Novel interactions of the 5-HT₂ₐ and related receptors with intracellular signalling proteins

The aim of this project is to further elucidate the pathways involved in the intracellular signalling mechanisms of the 5-HT₂ₐ and related receptors. The Gprotein coupled receptors (GPCRs) are named after their ability to interact with and signal through the trimeric G-proteins. The 5-hydroxytryptamine 2A receptor (5- HT₂ₐR) is a member ofthe group I family of rhodopsin-related GPCRs. The receptor is known to activate phospholipase C (PLC) via the heterotrimeric G proteins Gaq/n, but has been shown to also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner, that appears to be independent of Gq/n. The M3 muscarinic receptor, another member of the group I GPCRs, has also been shown to signal through both PLC (via Got) and the alternative pathway of PLD activation via ARF. In this thesis, it has been shown that both these receptors interact directly with members of the ADP-ribosylation Factor (ARF) family of small G-proteins. Not only is there evidence to show that these receptors activate PLD signalling through the ARF family of proteins, as shown by in vivo signalling assays, but it can also be shown that the receptors interact directly with ARF. The 5-FIT₂ₐ receptor associates with ARF1, and the third intracellular loop domain of the M3 muscarinic receptor associates with both ARF1 and ARF6, as shown by in vitro GST interaction assays.Experiments undertaken to elucidate the exact criteria for this interaction suggest that a complex of proteins involving GPy for the M3 muscarinic receptor, and arrestin for the 5-HT₂ₐ receptor. The GDP/GTP status of the ARF involved plays a role in the ability of this interaction to take place. The conserved N/DPxxY motif in transmembrane domain 7 (tm7) ofthe Group I GPCRs also seems to affect the ability of the receptor to signal through ARF. Thus changing this motif altered the binding of ARF isoforms to the 5-FIT₂ₐ receptor.The binding of novel interaction partners to the 5-HT₂ₐ receptor was also investigated, with the discovery ofthat the glial protein S100B bound to the carboxy terminal domain ofthe 5-HT₂ₐ receptor in a calcium dependent manner.These findings have implications for the investigation of the signalling pathways of these and other related Group I type GPCR

Host parasite interactions between Ichthyobodo necator (Henneguy 1883) and farmed Salmonids

The literature on Ichthyobodo necator is reviewed. The prevalence and intensity of Ichthyobodo infestations on farmed salmonids was investigated on three farms over a period of two years. The infestations were found to be markedly age dependent. Peak infestations and related mortalities occurred in the first eight weeks after first feeding. Both mortalities and infestations declined to zero shortly after this period with no chemotherapy. Ichthyobodo reappeared on 0+ and appeared for the first time on I+ fish after a drop of water temperatures to less than 10*C. Many of the 1+ fish had started to mature. It is suggested that some form of host defence mechanism operates which limits the Ichthyobodo infestations in farmed salmonids. The sequential pathology of Ichthyobodo infestations of the skin of 0+ and 1+ salmon and rainbow trout was studied. Areas of greatest shelter from water currents were found to be most commonly infested and no parasites were found attached to the epidermis on the head of the fish. The parasite caused hyperplasia of the malphigian cells and exhaustion of the goblet cells below infestations, followed by spongiosis of the underlying epidermis. The epidermal plaque then sloughed off leaving a single layer of cells attached to the basement membrane. Cell kinetic studies showed that Ichthyobodo caused the cells immediately below infestations to divide, a markedly different pattern from that of normal teleost epidermal cell proliferation. The possibility that the parasite secretes some form of digestive enzyme is postulated. In areas where sloughing had occurred, the remaining malphigian cells were seen to be in the process of division. Various endocrinological aspects of Ichthyobodo infestations were investigated. Three corticosteroids and one androgen were injected or implanted into 1 year old rainbow trout. Implantations of hydrocortisone led to very heavy ichthyobodo infestations. Radio immune assays showed that the level of cortisol and testosterone in the serum of implanted fish was similar to that which would occur when salmonids mature. There appears to be a clear link between cortisol levels in the serum and Ichthyobodo infestation. The host response to Ichthyobodo is discussed and it is concluded that cortisol may suppress the host's defence mechanism to Ichthyobodo

Feasibility study of commercial rainbow trout production in Lesotho

The objective of the study is to identify the technical, financial and economic feasibility of trout production in Lesotho. The approach adopted in this report is as follows: Chapter 1 provides a brief outline of the physical conditions in Lesotho with particular reference to factors relevant to trout production, Chapter 2 identifies the parameters which must be met in order to sustain trout production on an intensive/farm basis.. These criteria are then used to evaluate potential farm sites within Lesotho, Chapter 3 examines the current and potential markets for trout in Lesotho and South Africa, identifying the most feasible market areas which could be served, relevant price data and the likely share of markets which can be achieved, Chapter 4 draws together the findings of previous chapters in financial and economic analysis of hypothetical examples of trout farms. Hypothetical examples are used since no viable site for a farm was found to meet technical criteria, and Chapter 5 concludes the study with a brief reference to the Highland Water Project where in future, if proved feasible, trout production facilities in various forms could be attached