Terminally differentiating keratinocytes actively synthesize and accumulate lipids to maintain the production of lipid lamellae and an effective epidermal barrier. This thesis explored the role of steroidogenic acute regulatory (StAR) related-lipid transfer (START) proteins in the immortalized HaCaT keratinocyte cell line. Differing cell culture conditions were used to study differentiation of HaCaT keratinocytes, including the 'calcium switch' model, studied over a period of 21 days. Gene expression of five 'markers' of keratinocyte differentiation, KRTI, KRTIO, KRTI4, INVand LOR, was used to define progression of this process, revealing a dependency on calcium concentration, and increasing confluency. We then investigated gene expression of the START family of lipid trafficking proteins in HaCaT keratinocytes, compared with primary human keratinocytes. The same nine members of the family, STARDI, STARD2, STARD3, STARD4, STARD5, STARD7, STARDIO, STARD11 and STARDI2 were expressed in both cell types, although levels of STARD2, STARD5 and STARDI2 mRNA proved higher in HaCaT cells than in primary keratinocytes, and gene expression of STARD4 was lower in HaCaT cells compared with primary cells. During HaCaT differentiation, marked changes in gene expression of this family of proteins were noted, reflecting changes in lipid metabolism occurring during this process, the most notable being an increase in cholesterol mass after 21 days in cells exposed to I.4mM calcium compared with cells cultured in calcium depleted media. Steady state levels of mRNA encoding STARDI, D3, D2, D7, DID and Dll tended to increase during keratinocyte differentiation, while STARD4 and STARD5 showed a more complex pattern of expression, with decreased expression observed in the presence of I.4mM calcium. In order to investigate the function of the cytosolic cholesterol binding proteins, STARD4 and STARD5, in HaCaT cells, transient transfections were performed (48h) with pCMV6 vector encoding full length STARD4 or STARD5. Confocal microscopy confirmed the cytosolic location of both proteins, while in 3-D organotypic cultures STARD5 eo-localised with Keratin 10 rather than Loricrin, suggesting a locale in the spinous layer of these cultures. While both cholesterol trafficking proteins repressed cholesterol and cholesteryl ester biosynthesis from [I 4C] acetate, consistent with their proposed role as directional sterol transporters, these changes were associated with distinct gene expression patterns. Overexpression of ST ARD4 was associated with induction of Loricrin mRNA and protein, while STARD5 overexpression repressed gene expression of keratin 1, indicating that altered intracellular cholesterol transport can influence keratinocyte differentiation status. Further, ST ARD4 overexpression was associated with induction of SREBF2 and ABCG4, and repression of ABCAI, while overexpression of STARD5 resulted in induction of PPARD, PPARG and ABCAI, and repression of SREBF2 and LDLR. These findings confirm the impact of these cytosolic sterol transporters on keratinocyte lipid metabolism, and may suggest that ST ARD5 may be more efficient in this regard. In conclusion, manipulation of intracellular lipid transport proteins may therefore provide novel therapeutic strategies for the treatment of lipid-related skin disorders.

Previous studies have shown that supplementation of n-3 polyunsaturated fatty acids (PUFAs) has a beneficial effect on reducing the expression and activity of degradative enzymes and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases. The aims of this thesis was to use a well-established in vitro model of cartilage degradation to further these studies and to investigate how n-3 PUFAs effect the expression of inflammatory factors at a proteomic level and to use specific inhibitors to identify possible signalling pathways involved in cartilage metabolism. The results of this thesis research indicate that n-3 PUFAs abrogate IL-1-induced cyclooxygenase-2 (COX-2) mRNA expression, protein levels and activity, measured as PGE2 production, in both normal bovine and human osteoarthritis articular cartilage chondrocytes. These studies were followed by the use of a simple array system to analyse the expression of several marker genes from different signalling pathways after IL-1 exposure, plus or minus n-3 PUFA supplementation. This led us to identify three possible pathways involved in IL-1-induced cartilage catabolism and inflammation. These were analysed further with the use of specific inhibitors to ascertain whether the inhibition profiles were similar to those seen by n-3 PUFAs. Two main pathways, the extracellular signal-regulated kinase (ERK) pathway and NFkappaB pathway were identified. Further analysis using the ERK pathway inhibitor, U0126, showed that it decreased IL-1-induced glycosaminoglycan release from the tissue, endogenous aggrecanase activity, ADAMTS-4 (but not ADAMTS-5) mRNA levels, MMP-3 and MMP-13 mRNA levels, COX-2 message, protein levels and PGE2 production in a manner similar to that seen with n-3 PUFA supplementation. Collectively, these results suggest that n-3 PUFAs may be directing their effects through the ERK pathway.

Bovine chondrocyte cell cultures represent an experimental system that can be used to study arthritis in vitro and this was used in the work reported here. The relative effectiveness of different fatty acids in reducing inflammatory responses was studied using these cultures. Eicosapentaenoic acid (EPA) proved to be most effective n-3 PUFA compared to docosahexaenoic (DHA) or alpha-linolenic acid (ALA), in suppressing the levels of mRNA for pro-inflammatory proteins (COX-2, IL-1alpha, IL-1beta, TNF-1alpha), aggrecanases (ADAMTS-4 and ADAMTS-5) and matrix metalloproteinases (MMP-3 and MMP-13) in the bovine monolayer cultures which had been induced by IL-1alpha. Arachidonic acid (AA), an n-I6 PUFA, had no effect on these mRNA levels. Similarly, hydrolysed palm olein had no consistent affect, showing that neither of these fatty acid preparations could be regarded as anti-inflammatory. Microscopic examination of the cells in culture showed some evidence for destructive effects on IL-1alpha stimulation and this was reduced by EPA. Moreover, this was confirmed when GAG release was examined. The latter was increased by IL-1alpha exposure and this was reduced by n-3 PUFAs with EPA being the most effective. To increase the potential value of palm olein products, the n-3 PUFAs, alpha-linolenic acid and EPA were incorporated into palm olein through lipase-catalyzed interesterification reactions. Palm olein, which had been modified with EPA, was tested for its anti-inflammatory properties. It was found to reduce GAG release, and the levels of mRNA for various inflammatory proteins (COX-2, TNF-alpha, IL-1alpha, IL-1beta) and the proteinase ADAMTS-4. These results showed that it is possible to modify palm olein by interesterification to yield an oil with improved nutritional properties.

This thesis describes analytical work carried out to determine the stability and properties of lipid and peptide-lipid systems. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and tandem mass spectrometry (MSMS) analyses were carried out to establish a validated protocol for the complete identification of phospholipids, including the nature of the headgroup and acyl chains and the positions of the acyl chains on the glycerol backbone. Statistical differences were observed in the relative intensities of peaks corresponding to the neutral loss of the acyl chain from the sn-1 and sn-2 positions of POPE, POPC and OPPC, with a preferential cleavage of the chain from the sn-2 position of all three in the absence of added salt and a preferential cleavage at the sn-1 position in the presence of sodium or lithium ions. This knowledge was applied to the identification of unknown lipid mixtures both on the standard MALDI target plate and directly onto thin layer chromatography plates after separation. The above techniques, together with other analytical methods including thin layer chromatography and dynamic light scattering, were applied to the study and identification of lipids modified by actions such as hydrolysis and oxidation under conditions used for binding analyses of peptides and small molecules. Long-term analyses of samples containing synthetic melittin and liposomes showed that over time melittin both promotes the hydrolysis of liposomal lipids and is itself acylated. Analyses of the binding of a prototypical peptide (human neutrophil defensin HNP-2) to membranes, using methods that included dichroism and fluorescence spectroscopy, demonstrated that HNP-2 dimers act on lipid membranes via a carpet-type mechanism and allowed the rate of the formation of bound HNP-2 states to be determined. HNP-2 was modeled as a consecutive two-step process following pseudo-first order kinetics. The first step (membrane association) was rapid, with a half-life of around 0.5 minutes, while the second step (reorientation and partial insertion into the membrane) was slower by an order of magnitude.

A brief summary of the work described in this thesis is given below:- 1. The cholesterol side-chain cleavage reaction was investigated in preparations of porcine luteal mitochondria. The enzyme system was found to be similar to that studied in other steroidogenic tissues in that it was located in the mitochondrial subcellular fraction, was associated with cytochrome P450 and required NADPH as an electron donor. 2. A method for determining the oxygen content of incubation media was described; it was based on the stoichiometric oxidation of added NADH, catalysed by phenazine methosulphate. Using values for oxygen concentration obtained by this method, some respiratory characteristics of luteal mitochondrial preparations were investigated. Respiratory control and ADP:0 ratios of these preparations were found to be significantly lower than those measured for porcine liver mitochondrial preparations. 3. The efficiency of several tricarboxylic acid cycle intermediates as electron donors for cholesterol side-chain cleavage activity was investigated: citrate, isocitrate, succinate, fumarate and malate supported greater activity than NADPH. 4. Studies with respiratory inhibitors and uncoupling agents indicated that NAD+- linked substrates could donate electrons to the NADPH - cytochrome P450 reductase by an energy-independent pyridine nucleotide transhydrogenase. In contrast, succinate supported the reaction via an energy-dependent electron transfer pathway. Experimental evidence was presented which indicated that this latter route might not involve reduction of NADH. 5. In view of the natural abundance of cholesterolfatty acid esters in luteal tissues, it was thought relevant to investigate the utilization of such com¬ pounds by porcine luteal mitochondrial preparations. [4-14^C] cholesteryl oleate was shown to be hydrolysed, and the cholesterol thus liberated underwent side-chain cleavage. The capability of the fatty acid moiety to act as an electron donor for this reaction was also demonstrated. Palmitylcarnitine was shown to support high levels of cholesterol side-chain cleavage activity, via an energy dependent electron transfer process. 6. A schematic hypothesis of electron transfer pathways in porcine luteal mitochondria was presented. Its main features embodied electron transfer connections between the mitochondrial "respiratory" and "steroidogenic" chains at two different levels: (a) a reversible, non-energy dependent pyridine nucleotide transhydrogenase, and (b) an energy-dependent electron transfer route from reduced flavoprotein to the NADPHcytochrome P450 reductase. 7- As a speculation it was suggested that LH might stimulate cholesterol side-chain cleavage activity, and hence steroidogenesis, by promoting electron transfer from respiratory substrates to the NADPHcytochrome P450 reductase via these connections, at the expense of mitochondrial respiratory electron flow.

Cholesterol, along with a number of its derivatives and very small quantities of its precursors, is the only sterol which can be identified in extracts of mammalian tissue. Cholesterol is eliminated from the body in the form of bile acids, the degradation products of the steroid hormones and by way of excretion of neutral sterols in the faeces. The formation of the bile acids is the major quantitative pathway, . In the rat, the major bile acids which have been identified in bile are the dihydroxy acid, chenodeoxycholic acid and the trihydroxy acid, cholic acid. Small quantities of the muricholic acids (trihydroxy acids) have also been identified. There is no interconversion of chenodeoxycholic acid and cholic acid in the rat. The bile acids are excreted in bile as conjugates, mainly with taurine and are reabsorbed from the small intestine and returned to the liver in the portal blood. The re-excretion of the bile acid conjugates in bile thus establishes an enterohepatic circulation. The total biliary content of bile acid conjugates represents both those conjugates which have been reabsorbed from the small intestine and a small quantity which have been newly synthesised. Thus direct measurement of the concentration of bile acid conjugates does not provide an estimate of the bile acid synthesis rates.

(1) A cholesterol-7α-hydroxylase has been investigated in rat liver cell fractions; the enzyme is located in the endoplasmic reticulum (microsomes) and requires co-factors in the cytoplasm. (2) The enzyme was assayed "by following the metabolism of cholesterol-4-14C; analysis was effected by thin layer chromatography followed by liquid scintillation counting. (3) Cholesterol-7α.-hydroxylase was found to be sensitive to prolonged homogenisation of the liver, long incubation periods, etc.; stimulation of the activity was observed only in the presence of NADPM. (4) Preliminary studies showed that the enzyme was inhibited by carbon monoxide; it appeared that a carbon monoxide binding pigment may be involved in oxygen activation for the system. The enzyme is suggested to be a mixed function oxidase. (5) The conversion of cholesterol to 7α-hydroxycholesterol can be increased several fold by preventing the reabsorption of bile salts from the gut; the significance of this enzyme as a rate-controlling enzyme in the overall catabolism to bile acids is discussed. (6) Non-enzymic oxidation of cholesterol has been investigated in some detail in order to determine whether enzymic and non-enzymic cholesterol oxidation have any common characteristics. Evidence is presented to suggest that cholesterol can be oxidised in conditions which support peroxidation of unsaturated lipids.