Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

Asymmetric reactions of organosilicon compounds and properties of novel optically active organosilanes

Partial reduction of racemic methoxysilanes by 1:1 complexes of lithium aluminium hydride with optically active cinchona and ephedra alkaloids give optically active silanes and methoxysilanes. Optical yields depend on the groups attached to silicon and the alkaloid used but in some cases approach 50%, The method has been used to prepare novel optically active organosilanes, possessing an asymmetric silicon centre, which are either inaccessible by any of the other available routes or would require a time consuming preparation. Such compounds are of use in the study of the mechanism of substitutions at silicon. Attempts have been made to rationalize the results of the asymmetric reductions in terms of differences in sterio and electronic interactions in diastereoisomeric transition states. Circular dichroism and optical rotatory dispersion spectra have been obtained for the optically active products in an attempt to elucidate the absolute configurations of the novel asymmetric organosilanes. The results from these studies provide a useful addition to the data so far accumulated for asymmetrically perturbed aromatic chromophores. Nuclear magnetic resonanoe studies of diastereoisomaric (-)-menthoxysilanes show that these compounds possess resonances extremely useful in the determination of optical purities for asymmetric organosilanes which possess an aromatic group. The effect of variable temperature on the spectra has revealed evidence for the conformational preferences in these compounds. Other diastereoisomeric alkoxysilanes have been prepared and their n.m.r.spectra studied in the hope of establishing trends. Exploratory studies for other asymmetric reactions proceeding at silicon have proved unfruitful.

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5′-GGA GGG TCG CAT CGC-3′) as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5′-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5′-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2′-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5′-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2′-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs. © 2002 Published by Elsevier Science Inc.

The antioxidant potential of modified quercetins and quercetin-metal complexes

Quercetin is a naturally occurring polyphenol compound present in grapes, red wine, tea, apples and some vegetables. Like other flavonoids, it has been found to have antioxidant activity in studies in vitro, although there is still much debate about the bioavailability of flavonoids in the diet and their in vivo antioxidant activity. In general, it is thought that the antioxidant efficiency of polyphenols increases with increasing hydroxylation of the rings, but there have been few studies of other substitutions. We have prepared several derivatives of quercetin, to test the effect of modification on their antioxidant potential. Sodium salts of quercetin-5-sulfonate and quercetin-5,8-sulfonate, and transition metal complexes of quercetin-5-sulfonate were analysed for their total antioxidant potential using the FRAP assay, and compared to unmodified quercetin. It was found that quercetin-5-sulfonate complexes with Zn, Cu(II), Fe(II) and Mg were all significantly better antioxidants than quercetin, quercetin-5-sulfonate was comparable to quercetin, whereas the sodium salt of quercetin-5,8-sulfonate had a decreased total antioxidant potential. Kinetic studies of the FRAP reaction showed no significant differences between quercitin and any of the derivatives. The reaction of all the quercetins in the FRAP assay was found to be slower to reach completion than ascorbate, and appeared to have biphasic characteristics. These results suggest that transition metal ions may facilitate the transfer of electrons from the polyphenol ring system to the oxidant, while substitution with S03 is electron-withdrawing and destabilizes the ring system. This is important both for understanding the antioxidant ability of flavonoids, and for the design of novel antioxidant compounds. Further work is being carried out to assess the ability of the quercetin complexes to protect cultured cells from oxidative stress.

Morphological-compound dysgraphia in an aphasic patient:"A wild write through the lexicon"

We describe the case of a dysgraphic aphasic individual-S.G.W.-who, in writing to dictation, produced high rates of formally related errors consisting of both lexical substitutions and what we call morphological-compound errors involving legal or illegal combinations of morphemes. These errors were produced in the context of a minimal number of semantic errors. We could exclude problems with phonological discrimination and phonological short-term memory. We also excluded rapid decay of lexical information and/or weak activation of word forms and letter representations since S.G.W.'s spelling showed no effect of delay and no consistent length effects, but, instead, paradoxical complexity effects with segmental, lexical, and morphological errors that were more complex than the target. The case of S.G.W. strongly resembles that of another dysgraphic individual reported in the literature-D.W.-suggesting that this pattern of errors can be replicated across patients. In particular, both patients show unusual errors resulting in the production of neologistic compounds (e.g., "bed button" in response to "bed"). These patterns can be explained if we accept two claims: (a) Brain damage can produce both a reduction and an increase in lexical activation; and (b) there are direct connections between phonological and orthographic lexical representations (a third spelling route). We suggest that both patients are suffering from a difficulty of lexical selection resulting from excessive activation of formally related lexical representations. This hypothesis is strongly supported by S.G.W.'s worse performance in spelling to dictation than in written naming, which shows that a phonological input, activating a cohort of formally related lexical representations, increases selection difficulties. © 2014 Taylor & Francis.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

The hormone glucagon-like peptide-1(7-36)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the E-amino group in the side chain of the LyS26 residue and to combine this modification with substitutions of the Ala 8 residue, namely Val or amino-butyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal) 26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys-(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal β-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal) 26]GLP-1 and [Val8,Lys(pal) 26]GLP-1 did not demonstrate acute glucose-lowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability. Copyright © by Walter de Gruyter.

Greater CD8+ TCR heterogeneity and functional flexibility in HIV-2 compared to HIV-1 infection

Virus-specific CD8+ T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8+ T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8+ T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8 T cells was associated with an enhanced potential for CD8+ expansion and IFN- production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8+ T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8+ T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Synthesis, characterization and study of physical properties of novel M(n+1)AX(n) compounds

Mn+1AXn compounds, the ternary layered nanolaminates have gathered momentum in the last decade since its advent because of their unusual but exciting properties. These technologically important compounds combine some of the best properties of metals and ceramics. Like ceramics they are refractory, oxidation resistant, elastically stiff and relatively light. They also exhibit metallic properties like excellent machinability, thermal and electrical conductivity. This dissertation concentrates on the synthesis of germanium-based 211 Mn+1AXn compounds. The main objective of the research was to synthesize predominantly single phase samples of Cr2GeC, V2GeC and Ti2GeC. Another goal was to study the effect of solid substitutions on the M-site of Mn+1AXn compounds with Ge as an A-element. This study is in itself the first to demonstrate the synthesis of (Cr0.5V0.5)2GeC a novel Mn+1AXn compound. Scanning electron microscopy coupled with energy dispersive spectroscopy, x-ray diffraction and electron probe microanalysis were employed to confirm the presence of predominantly single phase samples of M2GeC compounds where M = Ti, V, Cr and (Cr 0.5V0.5). A large part of the dissertation also focuses on the effect of the compressibility on the Ge-based 211 Mn+1AXn compounds with the aid of diamond anvil cell and high energy synchrotron radiation. This study also concentrates on the stability of these compounds at high temperature and thereby determines its suitability as high temperature structural materials. In order to better understand the effect of substitutions on A-site of 211 Mn+1 AXn compounds under high pressure and high temperature, a comparison is made with previously reported 211 Mn+1AXn compounds with Al, Ga and S as A-site elements.

Structural flexibility and oxygen diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Ellipticine is a natural product which possesses multimodal anti-cancer activity. This thesis encompasses the synthesis and biological evaluation of novel ellipticine and isoellipticine derivatives as anti-cancer agents. Expanding on previous work within the group utilising vinylmagnesium bromide, derivatisation of the C5 position of ellipticine was accomplished by reaction of a key ketolactam intermediate with Grignard reagents. Corresponding attempts to introduce diverse substitution at the C11 position were unsuccessful, although one novel C11 derivative was produced using an alkyllithium reagent. A panel of novel ellipticinium salts encompassing a range of substitutions at the N2, C9 and N6 positions were prepared. Extensive derivatisation of the N10 position of isoellipticine was undertaken for the first time. Novel substitution in the form of acid and methyl ester functionalities were introduced at the C7 position of isoellipticine while novel C7 aldehyde and alcohol derivatives were synthesised. A large panel of isoellipticinium salts were prepared with conditions adjusted for the reactivity of the alkyl halide. Novel coupling reactions to increase the yield of isoellipticine were attempted but proved unsuccessful. A panel of 54 novel derivatives was prepared and a multimodal analysis of their anti-cancer activity was conducted. The NCI 60-human tumour cell lines screen was a primary source of information on the in vitro activity of compounds with derivatives found to exert potent anticancer effects, with mean GI50 values as low as 1.01 μM across the full range of cancer types and as low as 16 nM in individual cell lines. A second in vitro screen in collaboration with researchers in the University of Nantes identified derivatives which could potently inhibit growth in a p53 mutant NSCLC cell line. The cell cycle effects of a selected panel of isoellipticines were studied in leukaemia cell lines by researchers in the Department of Biochemistry and Cell Biology, UCC. Emerging from this, the therapeutic potential of one of the derivatives in AML was then assessed in vivo in an AML xenograft mouse model, with tumour weight reduced by a factor of 7 in treated mice relative to control.

Combined analysis of variation in core, accessory and regulatory genome regions provides a super-resolution view into the evolution of bacterial populations

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug–resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.