Organic chemistry is a valuable tool for studying enzyme mechanisms. Upon incubation with a specific enzyme, synthetic substrate analogues labeled with heavy atoms or carrying extra functional groups can provide mechanistic insights. In the present work, new compounds were synthesised in order to study the mechanism and substrate selectivity of two enzymes: human γ-butyrobetaine hydroxylase and bacterial carboxymethylproline synthase. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses the stereospecific hydroxylation of γ-butyrobetaine, the final step of L-carnitine (L-Car) biosynthesis in mammals. Substrate analogues were synthesised to probe BBOX specificity in vitro. Some of those unnatural substrates were oxidised by BBOX and the products identified using a range of analytical techniques. 3-(2,2,2-Trimethylhydrazinium)propionate (THP) is a clinically used BBOX inhibitor. Under standard assay conditions, THP was oxidised by BBOX. NMR studies have identified the products of this reaction to be malonic acid semialdehyde, formaldehyde, dimethylamine and 3-amino-4-(methylamino)butanoic acid. The formation of 3-amino-4-(methylamino)butanoic acid suggests that BBOX can catalyse a Stevens type rearrangement involving N-N bond cleavage and C-C bond formation. The proposed structures and mechanisms were confirmed by mass spectrometric and NMR analyses using [¹³C]-labeled THP as well as synthetic standards of both enantiomers of 3-amino-4-(methylamino)butanoic acid. Although the structure of the rearrangement product was confirmed, the stereochemistry remains unknown. Altogether, these studies revealed the unprecedented nature of a BBOX-catalysed C–C bond formation reaction upon THP oxidation and may inspire the design of improved inhibitors for BBOX and other 2OG oxygenases. Pectobacterium carotovorum CarB and Streptomyces cattleya ThnE are two carboxymethylproline synthases (CMPS) that catalyse an early step in carbapenem antibiotics biosynthesis. CMPS produces (2S,5S)-carboxymethylproline (t-CMP) from malonyl-CoA and L-glutamate semi-aldehyde. L-Glutamate semi-aldehyde exists in equilibrium with L-5-hydroxyproline and L-pyrroline-5-carboxylate in solution (collectively abbreviated L-GHP). Because of the high stereoselectivity of t-CMP formation and the growing interest in novel carbapenem antibiotics, CMPS is potentially an interesting biocatalyst. A series of L-GHP analogues were synthesised and tested as CMPS substrates in an attempt to produce unnatural t-CMP derivatives enzymatically. Methyl-substituted L-GHP analogues were accepted by CMPS and the t-CMP products could be further carried through to the corresponding bicyclic carbapenams using CarA, a β-lactam synthetase. These results demonstrate the versatility of the early carbapenem biosynthetic pathway and the possibility of introducing structural diversity using synthetic substrate analogues. A crystal structure of S. cattleya ThnE was obtained in complex with L-proline and coenzyme A, giving the first insight into substrate binding. This structural information will potentially allow further rational mutagenesis studies aiming to broaden the range of unnatural L-GHP analogues accepted by CMPS.

In the past decade, Staphylococcus aureus has developed two distinct vancomycin resistance mechanisms. First, the bacterium is capable of generating a thickened, poorly crosslinked cell wall that creates false targets. These targets cause vancomycin to bind at the periphery of the thickened peptidoglycan, allowing normal cell wall formation to continue at the cell membrane. Second, S. aureus has acquired genes from Enterococcus that encode an alternative stem peptide. The genes, known as van genes, alter the target of vancomycin, rendering vancomycin treatment ineffectual.In this work, we attempted to further characterize both mechanisms of vancomycin resistance. First, a potential link between up-regulated purine biosynthesis and increased vancomycin resistance due to a thickened cell wall was examined. Despite exploration of multiple mechanisms to increase purine levels within the cell, increased purine synthesis did not provide S. aureus with any advantage in the presence of vancomycin. However, during the investigation, purine biosynthesis in S. aureus was further characterized by confirming purr as the repressor of the purine pathway and demonstrating its sensitivity to mutation.Next, the relationship between homotypic oxacillin resistance and increased vancomycin resistance in the absence of the van genes was investigated. Vancomycin passage of two heterotypic methicillin resistant S. aureus (MRSA) caused these strains to convert to homotypic oxacillin resistance in the absence of oxacillin exposure. Additionally, conversion of heterotypic oxacillin resistant strains to homotypy by oxacillin passage increased strain survival in vancomycin. The SOS response was examined as the possible link between conversion to homotypic oxacillin resistance and increasing vancomycin resistance due to a thickened cell wall. The current study, however, detected no induction of the SOS response during vancomycin exposure.Lastly, the relationship between oxacillin resistance and vancomycin resistance due to the acquisition of the van genes was examined. In vitro and in vivo methods were utilized to determine the effectiveness of a combination of β-lactam antibiotics and vancomycin to treat vancomycin resistant S. aureus (VRSA) infections. Combination therapy provided a significant advantage over untreated control or either antibiotic alone in the rabbit model of endocarditis.

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

by Wong Kwan Po, Gary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 132-153). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.vi / Abbrevation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xiii / Table of Contents --- p.xiv / Chapter Chapte r One --- General Introduction --- p.1 / Chapter 1.1 --- Structures of PRL --- p.1 / Chapter 1.2 --- PRL receptor and its mechanism of action --- p.7 / Chapter 1.3 --- Biosynthesis of PRL --- p.11 / Chapter 1.4 --- Biological functions of PRL --- p.13 / Chapter 1.5 --- Organization and regulation of PRL gene --- p.16 / Chapter 1.6 --- Aims of this study --- p.25 / Chapter Chapter Two --- PCR Cloning of gfPRL Gene --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Buffers and Reagents --- p.27 / Chapter 2.2.2 --- Methods --- p.30 / Chapter 2.2.2.1 --- PCR of the 5'-flanking region of gfPRL gene --- p.30 / Chapter 2.2.2.2 --- Genomic PCR of gfPRL gene --- p.31 / Chapter 2.2.2.3 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.31 / Chapter 2.2.2.4 --- Agarose gel electrophoresis of DNA --- p.31 / Chapter 2.2.2.5 --- DNA radioactive labeling by random priming --- p.32 / Chapter 2.2.2.6 --- Vacuum transfer of DNA fragments to a nylon membrane --- p.32 / Chapter 2.2.2.7 --- Southern blot analysis --- p.33 / Chapter 2.2.2.8 --- Molecular Imager Analysis --- p.33 / Chapter 2.2.2.8 --- Phosphorylation of PCR amplified DNA --- p.34 / Chapter 2.2.2.9 --- Ligation of DNA fragment to linearized vector --- p.34 / Chapter 2.2.2.10 --- Preparation of Escherichia coli competent cells --- p.34 / Chapter 2.2.2.11 --- Bacterial transformation by heat stock --- p.35 / Chapter 2.2.2.12 --- Automated PCR sequencing with Sequencing Ready Reaction Kit --- p.35 / Chapter 2.2.2.13 --- Primer extension using reverse transcription --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Cloning of the 5'-flanking region of gfPRL gene --- p.38 / Chapter 2.3.2 --- PCR cloning of gfPRL gene --- p.43 / Chapter 2.3.3 --- Identification of the transcription initiation site --- p.47 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Sequence analysis of the gfPRL gene --- p.51 / Chapter 2.4.2 --- Analysis of the exon-intron boundaries --- p.53 / Chapter 2.4.3 --- Analysis of the 5'flanking region of gfPRL gene --- p.53 / Chapter 2.4.4 --- Identification of the transcription initiation site --- p.54 / Chapter 2.5 --- Conclusion --- p.54 / Chapter Chapter Three --- Promoter Analysis of the gfPRL Gene --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Preparation of Luciferase reporter constructs --- p.56 / Chapter 3.2.2 --- Preparation of frozen stock of culture cells --- p.56 / Chapter 3.2.3. --- Cell culture --- p.56 / Chapter 3.2.4 --- Transfection of mammalian cells for transient gene expression study --- p.57 / Chapter 3.2.5 --- Luciferase assay --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Tissue-specific transcription of gfPRL promoter --- p.58 / Chapter 3.3.2 --- Identification of regulatory regions of gfPRL gene promoter --- p.61 / Chapter 3.3.3 --- Inhibitory effect of DA on gfPRL promoter transcription activity --- p.63 / Chapter 3.3.4 --- GfPRL promoter sequences that specifically confer negative regulation by DA --- p.65 / Chapter 3.3.5 --- The action of TRH on gfPRL promoter --- p.67 / Chapter 3.3.6 --- Investigation of gfPRL promoter sequence responsiveness towards TRH --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Tissue-specific transcription of gfPRL promoter --- p.71 / Chapter 3.4.2 --- Identification of regulatory regions of goldfish prolactin gene promoter --- p.72 / Chapter 3.4.3 --- Dopamine inhibits gfPRL promoter activity --- p.73 / Chapter 3.4.4 --- TRH action on gfPRL promoter --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter Four --- Seasonal Study on gfPRL and gfGH expression --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and Methods --- p.81 / Chapter 4.2.1 --- Blood samples and radioimmunoassay --- p.81 / Chapter 4.2.2 --- Preparation of ribonuclease free reagents and apparatus --- p.81 / Chapter 4.2.3 --- Isolation of total RNA --- p.81 / Chapter 4.2.4 --- Formaldehyde agarose gel electrophoresis of RNA --- p.82 / Chapter 4.2.5 --- First strand cDNA synthesis --- p.82 / Chapter 4.2.6 --- RT-PCR --- p.83 / Chapter 4.2.7 --- Analysis of RT-PCR --- p.86 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Tissue-specific expression of gfPRL transcript --- p.88 / Chapter 4.3.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.90 / Chapter 4.3.3 --- Serum gfGH levels throughout the year of 2000 --- p.91 / Chapter 4.3.4 --- Serum gfPRL levels throughout the year of 2000 --- p.92 / Chapter 4.3.5 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.93 / Chapter 4.3.6 --- The variation of gfGHR mRNA in the liver throughout the reproductive cycle --- p.94 / Chapter 4.3.7 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.95 / Chapter 4.3.8 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- Tissue-specific expression of gfPRL transcript --- p.98 / Chapter 4.4.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.98 / Chapter 4.4.3 --- Serum gfGH and gfPRL level throughout the reproductive cycle --- p.99 / Chapter 4.4.4 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.100 / Chapter 4.4.5 --- The variation of gfGHR mRNA in the liver throughout ´Øthe reproductive cycle --- p.101 / Chapter 4.4.6 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.102 / Chapter 4.4.7 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.102 / Chapter 4.5 --- Conclusion --- p.105 / Chapter Chapter Five --- Recombinant gfPRL Production --- p.106 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.108 / Chapter 5.2.1 --- Buffers and Reagents --- p.108 / Chapter 5.2.2 --- Methods --- p.112 / Chapter 5.2.2.1 --- Recombinant protein expression --- p.112 / Chapter 5.2.2.2. --- Purification of the recombinant protein by XpressTM System Protein Purification (Invitrogen) --- p.112 / Chapter 5.2.2.3 --- SDS-PAGE preparation --- p.112 / Chapter 5.2.2.4 --- SDS-PAGE analysis of proteins --- p.113 / Chapter 5.2.2.5 --- Western blot analysis --- p.114 / Chapter 5.2.2.6 --- Protein refolding --- p.114 / Chapter 5.2.2.7 --- Alkaline Extraction --- p.115 / Chapter 5.2.2.8 --- Size Exclusion Chromatography --- p.115 / Chapter 5.2.2.9 --- ELISA analysis of the fractions --- p.115 / Chapter 5.2.2.10 --- Anion Exchange Chromatography --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Prokaryotic expression of recombinant gfPRL --- p.117 / Chapter 5.3.2 --- "Purification of reombinant gfPRL: SDS-PAGE, western blot and BCA analysis of purified recombinant gfPRL" --- p.119 / Chapter 5.3.3 --- Purification of native gfPRL and gfGH: Native hormone purification by size exclusion chromatography --- p.119 / Chapter 5.3.4 --- Native gfPRL purification by anion exchange chromatography --- p.122 / Chapter 5.3.5 --- Study the biological activity of refolded recombinant gfPRL --- p.126 / Chapter 5.4 --- Discussion --- p.127 / Chapter 5.4.1 --- Prokaryotic expression of recombinant gfPRL --- p.127 / Chapter 5.4.2 --- Purification of recombinant gfPRL --- p.128 / Chapter 5.4.3 --- Refolding of recombinant gfPRL --- p.129 / Chapter 5.4.4 --- Purification of native gfPRL --- p.130 / Chapter 5.4.5 --- Study the biological activity of recombinant gfPRL --- p.130 / Chapter 5.5 --- Conclusion --- p.131 / References --- p.132

Induced and activated as part of the innate immune response, the first line of defence against bacterial or viral infections, Inhibitor of Kappa-B Kinase ε (IKKε) triggers NF-κB and IFNβ signalling. Whilst not expressed at basal levels in healthy cells and tissue, the kinase is overexpressed in roughly 30% of human breast cancer cases, driving oncogenesis through aberrant activation of NF-κB. The impracticality of therapeutic targeting of NF-κB for cancer treatment has led to a requirement for greater understanding of IKKε's oncogenic potential to treat tumours driven by the kinase. Considering that IKKε alters cellular metabolism in dendritic cells, promoting aerobic glycolysis akin to the metabolic phenotype observed in cancer, it was hypothesised that the kinase would play a similar role in breast cancer. Using a Flp-In 293 model of IKKε induction and suppressing IKKε expression in a panel of breast cancer cell lines using siRNA, IKKε-dependent changes in cellular metabolism were characterised using labelled metabolite analysis. IKKε was found to induce serine biosynthesis, an important pathway in breast cancer development that supports glutamine-fuelling of the TCA cycle and contributes to one carbon metabolism to maintain redox balance. Promotion of serine biosynthesis occurred via a dual mechanism. Firstly, PSAT1, the second enzyme of the pathway, was found to be phosphorylated in an IKKε-dependent manner, promoting protein stabilisation. Secondly, an IKKε-dependent transcriptional upregulation of all three serine biosynthesis enzymes, PHGDH, PSAT1 and PSPH, was observed, induced by the inhibition of mitochondrial activity and the subsequent induction of ATF4-mediated mitochondria-to-nucleus retrograde signalling. These data demonstrate a previously uncharacterised mechanism of metabolic regulation by IKKε and highlight new potential therapeutic targets for the treatment of IKKε-driven breast cancer in the form of the enzymes of the serine biosynthesis pathway.

Integral membrane enzymes contribute a unique repertoire to the cell, as they are capable of synthesizing products from substrates of different chemical character at the membrane-water interface. Membrane-embedded enzymes are often responsible for the synthesis of important components of the cellular membrane and contribute to the structural integrity of the cell, maintenance of cellular homeostasis and signal transduction. One of the main focuses of Dr. Filippo Mancia’s laboratory is understanding how enzymes complete these functions by investigating, at an atomic level, the determinants of substrate binding and catalysis within the membrane and at the membrane surface. Here I will present my investigation of two such integral membrane enzyme systems, which are responsible for the synthesis and processing of membrane-embedded molecules in bacteria. Phosphatidylinositol-phosphate Synthase (PIPS) Phosphaitylinositol (PI) is an essential lipid component in mycobacteria, demonstrated by loss of viability when PI is reduced to 50% of wild-type levels. Phosphatidylinositol (PI) is required for the biosynthesis of key components of the cell wall, such as the glycolipids phosphatidylinositol-mannosides, lipomannan and lipoarabinomannan. For these molecules, PI serves as a common lipid anchor to the membrane. In Mycobacterium tuberculosis, the disease causing pathogen of tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. Therefore, the enzyme responsible for PI synthesis in this organism is a potential target for the development of anti-tuberculosis drugs. The defining step in phosphatidylinositol biosynthesis is catalyzed by a member of the CDP-alcohol phosphotransferase enzyme family. The enzyme uses CDP-diacylglycerol as the donor substrate, and either inositol in eukaryotes or inositol-phosphate in prokaryotes as the acceptor alcohol of the synthesis reaction. In prokaryotes, phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP-alcohol phosphotransferase family) catalyzes this reaction to yield phosphatidylinositol-phosphate, which is then dephosphorylated to PI by an uncharacterized enzyme. Structures of PIPS from Renibacterium salmoninarum (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the location of the acceptor site as well as molecular determinants of substrate specificity and catalysis of the enzyme. However, RsPIPS has low activity relative to PIPS from Mycobacterium tuberculosis (MtPIPS) and the two share only 40% protein sequence identity. Therefore, these initial structures have limited potential for meaningful homology modeling and drug design. Presented here are the structures of PIPS from Mycobacterium kansasii (MkPIPS), which is 86% identical to MtPIPS, in an apo state to 3.1 Å resolution, in a nucleotide-bound state to 3.5 Å resolution, and in a novel ligand-bound state to 2.6 Å resolution. This work provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens. RodA-PBP2 Complex The cell wall of most gram-negative and gram-positive bacteria (excluding atypical bacteria such as members of Mycoplasmataceae) is composed of peptidoglycan, a mesh of repeating carbohydrates (N-acetylmuramic acid, MurNAc, and N-acetylglucosamine, GlcNAc) cross-linked by small peptides. Peptidoglycan is essential for growth, division and viability of the organism. Any disruption of the biosynthesis of peptidoglycan, whether by genetic mutation, inhibition with antibiotics or degradation by lysozyme, results in bacterial cell lysis. Peptidoglycan helps maintain cell shape and serves as an anchor for accessory proteins and other cell wall components. As essential components of the cell wall, enzymes contributing to the peptidoglycan biosynthetic pathway can be exploited as antibiotic targets. After a hydrophilic peptidoglycan precursor (UDP-MurNAc-pentapeptide) is synthesized in the cytosol, it is attached to the lipid carrier undecaprenyl phosphate (UndP). The lipid-linked precursor (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide or Lipid I) is modified further to undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (Lipid II) by addition of a GlcNAc moiety. Lipid II is then flipped across the membrane to the periplasm where its sugars are polymerized to form the glycan strands of the peptidoglycan mesh. SEDS proteins, essential for maintaining bacterial processes that determine shape, elongation, cell division and sporulation, are integral membrane enzyme that have been implicated in this process as either Lipid II flippases, glycosyltransferases responsible for sugar polymerization, or both. SEDS proteins are also known to form a functional complex with type b penicillin-binding proteins (PBPs), which are known as transpeptidase enzymes, responsible for the crosslinking of peptides in the formation of the peptidoglycan mesh. Though structures of both RodA (a SEDS protein involved in bacterial growth and elongation) and type b PBPs are available, the interaction between the two proteins and their joint enzymatic activity is poorly characterized. Here, I present the preliminary structural characterization of a RodA-PBP2 protein complex by single-particle cryo-electron microscopy (cryo-EM). We hope this ongoing work will contribute to the understanding of these enzymes and to the development of antibiotics to combat antibiotic resistance.

Les flavan-3-ols appartiennent à la famille des polyphénols retrouvés dans diverses plantes et majoritairement dans le raisin. Ils jouent un rôle primordial dans les mécanismes de défense des plantes, influent sur les propriétés organoleptiques du vin et sont potentiellement bénéfiques au niveau de la santé humaine. La voie de biosynthèse des flavanols monomères est décrite dans la littérature. Cependant, les mécanismes de formation des proanthocyanidines (polymères) sont inconnus à ce jour. Des études ont montré que les flavanols glycosylés sont des intermédiaires potentiels dans la biosynthèse des PA et permettent le transport des unités de flavanols du cytoplasme vers la vacuole de la cellule, où a lieu la polymérisation. Un criblage global des flavanols glycosylés présents dans des raisins à trois stades de développement et dans des vins de différents cépages a été réalisé en analysant séparément les pellicules et les pépins de raisin par une méthode d’UPLC-DAD-ESI-MS. La teneur de ces composés dépend de paramètres de type variété du raisin, tissu biologique étudié et stade de maturité. La présence de dimères de flavanol glycosylés a été montrée pour la première fois dans le raisin. Grâce à l’hémisynthèse de la (+)-catéchine 4’-O--glucoside et 7-O--glucoside, certains monomères ont été caractérisés comme appartenant à la classe des -glucosides. Une étude quantitative a montré l’évolution des flavanols glycosylés au cours du développement de la baie de raisin (pépins et pellicules séparés) provenant de trois cépages différents. Les monomères et dimères d’ (épi) catéchine diglycosylés ont été découverts pour la première fois et uniquement dans les pépins. Une diminution en monomères d’ (épi) catéchine monoglycosylés a été observée dans la pellicule au cours de la maturation du raisin. Les dimères d’ (épi) catéchine monoglycosylés s’accumulent un peu après le stade de véraison et diminuent ensuite à maturité. Les monomères et dimères d’ (épi) catéchine diglycosylés s’accumulent dans les pépins au cours de la maturation. Finalement, l’évolution des teneurs en flavanols mono et diglycosylés au cours de microvinifications a été étudiée. On observe des profils d’extraction similaires pour les deux variétés utilisées (Grenache et Syrah). La quantité totale des différentes familles de composés augmente au cours de la vinification, et ensuite diminue en fin de FA. Certains composés sont dégradés préférentiellement, suggérant la présence d’activités glycosidases spécifiques du raisin ou de la levure. / Flavan-3-ols belong to a group of polyphenols present in a wide variety of plants, and particularly in grapeberries. They play an important role in defense mechanisms in plants, have a significant impact on wine organoleptic properties; and their beneficial effects on human health may help to protect against chronic diseases such as atherosclerosis. The sequence of common flavanol monomer biosynthesis is widely described in the literature, but the formation mechanisms of proanthocyanidins (PA) remain unknown. Studies show that flavanol glycosides are potential intermediates in PA polymerization and have transporter roles of monomeric units from cytoplasm to vacuole in cell, where polymerization takes place. Global screening of grapeberry flavanol glycosides were carried out at three stages of grape development and in wines of different varieties; skin and seeds were measured separately using an UPLC-DAD-ESI-MS method. The composition of the target isomers depends on different parameters such as tissue type or stage of development. The presence epi catechin monoglycoside is reported here for the first time in grapes. Using (+)-catechin 4’-O--glucoside and 7-O--glucoside hemisynthesis, several monomers were shown to -glucosides. Quantitative analysis demonstrates the evolution of flavanol glycosides in both skin and seeds during the development of three grapevine varieties. For the first time monomeric and dimeric (epi) catechin diglycosides were revealed and shown accumulate only in grape seeds during ripening. A reduction in the concentration of monomeric (epi) catechin monoglycoside was observed during grape skin development. Dimeric (epi) catechin monoglycosides accumulate after veraison and then decrease at the end of grape ripening. The extraction profiles of flavanol glycosides during red grape fermentation showed similar evolution patterns for both varieties used. The total concentration of different compound families increases during winemaking, and then decreases at the end of fermentation. Degradation of specific compounds was observed at the end of fermentation which may be explained by the activity of glycosidases from grape extracts released during fermentation and pressing.

Lolines are specialized metabolites produced by endophytic fungi, such as Neotyphodium and Epichloë species, that are in symbiotic relationships with cool-season grasses. Lolines are vital for the survival of the grasses because their insecticidal and antifeedant properties protect the plant from insect herbivory. Although lolines have various bioactivities, they do not have any concomitant antimammalian activities. Lolines have complex structures that are unique among naturally occurring pyrrolizidine alkaloids. Lolines have four contiguous stereocenters, and they contain an ether bridge connecting C(2) and C(7) of the pyrrolizidine ring. An ether bridge connecting bridgehead C atoms is unusual in natural products and leads to interesting questions about the biosynthesis of lolines in fungal endophytes. Dr. Pan, who was a graduate student in Dr. Schardl Lab at University of Kentucky, isolated a novel metabolite, 1-exo-acetamidopyrrolizidine (AcAP). She observed that AcAP was accumulating in naturally occurring and artificial lolO mutants. I synthesized an authentic sample of (±)-AcAP and compared it spectroscopically with AcAP isolated from a lolO mutant to determine the structure and stereochemistry of the natural product. I was also able to grow crystals of synthetic (±)-AcAP, X-ray analysis of which further supported our structure assignment. There were two possible explanations for the fact that a missing or nonfunctional LolO led to the accumulation of AcAP: that AcAP was the actual substrate of LolO, or that it was a shunt product derived from the real substrate of LolO, 1-exo-aminopyrrolizidine (AP), and that was produced only when LolO was not available to oxidize AP. To distinguish between the two hypotheses, I synthesized 2´,2´,2´,3-[2H4]-AcAP. Dr. Pan used this material to confirm that AcAP was an intermediate in loline alkaloid biosynthesis, not a shunt product. To determine the product of LolO acting on AcAP, Dr. Pan expressed LolO in yeast (Saccharomyces cerevisiae). When Dr. Pan fed AcAP (synthesized by me) to the modified organism, it produced NANL, suggesting that LolO catalyzed two C–H activations of AcAP and the formation of both C–O bonds of the ether bridge in NANL, a highly unusual transformation. Dr. Chang then cloned, expressed, and purified LolO and incubated it with (±)-AcAP, 2-oxoglutarate, and O2. He observed the production of NANL, further confirming the function of LolO. Dr. Chang also observed an intermediate, which we tentatively identified as 2-hydroxy-AcAP. In order to determine whether the initial hydroxylation of AcAP catalyzed by LolO occurred at C(2) or C(7), I prepared (±)-7,7-[2H2]- and (±)-2,2,8-[2H3]-AcAP. When Dr. Pan measured the rate of LolO-catalyzed hydroxylation of these substrates under conditions under which only one C–H activation would occur, she observed a very large kinetic isotope effect when C(2) was deuterated, but not when C(7) was deuterated, establishing that the initial hydroxylation of AcAP occurred at the C(2) position. In order to determine the stereochemical course of C–H bond oxidation by LolO at C(2) and C(7) of AcAP, I synthesized trans- and cis-3-[2H]-Pro and (2S,3R)-3-[2H]- and (2S,3S)-2,3-[2H2]-Asp. Feeding experiments with these substrates carried out by both Dr. Pan (Pro) and me (Asp) showed that at both the C(2) and C(7) positions of AcAP, LolO abstracted the endo H atoms during ether bridge formation. In summary, feeding experiments with deuterated (±)-AcAP derivatives and its amino acid precursors have shown that AcAP is an intermediate in loline biosynthesis. We have shown that LolO catalyzes the four-electron oxidation of AcAP at the endo C(2) position first and then the endo C(7) position to give NANL.

Chan, Ming Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-106). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Aromatase --- p.1 / Chapter 1.2 --- Tissue Specific Promoter for Aromatase Expression --- p.4 / Chapter 1.3 --- Signaling Pathway --- p.7 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.9 / Chapter 2.1 --- Chemicals And Materials --- p.9 / Chapter 2.2 --- Mammalian Cell Culture --- p.9 / Chapter 2.2.1 --- Maintenance of Cells --- p.10 / Chapter 2.2.2 --- Preparation of Cells Stock --- p.10 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.11 / Chapter 2.3 --- Tritiated Water Release Assay --- p.11 / Chapter 2.3.1 --- Aromatase Activity in Intact Cell --- p.11 / Chapter 2.3.2 --- Aromatase Assay on Recombinant Supersomes --- p.12 / Chapter 2.4 --- RNA Isolation and cDNA Synthesis --- p.13 / Chapter 2.5 --- Semi-Quantitative PCR Reaction --- p.13 / Chapter 2.6 --- Quantitative Real Time PCR Using Taqman Probe --- p.15 / Chapter 2.7 --- Western Blotting --- p.17 / Chapter 2.8 --- Measurement of Promoter Activity --- p.18 / Chapter 2.8.1 --- Plasmid Preparation --- p.18 / Chapter 2.8.2 --- Transient Transfection and Dual Luciferase Assay --- p.18 / Chapter 2.9 --- Statistical Methods --- p.19 / Chapter CHAPTER 3 --- Genistein up-regulate aromatase in Estrogen receptor alpha-transfected HepG2 cells --- p.21 / Chapter 3.1 --- Introduction --- p.21 / Chapter 3.1.1 --- Cardiovascular Disease (CVD) --- p.21 / Chapter 3.1.2 --- Phytoestrogen --- p.21 / Chapter 3.1.3 --- Estrogen Receptor --- p.24 / Chapter 3.1.4 --- Protective Mechanism Against CVD Protection --- p.25 / Chapter 3.1.5 --- Effects of genistein on LDL Receptor and Apolipoprotein A-I --- p.26 / Chapter 3.1.6 --- Effects of estradiol on LDL Receptor and Apolipoprotein A-I　 --- p.26 / Chapter 3.1.7 --- Aim of study and hypothesis --- p.27 / Chapter 3.2 --- Result --- p.29 / Chapter 3.2.1 --- ERa increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.2 --- Genistein increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.3 --- Differential Effect of MAP kinase Inhibitors --- p.35 / Chapter 3.2.4 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in ERa-transfected HepG2 cells" --- p.35 / Chapter 3.2.5 --- Genistein Increased Aromatase Protein Expression in ERa-transfected HepG2 cells --- p.38 / Chapter 3.2.6 --- Genistein Induced Aromatase mRNA Expression Attributed to Induction of Exon ̐ơ.1 Expression --- p.40 / Chapter 3.2.7 --- Genistein Induced Promoter 1.1 Transcriptional Activity in ERa- transfected HepG2 cells --- p.44 / Chapter 3.2.8 --- Genistein Increased ERE and AP-1 Reporter Activity Through Interaction with ERa --- p.47 / Chapter 3.3 --- Discussion --- p.51 / Chapter CHAPTER 4 --- "Effect of 2,3,7,8-tetrachlorodibenzo- para-TCDD (TCDD) on aromatase in MCF-7 cells" --- p.54 / Chapter 4.1 --- Introduction --- p.54 / Chapter 4.1.1 --- Breast Cancer --- p.54 / Chapter 4.1.2 --- TCDD --- p.54 / Chapter 4.1.3 --- CYP Enzymes --- p.55 / Chapter 4.1.4 --- TCDD and Breast Cancer --- p.56 / Chapter 4.1.5 --- Aim of Study --- p.56 / Chapter 4.2 --- Result --- p.57 / Chapter 4.2.1 --- Effect of TCDD on Aromatase Activity in Different Cell Lines --- p.57 / Chapter 4.2.2 --- TCDD Increased Aromatase Activity in MCF-7 Cells --- p.62 / Chapter 4.2.3 --- Effect of TCDD on Human CYP 19 Recombinant Supersomes® and MCF-7aro Cells --- p.66 / Chapter 4.2.4 --- TCDD Increased Aromatase Protein Expression in MCF-7 Cells --- p.66 / Chapter 4.2.5 --- Effect of TCDD in Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.6 --- Effect of TCDD in CYP 19 Promoter and AP-1 Promoter Activity in MCF-7 Cells --- p.70 / Chapter 4.2.7 --- Effect of TCDD in CYP 19 mRNA Half-life --- p.75 / Chapter 4.2.8 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in MCF-7 Cells" --- p.78 / Chapter 4.2.9 --- TCDD induced ERK1/2 Activation --- p.78 / Chapter 4.2.10 --- Induction of aromatase activity in MCF-7erk cells --- p.78 / Chapter 4.3 --- Discussion --- p.87 / Chapter CHAPTER 5 --- Summary --- p.90 / BIBLIOGRAPHY --- p.92