Leung Yiu Fai Ivan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 41-46). / Acknowledgements --- p.ii / Table of contents --- p.iii / List of tables --- p.vi / List of figures --- p.vii / List of Abbreviations --- p.xii / Abstract --- p.xiii / Chapter I. --- INTRODUCTION --- p.1 / Chapter A. --- Current knowledge on carotenoids --- p.1 / Chapter 1. --- Chemistry --- p.1 / Chapter 2. --- Occurrence --- p.3 / Chapter 3. --- Metabolism --- p.6 / Chapter 4. --- Biological function --- p.8 / Chapter B. --- Statement of the problem --- p.10 / Chapter II. --- MATERIAL AND METHODS --- p.11 / Chapter A. --- Materials --- p.11 / Chapter 1. --- Animals --- p.11 / Chapter 2. --- Human samples --- p.11 / Chapter 3. --- Chemicals --- p.12 / Chapter 4. --- HPLC Apparatus --- p.12 / Chapter B. --- Methods --- p.14 / Chapter 1. --- Animal specimens --- p.14 / Chapter (a) --- Carotenoid supplement --- p.14 / Chapter (b) --- Tissue preparation --- p.14 / Chapter 2. --- Human specimens --- p.15 / Chapter 3. --- Extraction of lipid component --- p.15 / Chapter (a) --- Retina --- p.15 / Chapter (b) --- Serum or subretinal fluid --- p.16 / Chapter (c) --- Liver --- p.16 / Chapter 4. --- Analytical methods --- p.17 / Chapter (a) --- Isocratic elution --- p.17 / Chapter (b) --- Gradient elution --- p.17 / Chapter III. --- RESULTS --- p.18 / Chapter A. --- Selection of chromatographic method for carotenoid analysis --- p.18 / Chapter 1. --- Effect of dioxane concentration on the retention time of carotenoids --- p.18 / Chapter (a) --- Gradient elution --- p.18 / Chapter (b) --- Isocratic elution --- p.18 / Chapter 2. --- Chromatograms of carotenoids and retinoids of a selected method for routine analysis --- p.19 / Chapter (a) --- Carotenoids --- p.20 / Chapter (b) --- Retinoids --- p.21 / Chapter B. --- Application of the selected method to study carotenoid in human and rats --- p.22 / Chapter 1. --- Study in human serum and subretinal fluid --- p.22 / Chapter (a) --- Serum --- p.22 / Chapter (b) --- Subretinal fluid --- p.22 / Chapter 2. --- Study in rats tissues --- p.24 / Chapter (a) --- Liver --- p.24 / Chapter (b) --- Serum --- p.26 / Chapter (c) --- Retina --- p.28 / Chapter C. --- Influence of dietary carotenoids on retinol concentration in rats --- p.31 / Chapter 1 --- Serum --- p.31 / Chapter 2 --- Retina --- p.31 / Chapter IV. --- DISCUSSION --- p.32 / Chapter A. --- Chromatographic analysis of carotenoids and retinoids --- p.32 / Chapter B. --- Carotenoid study in human and rats --- p.33 / Chapter 1. --- Carotenoids in human tissues --- p.33 / Chapter (a) --- Serum --- p.33 / Chapter (b) --- Subretinal fluid --- p.34 / Chapter 2. --- Dietary supplement of carotenoids to rats --- p.35 / Chapter (a) --- Choice of animal and types of carotenoids --- p.35 / Chapter (b) --- Carotenoids uptake into rats tissues --- p.36 / Chapter (c) --- Effect of dietary carotenoids on retinol concentration in rat tissues --- p.38 / Chapter V. --- CONCLUSION --- p.39 / Chapter VI. --- REFERENCES --- p.41 / Chapter VII. --- TABLES --- p.47 / Chapter VIII. --- FIGURES --- p.56

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Stagonospora nodorum is a necrotrophic fungal pathogen, and the causal agent of stagonospora nodorum blotch of wheat. Despite the economic importance of this disease, the molecular basis of the pathosystem is poorly understood. The aim of this study was to investigate the interaction between metabolism and infection in this pathosystem, with particular reference to the metabolism of mannitol. In common with many fungi, the main metabolite produced by S. nodorum is the acyclic hexitol mannitol. Among the previously suggested roles for this compound is a role in pathogenicity. The metabolism of mannitol has been hypothesised as occurring in a cycle involving the enzymes mannitol 2-dehydrogenase (Mdh1) and mannitol 1-phosphate 5 dehydrogenase (Mpd1). A strain was created harbouring disruption constructs for both of these genes. The double mutant was unable to synthesise or catabolise mannitol, and was unable to sporulate. Addition of exogenous mannitol completely restored in vitro sporulation, and partially restored in planta sporulation. This demonstrated an essential role for mannitol in asexual sporulation. This is the first demonstrated role for this compound. A 13C NMR study of the wild type strain, the mdh1 and mpd1 single mutants, and mpd1mdh1 double mutant was undertaken to investigate carbon utilisation and cycling. Disruption of Mpd1 significantly altered the metabolite profile with the mpd1 mutants producing trehalose and glycerol in place of mannitol. Labelling patterns in the double mutant showed that scrambling of label can be explained by the triosephosphate isomerase triangle and pentose phosphate pathway. This suggests the contribution of mannitol to label scrambling has been overstated in previous studies. The evidence did not support the metabolism of mannitol in S. nodorum as occurring in a cycle, but rather as two separate pathways. A GC-MS analysis of diseased and non-diseased tissue from infected leaves, compared to non-infected and mock-inoculated leaves, could not detect any metabolites associated with a systemic host reaction to pathogen attack.

The myotome is a segmented skeletal muscle developing along the axis and is the first muscle to differentiate in every vertebrate. While fish and tadpole myotomes persist during development, myotomes of amniote embryos disappear during embryogenesis and are replaced by the long and complex epaxial muscles. Whereas the initial development of the myotome has been intensely investigated, very little is known about the fate of the myotome and the morphogenesis of the epaxial muscles in mammals. This study firstly examined epaxial muscle morphogenesis. Myotomal fibres and cell death in muscle fibres were followed by immunohistochemistry during rat embryogenesis. Results showed that the morphogenesis of epaxial muscles occurs through the movement of the differentiated myotomal muscle fibres rather than by de novo fusion of myoblasts after apoptosis of the initial myotomal myofibres. The myotomal muscle masses undergo progressive transformation and segregation that result in the formation of the distinct groups of epaxial muscles. Next, the mechanisms of epaxial muscle morphogenesis were investigated in rat embryos, by following muscle progenitor cells expressing the transcription factors Pax7 and Pax3 during epaxial muscle morphogenesis using immunohistochemistry. This demonstrated that the myoblasts responsible for epaxial muscle growth derive from a population of progenitors mingled within the epaxial muscle masses as they segregate from the myotome. No migration of precursors is involved. Transgenic ScxGFP mouse embryos, carrying a marker green fluorescent protein under the control of scleraxis (a transcription factor specific to tendons and muscle connective tissues), permitted the tracing of the connective tissues during myotome transformation. Results strongly suggest that connective tissues associated with epaxial myofibres could be actively involved in creating the displacement of the myotomal myofibres during the transformation process. Finally, to test whether the mammalian myotome has a function as a neurally-controlled muscle during development, innervation of the myotome was studied using immunohistochemistry in comparison with the innervation of the forelimb muscles of rat embryos. The results were striking, showing that whereas the migratory limb muscles are contacted by nerves from the beginning of their differentiation, the myotome differentiates and then develops over more than two days without nerves. As revealed by the appearance of acetylcholine receptors clusters, functional innervation only occurs in the epaxial muscles when the myotome has started its transformation. The true mammalian myotome is therefore never innervated and seems to have lost its role as a neurally-controlled muscle in contrast to the myotomal muscle of fish and amphibian tadpoles. Overall the results indicate that the development of the epaxial muscles is strikingly different from that of the muscles originating from migratory myoblasts. Contrary to the migratory muscles, the myotome develops in the absence of nerves and its differentiated muscle fibres are transformed in position and orientation to create the epaxial muscles. The development of mammalian epaxial muscles upon a template of embryonic muscle resembles the development of some adult muscles in Drosophila, developing from the larval muscles. This suggests that the mammalian myotome could be of a larval nature but with the loss of innervation.

Amendements: leaves 252-254. Copies of author's previously published articles inserted. Bibliography: leaves 226-251. The CYP2C19 and CYP2D6 genetic polymorphisms control the oxidative metabolism of many different drug classes. Populations are separated into groups of extensive metabolisers (EM), poor metabolisers (PM), and in the case of CYP2D6, ultra-rapid metabolisers (UM). In vitro studies using human liver microsomes were conducted to examine the kinetics of the oxidative metabolism of flunitrazepam, and which CYP450 enzymes mediate the oxidative metabolism of flunitrazepam, (S)-mephenytoin and proguanil.

N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor.

The production of reactive oxygen species by mitochondria is implicated in mitochondrial dysfunction associated with a range of diseases and ageing. In addition, reactive oxygen species produced by mitochondria are involved in redox signalling pathways that modulate a number of cell processes. Mitochondria targeted antioxidants comprised of an antioxidant moiety linked to a lipophilic triphenylphosphonium cation have recently been used to decrease oxidative damage to mitochondria and to investigate the involvement of mitochondrial reactive oxygen species in redox signalling. These lipophilic cations are selectively accumulated by mitochondria within cells due to the mitochondria membrane potential. This thesis presents the synthesis and characterization of mitochondria targeted membrane uncoupler, cyclic nitroxide and alkyl thionitrite derivatives, all of which had the potential to influence reactive oxygen species. The biological analysis of these compounds is also presented. A triphenylphosphonium derivative of the membrane uncoupler 2,4-dinitrophenol (DNP) was anticipated to act as a self regulating protonophore. The DNP moiety would influence the scale of the membrane potential while the triphenylphosphonium cation would respond to the membrane potential. These two factors would combine so that as the membrane potential was dissipated by the uncoupler, the phosphonium cation would be released from the mitochondria and the effect of the uncoupler would thereby be nullified until the membrane potential had increased again. The compound was prepared by nitration of 3-(4-hydroxyphenyl)propyl triphenylphosphonium bromide. An untargeted derivative was also prepared by nitration of 3-(4-hydroxyphenyl)-1-propanol. Unfortunately, while this compound had appropriate acidity and lipophilicity to act as a membrane uncoupler, and did enter mitochondria in response to the membrane potential, it did not act as an uncoupler. A chemically stable targeted cyclic nitroxide based on Tempol was prepared following literature procedure, although other synthetic routes were also trialled. This compound was shown to concentrate in mitochondria in response to the membrane potential, was reduced by ubiquinol of the coenzyme Q pool, acted as a superoxide dismutase mimetic, and protected membranes against lipid peroxidation. A mitochondria targeted thionitrite or nitric oxide (NO) donor was anticipated to exhibit an effect on respiration at low oxygen concentrations as the released NO interacted with aspects of the respiratory chain. The alkyl thionitrites were synthesised from appropriate thiol precursors, several of which were prepared. Two targeted alkyl thionitrites were prepared with primary or tertiary carbon arrays next to the thionitrite functionality. Another targeted thionitrite, based on S-nitroso-N-acetylpenicillamine (SNAP), was also prepared. These compounds were difficult to characterise because of issues surrounding their stability. However, modified high resolution positive ion electrospray mass spectrometry in combination with HPLC and NMR was used to identify the compounds and to gauge the purity of the samples. Initial biological investigations verified that the primary alkylthionitrite derivative accumulated in mitochondria, released NO, and had an effect on respiration at low oxygen concentrations.