Broiler chicken has been bred for meat production and is characterised with a very fast skeletal muscle growth rate, whilst the layer type have been bred for the production of eggs. This study aimed to understand how intense breeding programmes have developed commercial meat type chickens that have resulted in a fast muscle growth phenotype by a comparative analysis of gene expression between fast-growing broiler and slow-growing layer type chicken. Two chicken trials were carried out. In Trial 1 from fast-growing broiler Ross 308 genotype (R) fast-growing breast Pectoralis major (RPM) and slow-growing leg Peroneus tertius (RPT) muscles were collected at day 14, 36 and 42 post-hatch. In Trial 2, from fast-growing broiler Ross 308 (R) or slow growing layer Hy-Line (H) Pectoralis major (either RPM or HPM) and Peroneus tertius (either RPT or HPM) were collected at day 4, 14, 23, 35 and 42 post-hatch. In Trial 1, there was a muscle type x age interaction in muscle weights with the RPM having the highest value at day 43 (P < 0.001). This effect was also reflected in the protein content with RPM having more protein than RPT. However, RPT had a higher DNA content per unit tissue weight (muscle type x age interaction, P=0.003) with highest value seen at day 43. This suggests that muscle cells of RPM are bigger than those in RPT and that as a result they contain more protein, potentially reflecting RPM hypertrophy compare to RPT. In Trial 1, genes associated with glycolysis, GAPDH and -enolase, were significantly higher in the faster growing RPM (P < 0.05). Genes involved in serine biosynthesis PHGDH, PSAT and PSPH, as well as P70S6K involved in protein translation, also had a significantly higher expression in the RPM when compared to the RPT (P < 0.05) indicating a greater rate of protein synthesis in the faster growing RPM. Expression of genes associated with smaller muscles (myostatin) or protein degradation (calpastatin, the specific endogenous inhibitor of calpain proteinases) were not different between muscles. LIM domain proteins, CSRP3 and FHL2, had a higher expression in the slower growing muscle, indicating the possibility of those two genes being negative regulators of muscle growth, or may be involved in the upregulation of muscle regenerative physiological processes, as a result of the strain on the leg muscles induced by physical activities during locomotion. In Trial 2 there was a three-way interaction between genotype, muscle type and age in muscle growth (P < 0.001) with the highest value seen at day 42 in the RPM. There was a 3-way interaction between genotype, muscle type and age in the expression of -enolase (P=0.036) with the highest value seen at day 42 in the HPM. For -Enolase mRNA expression, there was a separate genotype and a muscle type effect (both P < 0.001) with HPM and HPT been higher than RPM and RPT, and PM muscles having a higher expression than PT muscles in both genotypes. When the serine synthesis pathway was examined, there was a genotype x age interaction (P=0.046) for PSAT and PSPH gene expression, with the highest expression at day 35 in both muscles of the Ross 308 genotype for the former, whilst the latter had the highest expression in the RPM at day 35. For ASNS mRNA expression there were significant genotype (P=0.004) and muscle type effects (P=0.014), with the Ross 308 genotype having the higher expression and the PM being greater than PT. For P70S6K mRNA expression, PM was higher than PT (P=0.012). For P70S6K total protein there was a significant muscle type x age interaction (P=0.049), RPM and HPM being higher than RPT and HPT at days 14 and 35. For calpain activity only at day 35 was there significant genotype x isoform interaction (P < 0.001), with the Hy-Line genotype having a higher micro and milli calpain activity than the Ross 308 genotypes, irrespective of muscle. For trypsin, chymotrypsin and caspase – like activities of the proteasome, there was a significant genotype effects (P < 0.001). In all three assays Ross 308 muscles had a higher activity when compared muscles from Hy-Line at both time points. For the ubiquitin ligases there was a borderline muscle type x time interaction (P=0.05) in the expression of MAFbx mRNA, with the RPT and HPT having a higher expression than the RPM and HPM respectively at day 14. For MuRF1 mRNA there was a significant genotype x age interaction (P < 0.001) with the HPM and HPT having a higher expression than the RPM and RPT at day 14 and 35. For CSRP3 there was a genotype x age interaction (P < 0.001), with the highest expression seen in the HPT at day 42. There was also muscle type effect within genotypes with the RPT and HPT having a higher expression than the RPM and HPM (P < 0.001). Trial 2 indicated that there is an increase in protein synthesis which leads to clear increase in protein accretion in faster growing chicken muscles, thereby supporting the wealth of literature detailing protein turnover in chicken skeletal muscles. There also appears to be an interaction between protein synthesis and degradation, however in most cases protein synthesis seems to be more dynamic and these changes seem to appear around day 35. The novel findings of this study were the observed increase in the expression genes that could limit the synthetic capacity of non-essential amino acid (non-EEA) in fast growing muscles.

Spleen tyrosine kinase (Syk) is an enzyme which plays a prominent role in IgE-dependent signal transduction in type 1 hypersensitivity reactions. Due to the important role of Syk in the early signalling cascade in mast cells and basophils for inducing allergic reaction, it could be a suitable target for the treatment of allergy. In this project, the efficacy of Syk gene silencing in the inhibition of mast cell degranulation and cytokine induction by small interfering RNA (siRNA) in vitro was evaluated. The evaluation of the inhibition of Syk function was assessed by two novel rat basophil leukaemia (RBL-2H3) derived reporter cell lines which are Neuropeptide Y-Red Fluorescent Protein fusion (NPY–mRFP) and Nuclear factor of activated T-cells-DsRed (NFAT-DsRed) reporter cell lines. In NPY–mRFP reporter cells, red fluorescent protein (RFP) is targeted and stored in the granules and released into the medium during degranulation upon appropriate IgE-dependent stimulation of the cells. In NFAT-DsRed reporter cell, DsRed can be expressed upon appropriate IgE-dependent stimulation of the cells resulting in translocation of NFAT from the cytosol to the nucleus and reporter gene expression. Novel cationic polymers in this project were evaluated for the delivery of siRNA. The first polymer was AGMA29, an amphoteric linear polyamidoamine polymer which has been reported to be useful for gene delivery and with a low toxicity. In addition, two modified poly(glycerol adipate) (PGA) variants were used. PGA is a polyester which can be derivatised with a variety of substituents to make cationic polymers. Derived polymers PGA-50% lysine (PGA-50% Lys) and PGA-20% histidine (PGA-20% His) were evaluated in this work to determine the size, efficacy and toxicity of polyplexes used in the Syk siRNA delivery. Using optimised polyplex resulted in the knockdown of the Syk function in the release of RFP in NPY–mRFP and the DsRed induction in NFAT-DsRed reporter cell lines, optimal calcium concentration and media used were determined. A new method was established for measuring the release of RFP in the NPY–mRFP cells, which could be used for detecting allergic reactions. The results showed that the polyplexes could be used for transfection when they were prepared in deionised water. 5 RU/Nt of AGMA29/Syk siRNA H polyplex resulted in a 31% decrease in the release of RFP in NPY-mRFP cell lines in comparison to untreated cell or scrambled siRNA. PGA-50% Lys/Syk siRNA H at 10AA/Nt ratio resulted in a 31% decrease in the release of RFP in NPY-mRFP cell lines in comparison to untreated cell and 24% in comparison to scrambled siRNA. The final finding from evaluation of Syk mRNA by RT-qPCR showed that the Syk mRNA knockdown at 5 RU/Nt of AGMA29/siRNA H was low (~26%) after 48 hr of the transfection. Interestingly, almost complete knockdown of Syk mRNA by PGA-50% Lys/Syk siRNA H was achieved at 5,10, and 20 AA/Nt ratios. This work therefore contributes to the exploration of a novel polyester based polymer for siRNA gene therapy and compares it with a polyamidoamine based polymer. From the results obtained it is concluded that the novel polyester based polymer for siRNA delivery is safe, effective in the transfection of hard to transfect cells and could be used for future siRNA in vivo gene silencing applications.

The functional significance of G protein coupled receptor (GPCR) dimerisation remains debatable partly due to the inability to assign pharmacological properties directly to defined dimers. To address this problem, this thesis uses bimolecular fluorescence complementation (BiFC), whereby protein-protein interactions are identified by the fluorescence generated from the association of complementary fluorescent protein fragment tags. The irreversibility of BiFC constrains receptor complexes of precise composition. The 4 Y receptor family members were chosen as model GPCRs as their relative β-arrestin recruitment and regulation by endocytosis remains questionable. BiFC based high content imaging assays quantified the pharmacology of β-arrestin2 recruitment to Y receptors, which correlated with their agonist induced endocytosis (Chapter 3). Targeted mutagenesis, showed key intracellular receptor domains shared in arrestin recruitment and internalisation. Fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy measured the diffusion of fluorescent Y receptor complexes and showed agonist stimulation slowed receptor motility (Chapter 4). A novel BiFC system allowed the correlation of this slowed motility with the behaviour of defined Y receptor/arrestin signalling complexes. BiFC also constrained Y1 receptors or β2-adrenoceptors as dimers of precise composition. Quantitative platereader imaging, measured BiFC dimer internalisation as an indirect readout of β-arrestin recruitment and dimer function (Chapters 5 and 6). Selective mutagenesis showed occupation of a single ligand binding site and the presence of one phosphorylated C terminal domain was sufficient, implying symmetrical binding of β-arrestin to Y1 receptor dimers. Finally Y1/Y5 receptor BiFC heterodimers showed modified pharmacology not evident for other heterodimer combinations. The most striking alterations were switching of Y5 selective antagonists from surmountable to insurmountable antagonism and the ineffectiveness of Y1 antagonists at inhibiting Y1/Y5 dimer responses, suggesting allosteric interactions between the respective protomers. Previous anti-obesity therapies targeting either Y1 or Y5 subtypes have lacked long term efficacy. However novel responses of the Y1/Y5 dimer, and the use of BiFC to screen for selective antagonism, may help identify future treatments for obesity.

The human ABCG2 (second member of ABC transporter G-subfamily) is an important ATP-dependent exporter in the body with broad substrate specificity including xenobiotics (e.g. anticancer agents) and endogenous compounds (e.g. sterols and lipids). ABCG2 was first discovered in a multidrug resistant breast cancer cell line and it is suggested to cause resistance to chemotherapy in certain cancers such as acute myeloid leukaemia and small cell lung cancer. Physiologically, ABCG2 is found in the protective sanctuary sites of the body, for instance the gut and blood-brain-barrier, affecting pharmacokinetics and treatment efficacies of small molecule drugs. Structurally, the polypeptide chain of ABCG2 contains a single nucleotide binding domain and a single transmembrane domain, which is half the number of domains required for a fully functional ABC transporter. Although many have suggested that ABCG2 function as dimer or higher order oligomer, studies so far have been unable to convincingly address the oligomeric state of ABCG2. We aim to bridge this knowledge gap by resolving the oligomerisation of ABCG2 using fluorescence techniques in mammalian cells. The expression and function of fluorescent proteins tagged ABCG2 were verified using confocal imaging and fluorescence accumulation assays, prior to the fluorescence studies. As the membrane dynamics of ABCG2 are unknown, we first measured the diffusion of ABCG2 in live HEK293T cells using fluorescence recovery after photobleaching (FRAP) microscopy, in comparison to membrane localised fluorescent proteins and a full length (i.e 4 domain) ABC transporter (ABCC4). We also demonstrated oligomerisation of ABCG2 by measuring a specific increase in fluorescence resonance energy transfer (FRET) efficiency between CFP- and YFP-tagged ABCG2 expressed in live HEK293T cells in comparison to non-specific control interactions, including with the adenosine A3 receptor. Subsequently, we employed high resolution and single particle fluorescence techniques to resolve the oligomeric organisation of ABCG2. First, fluorescence fluctuations of tagged ABCG2 within a confocal volume, positioned on the upper plasma membrane, were measured using fluorescence correlation spectroscopy (FCS) at “single molecule” resolution. Photon counting histogram (PCH) analysis of the FCS measurements was performed to determine the molecular brightness of the fluorescent species detected within the confocal volume. Using CD86 and CD28 as monomer and oligomer controls respectively, PCH analysis demonstrated higher order oligomer formation of ABCG2, with increased brightness (up to 4-fold) observed for both ABCG2 and CD28, compared to CD86. For validation of the oligomeric organisation of ABCG2, we acquired a series of single particle photobleaching images of cells expressing fluorescent protein tagged ABCG2 using total internal reflection fluorescence (TIRF) microscopy at the lower plasa membrane, and employed a step detection algorithm to identify the number of photobleaching steps within the distinguished fluorescent spots. Statistical modelling of the photobleaching step frequency histogram provided credible evidence of tetrameric organisation of ABCG2 in the plasma membrane. The findings and methodology presented in this study have provided further insights into the membrane dynamics and oligomerisation of ABCG2. This could lead to future studies to explore new pharmacological avenues that target the oligomerisation interfaces of ABCG2.

Precise and complete replication of the genome is essential for a cell. Chromosome replication follows a defined temporal order, depending on the efficiency and timing of the replication origins. However, the mechanism regulating origin activity has not been properly explained to date. Yeast replication origins are very well characterized and well studied. Genome wide replication in yeast was detailed through deep sequencing in various studies. In yeast, there are multiple replication origins for the complete replication of the genome. In Saccharomyces cerevisiae, there are ~400 replication origins, which are also referred to as Autonomously Replicating Sequences. Replication origins have varied levels of activity and varying times of activation. There are many lines of evidences, which suggest that the origins function differently in mitotic and meiotic cell cycles. It was thought that same origins function both during mitosis and meiosis. However, there is a difference in the replication timings of both the cell cycles, the reason for which is not known. Meiotic cell cycle is longer than the mitotic cell cycle. By using the plasmid-based assays, specific origins were selected and origin activity was analyzed during mitosis and meiosis to see if individual origins show any differences in origin activity. For all the origins tested, the meiotic activity was found to be less than the mitotic activity, which provides a possible explanation for a longer pre-meiotic S phase. Most of the confirmed yeast replication origins are present in the intergenic regions of the chromosome. Due to the presence of majority of replication origins in the intergenic regions and not on the genes, it was thought that the gene transcription might be detrimental to origin activity, hence not supporting the existence of an origin on a gene. Careful analysis of genome wide replication data along with plasmid based ARS assays confirmed that a few replication origins are present within genes. Assays were preformed to study the relation between transcription and origin activity both during mitosis and meiosis. Mitotic origin activity was shown to have no known affect from gene transcription. However, due to some unknown technical faults or other reasons, assays to find out transcription and meiotic activity were not successful.

Active immunisation against GnRH inhibits reproductive function by inducing a hypogonadotropic condition associated with gonadal atrophy. Despite economic, ethical and environmental advantages of GnRH immunisation in cattle over conventional castration methods, the technology has not yet been commercially adopted. Primarily because of the requirement for numerous booster vaccinations because of the reversibility of physiological effects, the commercial efficacy of immunocastration is currently poor. However, neonatal GnRH immunisation in sheep can result in a permanent suppression of reproduction (Brown et al., 1994; 1995; Clarke et al., 1998). These findings and a study in pigs (Molenaar et al., 1993) indicate that, the hypothalamic/pituitary gland unit (HPU) may be particularly susceptible to GnRH antibodies during a specific window of development in the pre-pubertal animal, but no long-term studies in cattle have been conducted. Therefore the primary objective of this project was to determine the effect of neonatal immunisation against GnRH in cattle. Beef cross bull (n=9; Chapter 3) and heifer calves (n=9; Chapter 4) were vaccinated against a newly developed (Pfizer®) GnRH construct vaccine at -2, 6 and 13 weeks of age. Nine calves of each sex served as negative controls, receiving saline injections only. The GnRH vaccine had proved effective (Dr. A.R. Peters, personnel communication 2000) in inducing immune responses and reducing variation between animals in unpublished industrial studies, compared to earlier vaccines, and hence was reasoned to be capable of raising GnRH antibodies despite the relative immaturity of the neonatal immune system. Following vaccination, circulating GnRH antibodies and reproductive hormones, such as FSH (Chapters 3 and 4), testosterone (Chapter 3), progesterone (to assess onset of puberty) and oestradiol (Chapter 4) were measured and additional intensive serial bleeds were carried out to assess LH parameters up to and beyond puberty (puberty defined by testes circumference in bulls). Gonadal (antral follicles and testes growth) and accessory gland development was quantified throughout the trial using ultrasound scanning. Sexual behaviour (Chapter 3) was studied from 38 weeks of age, while an assessment of sperm quality (Chapter 3), and anabolic response to vaccination was also performed post-mortem (Chapters 3 and 4). GnRH immunisation in neonatal calves did not permanently impair reproduction. A temporary suppression in reproductive function was evident through the disruption of pituitary gland function, as indicated by a reduction of LH pulse amplitude and mean plasma LH concentrations (Chapters 3 and 4). In addition, a reduction in medium- sized follicle numbers, testes growth, plasma testosterone concentration, vesicular gland length and juvenile aggression occurred. Some beneficial anabolic effects were observed e.g., carcass composition grades. Changes all occurred subsequent to increased GnRH antibody titres in immunised cattle. Despite some evidence of prolonged effects on LH amplitude and circulating testosterone after anti-GnRH titres had dissipated, all inhibited parameters, except carcass quality, returned to levels comparable to control animals by 72 weeks of age. No treatment effects on FSH concentrations, large follicle numbers, reproductive tracts (post mortem) or peri- and post-pubertal behaviours were observed following treatment. Sperm morphological abnormalities tended to be more prevalent in GnRH immunised bulls. A significant increase in GnRH antibody titres occurred at -23 weeks of age (Chapter 4), this may have been a rebound in antibody titre, possibly caused by an anti-idiotype immune response (antibody response to GnRH antibodies), or due to significant maturational changes in immune function at this time causing a delayed response to vaccination. Alternatively a novel "auto-immune" response may have been detected, which if confirmed/repeatable might be incorporated into an immunisation protocol to act as a "self-booster". However, no previous reports of such an event have been published and further investigation is urgently required. A more prolonged or permanent suppression of reproductive function may be possible following an earlier, greater and more sustained elevation of antibody titres during the neonatal period. Further development of GnRH vaccines and/or protocols (prime-boost, cytokine modulation vaccines, concomitant passive and active immunisation and pregnant cow GnRH vaccination), and studies of performance and GnRH antibody mechanism(s) of action in cattle are required. Chapters 3 and 4 provide a comprehensive study on pubertal development and neonatal GnRH vaccination, thus contributing significantly to knowledge in these fields. Currently, the vaccine used in this trial may be used to delay puberty in older calves or transiently suppress reproductive function to aid management. The economical viability of animal production systems such as beef and lamb are closely related to rates of reproduction. The Fec B gene in ewes increases ovulation rate and litter size, possibly through the development of precocious follicles, which can switch their primary dependence from FSH to LH. As a result, more follicles are selected to continue growth to an ovulatory size. The precise mechanisms by which these processes occur have recently been shown to involve oocyte follicle interactions (see section 1.1.5). Follicle development is modulated by GHIIGF and inhibin, however attempts to increase follicular development and ovulation through active inhibin immunisation alone have been variable and hence not commercially attractive. To develop successful protocols to induce twin ovulations in cows· and ewes, without superovulation, a clearer, more details understanding of follicullogenesis is required. The objective of the current study was to better understand these mechanisms through investigating interactions of GH/GF and inhibin in the ovary, follicle development, steroidogenesis, and receptor populations using an anoestrous sheep model. Spring born Mule x Charolais ewe lambs were actively immunised (n=8) against porcine inhibin α-C 1-26 peptide conjugated to KLH in NUFCA (primary and 3 boosters (NUFA», while 8 served as negative controls. Seven days following the final booster, the ewes were subdivided to give four groups: (1) controls + saline (n=4); (2) controls + rbGH (4ml s.c; 1mg. mr1; n=4); (3) inhibin immunised + saline (n=4); and (4) immunised + rbGH (n=4). Recombinant bovine growth hormone (rbGH) was given (Lm.) for 6 days. On day 4 GnRH (Receptal®; 1 ml) was injected s.c, to all animals to initiate the beginning of a new follicular wave. Blood samples were collected fortnightly to measure inhibin antibody titres, IGF-I, FSH and steroids. On the seventh day ensuing slaughter serum antibodies and ovaries were harvested. Left ovaries were intended for ISH (mRNA for P450arom) and/or immunohistochemical analysis. Follicles from right ovaries were dissected out, counted, measured and cultured in M199 at 37°C for 2 hours. Culture media was then assayed for oestradiol. Follicle shells were stored at -180°C for LH receptor binding studies. This work reports on the influence of different treatments on follicle populations. All immunised animals produced antibodies, which bound to 1251-inhibin. Using ANOVA to compare treatments it was observed that, Inhibin immunisation significantly (P<O.05) increased the number of follicles >3.5mm in diameter, but did not affect the smaller <3.5mm population. In contrast, rbGH administration led to a significant (P<O.05) elevation of follicles <3.5mm, without increasing the >3.5mm follicle numbers. These findings are in agreement with previous research. The molecular studies of left ovaries are not presented herein as due to time constraints the work was not completed and is currently on going. In conclusion, additional results of this study are required to meet the objectives of the experiment. Further research is required on dominant follicle selection if superovulatory programmes in both livestock and humans are to be more precisely controlled and readily accepted.

The findings of this project have added to the pool of information reported in the literature regarding the application of the nasal route for the delivery of insulin and other peptide drugs. The preliminary studies reported in this project were apparently the first studies performed to investigate the potential use of chitosan in nasal delivery systems. Nasal delivery systems were investigated in rat and sheep models. The efficacy of chitosan as a nasal absorption enhancer for insulin was compared to that of several other compounds which had been reported in the literature to enhance nasasl [i.e. nasal] drug absorption. Erythrocyte haemolysis studies were also performed to evaluate the membrane damaging effects of the various compounds tested. The grade of chitosan predominantly used was a medium viscosity glutamate salt (MVCSN) which was 82% deacetylated and had a molecular weight of about 162,000. Other grades of chitosan of similar degree of deacetylation were also investigated for comparison with MVCSN (low viscosity grades of chitosan glutamate (LVCSN) and lactate (CSN lactate), medium viscosity chitosan hydrochloride (CSN HC1) and high viscosity chitosan base (HVCSN)). The efficacy of chitosan in enhancing the nasal absorption of both insulin and salmon calcitonin, used as an alternative peptide, was demonstrated in rat and sheep models. Nasal insulin delivery systems were extensively investigated in rat and sheep models. In the rat model, insulin / LVCSN formulations at pH -4 were more effective than formulations at pH -7 in enhancing intranasal insulin absorption which was assessed indirectly from the degree of hypoglycaemia following dose administration. The reduced absorption in the latter formulation which was in the form of a suspension was attributed to complex formation between insulin and LVCSN. In the rat model, the absorption enhancing efficacy of MVCSN was second only to that of LPC. This was encouraging in view of the severe membrane damaging effects that LPC solutions have been shown to cause. In contrast, chitosan solutions have been shown to be relatively non-toxic to biomembranes. In the sheep model, a formulation incorporating MVCSN was much more effective than a formulation containing LPC in promoting nasal insulin absorption. These differences were attributed to the animal models used to investigate nasal absorption. The degree of nasal absorption enhancement was improved by increasing the solution concentration of MVCSN until an optimal concentration was attained (approximately 0.5% and 0.35% in rat and sheep models, respectively). Further evaluation of nasal insulin / chitosan formulations in sheep, suggested that the formulation concentration of chitosan was important for its absorption enhancing efficacy and at optimal chitosan concentration nasal insulin absorption was limited by the dose concentration of insulin. In both rat and sheep models, the nasal administration of hypotonic or isotonic formulations of insulin with chitosan did not influence the degree of nasal absorption enhancement attained. However, in rats, a hypertonic formulation was shown to further improve nasal insulin absorption which was attributed to the combined effects of the chitosan and the increased tonicity of the formulation on the nasal membrane. The grade of chitosan used in the nasal absorption studies appeared to influence the degree of absorption enhancement obtained. In the rat model there was no difference in the absorption enhancing efficacy of CSN lactate and MVCSN although the performance of HVCSN was marginally reduced. In contrast, in the sheep model, MVCSN was more effective than LVCSN and CSN lacate in enhancing nasal insulin absorption although there was no difference in the performance of MVCSN and CSN HCL. In studies in the rat, MVCSN was shown to have a transient effect on the permeability of the nasal mucosa to insulin which lasted about 30 minutes. This supports the claims that chitosan is non-damaging to the nasal mucosa. Erythrocyte haemolysis studies showed that MVCSN was non-damaging to rat erythrocyte membranes at concentrations which were higher than the concentrations used in nasal absorption studies. This was encouraging since the other compounds investigated for comparison with chitosan in this project were shown to be potent haemolytic agents at concentrations which were much lower than the concentrations which were effective for nasal absorption enhancement. MVCSN was less damaging to erythrocyte membranes than the other grades of chitosan tested. This project demonstrated that chitosan enhanced the nasal absorption of insulin in rat and sheep models. In the sheep model the bioavailability of nasal insulin, relative to the subcutaneous route, was generally less than 5%. However, the hypoglycaemia which followed nasal insulin / chitosan dose administration was encouraging and a similar degree of efficacy in humans could be feasible for the therapeutic application of nasal insulin.

The term conjugated linoleic acid (CLA) refers to a range of geometric and positional isomers of linoleic acid. Recent research suggests a variety of potential health benefits with consumption of dietary CLA. These include a reduction in body fat deposition that has been demonstrated in a number of monogastric species including the mouse, rat, hamster and pig. This thesis describes the effects of CLA on lipid metabolism in sheep, where CLA may be useful in reducing carcass fat and improving fatty acid profile. The results are contrasted with those in the hamster, a model monogastric species, previously shown to respond to CLA. Ovine adipose tissue metabolism was studied in explants maintained in culture and incubated with a mixture of CLA isomers and individual isomers. Total lipogenesis, and the formation of saturated and monounsaturated fatty acids were examined. Results show no effect of CLA (mixed or individual isomers) on total lipogenesis or desaturation of fatty acids. Furthermore, there was no effect on mRNA concentration for acetyl coenzyme A carboxylase (ACC) or stearoyl coenzyme A desaturase (SCD). The effect of feeding CLA (protected from rumen degradation) to sheep, on lipogenic gene expression was then investigated. While there was no evidence of a decrease in total fat deposition, there was a decrease in the proportion of monounsaturated fatty acids in the tissues. As there was no effect on SCD mRNA levels, it appears likely that CLA inhibits SCD activity rather than affecting gene expression. A further feeding study was undertaken in Golden Syrian hamsters. As in the sheep, CLA feeding reduced the monounsaturated fatty acid content of the tissues but did not change the SCD mRNA concentration in adipose tissue or liver. This further supports the suggestion that CLA directly inhibits SCD activity. Unlike the sheep, there was an overall decrease, of approximately 10%, in total carcass fat. However, paradoxically, there was an increase in adipose tissue ACC and fatty acid synthase (FAS) mRNA concentrations. Thus, suppression of lipogenic enzyme expression does not appear to be the mechanism by which CLA reduces fat deposition.

The initial aims of this investigation were to develop a reliable assay system for measuring serum 1,Z5-dihydroxyvitamin D [1,Z5(OH)ZD] concentrations and to establish a normal range in young healthy adults. Compared with young healthy individuals, the elderly population are indeed vitamin D deficient. Vitamin D deficiency was also demonstrated in a group of elderly osteomalacic patients. Slight improvements in osteomalacia was achieved by one month of treatment with vitamin D3' or alphacalcidiol with or without calcium supplements. The improvements were small and occured slowly. A significant increase in the strength of bone seems unlikely to occur in the short term. On the present evidence the combination of alphacalcidiol and calcium supplements seems no better than vitamin D3 or alphacalcidiol alone although it may require closer monitoring to avoid hypercalcaemia. In a group of elderly patients with osteoporosis and femoral neck fracture (FNF), serum osteocalcin concentrations rose significantly in the first week after fracture fixation. The change in osteocalcin correlated well (p < 0.001) with the change in serum 1,Z5(OH)ZD concentration. Histomorphometric measurements of the extent of osteoid correlated better with osteocalcin than alkaline phosphatase. Serum concentrations of 1,Z5(OH)ZD were also reduced in elderly patients with FNF irrespective of the presence of osteomalacia and therefore cannot be used as a screening test for osteomalacia in this patient group. Reduction of 1,Z5(OH)ZD was not due to a reduction in vitamin D binding protein. It is suggested that the low rate of bone turnover in these elderly patients reduces the requirement of vitamin D. Of the ten elderly patients who had underwent laryngo pharyngeal surgery all developed hypocalcaemia. This immediate post-operative decrease, due to a rapid reduction in circulating PTH concentrations, lead to an overall increase in urinary calcium excretion. Serum concentration of 1,25(OH)2D also fell postoperatively thus potentiating the hypocalcaemic state in these patients. Thus, it is important to give parenteral feeding supplemented with calcium and vitamin D, preferably alphacalcidiol. If delayed then profound as well as prolonged hypocalcaemia can occur. The human osteosarcoma cell 20S metabolised 25(OH)D3 in a substrate concentration and time dependent manner to produce products which were secreted into the extracellular medium. These products eluted from HPLC with a retention time coincident with 24,25(OH)2D3 and exhibited an UV absorption spectrum characteristic of a vitamin D sterol. Mass spectroscopy analysis indicated at least two products were synthesised by the cells. One was identical to 24,25(OH)2D3; the other appeared to be an unsaturated trihydroxylated derivative of vitamin D3.

The unlimited replicative potential of cells is one of the hallmarks of cancer. Telomeres, DNA structures found at the ends of chromosomes have attracted a great deal of interest in recent years as potential anti-cancer drug targets since they play an important role in cancer cell immortality. The repetitive TTAGGG sequences of telomeres are complexed to a group of six indispensible proteins, one of which is the protection of telomeres 1 (POT1) protein. This specialised protein binds to a ten nucleotide single stranded DNA sequence at the ends of chromosomes and plays an important role in telomere capping and length regulation. It has recently been proposed that the key function of POT1 is to suppress a potent DNA damage response at telomeres thereby protecting chromosome tips from being recognised as sites of DNA damage. Deletion of POT1 from telomeres in a variety of organisms including humans results in cytogenetic aberrations, senescence and cell death. These results indicate that POT1 is an integral telomere end-protection protein which is necessary for continued cellular proliferation and therefore POT1 is becoming a promising new target in cancer. Using a structure-based approach, several small molecule inhibitors of POT1 have been designed to affect telomere integrity by disrupting the binding interaction of human POT1 with its target DNA sequence thereby driving cancer cells into senescence/apoptosis. Using a range of computational tools, a suitable drug binding pocket in POT1 has been identified and the de novo design of a specific class of POT1 inhibitor was completed. Using this novel scaffold, a small focussed library of hit-like compounds were synthesised and screened in a new POT1 fluorescence polarisation displacement assay developed by scientists at the University of Nottingham. In total, over 90 small molecule inhibitors based on two different scaffolds: pyrido[1,2-a]pyrimidines and sulfathiazoles have been synthesized with some inhibitors effectively decreasing POT1-DNA binding between 10-54% at 100μM ligand concentration. The biological results have established that electron-withdrawing substituents on the pendent phenyl ring of the pyrimidine core are essential for strong binding. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of cancer.