domain of rat ASIC 1-4 were trapped. Many of these proteins were involved in trafficking of ion channels, G protein pathways, endocytosis, protein ubiquitination, or cell adhesion, suggesting potentially novel roles and regulatory mechanisms for ASIC channels. The annexin II light chain pll was found to specifically interact with ASIC la, and to promote its functional expression at the plasma membrane, as shown by immunocytochemistry, cell surface protein biotinylation and electrophysiology. ASIC4 was shown to decrease the protein level of other ASIC subunits, and to downregulate ASIC la-mediated currents. Preliminary data suggest that this effect may be due to an involvement of ASIC4 in ubiquitination pathways. Overall, this work has led to the identification of interacting proteins that regulate ASICs and suggested novel functions for ASIC4.

Lines selected high or low on lean body mass (P-Lines) or on carcass fat (F-Lines) for more than 30 generations were treated with recombinant bovine growth hormone (GH) to obtain information about the effects of selection on GH levels and responsiveness. All lines responded to GH with an increase in final body weight. In the lean mass selected lines the high line increased more than the low, but the increase relative to initial weight was similar in both lines (10-15% ). GH did not have a significant effect on gonadal fat percentage. Further investigations involved the lean mass selected lines only. Weights were recorded on a daily basis from birth to weaning at 21 days, to investigate growth before mice become responsive to GH at 14 days. The high line had a mean litter size approximately twice that of the low line, and a mean birth weight approximately 50% greater than the low line. The difference in total litter weight at birth is greater (3 to 4 fold) than the difference in adult body weight (approximately 3 fold) suggesting that there may be between line differences in individual effects on <i>in utero</i> growth rates as well as differences due to maternal effects. Also an increase in the rate of gain was observed for both lines at approximately 18 days of age. An increase was also observed in GH deficient <i>little</i> dwarf mice from the high P-Line, and hence is not due to GH. The increase in the rate of weight gain is greater in the wild type mice than in the <i>little</i> mice suggesting GH acts to magnify the effects of whatever causes the increase. Plasma GH levels were assayed, the high P-Line had lower levels of GH at 4, 5 and 7 weeks of age than the low P-Line. As IGF-I levels become GH inducible at about 14 days of age and the lines experience an increase in the rate of weight gain, which is greater in the high line than in the low line, at about 18 days it appeared possible that IGF-I levels differed between the lines.

The aim of this project was to create an inducible thyroxine-deficient mouse model which could be used to investigate the role of thyroxine in gene expression. This was achieved by using the HSV1-tk ablation technique. Transgenic mice were produced containing 3.1kb of the bovine thyroglobulin promoter linked to 1.8kb of the HSV1-tk gene. Expression of the transgene was detected in the thyroid and testes. Transcription of the transgene in transgenic thyroid tissue was correctly initiated at the cap site with 4.5mg/day of an anti-herpetic agent, such as ganciclovir, resulted in a deduction in the number of thyroid follicle cells within 3 days and their complete absence after 7 days. After 14 days of treatment the mice lacked circulating thyroid hormones, the HSV1-TK activity of the thyroid rudiments was comparable to non-transgenic controls and the total soluble protein remaining was approximately 20% of controls. The thyroid gland comprises two cell types, thryoxine producing thyrocytes and calcitonin producing C-cells. The gland is also in intimate contact with the parathyroid gland which secretes parathyroid hormone. The expression of the transgene was restricted to the thyroid follicle epithelial cells. Transgenic ablation resulted in the precise removal of HSV1-TK expressing cells with no secondary effect on either the C-cells or the parathyroid gland. My results also demonstrate that the normal function of the C-cells and parathyroid gland are not dependent on thyroid hormones. When treatment with ganciclovir was terminated no recovery of thyroid hormones in the circulation or HSV1-TK activity in the thyroid rudiment were observed (for up to 113 days). This suggests that young adult mice do not contain a non-differentiated stem cell that is capable of repopulating the follicle cells of the thyroid. The mouse major urinary proteins (MUP) genes in the liver are regulated by thyroid hormones and growth hormone. I investigated the role of thyroid hormones on the expression of resident MUP genes and also a MUP transgene and demonstrated that hepatic MUP gene expression is absolutely dependent on the presence of thyroid hormones.

No description available.

No description available.

No description available.

No description available.

Since its discovery in 1994, the adipose tissue hormone leptin has been well established as a key regulator of energy balance in mammals. However, little is known about the molecular evolution of the hormone and its function in non-mammalian vertebrates. This project builds on the recent identification of leptin in an amphibian, the tiger salamander, to investigate the leptin signalling system in a laboratory salamander, the axolotl. The overall aim of the project was to obtain cDNA sequences of the axolotl leptin and leptin receptor (LEPR) genes, to analyse their expression and to study their expression due to nutritional state. Cloning the axolotl LEPR was a key component of the work because no sequence information was previously available. Semi-degenerate primers were used to clone a 248 bp fragment of the LEPR, which shared 62% identity with human leptin at the amino acid level. Attempts to obtain the full-length cDNA sequence were unsuccessful. However, the sequence grouped in proximity to a Xenopus LEPR in a phylogenetic tree, and Northern hybridization revealed a transcript size of approximately 3 kb, which corresponded with that of other vertebrate LEPRs. To establish the expression pattern of leptin and the LEPR between tissues, quantitative real-time PCR was performed in two different age groups of animals. In adults, the highest expression of leptin was observed in the fat, brain and heart whereas in juveniles leptin expression was significantly higher in the fat body compared to all other tissues. The highest expression of LEPR was found in the brain and skeletal muscle. These findings agree with the main sites of leptin and LEPR expression in mammals, Xenopus, and fish providing further evidence that the gene fragments cloned represents the axolotl leptin and LEPR. In order to understand the possible role(s) of leptin in the regulation of food intake and energy metabolism in amphibians, changes in leptin and LEPR expression due to nutritional state were investigated. Short-term fasting did not result in any significant changes in leptin expression in the fasted animals, nevertheless it showed a tendency towards a lower leptin and LEPR expression of fasted axolotls. These findings indicate that the regulation of leptin expression by nutritional state more closely resemble the situation in other ectotherms such as teleost fish. This work provides the opportunity to explore how the physiological functions of leptin have changed during evolutionary history.

No description available.

For many years, ovariectomy has been known to cause cessation of oestrus and modification of secondary sex characteristics in animals. In 1913, Fellner observed that transient oestrous symptoms could he produced by injecting an ovariectomized animal with extracts of ovaries. These oestrous symptoms could be followed in the rat by observing histological changes in the vaginal epithelium and in 1923 this keratinization of the vaginal wall was used in the development by Allen and Doisy (1923) of a quantitative test for oestrogenic substances. Four years later, using this method of bioassay, Ascheim and Zondek (1927) discovered that extracts of human pregnancy urine possessed much greater oestrogenic activity than did ovarian extracts. This observation led to a closer examination of pregnancy urine and within two years the first crystalline oestrogenic substance, oestrone (oestra-1:3:5-triene-5-ol-17-one), was isolated from urine independently by two groups of workers - Doisy, Veler and Thayer (1929) and Butenandt (1929). In the following year, oestriol (oestra-1:3:5-triene-3:16α:17β-triol) was isolated from the same source first by Marrian (1930) and soon after by Doisy, Thayer, Levin and Curtis (1930), In 1933, Schwenk and Hildebrandt succeeded in reducing oestrone to an oestradiol (later shown to be oestradiol-17β (oestra-1:5:5-triene-3:17β-diol)) which was found to possess higher oestrogenic activity than either oestrone or oestriol. As a result of this finding, oestradiol-17β was adopted as the probable active principle secreted by the ovaries. Although this oestrogen was isolated from pregnant mares' urine (Wintersteiner, Schwehk and Whitman, 1935) and sows' ovaries (MacCorquodale, Thayer and Doisy, 1936) it was not until 1939 that Smith, Smith, Huffman, MacCorquodale, Thayer and Doisy isolated it from human pregnancy urine. Between 1939 and 1953 no further metabolites of oestrogen metabolism were isolated from urine although a number of investigators reported the presence in various urine extracts of unknown substances whose chemical and physical properties suggested that they might prove to be derivatives of known oestrogens (Pincus and Pearlman, 1943; Serchi, 1952; Zondek and Finkelstein, 1952; Migeon, 1953; Braunsberg, Stern and Swyer, 1954). Only one of these led to the actual isolation of an oestrogen metabolite, viz. 16-oxo-oestrone (oestra-1:3:5-triene-3-ol-16:17-dione) which Serchi (1953) obtained in crystalline form from the urine of non-pregnant women.