Human embryogenesis and ensuing adult homeostasis are directed by the complex interplay of a wide range of exquisitely controlled signalling molecules and pathways. All-trans-retinoic acid (ATRA) is one such important hormone-like compound that regulates a wide range of processes. The endogenous effects of ATRA have the potential to be translated into treatment for numerous clinical indications; however administration at efficacious concentrations is associated with severe side effects. Consequently, a large group of synthetic molecules - known as retinoids - that are structurally and/or functionally analogous to ATRA have been prepared and tested in vitro in the search for a panacea and for use as pharmacological tools to elucidate the retinoid molecular pathway. A small library of stable synthetic retinoids was prepared and their biological activity investigated using TERA2.cl.SP12 human embryonal carcinoma (EC) stem cells. Three compounds, CEB16, CEB17 and CEB18 were found to inhibit cellular proliferation and induce neural and non-neural differentiation. These effects were thoroughly characterised and quantified by monitoring phenotypic changes and the expression of established antigenic markers. Compared to the ability of ATRA and its geometric isomers 9-cis-retinoic acid and 13-cis-retinoic acid, the order of efficacy of induction of neural differentiation was found to be: 13-cis-retinoic acid>9-cis-retinoic acid=ATRA>CEB 18>CEB 17>>CEB 16. The molecular mechanism of natural and synthetic retinoid action during TERA2.cl.SP12 differentiation was investigated by performing a detailed temporal analysis of the gene expression of eleven transcripts involved in retinoid transport, activation and metabolism. To date, limited data have been published on the effects of synthetic retinoids on the retinoid pathway during differentiation of human cells and no human studies have examined the activity of synthetic retinoids on the HOX genes. All of the retinoic acid inducible genes examined in this study were found to be modulated in TERA2.cl.SP12 cells in response to both ATRA and two isomeric synthetic retinoids, CEB16 and CEB17, albeit relatively slowly compared to other cell lines. These compounds are therefore believed to activate the same pathway as the natural metabolite in these cells and that this is responsible for at least some of the observed effects. Genes were regulated in a concentration and retinoid dependent manner and this modulation was often multi-phasic demonstrating the complex behaviour of the retinoid system. Interestingly, ATRA was not the most effective inductive agent in all gene analyses. For example 10 μM CEB17 induced up-regulation of RAR-ß more strongly than 10 μm ATRA and 10 μM CEB16 induced the greatest increase in RAR-y transcripts. Further experimentation is required to confirm an apparent relationship between the timing of addition of inductive agent and the expression pattern of several genes. This behaviour highlights the importance of retinoid degradation in culture, which is often overlooked. The three synthetic compounds described extensively herein should be more stable, and thus may be suitable and convenient alternatives for molecular biologists to use in place of ATRA.

BACKGROUND: Vitamin D deficiency is becoming a worldwide public health concern as it increases the risk of many diseases and disorders. Little is known about the health outcomes of such deficiency in the South Asian population, in which it is more pronounced. The aim of this thesis was therefore to investigate the ethnic differences between South Asians and Caucasians with regard to the effect of vitamin D status on osteoporosis and cardiovascular disease risk. Furthermore, the study aimed to determine the extent of the problem and the genetic or social reasons behind it. METHODS: The research involved three sub-projects; the first of which undertook further analysis of the data from the original Vitamin D, Food Intake, Nutrition and Exposure to Sunlight in Southern England (D-FINES) study. Subjects from D- FINES were then re- invited to take part in the second part of this research, which measured volumetric bone density of the tibia and radius as using (pQCT)estimates of bone health. General health and biomarkers of cardiovascular disease and osteoporosis were assessed by standard clinical biochemical blood assays. The final stage utilised an interventional study design to test the efficiency of vitamin D production in both ethnic groups following controlled exposure to ultraviolet (UV) radiation.

Vitamin B 12 (cobalamin) is of key medical and commercial importance. It is one of the eight B vitamins that are only produced by prokaryotes, but required in the diet by humans. It primarily affects DNA synthesis and regulation, but is also involved in fany acid synthesis and energy production. Deficiencies in cobalamin can lead to anaemia, sensory or motor deficiencies, fatigue, depression, dementia and other psychiatric problems. Commercially over 10 tonnes of cobalamin are produced every year, therefore understanding the pathway to make the process cost efficient is of significant importance. The de novo synthesis of cobalamin can proceed via two different routes, the aerobic and anaerobic pathway. and requires approximately thirty enzyme mediated steps. The aerobic pathway inserts the cobalt ion into the tetrapyrrole framework late in the pathway and CobR, the cobalt reductase, reduces cobalt to form a tighter complex within the molecule. CobR from Brucella melitensis was previously shown by NMR to be thermostable for two weeks at temperatures exceeding 60°C. Biophysical characterisation and NMR analysis were used to determine whether enhanced thermostability in CobR was required for function. A comparison of Brucella melitensis CobR with a homologue from Sinorhizobium melilol; as well as creating seven single point mutants of Brucella melitensis CobR provided important standpoints to test whether CobR thermostability underpinned function. The production of the Brucella melitensis CobR mutants revealed salt bridges and beta barrel components as important for enzyme stability. However the largest contributing factor was availability and binding of the cofactor, FAD. It was shown by differential scanning calorimetry there was an increased thermal stability when increasing the concentration of excess FAD. If the cofactor is stripped from the enzyme completely the protein aggregates, and it was also shown by production of a double mutant, that lowering the binding constant of the cofactor reduced the thennostability that produced a coordinated loss of catalytic activity that was only 10% of the wild type enzyme. The study of the pathway intermediates of tetrapyrrole biosynthesis have often proved challenging for several reasons including the complexity of synthesis, compound instability and the relatively low levels of each intermediate produced. The second focus of this thesis utilises a new method, devised in the Warren laboratory at Kent, that uses pathway enzymes as 'traps' to enable isolation, stabilization and analysis of these intennediates for the first time. The 'trap' method allows pathway intermediates to be produced that are stable for NMR analysis. Precorrin 7 (PC-7), precorrin-8 (PC·8), and hydrogenobyrinic acid (HBA), were produced without isotopic enrichment and characterised by NMR experiments that were acquired on a Broker AV3 using a QCI·F cryoprobe at 14.1 Tesla (600 MHz lH). Analysis of the data confirmed the seventh methylation position of PC-7 was at position CIS on the tetrapyrrole framework as well as highlighting major and minor forms that exist due to tautomeric interconversion. Assignment of PC·8 and HBA was also successfully completed. An additional study was completed to confirm binding of PC·6B (precorrin.6B). PC·7, PC·8 and HBA to Brucella melitensis CobE, a protein believed to playa crucial scavenging role in the vitamin Bu aerobic pathway.

Η ασιτρετίνη, τα ψωραλένια και η μινοξιδίλη αποτελούν φάρμακα επιλογής για την αντιμετώπιση δερματικών ασθενειών (ψωρίαση, καρκίνος δέρματος, λεύκη, ανδρογενής αλωπεκία). Στο πλαίσιο της παρούσας διδακτορικής διατριβής αναπτύχθηκαν συγκλίνουσες συνθετικές μεθοδολογίες για την ολική σύνθεση αναλόγων της ασιτρετίνης με μεταβολές στο λιπόφιλο τμήμα της. Επιπλέον, τροποποιήθηκαν με χημικό τρόπο ψωραλένια, όπως το τριοξαλένιο, το μπερκαπτένιο και το ξανθοτοξένιο και συντέθηκε μια πληθώρα υβριδικών αναλόγων και συζευγμάτων τους με όξινα ρετινοειδή. Τέλος, αναπτύχθηκαν μεθοδολογίες για την ολική σύνθεση αναλόγων και συζευγμάτων της μινοξιδίλης με πολυαμίνες και άλλα βιοδραστικά μόρια. / Acitretin, Psoralens and Minoxidil are the drug of choice for the treatment of several dermatological disorders, such as psoriasis, vitiligo, cancer and adrogenic alopecia. In the context of the present thesis we developed efficient convergent synthetic methodologies for the total syntheses of acitretin analogs, incorporating changes in the lipophilic part. Moreover, psoralens, such as trioxsalen, bergapten and xanthotoxin, were chemically modified, in order to synthesize, hybrid analogs and conjugates with acidic retinoids. Finally, we developed efficient synthetic methodologies for the total synthesis of analogs and conjugates of Minoxidil with polyamines and other molecules with biological interest.