Dissertations / Theses on the topic 'Carbohydrate receptors'

The macrotricyclic system as a receptor for carbohydrates in organic as well as aqueous media has been extensively studied within the Davis group. The hydrogen bonding and CH-π interactions are the two main non covalent interactions operating for the recognition of carbohydrates in our system. In principle higher affinities could be achieved by enhancing either of these types of interactions. The possibility of enhancing H-bonding interactions with host-guest was explored by the introduction of heterocyclic linkers in the 'temple' receptors. Macrocycle with pyrrole linker 132 and pyridine linker 131 (Figure i) were synthesised and studied for their affinities with different carbohydrate substrates. Pyrrole macrotricycle 132 bound D- Glucose selectively over other substrates with K; = 18 M-1. However pyridine macrotricycle 131 did not show selectivity and higher affinities with any substrates. Figure i. Macrotricycle with pyridine linker 131 and pyrrole linker 132. In collaboration with Roelens group in Firenze, Italy, a series of water soluble C3-symmetric receptors were synthesized and studied for their affinity for carbohydrates. Unfortunately, these series of receptors did not bind the substrates strong enough to be detected by lH NMR titration studies.

The purpose with this study was to investigate the effect of a carbohydrate (CHO) mouth rinse protocol on simulated cross country (XC) sprint. The primaryaim was to investigate ifa 10 secCHO solutionmouth rinsingprotocolimprovedthe finish timein a simulated cross country (XC) sprint(800 meters for female and 1000 meters for male) compared with control (CON).The second aim was to examif post-finish blood lactateconcentrationwouldbe lowercompared with CONwith mouth rinsing. Seven participants(four males and three females)completed four simulated sprints, two CON and two experimentalrinse protocols, CHO solution and placebo (PLA).Time to completion was not different with CHO compared with CON or PLA (203.0 ±16.5 sec, 202.3 ± 15.7 sec, 203.3 ± 14.6respectively.p >.05).Mean power output was CON 264 ± 73, PLA 258 ± 65, CHO 261± 70.Blood lactateat 15 minweresimilar between CHO, CON and and PLA(9.9 ± 3.9 mmol 7.6 ± 4.0 mmol, 10.1 ± 3.7 mmol respectively.p >.05).We concludethat mouth rinsing a CHO solutionfor 10 sechave no effect on high intensity exercise with duration lasting between 3 and 4 minutes.

Clostridium difficile causes pseudomembranous colitis in humans and a similar ileocecitis in hamsters. This organism can colonize the intestines after antibiotic therapy disrupts the normal intestinal microflora. Once established in the intestines, the organism causes disease by producing two toxins, designated toxin A and toxin B. Only toxin A is active on intestinal epithelium, thus toxin A is the cause of the initial tissue damage in the intestines. In order for a toxin to affect a cell, it must first bind to the cell. Toxin A has been shown to bind to Galα 1-3Galβ 1-4GlcNAc on the intestinal epithelium of hamsters. I provide evidence that toxin A can use this trisaccharide as a functional receptor on cell lines, and that the expression of the carbohydrate receptor increases the sensitivity of the cells to toxin A Furthermore, the intestinal epithelium of infant hamsters bound less toxin A at 37C than did the adult tissue, and infants are less sensitive to the disease caused by C. difficile than are adults. This provides further evidence that the activity of toxin A is increased by the binding of the toxin to Galα 1-3Galβ 1-4GlcNAc. Even though Galα 1-3Galβ 1-4GlcNAc was a receptor for toxin A on animal cells, it probably is not a receptor for toxin A in humans, because people do not normally express this carbohydrate. Instead, I found that toxin A bound to the carbohydrate antigens designated I, X, and Y, which are present on the intestinal epithelium of humans. These carbohydrates could be receptors for toxin A The possible significance of these receptors is discussed.
Ph. D.

L'infection au virus de l'hépatite B (VHB) est étroitement liée au métabolisme énergétique hépatique. La réplication du virus est contrôlée en principal par des facteurs de transcription et récepteurs nucléaires tels que PPARa, HNF4a et Foxül, impliqués dans ce métabolisme. Ainsi, la réplication du virus est augmentée par la privation de nutriments et le stress énergétique en modèles cellulaires, et par le jeûne, en modèles murins. PGC-la, un régulateur majeur de la réponse métabolique adaptative au jeûne, est impliqué dans l'augmentation de la transcription du VHB par son interaction avec plusieurs facteurs de transcription. Il est connu que le récepteur des acides biliaires, FXRa, qui est capable d'activer le promoteur de Core du VHB, est co-activaté par PGC-la. Un autre acteur important dans l'adaptation métabolique à la privation d'énergie est la protéine déacétylase SIRTl. Lorsqu'il est activé, SIRTl hépatique est capable de désacétyler et activer autant PGC-la que FXRa. Ces données nous ont amenés à émettre l'hypothèse que SIRTl pourrait coopérer avec FXRa et PGC-la pour augmenter la transcription du VHB. Dans un premier travail, nous avons donc étudié le rôle de la coopération de ces trois facteurs métaboliques dans la réplication du virus. Ça nous a permis de décrire un nouveau réseau métabolique, composé de FXRa, PGC-la et SIRTl, qui régule l'activité transcriptionnelle du VHB. Nous avons montré que SIRTl augmente l'activité du promoteur de Core par l'intermède d'autre facteurs, parmi lesquels, FXRa. Nous avons en outre observé que la fonction de déacétylase de SIRTl était nécessaire pour l'amplification de l'effet de FXRa sur VHB promoteur de Core. Une autre cible de SIRTl, connue pour son activité co-activatrice sur FXRa, est PGC-la. Grâce à une série d'expériences de surexpression et suppression, nous avons montré que non seulement la co-activation de FXRa par PGC-la est potentialisée par SIRTl, mais la présence de PGC-la est nécessaire pour l'effet de SIRTl sur l'activation du promoteur de Core VHB induite par FXRa. Ces données suggèrent que FXRa, PGC-la et SIRTl coopèrent dans la modulation de l'activité transcriptionnelle du promoteur de Core. Nous avons ensuite confirmé nos observations initiales et avons montré que l'activation de l'axe SIRTl/PGC-la/FXRa induit la transcription de l'ARN de VHB dans des lignées cellulaires d'origine hépatique et non-hépatique. Ces résultats renforcent l'idée que la réplication du VHB peut être modulée en fonction de l'état nutritionnel. Les rapports des études précédentes menées in vitro et sur des modèles animaux suggèrent que la transcription du VHB est contrôlée de la même manière que les gènes de la néoglucogenèse. Notre hypothèse a été que chez l'homme, la réplication du VHB montrerait des fluctuations diurnes, selon les périodes de la journée de jeûne et de réalimentation. Le but de la deuxième étude a été donc de déterminer si la charge viral du VHB plasmatique montre des variations importantes tout au long du nichthemeron chez les patients chroniquement infectés par VHB, avec une réplication virale active [etc...]
Hepatitis B virus (HBV) infection is tightly linked with hepatic fuel metabolism. HBV replication depends on the activity of several liver-enriched nuclear receptors and transcription factors, such as PPARa, HNF4a, and Fox01, involved in the metabolic adaptive response to fasting. In the first part of our work, we identified a metabolic subnetwork that enhances the activity of HBV core promoter. FXRa (NR1H4), PPAR gamma coactivator 1a and SIRT1, the members of this regulatory axis, cooperate to increase HBV transcription. The three molecules are themselves key factors of liver metabolism, linking HBV replication to complex metabolic cues, such as energy status and nutrient availability during the fasting-refeeding cycles. We then observed the existence of a circadian cycle of HBV replication in humans, underlining the role of nutrient availability in the modulation of HBV replication, previously predicted by experimental models. The second part of the work focused on the plasma cell-free nucleic acids as potential biomarkers in chronic viral hepatitis. Due to the multiple links between HBV replication and cellular factors involved in fuel metabolism, we hypothesized that plasma mRNAs corresponding to these factors may constitute potential biomarkers for chronic hepatitis B. We successfully detected more than 30 plasma mRNA sequences corresponding to enzymes, transporters, nuclear receptors and transcription factors involved in fatty acids synthesis and oxidation, cholesterol synthesis, transport and excretion, and energy sensing and expenditure. The circadian variation and the multiple correlations in the expression patterns of these plasma transcripts are similar to those previously described in cells both in vitro and in vivo. This suggests that cell- free mRNAs may provide a "virtual biopsy" of the transcriptional status of the organism. Moreover, we found significant differences in the plasma mRNA profiles of HBV carriers compared with healthy controls, similar to those found in experimental models of infection, suggesting that these transcripts may also serve as biomarkers of liver disease. Further research is warranted to shed new light on the complex relationship between HBV life cycle and host lipid-carbohydrate-fuel metabolism and may lead to the identification of both actionable targets in antiviral therapy, and putative biomarkers in chronic hepatitis B

Les foldamères d'oligoamides aromatiques sont des structures abiotiques qui se replient en structures secondaires telles que des hélices et des feuillets que l'on peut rencontrer dans les protéines. Il est possible de contrôler leurs architectures pour qu'elles adoptent des conformations en hélices, capsules ou cônes, en tirant parti de leur rigidité, et créer au sein de ces edifices des interactions non covalentes capables de lier sélectivement des molécules invitées. Ces propriétés ouvrent la voie vers la conception d'objets moléculaires avancées capables de réaliser des fonctions complexes, telles que le transport moléculaire et la reconnaissance sélective d'invités. Ce travail de thèse se concentre sur deux de ces systèmes : (1) la caractérisation des mouvements en cascade interdépendants entre un macrocycle et une hélice le long d'un axe dans une machine moléculaire de type foldarotaxane, dont les stimuli sont le pH et la concentration, et (2) le développement d’un récepteur en forme de cône hélicoïdal à double brins pour la reconnaissance de glucides, répondant aux limitations des capsules traditionnelles construites par des foldamères. (1) Un foldarotaxane se forme par l'auto-assemblage d'un foldamère hélicoïdal et d'un macrocycle autour d'un axe moléculaire en forme de haltère. L'axe comporte deux stations pour le macrocycle, l'une plus attractive à pH bas et l'autre à pH élevé. L'hélice possède également deux sites de liaison de longueurs différentes. La station la plus attractive se situe près du second site du macrocycle, entraînant ainsi une compétition entre le macrocycle et l'hélice pour cette position à pH élevé. Dans ce travail, il est exposé comment, sous l'influence du pH, le macrocycle induit le mouvement de l'hélice sur la tige, lorsque l'on se trouve à faibles concentrations, tandis qu'à des concentrations plus élevées, la dissociation et le réassemblage rapides de l'hélice conduisent à une navette supramoléculaire compartimentée. L'hélice et le macrocycle peuvent se retrouver piégés dans des régions de l'axe où ils ont une faible affinité. (2) Les capsules de foldamères oligoamides aromatiques offrent une complémentarité de formes précise pour la reconnaissance de substrat et limitent l'exposition de l'invité au solvant. Cette encapsulation restreint toutefois leurs applications telles que la liaison de molécules plus grandes (par exemple, des protéines glycosylées) ou les transformations chimiques sélectives. Pour remédier à cela, nous avons développé un récepteur en forme de cône hélicoïdal à deux brins moléculaires en modifiant la conception de la capsule et en empêchant la dimérisation des cônes hélicoïdaux simples. Ce nouveau récepteur, incorporant une unité en coude dans le squelette même du foldamère, a été synthétisé et son affinité de liaison ainsi que sa sélectivité envers divers monosaccharides et disaccharides ont été testé
Aromatic oligoamide foldamers are abiotic backbones that fold into secondary structures such as helices and sheets, similar to proteins. They can adopt defined conformations such as helices, capsules, and cones, leveraging their rigidity, torsional flexibility, and noncovalent interactions to selectively bind specific guests. These versatile properties open pathways for designing advanced molecular architectures that can perform complex functions, such as molecular shuttling and selective guest recognition. This work focuses on two such systems: (1) characterizing the interdependent cascading motions between a macrocycle and helix along a thread in a foldarotaxane-molecular machine under pH stimuli and variable concentration, and (2) developing a double helical cone-shaped receptor for carbohydrate binding, addressing the limitations of traditional foldamer capsules. (1) A foldarotaxane forms through the self-assembly of a helical foldamer and a macrocycle around a dumbbell-shaped molecular axle. The axle contains two stations for the macrocycle, one of which is more attractive at low pH, while the other becomes favoured at high pH. Similarly, the helix has two binding stations of different lengths, and the more attractive station is located near the second macrocycle station, creating competition between the helix and macrocycle at higher pH. Under low concentrations and pH stimuli, the macrocycle translates the helix along the axle. At higher concentrations, faster helix disassembly and reassembly lead to a compartmentalized supramolecular shuttle where the helix and macrocycle can be trapped in regions of lower affinity. (2) While aromatic oligoamide foldamer capsules offer precise shape complementarity, they limit guest exposure to the solvent, restricting applications such as binding larger molecules (e.g., glycosylated proteins) or selective chemical transformations. To address this, we developed a double helical cone-like receptor by modifying the capsule design and preventing the dimerization of single helical cones. The new receptor, incorporating a turn unit in the foldamer backbone, was tested for its binding affinity and selectivity toward various monosaccharides and disaccharides

Boron acids are very useful in many organic syntheses, particularly as catalysts or participating agents. Their catalytic properties are featured in common functional group transformations including amidations, esterifications and cycloadditions. However, boron acids are more commonly known for having a binding affinity for vicinal diols and polyols. The reversible, covalent interaction between boron and diols is a natural phenomenon important to several biological processes. Chemists have worked over several centuries to understand these complexes. This covalent interaction that occurs between the boron acid and cis-diols has been exploited in an attempt to target specific polyols, with the design and synthesis of carbohydrate sensors as well as inhibitors of enzymes and artificial lectins. The first part of this thesis provides a literature overview of the potential application of boron-based receptors as both diagnostic tools and drug delivery-agents in diseases. Our aim was to utilise boron acids affinity towards diols as an accelerant to facilitate the formation of novel receptors for cell-surface carbohydrates. Within some disease states, particular aberrant carbohydrates become potential sensor targets for disease detection. Specifically, the Mycobacteria cell wall contains bacteria unique structural fucose and galactose residues that could be detectable via a boron-based carbohydrate sensor.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Antígenos de alta massa molecular (PI) do extrato de Ascaris suum exercem efeito supressor sobre a resposta imune a antígenos heterólogos. PI atua diretamente sobre as DCs, diminuindo a expressão das moléculas coestimuladoras, MHC de classe II e assim, a proliferação de linfócitos T. Esse efeito é independente de TLR2, TLR4 e da molécula MyD88. Nesse trabalho estudamos a participação dos receptores DC-SIGN e MR, na modulação da atividade das DCs. PI contém oligossacarídeos N-ligados com cadeias de alta manose e do tipo complexa e contém resíduos de fosforilcolina. Os componentes do PI contendo as cadeias glicosídicas N-ligadas inibem a maturação de DCs incubadas com LPS. Receptores DC-SIGN e MR estão envolvidos no reconhecimento e internalização dos componentes do PI pelas DCs. O bloqueio desses receptores foi capaz de abolir o efeito inibitório do PI sobre as DCs e a resposta proliferativa de linfócitos T in vitro. Portanto, os resultados mostram a participação do DC-SIGN e MR no reconhecimento de componentes glicosilados do PI e na sua ação imunomoduladora.
High molecular weight components (PI) of Ascaris suum extract exert suppressive effect on the immune response to OVA. PI exert direct effect on DCs, decreasing the T lymphocyte proliferation. This effect is independent of TLR2 and 4 as well as MyD88 molecule. In this work we studied the glycoconjugates in PI and the participation of DC-SIGN and MR, in the modulation of the functional activity of DCs. PI components contain high mannose- and complex-type N-linked oligosaccharides and phosphorylcholine residues. PI components containing N-linked glycans inhibited the DCs maturation induced by LPS. The previous incubation of DCs with mannan, anti-DC-SIGN and anti-MR antibodies abolished the modulatory effect of PI on the DCs maturation. It was also observed that the blockage of DC-SIGN and MR in DCs reversed the inhibitory effect of PI in the in vitro T cells proliferative response. Taking together these results show the involvement of DC-SIGN and MR in the recognition of glycosylated components of PI by DCs and in its modulatory effect.

The current thesis presents work on the structure and dynamics of oligosaccharides and polysaccharides as well as the free energetics of carbohydrate-protein interactions. By applying various computational tools such as molecular dynamics simulation, our in-house fast sugar structure prediction software, replica exchange molecular dynamics, homology modeling, umbrella sampling, steered molecular dynamics as well as the thermodynamic integration formalism, we have been able to study the role of water on the surface of homopolysaccharides as well as complex oligosachharides, we have been able to produce a prediction of the bound structure of triantennary oligosaccride on the asialoglycoprotein receptor, we have been able to estimate the free energy of binding of ManΑ1→2Man to the HIV-1 inactivating protein, Cyanovirin-N as well as the relative binding free energies of mutants of Cyanovirin-N to the same ligand.

ABSTRACT Carbohydrates in various forms play vital roles in numerous critical biological processes including cell-cell adhesion and communication, embryo development, immune response, etc. Fluorescent sensors for such carbohydrates have a wide range of potential applications including glucose concentration determination, cell labeling and targeting based on carbohydrate biomarkers, as in vitro diagnostic tools, and biomarker-directed cellular imaging. Our group has been interested in the design and synthesis of multi-boronic acid compounds with well-defined three-dimensional scaffolding for the specific recognition of selected carbohydrate biomarkers. Aberrant expression of carbohydrate antigens such as sialyl Lewis X (sLex), sialyl Lewis A (sLea), Lewis X (Lex), and Lewis Y (Ley) have been associated with tumor formation and metastasis in various cancer types.1-4 As such, for our initial design, we have selected sialyl Lewis X (sLex) as our potential target due to implication in the development of liver and colon cancer.5, 6 Herein, we describe the design, synthesis and evaluation of four such compounds, each having about ten linear steps in its synthesis. In addition to the design of fluorescent probes for cell surface carbohydrates, we also have designed lipophilic boronic acid derivatives as potential fusogenic agents. Due to boronic acid¡¯s ability to bind to 1,2 and 1,3 cis diols, we hypothesize that the aliphatic chain should be able to insert into lipid cellular membrane and the boronic acid units should allow for the ¡°attachment to neighboring cells¡± through complexation with cell surface glycans. Such interactions should allow the boronic acid compounds to bring two or more cells together for fusion. Herein, we have described the methodologies of the design of such compounds. INDEX WORDS: Boronic acid, sialyl Lewis X probe, boronolectin, fluorescence, sensor, cell-cell fusion, fusogen, immunotherapy.

Cell surface carbohydrates play an important role in a wide variety of biological processes such as inflammation, tumor metastasis, and viral and bacterial infection. The goal of our research has been two-fold. The first objective was the synthesis of antimycobacterial compounds. A mannose containing tetrasaccharide from the mannan core of lipoarabinomannan (LAM) of Mycobacterium tuberculosis has been synthesized using α-D-methylmannopyranoside as starting material and Koeings-Knorr reactions to couple saccharides. The synthesis was completed in nine steps and in 14% total yield. This compound should be useful in competitive inhibition studies with macrophages or as an immunological marker. We have successfully synthesized nonsulfated mimics of the aminosterol antibiotic from 5α-cholestan-3-one in two steps in 40-70% total yield. The critical step in this synthesis is the addition of the boronic acid functional group using 2-o-formylphenylboronic acid. It is hypothesized that the addition of boronic acids will improve the antibacterial and anti-angiogenic activity of these compounds. The second objective was the synthesis of a simple fluorescent receptor for simple carbohydrates. A receptor using anthracene as the fluorophore has been completed demonstrating an improved yield over previous methods. This receptor is the first to show selectivity for myo-inositol over other saccharides.

Intestinal microbiota play an important role in determining susceptibility to obesity. Their ability to ferment non-digestible carbohydrates, to produce short-chain fatty acids (SCFAs), is thought to be important in influencing host metabolism. However, the underlying mechanisms are poorly understood. Using mice that lack the short-chain fatty acid receptor; free fatty acid receptor 2 (FFAR2), it was found that this receptor is essential for transducing the effects of fermentable carbohydrate to reduce food intake and prevent body weight gain. These beneficial metabolic effects were associated with an increase in the anorectic gut hormone peptide YY (PYY) due to an FFAR2-dependent increase in the density of PYY-expressing cells within the proximal colon. Inulin supplementation also increased GLP-1 cell density in the proximal colon, resulting in increased circulating GLP-1 concentrations. However, this was not dependent on expression of FFAR2. Further investigation revealed that the increase in PYY cell density was associated with increased expression of the paired-box transcription factor Pax4 known to function in terminal differentiation of PYY cells. It is, therefore, apparent that SCFAs, produced by microbial fermentation of inulin, act via FFAR2 to increase Pax4 expression and thereby enhance PYY cell proliferation. The ability of SCFAs to influence enteroendocrine cell fate and potentiate anorectic gut-brain signalling represents a paradigm shift in the understanding of nutrient sensing and highlights the potential for cellular engineering approaches for the treatment obesity.

In multicellular organism, bioenergetic metabolism is strictly regulated toward efficient generation of ATP. However, in certain situations, such as in limiting oxygen or in the rapidly proliferating system like growing juvenile or cancer cells, organisms apply the metabolic strategy that favors the production of biomass (e.g., nucleotides, amino acids, and lipids) over efficiency of ATP generation. The conserved estrogen-related receptors (ERRs) are master regulators in controlling metabolic homeostasis, and good candidates for mediating the metabolic transition induced by hypoxia and development. First, we investigate how dERR influences hypoxic adaptation in Drosophila melanogaster. We find that dERR is required for a competent hypoxic response alone, or together with hypoxia inducible factor (HIF), which is the main transcription factor modulating the hypoxic adaptation. We show that dERR binds to dHIFα and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFα in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including up-regulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. Secondly, we examine how dERR modulates metabolic transition toward the fatty acid oxidation at late L3 larva stage. We show that dERR is essential for the expression of an uncharacterized long-chain-fatty-acid acyl-CoA synthetase, CG4500, which is subject to induction by starvation. Furthermore, late L3 larvae of dERR mutants exhibit altered lipid profiles with elevated medium-chain and long-chain fatty acids. Together, with the previous finding that ERR directs an early switch toward glycolysis in the embryo, our studies indicate that ERR is a master regulator of programmed metabolic shifts through Drosophila development.

A novel class of potential positron emission tomography (PET) radiotracers for imaging aminopeptidase N (also known as APN or CD13) and cannabinoid type 1 (CB1) receptors were designed and synthesised with an efficient chemical strategy. Both targets have remarkable diagnostic and therapeutic potential, in fact the CD13 receptors are over-expressed during tumour angiogenesis and the CB1 receptors are highly expressed in the brain playing important functions in several pathophysiological processes. The target compounds were obtained by means of oxime-bio-conjugation between fluoro-deoxy-carbohydrates, used as prosthetic groups, and hydroxylamine-functionalised cyclic NGR (asparagine-glycine-arginine) motif sequences for CD13 receptor and rimonabant-type pyrazoles for the CB1 receptor. In particular, aminooxy-cyclic NGR peptides were conjugated with the novel prosthetic group 5-FDR (5-fluoro-5-deoxy-D-ribose) and the aminooxy- pyrazole-type cannabinoid molecules were conjugated with both 5-FDR and with FDG (2-fluoro-2-deoxy-D-glucose). 5-FDR proved to be superior to FDG, as the bioconjugation reaction occurred in milder conditions (room temperature vs 100 °C) and at faster rate. Furthermore, we observed that the rate of the oxime bond formation depends on the solubility of the aminooxy-functionalized core used. In fact, the bioconjugation with hydrophilic cyclic aminooxy-NGR peptides was faster than in the case of lipophilic aminooxy-pyrazoles (10 min vs 20-30 min). The receptor affinity is decreased in the case of the CB1 receptors after conjugation with the fluoro-carbohydrates. This is not observed with the conjugated NGR peptides, which maintain similar affinity for the CD13 receptor compared with the unconjugated NGR. In conclusion, we have developed an efficient strategy for the synthesis of a novel class of CD13 ligands, which may be also produced in radiofluorinated form, and explored a novel bioconjugation strategy for CB1 receptor ligands. Both may have important applications in the development of PET tracers.

La dérégulation du métabolisme glucidique conduisant au développement d’une hyperglycémie est classiquement associée aux maladies métaboliques, telles que le diabète de type 2 et l’obésité. Le foie est un organe clé dans le contrôle de l’homéostasie glucidique. Ainsi, lors d’un état post-prandial (après un repas), il utilise le glucose pour produire de l’énergie par la voie de la glycolyse, mais surtout stocke l’excès de glucose sous forme de glycogène par la voie de synthèse du glycogène et l’excès d’énergie sous forme d’acides gras par la voie de la lipogenèse. Ces voies sont sous le contrôle des hormones insuline et glucagon qui, en fonction des changements nutritionnels, régulent respectivement l’utilisation (glycolyse) et la production (néoglucogenèse) de glucose en induisant l’expression des enzymes de ces voies métaboliques. Plus récemment, il a été montré que les voies de la glycolyse et de la lipogenèse sont également régulées par le glucose qui active le facteur de transcription ChREBP (Carbohydrate Response Element Binding Protein) et induit de ce fait l’expression des gènes de la glycolyse, tels que la LPK (Liver Pyruvate Kinase), et de la lipogenèse, tels que FAS (Fatty Acid Synthase) et ACC1 (Acetyl-CoenzymeA Carboxylase 1). Le récepteur nucléaire Farnesoid X Receptor (FXR), un facteur de transcription activé par des ligands, en plus de son rôle très important dans la régulation des acides biliaires et des lipides, contrôle aussi le métabolisme glucidique dans le foie. Ainsi, FXR inhibe l’expression des gènes des voies de la glycolyse et de la lipogenèse, probablement en interférant avec le facteur de transcription ChREBP, comme le propose une étude récente. Les objectifs de ma thèse ont été de caractériser deux lignées hépatocytaires humaines IHH (Immortalized Human Hepatocytes) et HepaRG d’un point de vue métabolique et d’étudier les mécanismes moléculaires d’interférence du récepteur nucléaire FXR avec l’activité du facteur ChREBP dans ces deux lignées
Glucose metabolism dysreglation leads to the developpment of hyperglaecemia and is classically associated with metabolic diseases such as Type II diabetes or obesity. The liver is a key organ in the control of glucose homeostasis. Indeed, at a post-prandial state (after a meal), it utilizes glucose to produce energy by the glycolysis pathway, but mostly stores the glucose excess as glycogen by the glycogenesis pathway and the energie excess as fatty acids by the lipogenesis pathway. These pathways are controlled by insulin and glucagon hormones which, in response ton nutritional changes, regulate respectively the utilization (glycolysis) and the production (gluconeogenesis) of glucose by inducing the expression of enzymes involved in these pathways. More recently, it has been shown that glycolyisis and lipogenesis are also regulated by glucose who activates the transcription factor ChREBP (Carbohydrate Response Element Binding Protein) and therefore induces the expression of glycolytic genes, such as LPK (Liver Pyruvate Kinase) and lipogenic genes, such as FAS (Fatty Acid Synthase) and ACC1 (Acetyl-CoenzymeA Carboxylase 1). Nuclear receptor Farnesoid X Receptor, a transcription factor activated by ligands, besides its role in the regulation of bile acids and lipids, also controls the glucose metabolism in liver. Thus, FXR inhibit the expression of genes involved in glycolysis and lipogenesis, probably by interfering with the transcription factor ChREBP, as it has been suggested by a recent study The objectifs of my thesis were to characterize two human hepatocyte cell lines IHH ( (Immortalized Human Hepatocytes) and HepaRG from a metabolic point of view and to study the molecular mecanisms involved in the interference of FXR with the activity of ChREBP in these two cell lines

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses, which have a lipid bilayer that surrounds their nucleocapsids, entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. For many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion. However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution of functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes, mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells, and therefore, is an important aspect of virus infection and disease progression. Despite the conservation of receptor binding activity among members of the Paramyxovirinaesubfamily, for most of these viruses, including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virus-specific interaction between the two glycoproteins. Thus, one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process. Therefore, the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN-F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region of HN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines F-specificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN-F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface. As an initial probe of the role of the HN stalk in mediating the interaction with F, N-glycans were individually added at several positions in the region. N-glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HN-F interaction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F. To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head of HN (residues 124-152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carrying this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein. Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F-interactive domain in the stalk of HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95); a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction. The second goal of this research was to address the HN-F interaction from the perspective of the F protein by investigating the relationship between receptor binding, the HN-F interaction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN, enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F, a 2.5-fold increase in fusion was observed. However, similar to wt F, an interaction between L289A-F and the IR-mutated HN proteins was not detected. These results imply that the attachment function of HN, as well as the conformational change in L289A-F, are necessary for the enhanced level of fusion exhibited by HN proteins co-expressed with L289A-F. Indeed, two MAbs detected a conformational difference between L289A-F and the wt F protein. These findings support the idea that the L289A substitution converts F to a form that is less dependent on an interaction with HN for conversion to the fusion-active form. The last goal of this research was to address the cellular site of the HN-F interaction, still a controversial issue based on conflicting data from studies of different paramyxoviruses, using various approaches. This is a particular point of interest, as it speaks to the mechanism by which the HN-F interaction regulates fusion. Thus, NDV HN and F were successfully retained intracellularly with a multiple arginine or KK motif, respectively. The results of Endoglycosidase H resistance and F cleavage studies indicate that the mutated proteins, HN-ER and F-ER, are retained in a compartment prior to the medial-Golgi apparatus and that they are unable to interact with a high enough affinity to co-retain or even cause reduced transport of their wt partner glycoproteins. This is consistent with the HN-F interaction occurring at the cell surface, possibly triggered by receptor binding. In conclusion, this thesis presents evidence to argue that the IR in the stalk of the NDV HN protein directly mediates the interaction with the F protein that is necessary for fusion. Overall, the data presented in this thesis extend the current knowledge of the mechanism by which the paramyxovirus attachment protein can trigger the F protein to initiate membrane fusion. A clear understanding of this process has the potential to identify new anti-viral strategies, such as small molecule inhibitors, aimed at controlling paramyxovirus infection by interfering with early steps in the virus infection cycle.

The primary goal of my thesis research was to characterize the basis for the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in primary culture diabetic cardiomyocytes. Toward this goal, I have investigated several key regulatory sites in this signaling pathway which could promote the hypersensitive activation of phosphorylase. Specifically, I investigated (1) which adrenergic receptors are involved in mediating the hypersensitive response of glycogen phosphorylase to epinephrine stimulation; (2) whether the presence of fatty acid metabolites affects phosphorylase activation; (3) whether the hypersensitive response of phosphorylase results from altered signal transduction through the β-adrenergic receptor system or from a post-receptor defect; and (4) the potential role for phosphorylase kinase in mediating the hypersensitive response of phosphorylase to catecholamine stimulation. The basis for adrenergic receptor mediation of the catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation which was apparent 3 hours after cell isolation and was further enhanced upon maintenance of the myocytes in culture for 24 hours. Normal cells initially lacked the hypersensitive response to epinephrine stimulation although upon maintenance of these cells in culture for 24 hours, the hypersensitive response was acquired in vitro. To assess alpha- and beta- adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a β-receptor antagonist, prior to direct α1receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3 or 24 hour cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol whereas prazosin, an α-receptor antagonist, was unsuccessful. This data suggests that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through β-adrenergic receptor activation. Since the accumulation of various fatty acid metabolites can affect certain enzymes and signal transduction pathways within the cell, the potential effect of various fatty acid metabolites on phosphorylase activation was investigated. To determine the potential effects of fatty acid metabolites on phosphorylase activation in cultured cardiomyocytes, normal and alloxan-diabetic cells were incubated with either carnitine or palmitoylcarnitine prior to stimulation with epinephrine. Pretreatment of cardiomyocytes with or without carnitine or palmitoylcarnitine for 3 or 24 hours before epinephrine stimulation failed to alter phosphorylase activation. The addition of exogenous carnitine in the absence and presence of insulin was also unsuccessful in attenuating the hypersensitive phosphorylase activation response in 3 and 24 hour, normal and alloxan-diabetic derived cardiomyocytes. To determine if carnitine palmitoyltransferase 1 (CPT-1) activity was responsible for the hypersensitive response of phosphorylase in the diabetic myocytes, both normal and diabetic myocytes were maintained for 3 and 24 hours in the absence and presence of etomoxir, a CPT-1 inhibitor. Subsequent activation of phosphorylase by epinephrine in normal and diabetic myocytes was unaltered in the presence of etomoxir. Collectively, these data fail to support a critical role for fatty acid metabolite involvement in the hypersensitive activation of glycogen phosphorylase in acute, alloxan-diabetic cardiomyocytes. To assess potential G-protein involvement in the response, normal and diabetic-derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylyl cyclase activation, the cells were challenged with forskolin. After 3 hours in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hours in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes. This response, which is present in alloxan-diabetic cells, and is induced in vitroin normal cardiomyocytes, is primarily due to a defect at a post-receptor site. To assess the role of phosphorylase kinase in the hypersensitive activation of glycogen phosphorylase in the diabetic heart, phosphorylase kinase activity was measured initially in perfused hearts (to optimize the assay parameters) and subsequently in primary culture cardiomyocytes. Results from these experiments demonstrate that the present method for measuring phosphorylase kinase activity is a reliable indicator of the enzyme's activity in the heart, although the assay conditions must be further optimized before this system can be applied to the measurement of phosphorylase kinase activity in primary cultured cardiomyocytes.

abstract: Small molecules have proven to be very important tools for exploration of biological systems including diagnosis and treatment of lethal diseases like cancer. Fluorescent probes have been extensively used to further amplify the utilization of small molecules. The manipulation of naturally occurring biological targets with the help of synthetic compounds is the focus of the work described in this thesis. Bleomycins (BLMs) are a class of water soluble, glycopeptide-derived antitumor antibiotics consisting of a structurally complicated unnatural hexapeptide and a disaccharide, clinically used as an anticancer chemotherapeutic agent at an exceptionally low therapeutic dose. The efficiency of BLM is likely achieved both by selective localization within tumor cells and selective binding to DNA followed by efficient double-strand cleavage. The disaccharide moiety is responsible for the tumor cell targeting properties of BLM. A recent study showed that both BLM and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Work presented here describes the synthesis of the fluorescent carbohydrate conjugates. A number of dye-labeled modified disaccharides and monosaccharides were synthesized to study the nature of the participation of the carbamoyl moiety in the mechanism of tumor cell recognition and uptake by BLM saccharides. It was demonstrated that the carbamoylmannose moiety of BLM is the smallest structural entity capable for the cellular targeting and internalization, and the carbamoyl functionality is indispensible for tumor cell targeting. It was also confirmed that BLM is a modular molecule, composed of a tumor cell targeting moiety (the saccharide) attached to a cytotoxic DNA cleaving domain (the BLM aglycone). These finding encouraged us to further synthesize carbohydrate probes for PET imaging and to conjugate the saccharide moiety with cytotoxins for targeted delivery to tumor cells. The misacylated suppressor tRNA technique has enabled the site-specific incorporation of noncanonical amino acids into proteins. The focus of the present work was the synthesis of unnatural lysine analogues with nucleophilic properties for incorporation at position 72 of the lyase domain of human DNA polymerase beta, a multifunctional enzyme with dRP lyase and polymerase activity.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2014

Carbohydrate–protein interactions play vital roles in various biochemical processes such as signal transduction and cell surface recognition events. The clustering of carbohydrates into dense domains such as lipid rafts regulates recognition by multivalent receptors (i.e. lectins). These regions are known to play important roles in biological processes such as cellular transduction and trafficking. In order to characterize the clustering of glycans on cell surfaces, detection of domains with high carbohydrate density is of great interest. In this thesis, we present the work based on a modular strategy to design and synthesize boronic acid-based carbohydrate receptors, which are termed as boronolectins because of their similarly in functions with lectins, in order to understand the molecular basis of carbohydrate–protein interactions. These receptors will then be employed for binding studies with carbohydrate based guests and catechol derivatized diol target molecules in order to study the binding interactions between the boronic acid receptors and diol moieties present in guest molecules. A second project that is described in this thesis is based on developing diacylglycerol-based lipid probes, which could be employed for studying protein-lipid binding interactions. Due to involvement of protein-lipid binding interactions in the onset of various pathophysiological conditions, it is of paramount importance to investigate these interactions at the molecular level. DAG (diacylglycerol) represents an important class of signaling lipids and members of the Protein kinase C (PKC) family are described as the main responsive receptors of DAG. PKCs are known to be involved in tumorigenesis. In order to elucidate the exact correlation between PKC activity and carcinogenesis, it will be beneficial to design and synthesize DAG-based lipid analogs. In this thesis, hence, a modular strategy to design and synthesize a class of DAG-based lipid analogs by appending reporter groups such as polyaromatic fluorophores in the sn-1 acyl chains via the traceless Staudinger ligation is described. In both the projects, the synthetic strategy adopted is based on a modular design in order to generate a common scaffold which undergoes modification at the last step to generate a class of fluorophore tagged analogs to be employed for studies.

Lectin-saccharide interactions are involved in many biological processes essential for the survival and proper function of multicellular organisms. C-type lectin-like receptors, predominantly expressed by cells of the innate immune system, recognize saccharide structures on microbes and also aberrant glycosylation pattern of cancer cells. The NKR-P1 receptor family was among the first natural killer (NK) receptor families that were identified, however ligands for some of members remain still elusive. Recently, publications describing N-acetylglucosamine-terminated oligosaccharide structures as possible ligands for NKR-P1 receptor have been subjects for correction/retractions after investigation of the Ethical Committee of the Institute of Microbiology, ASCR, v. v. i. and Charles University in Prague. Re-evaluation of glycodendrimer effect, particularly effect of N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P), revealed mostly indirect role of NK cells on modulation of immune responses. Properly folded soluble recombinant rat NKR-P1A and mouse NKR-P1C lack binding activity to neoglycoproteins modified with GlcNAc-terminated structures. Moreover, new possible target cell populations (NKT cells and macrophages) for saccharide binding were identified.

Thesis (M. Sc.)--University of Alberta, 2009.
Title from pdf file main screen (viewed on Jan. 11, 2010). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Chemistry, University of Alberta." Includes bibliographical references.

碩士
國立交通大學
分子醫學與生物工程研究所
103
Carbohydrate metabolism is an important biochemical reaction that provides energy and builds functional materials in various life forms. Carbohydrate is the preferred carbon source over lipid, peptide, organic acids, and alcohol. Carbohydrate metabolism in Escherichia coli, a well-established model in research of microorganisms, is mainly controlled by cyclic adenosine monophosphate (cAMP) and its receptor protein (CRP) complex cAMP–CRP. The cAMP–CRP can form a mechanism known as “catabolite repression,” which allows E. coli to rapidly select different carbohydrate sources. CRP can form a close loop called “feed-forward loop (FFL)” with CRP-mediated genes and CRP-mediated transcription factors (TFs). An FFL demonstrates significant regulatory patterns and can be detected in many types of gene mediation and in carbohydrate metabolism. Our previous study demonstrated that in E. coli carbohydrate degradation, more than 70% genes were mediated by CRP; this percentage is similar to that in carbohydrate transportation. In addition, 50% of these genes can form FFLs with CRPs and their TFs. Therefore, we aim to determine the FFL relationship between degradation and transportation, and have discovered the relation between carbonhydrate metabolism and the numbers of FFLs. To precisely determine the FFL mechanism, we selected the maltose degradation system, which the CRP-mediated FFL remained unknown. In this case, CRP can activate malT to facilitate the utilization of maltose by E. coli but repress the degradation genes malZ and malPQ. In maltose diauxic growth test, malT without CRP mediated had delayed growth rate. After studying the FFL, we hypothesize that E. coli can adapt to environmental shifts with specific gene regulation.

abstract: ABSTRACT Manipulation of biological targets using synthetic or naturally occurring organic compounds has been the focal point of medicinal chemistry. The work described herein centers on the synthesis of organic small molecules that are targeted either to cell surface receptors, to the ribosomal catalytic center or to human immunodeficiency virus reverse transcriptase. Bleomycins (BLMs) are a family of naturally occurring glycopeptidic antitumor agents with an inherent selectivity towards cancer cells. DeglycoBLM, which lacks the sugar moiety of bleomycin, has much lower cytotoxicity in cellular assays. A recent study using microbbuble conjugates of BLM and deglycoBLM showed that BLM was able to selectively bind to breast cancer cells, whereas the deglyco analogue was unable to target either the cancer or normal cells. This prompted us to further investigate the role of the carbohydrate moiety in bleomycin. Fluorescent conjugates of BLM, deglycoBLM and the BLM carbohydrate were studied for their ability to target cancer cells. Work presented here describes the synthesis of the fluorescent carbohydrate conjugate. Cell culture assays showed that the sugar moiety was able to selectively target various cancer cells. A second conjugate was prepared to study the importance of the C-3 carbamoyl group present on the mannose residue of the carbohydrate. Three additional fluorescent probes were prepared to improve the uptake of this carbohydrate moiety into cancer cells. Encouraged by the results from the fluorescence experiments, the sugar moiety was conjugated to a cytotoxic molecule to selectively deliver this drug into cancer cells. The nonsense codon suppression technique has enabled researchers to site specifically incorporate noncanonical amino acids into proteins. The amino acids successfully incorporated this way are mostly α-L-amino acids. The non-α-L-amino acids are not utilized as substrates by ribosome catalytic center. Hoping that mutations near the ribosome peptidyltransferase site might alleviate its bias towards α-L-amino acids, a library of modified ribosomes was generated. Analogues of the naturally occurring antibiotic puromycin were used to select promising candidates that would allow incorporation of non-α-L-amino acids into proteins. Syntheses of three different puromycin analogues are described here. The reverse transcriptase enzyme from HIV-1 (HIV-1 RT) has been a popular target of HIV therapeutic agents due to its crucial role in viral replication. The 4-chlorophenyl hydrazone of mesoxalic acid (CPHM) was identified in a screen designed to find inhibitors of strand transfer reactions catalyzed by HIV-1 RT. Our collaborators designed several analogues of CPHM with different substituents on the aromatic ring using molecular docking simulations. Work presented here describes the synthesis of eight different analogues of CPHM.
Dissertation/Thesis
Ph.D. Chemistry 2013

Tese de doutoramento, Química (Química Orgânica), Universidade de Lisboa, Faculdade de Ciências, 2010.
Inspired by the role of carbohydrates as natural scaffolds, we exploited the sugar skeleton to generate new libraries of polyfunctionalized compounds as GABAA receptor ligands. Hybrids of benzodiazepines, γ-butyrolactone and -lactam derivatives, and a GABA analogue were developed. The incorporated sugar moiety offered the possibility of diverse and controlled functionalization, modulating physicochemical and structural properties, namely solubility and rigidity, and consequently biological activity of the synthesized scaffolds. 1,4-Benzodiazepine-2,5-dione scaffolds (i and iv) derived from spiro bicyclic D- or Lproline analogues (ii and v), containing a D- or a L-fructose moiety were synthesized in the present work. The D-proline analogue ii was prepared in a sixteen-step synthesis in 24% overall yield, adopting a methodology which used D-fructose as starting material and 3-C-(3,4,6-tri-O-benzyl-α-D-fructofuranos-2-yl)propene iii as key intermediate. Instead, the L-fructose moiety, required for the preparation of the corresponding L-proline analogue v, was obtained using a new synthetic pathway. The key intermediate of this synthesis, the 3-C-(3,4,6-tri-O-benzyl-α-L-fructofuranos-2- yl)propene vi, was effectively obtained from L-arabinose through a seven steps pathway in 13.5% overall yield. This starting material embodies the furanose ring with the appropriate configuration. Hence, a one carbon chain elongation at the anomeric position lead to the desired L-fructose derivative, compound vi. For that purpose, oxidation to arabinonolactone, formyl group introduction via a dithiated intermediate and reduction to the primary alcohol were performed. The subsequent eleven-step synthesis afforded the new spiro bicyclic L-proline analogue v in 24% overall yield. In addition, a procedure previously reported for L-fructose synthesis was also used to obtain 3-C-(3,4,6-tri-O-benzyl-α-L-fructofuranos-2-yl)propene vi. Starting from L-sorbose, diisopropylidene protection, mesylation, selective deprotection, oxirane formation and opening led to inversion of configuration at C-3 and C-4 affording the stereochemistry of L-fructose. The key intermediate vi was obtained by this eight-step synthesis with a 8.5% overall yield. Molecular modelling calculations, NMR studies at variable temperature (dynamic NMR–DNMR) and preliminary biological studies were also performed on these compound libraries of enantiomeric fructose–proline–benzodiazepine derivatives (i and iv). The proline moiety linked to the fructose derivative through a spiro junction and consequent fusion of this bicyclic structure to the benzodiazepine ring promoted high conformational rigidity to the hybrid scaffold. Therefore, the conformational equilibrium normally occurring with (P) and (M) benzodiazepine conformers was not observed. The benzodiazepine derivatives synthesized adopted a rigid conformation in which the C-11a substituent was always pseudo-equatorial. The diverse and controlled functionalization of these molecules (i and iv) was achieved using different isatoic anhydride types which are commercially available. Coupling of D- or L-proline analogues, ii and v, respectively, with the suitably functionalized isatoic anhydride, afforded the desired sugar-based pyrrolobenzodiazepine library (i and iv). These compounds weres functionalized not only with electronegative substituents, such as –Cl, –Br and –NO2, but also with –NH2 at position seven, while at position ten the presence of –CH3 is expected to give a more appropriate binding affinity, as suggested by reported QSAR studies. The ability of these compounds to displace the [3H]flunitrazepam from the GABAA receptor was measured with a competition binding assay using rat cortical membrane. It was observed that substitution on both positions are crucial for binding affinities, being particularly effective –NO2 and –NH2 groups. The free –OH or –CH2Ph groups on the sugar offered the possibility to balance hydrophilic/lipophilic for tuning the pharmacokinetic properties and the binding affinities of the potential drug. As expected, water soluble sugar-based pyrrolobenzodiazepine derivatives, i.e. with –OH groups, presented highest binding affinities. The effect of conformational changes in the 1,4-benzodiazepine-2,5-dione ring and of different substituents allowed an evaluation of binding affinities at the benzodiazepine site on the GABAA receptor. Additionally, was developed the synthesis of β-disubstituted D-fructose-based γ-butyrolactone vii and γ-butyrolactam analogues viii, and that of a lipophilic D-fructose-based GABA analogue ix, where the pharmacophore is engineered into the carbohydrate scaffold through a spiro junction. The ability to bind GABAA receptor, using a radioligand binding technique, was evaluated for all these compounds. GABA lactones vii were synthesized via the key intermediate 3-C-(3,4,6-tri-O-benzyl-α-Dfructofuranos- 2-yl)propene iii, while γ-butyrolactams viii and GABA analogue ix took advantage of the allyl group and an amino functionality replacing the hydroxyl group in iii. The fructose moiety acted as versatile scaffold, rich in stereochemistry and with a relatively rigid skeleton. Thus, additional hydroxyl derivatization was used to increase lipophilicity, as well as to modulate the activity of pharmacophores or the receptor specificity, since benzyl groups could facilitate blood-brain barrier (BBB) crossing, which is one of the main issues to be addressed for central nervous system (CNS) directed drugs.
Os hidratos de carbono constituem uma família única de compostos polifuncionais com primordial importância em química e biologia. Funcionalidade, quiralidade e diversidade estrutural são algumas das suas características mais relevantes. Neste contexto, dirigimos esta investigação para a síntese de novas estruturas que possuem unidades monossacarídicas com rigidez estrutural e ainda uma complexidade e diversidade tais que permitiram a construção de novas bibliotecas de compostos bioactivos. Inspirados pela proeminente utilidade dos hidratos de carbono como scaffolds em química medicinal, foram sintetizadas duas bibliotecas de potenciais ligandos do receptor GABAA, geradas a partir da D-fructose e de um derivado da L-fructose. A primeira biblioteca de compostos inclui várias estruturas análogas às benzodiazepinas e a segunda contém γ-butirolactonas, γ-butirolactamas e um análogo do ácido gama-aminobutírico (GABA). Todos estes compostos foram submetidos a ensaios biológicos, nomeadamente utilizando a técnica que investiga a competição entre o composto e o radioligando correspondente relativamente à afinidade para a ligação ao receptor no seu local específico. O GABA é um neurotransmissor inibidor do sistema nervoso central. Os seus efeitos resultam principalmente da sua ligação ao receptor GABAA, que constitui uma estrutura macromolecular em volta de um canal iónico permeável ao cloreto. A fixação do GABA ao receptor GABAA causa um aumento da condutância da membrana celular ao cloreto, que habitualmente existe em maior concentração no exterior do que no interior da célula. O movimento de aniões para dentro da célula aumenta a diferença de potencial entre a face externa e a interna da membrana celular e reduz a excitabilidade neuronal. O GABA funciona como uma molécula reguladora de efeitos tais como ansiedade, depressão, insónias, convulsões, tensão ou relaxamento muscular, actividade epiléptica, memória, euforia e disforia. O receptor GABAA tem locais específicos de ligação para distintas moléculas que influenciam a actividade do GABA, tais como benzodiazepinas, barbitúricos, etanol, esteróides, etc. As benzodiazepinas são um grupo de fármacos ansiolíticos utilizados no tratamento sintomático da ansiedade, insónia, depressão e distúrbios mentais em geral. Estas moléculas ligam-se a um local próprio, denominado receptor das benzodiazepinas, e aumentam a afinidade do GABA para o receptor GABAA. Os barbitúricos, por exemplo, fixam-se também ao receptor GABAA e provocam uma abertura prolongada do canal do cloreto. Diferem das benzodiazepinas porque a abertura do canal não exige a presença de GABA, ao contrário das benzodiazepinas que se limitam a potenciar o efeito do GABA endógeno. Além disso a abertura é mais prolongada, enquanto que as benzodiazepinas aumentam a frequência das aberturas sem prolongarem a duração de cada uma delas. no presente trabalho é apresentada a síntese de uma biblioteca de enantiómeros análogos das benzodiazepinas, i e iv, desenvolvidos a partir da D-frutose e de um derivado da L-frutose, respectivamente. A estratégia adoptada centrou-se na síntese de um novo análogo da D- e da L-prolina, ii e v, respectivamente, com uma estrutura bicíclica espiro que contém a unidade sacarídica. A junção espiro reduziu significativamente a flexibilidade molecular, induzindo restrições conformacionais ao anel da prolina e consequentemente a toda a estrutura da molécula final (comprovado por NMR a temperaturas variáveis e estudos de modelação molecular). A escolha do monossacárido frutose resultou do facto deste açúcar oferecer potencialidades interessantes devido à presença de grupos hidroximetilo (em C-1 e C- 6) e devido à reactividade única do seu carbono anomérico. A síntese de derivados protegidos da frutose baseia-se na ramificação no carbono C-2 (iii e vi) e posterior reacção de ciclização entre uma função amina e um grupo alilo ligado à posição anomérica dando origem ao anel de pirrolidina seguidamente transformado no aminoácido ii e v. O plano de síntese para o análogo da D-proline ii, envolveu a metilação da posição anomérica e benzilação das restantes posições utilizando a metodologia clássica (NaH, BnBr, DMF). Seguidamente foi introduzido um grupo alilo na posição anomérica através da formação de uma ligação C-C com aliltrimetilsilano mediada por um ácido de Lewis. A reacção dá-se geralmente por intermédio de um ião oxocarbénio cíclico, o qual sofre um ataque nucleófilo estereoquimicamente controlado, conduzindo a uma elevada estereoselectividade tanto em anéis de piranose como em anéis de furanose. Consequentemente, o tratamento de 1,3,4,6-tetra-O-benzilfrutofuranósido de metilo com aliltrimetilsilano na presença de eterato de trifluoreto de boro originou 3-C-(1,3,4,6-tetra-O-benzilfrutofuranos-2-il)propeno. A relação espacial entre a ligação dupla e o oxigénio do grupo benziloxi do carbono C-1 favorece a ocorrência da ciclização 5-exo por tratamento com iodo, desprotegendo selectivamente o grupo hidroxilo primário de C-1. A introdução da amina foi realizada através da oxidação a aldeído do álcool primário, formação de oxima e redução com hidreto de alumínio e lítio. Uma segunda iodociclização, desta vez envolvendo a função amina, protegida com um grupo carbamato, e o grupo alilo, conduziu à formação do anel pirrolidina. Tratamento com hidróxido de sódio permitiu, através da formação de uma oxazolidinona intermediária, obter o composto que possui a função álcool primário e a estereoquímica desejada. A sua oxidação conduziu à síntese do aminoácido prolina. O plano de síntese do análogo da L-proline v envolveu a preparação de um derivado da L-frutose, uma vez que este açúcar não natural é comercializado a um preço bastante elevado. Foram desenvolvidas duas metodologias para a formação do intermediáriochave, o composto 3-C-(3,4,6-tetra-O-benzil-α-L-frutofuranos-2-il)propeno, uma das quais se baseou num procedimento descrito em literatura para a síntese da L-frutose, o qual usa a L-sorbose como composto de partida. Esta estratégia baseia-se na inversão da configuração das posições três e quatro para obter assim o intermediáriochave com a configuração do açúcar desejada. A estratégia alternativa desenvolvida partiu da L-arabinose, a qual possui a estereoquímica pretendida, resultou no alongamento da cadeia carbonada em C-1. Esta síntese é mais directa e permite obter a molécula-alvo com um rendimento ligeiramente superior (13,5% rendimento total) do que a utilizada inicialmente (8,5% rendimento total). O uso do aminoácido prolina na produção de pirrolobenzodiazepinas foi estabelecido como sendo um dos processos sintéticos mais eficazes e rentáveis, através do uso de anidrido isatóico em DMF e refluxo. O reagente anidrido isatóico pode ser diversamente substituído permitindo a obtenção de uma variada biblioteca de compostos, nomeadamente benzodiazepinas substituídas na posição sete com halogéneos (Br e Cl), grupos amina ou nitroílo e metiladas em N (posição dez). O nosso contributo singular na estrutura das benzodiazepinas consistiu na incorporação de um açúcar de modo a funcionalizar os grupos hidroxilo e modular as propriedades físico-químicas e estruturais, nomeadamente a solubilidade e a rigidez, e consequentemente a actividade biológica do potencial fármaco. A síntese destas estruturas rígidas teve como objectivo, não só permitir o estudo da relação estrutura-actividade, mas também uma análise acurada da especificidade conformacional do local de ligação no receptor das benzodiazepinas. Conforme descrito na literatura, o anel diazepina das benzodiazepinas tem um equilíbrio conformacional entre os confórmeros (M) e (P) e a ligação das mesmas ao receptor é dependente da conformação adoptada por estas. Embora existam estudos prévios que demonstraram que o receptor é favorável à conformação (M), a investigação desenvolvida no âmbito desta tese, utilizando enantiómeros conformacionalmente rígidos no anel diazepam, permitiu concluir que ambas as conformações apresentam actividades biológicas semelhantes para estes compostos. As actividades biológicas determinadas demonstraram que a contribuição mais significativa para a ligação específica ao receptor provém dos substituintes no anel do benzeno, nomeadamente os grupos amina e nitroílo (posição sete), bem como a N-metilação. Os grupos hidroxilo do açúcar também contribuíram para um aumento da actividade biológica. Paralelamente a estes análogos das benzodiazepinas, desenvolvemos uma segunda biblioteca de compostos, igualmente potenciais ligandos do receptor GABAA. Foram sintetizadas γ-butirolactonas e γ-butirolactamas β-disubstituídas, vii e viii, respectivamente, e um derivado do GABA ix. Estas estruturas bicíclicas possuem uma junção espiro com um derivado da D-frutose. Os resultados de estudos farmacológicos, envolvendo radioligandos através de ensaios de competição entre o composto e o muscimol marcado, indicaram que as lactamas são os compostos com maior actividade biológica, seguidas das lactonas. O açúcar mostrou ser uma estrutura esteroquimicamente não impeditiva para a ligação ao receptor e até mesmo os compostos benzilados tiveram actividades biológicas interessantes, o que reforça a importância de estruturas lipofílicas para atravessar a barreira hemato-encefálica (BHE). Em resumo podemos assumir que o trabalho desenvolvido conduziu ao desenvolvimento de novas estruturas glucídicas para a produção de novas bibliotecas de compostos com interesse farmacológico, que são excelentes candidatos para investigações de estrutura-actividade no receptor GABAA.

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens.
Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens.
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Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009