Rozprawy doktorskie na temat „Oligomers”

Nowadays, the emerging role of amyloid-β peptide (Aβ) oligomers in Alzheimer’s disease (AD) is widely accepted, putting aside the old idea that fibrils are the primary entities responsible for the onset of the disease. Recent studies indeed show that the level of soluble Aβ oligomeric forms better correlates with the progression of the disease than the level of fibrillar forms.

Using conditions which yield characteristic Aβ42 oligomers or fibrils, we studied the secondary structure of these species by ATR (attenuated total reflection)-FTIR (Fourier transform infrared) spectroscopy. Whereas fibrillar Aβ was organized in a parallel β-sheet conformation, oligomeric Aβ displayed distinct spectral features attributed to an antiparallel β-sheet structure. Antiparallel β-sheet structure may thus be a structural signature of oligomeric Aβ. Moreover, we noted striking spectral similarities between Aβ oligomers and a bacterial pore-forming protein, OmpF.

Apolipoprotein E (apoE) isoforms are strongly linked to Alzheimer’s disease, with the E4 isoform being the most recognized genetic risk factor so far. Nevertheless, the involvement of apoE4 in AD remains confusing. We evaluated the influence of apoE isoforms on Aβ aggregation in vitro. Comparing Aβ controls with Aβ incubated either with the apoE3 or apoE4 isoform, we observed a sharp reduction of the Aβ fibrillar content, whereas the oligomeric content was increased upon incubation with the pathological isoform apoE4. These data suggest that apoE4 binds and blocks Aβ in its oligomeric conformation, inhibiting further formation of less toxic fibrillar forms of Aβ. The enhanced interaction of apoE4 with Aβ oligomers could arise from its reported unique propensity to form a molten globule state, unlike the other isoforms of apoE. While previous studies mostly correlated E4 with fibrils, our data underline a correlation between apoE4 and Aβ oligomers. Our work reconciles apoE4 with the new amyloid cascade hypothesis and brings support to studies whose therapeutic strategy aims at designing inhibitors of the apoE/Aβ interaction./

Le rôle central des espèces oligomèriques du peptide amyloïde bêta (Aβ) dans la maladie d’Alzheimer est de plus en plus reconnu actuellement, mettant de côté l’ancien concept selon lequel les espèces fibrillaires sont les entités responsables du développement de la maladie. Des études récentes montrent en effet que le taux d’oligomères semble bien mieux corrélé à la progression de la maladie que le taux de fibrilles.

A l’aide de protocoles bien établis permettant de former des oligomères ou des fibrilles d’Aβ42 in vitro, nous avons étudié la structure secondaire de ces espèces par spectroscopie infrarouge en réflexion totale atténuée. Alors que les fibrilles présentaient une conformation en feuillets β parallèles, les oligomères quant à eux, ont révélé des caractéristiques spectrales distinctes, attribuées à du feuillet β antiparallèle. Cette structure en feuillets β antiparallèles pourrait donc représenter une signature structurale typique des espèces oligomèriques d’Aβ. De plus, nous avons observé de frappantes similarités spectrales entre les oligomères d’Aβ et une protéine bactérienne formant des pores, l’OmpF.

Les isoformes de l’apolipoprotéine E (apoE) sont fortement impliquées dans la maladie d’Alzheimer et plus particulièrement, l’isoforme E4 qui est actuellement reconnue comme étant le plus important facteur de risque d’origine génétique. Néanmoins, le rôle précis joué par l’apoE4 dans la maladie est encore mal connu. Nous avons étudié l’influence des isoformes de l’apoE sur l’agrégation du peptide amyloïde in vitro. En comparant des échantillons contrôle d’Aβ avec des échantillons incubés en présence d’apoE3 ou d’apoE4, nous avons observé une nette réduction de la quantité de fibrilles ainsi qu’une augmentation concomitante de la proportion d’oligomères lors de l’incubation avec l’isoforme pathologique E4. Ces résultats suggèrent que l’apoE4 interagit avec Aβ et le bloque dans sa conformation oligomèrique, inhibant ainsi le processus d’agrégation et la formation de fibrilles, espèces moins toxiques. Cette plus forte interaction entre l’apoE4 et les oligomères d’Aβ pourrait s’expliquer par la propriété unique de l’apoE4 à former un état intermédiaire ‘molten globule’, ce qui n’est pas le cas des autres isoformes. Tandis que d’anciennes études ont corrélé l’apoE4 principalement avec les fibrilles, nos résultats mettent en évidence un lien entre l’apoE4 et les oligomères d’Aβ, respectivement l’isoforme pathologique et les espèces les plus toxiques du peptide. Ce travail réconcilie donc l’apoE4 avec la nouvelle hypothèse de la « cascade amyloïde » et soutient les études thérapeutiques visant à mettre au point des inhibiteurs spécifiques de l’interaction apoE/Aβ.

Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

A low molecular weight fraction was obtained from a mass polymerised PVC resin by using diethyl ether Soxhlet extraction followed by either preparative gel filtration or solvent fractionation. A gas chromatography - mass spectroscopy (GC-MS) analysis of this fraction revealed that, in addition to vinyl chloride (VC) oligomers, it contained a large number of other compounds, in particular a large concentration of phthalates. By using adsorption liquid chromatography it was possible to remove the phthalates, along with other contaminants having a similar or greater polarity, from the low molecular weight PVC fraction.

The nucleic acids contain chemical information in the form of a sequence of bases. This information content is expressed through sequence selective duplex formation and template directed synthesis. To date, the only programmable artificial information molecules that can truly rival the nucleic acids, in terms of their function, are structurally very similar to the nucleic acids. This thesis describes a synthetic approach to duplex forming hydrogen bonding oligomers that contain information in the form of a sequence of H-bond donor and acceptor groups, in the pursuit of a programmable material that is orthogonal to the nucleic acids. Chapter 1 is a literature review of natural and synthetic information molecules and their applications in nanotechnology, including an overview of the structurally reengineered versions of the nucleic acids, foldamers, template polymerisation and synthetic duplexes. The review highlights the absence of totally synthetic information oligomers, that are orthogonal to the nucleic acids, and Chapter 2 sets out the aims of this thesis, which is to address this gap. Chapter 3 describes the synthesis of oligomers equipped with phenol H-bond donors and phosphine oxide H-bond acceptors. Through cooperative H-bond formation, these oligomers form double stranded complexes, which were characterised by NMR titrations and thermal denaturation experiments. For each additional H-bond there is an order of magnitude increase in association constant. In Chapter 4 it is demonstrated that the modular design for the oligomers in Chapter 3 represents a general strategy to synthetic information oligomers. Two new classes of H-bond acceptor oligomer were synthesised, bearing pyridine and pyridine N-oxide groups. Both these systems also exhibit cooperative duplex formation with H-bond donor oligomers, which were characterised by NMR titration. Chapter 5 examines the ability of mixed sequence 3-mers formed of H-bond donors (phenol) and H-bond acceptors (pyridine N-oxide) to form duplexes in a sequence selective manner. All 8 combinations of donor and acceptor were synthesised, and NMR titrations were used to measure the association constants for each pairwise combination of oligomers. Sequence matched duplexes generally have the highest association constants, but there are some anomalies.

Parkinson disease (PD) is the main neurodegenerative disease that involves motor symptoms. About 1% of population above 65 years is affected by PD. Main symptoms are bradikinesia, resting tremor, postural instability, muscle rigidity, and sometimes, cognitive problems and personality. Neuropathological features of PD are neuronal death in the substantia nigra pars compacta and formation of cytoplasmatic inclusion, named Lewy bodies, constituted by fibrillar form of α-synuclein (aS). aS is a 140 amino acid protein, whose structure and function is yet not well defined. As a consequence of specific genetic mutations or environmental factors, it undergoes aggregation and forms amyloid fibrils. It is highly expressed in neuronal pre-synaptic nerve terminals. Its sequence is characterized by an amphipathic lysine-rich amino terminus, which governs binding to lipids and interactions with membranes and contains seven imperfect repetition of the sequence KTKEGV; by a hydrophobic central region (NAC, non-amyloid component), responsible for protein aggregation and α-sheet formation and a highly acidic C-terminal, rich in Pro and acidic residues. Overexpression of aS and mutations in its gene are associated with a premature development of PD. Mechanism that make aS a toxic protein has not yet been well clarified, but it seems clear that oligomeric forms, and not the final fibrillar forms, are the main responsible for the pathogenesis of PD. The project of this thesis focuses on the characterization of oligomers of aS that form in the presence of docosahexaenoic acid (DHA), and their interaction with membrane, to understand the mechanism of toxicity. DHA is one of the most abundant fatty acids in neuronal membrane and it has been correlated to PD. It has been demonstrated that dopaminergic cell cultures exposed to PUFAs accumulate soluble cytotoxic aS oligomers (Assayag et al., 2007). Indeed, aS seems to be involved in fatty acids metabolism (Golovko et al., 2007). Moreover, it was reported that, in PD patients, DHA concentration is enhanced in those area affected by aS inclusions. In vivo studies demonstrated that a DHA enriched diet enhances formation of aS oligomers (Sharon et al., 2003). In previous studies in this laboratory, it was analyzed the aggregation process of aS in the presence of DHA using different protein to DHA molar ratios (De Franceschi et al., 2009; 2011). Oligomers obtained in these conditions were characterized from a morphological point of view (De Franceschi et al., 2011). The presence of DHA (50:1 lipid:protein molar ratio) leads to the formation of stable oligomers, off-pathway in the aggregation process of aS, that have significant toxic activity on cells, suggesting that they are potentially relevant in the pathogenesis of PD (De Franceschi et al., 2011). In the first part of this thesis a characterization of oligomers have been conducted using several biophysical methods, since these oligomers are sufficiently stable to allow the use of these techniques. In particular, transmission electron (TEM) and atomic force (AFM) microscopy were used to study oligomers morphology and dimension. The secondary structure was evaluated by circular dichroism (CD). This spectroscopic analysis reveals that oligomers have a partial α-helix structure, in contrast with the majority of oligomeric species described in literature. It was analyzed also the ability of oligomers to interact with membrane, using liposomes of different size and composition and cell cultures. Interaction of oligomers with membrane, analyzed by CD measurements and leakage assays, causes the leakage of small molecule, demonstrating their ability to destabilize membranes. Oligomers activity was tested also on dopaminergic cell culture that showed an altered permeabilization after treatment. To determine the mechanism by which oligomers cause membrane permeabilization, different tests were performed. Initially, dynamic light scattering (DLS) and TEM allow to exclude a detergent-like effect. Moreover, aggregation studies and planar lipid membrane (PLM) measurements lead to hypothesize a toxicity mechanism that depends on the formation of a transient aperture or on the enhancement of flip-flop. This part of the thesis is object of a publication (Fecchio et al., 2013). Another aspect faced in this thesis is the study of chemical modification occurring on oligomers after exposition to DHA. Different chemical modification were evidenced by mass spectrometry: carbonylation and the formation of adduct with DHA at the level of His50. To deepen the role of this residue in the interaction with FA, it was used an aS variant, H50Q, that has recently been linked to familiar form of PD. Finally, also the interaction with DHA of other pathological variants of aS (A30P, E46K, A53T) was studied. In particular their secondary structure and oligomerization in the presence of DHA were analyzed, in comparison with results obtained with aS. In conclusion, this study supplied further information about structure and activity of oligomeric species that are potentially relevant in PD pathogenesis. These data can be compared to oligomers produced in different conditions or formed by different amyloidogenic proteins: this knowledge would be fundamental to the development of therapeutic agent that would prevent or defeat these kind of debilitative diseases
Il morbo di Parkinson (PD) è la principale malattia neurodegenerativa riguardante la funzionalità motoria. L'1% della popolazione sopra i 65 anni è affetto da questa malattia. I sintomi principali sono bradichinesia, tremore a riposo, instabilità posturale, rigidità muscolare e, talvolta, problemi cognitivi e della personalità . Le principali caratteristiche neuropatologiche del PD sono la morte dei neuroni dopaminergici a livello della substantia nigra pars compacta e la formazione di corpi d'inclusione citoplasmatici composti da aggregati proteici fibrillari di tipo amiloide, Lewy bodies (LBs), il cui costituente principale è α-sinucleina (aS) (Spillantini et al., 1998). aS è proteina di 140 amminoacidi, natively unfolded, la cui funzione, nonostante il suo ruolo chiave nel PD, non è ancora completamente chiarita. E' espressa in livelli alti nel sistema nervoso centrale ed è abbondante nei terminali presinaptici neuronali. Strutturalmente è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale con numerosi residui acidi. La sovraespressione di aS e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Il meccanismo con cui un cambiamento nella struttura e nell'espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito, ma è sempre pi๠accreditata l'ipotesi che siano le forme oligomeriche e non gli aggregati finali fibrillari ad essere responsabili della malattia. Il progetto di questa Tesi riguarda la caratterizzazione di oligomeri tossici di α-sinucleina (aS) ottenuti in presenza di acido docosaesaenoico (DHA) e lo studio della loro interazione con membrane lipidiche, allo scopo di comprendere il meccanismo con cui esercitano la loro tossicità . Il DHA è uno dei principali acido grassi cerebrali, strettamente correlato al PD e ad aS. L'esposizione di colture cellulari dopaminergiche a PUFAs porta all'accumulo di oligomeri solubili di aS, responsabili della citotossicità associata alla neurodegenerazione (Assayag et al., 2007). Esistono poi evidenze dell'implicazione di aS nella regolazione del metabolismo degli acidi grassi (Golovko et al., 2007). Inoltre è stato osservato che, nei pazienti affetti da PD, la presenza di DHA è maggiore nelle aree cerebrali contenenti inclusioni di aS. Studi in vivo, infine, hanno dimostrato che il DHA induce la formazione di oligomeri di aS (Sharon et al., 2003). In precedenti studi condotti nel laboratorio dove è stata svolta questa Tesi è stato analizzato il processo di aggregazione di aS in presenza di DHA, utilizzando due diversi rapporti molari proteina/acido grasso) (De Franceschi et al., 2009) e gli aggregati proteici ottenuti in queste condizioni sono stati caratterizzati da un punto di vista morfologico e strutturale (De Franceschi et al., 2011). E' stato osservato che la presenza di DHA in rapporto molare 50:1 rispetto alla proteina, porta alla formazione di oligomeri stabili, off-pathway nel processo di fibrillazione, che presentano una significativa attività tossica sulle cellule rispetto al monomero di aS. Nella prima parte di questa ricerca è stata condotta una caratterizzazione di queste specie oligomeriche che sono sufficientemente stabili nel tempo da consentire l'uso di diverse tecniche biofisiche. In particolare gli oligomeri sono stati analizzati mediante microscopia elettronica a trasmissione (TEM) e a forza atomica (AFM), per studiare le dimensioni e la morfologia. Il tipo di struttura secondaria è stata valutata mediante dicroismo circolare che ha dimostrato un'altra peculiarità di questi oligomeri, ovvero la presenza di struttura parzialmente in α-elica, diversamente dalla maggior parte degli oligomeri descritti in letteratura. E' stata analizzata anche la capacità degli oligomeri di interagire con le membrane, utilizzando liposomi di diversa dimensione e diversa composizione e colture cellulari. L'interazione tra gli oligomeri e i liposomi, studiata mediante CD e saggi di leakage, causa il rilascio di piccole molecole interne alle vescicole, dimostrando così un loro effetto destabilizzante sulle membrane. L'attività degli oligomeri è stata anche testata su cellule in coltura che mostrano un'alterata permeabilità in loro presenza. Per determinare quale sia il meccanismo di destabilizzazione degli oligomeri, sono stati eseguiti vari saggi. Si è dimostrato tramite dynamic light scattering (DLS) e TEM che le vescicole, in seguito al legame con gli oligomeri, non vengono distrutte. Inoltre mediante studi di aggregazione e analisi su planar lipid membrane portano a ipotizzare un meccanismo di tossicità dovuto alla formazione di un'apertura transiente o un aumento di flip-flop a livello delle membrane. I risultati di questa parte di tesi sono oggetto di una pubblicazione (Fecchio et al., 2013). Un altro aspetto che è stato approfondito in questo lavoro di Tesi è lo studio degli oligomeri da un punto di vista chimico, allo scopo di caratterizzare le modifiche chimiche presenti sulla sequenza della proteina in seguito all'esposizione al DHA. Sono state evidenziate diverse modifiche mediante spettrometria di massa tra cui carbonilazioni e presenza di addotti. Quest'ultimo tipo di modifica in particolare avviene a livello dell'istidina in posizione 50. Per approfondire il ruolo di questo aminoacido nell'interazione con gli acidi grassi è stato studiato il mutante H50Q di aS. Questa proteina modificata è tra l'altro responsabile di forme familiari del PD. Infine è stata studiata anche l'interazione di altre varianti patologiche di aS associate a PD, A30P, E46K e A53T con DHA. In particolare, è stata analizzata la loro struttura e la loro tendenza ad aggregare in presenza di DHA, nonchè la loro capacità di formare oligomeri, in confronto ai risultati ottenuti con aS. In conclusione questo studio ha permesso di fornire maggiori informazioni sulla struttura e di studiare l'attività di specie oligomeriche che sono potenzialmente molto rilevanti per la patogenesi del PD. La struttura e l'attività di questi oligomeri potrà essere confrontata con quelle di oligomeri prodotti in diverse condizioni sperimentali o di oligomeri prodotti da altre proteine amiloidogeniche. Questa conoscenza è fondamentale per sviluppare agenti terapeutici che prevengano o debellino queste malattie debilitanti ed in continuo aumento

Large scale Soxhlet extraction and fractional precipitation have been utilised to provide low molecular weight extracts from poly(vinyl chloride) (PVC), polystyrene and polyethylene. These extracts were then resolved into their constituent oligomers by high performance gel permeation chromatography and high performance liquid chromatography.

Nucleic acids store genetic information in the sequence of nucleobases. Through duplex formation and template directed synthesis, the information stored in nucleic acids determines their three-dimensional structure and function. Nucleic acids are essential molecules for biological processes and have been used in nanotechnology. Modified nucleic acids have been synthesised that still form duplexes and can be tolerated by enzymes, suggesting that it is possible to construct a synthetic system comparable to nucleic acids, orthogonal to nucleic acids. This thesis describes the synthesis of a new class of synthetic information molecule, characterisation of the duplex forming properties, and attempts at templated oligomerisation reactions. The new synthetic information molecule is based on the phenylacetylene oligomer framework developed by Moore and co-workers. Recognition was achieved via a base-pair that is made from a single point high affinity H-bond, with phenol as the H-bond donor (D) and phosphine oxide as the H-bond acceptor (A). The Sonogashira coupling was used to construct the phenylacetylene oligomer backbone. The AA, DD and AD 2-mers were synthesised and complementary 2-mers showed cooperative duplex formation. No intramolecular H-bonding due to folding was observed in the AD mixed 2-mer. Longer oligomers were synthesised using a method of oligomerisation and chromatographic separation by reverse-phase preparatory HPLC. Homo-oligomers up to the 7-mer were isolated and binding studies between complementary all donor, all acceptor homo-oligomers showed increasing duplex stability with each additional recognition unit in the oligomer chain. Oligomers containing both acceptor and donor recognition modules in the same chain were synthesised and NMR dilution studies were used to investigate their ability to fold. Preliminary experiments were carried out to evaluate the ability of these information molecules to template oligomerisation reactions, but when reactions were carried out at concentrations low enough for a significant template effect, no coupling reactions were observed.

This thesis looks at the synthesis of hybrid polyoxometalates (POMs), a family of polyoxometalate compounds grafted to organic moieties. Focussing particularly on the Mn-Anderson hybrid, it looks at new ways of isolating and purifying them and at how the organic component can be used to alter their crystal structure or to link multiple Mn-Anderson cores together to form short oligomer chains. In the first section, two Mn-Anderson hybrids are prepared with large aromatic organic moieties, as a continuation of earlier work looking at how aromatic organic components can influence the structure of Mn-Anderson compounds in the solid state. The two compounds, one based on an anthracene moiety (three fused aromatic rings) and another based on a tetraphenylphthalic moiety (five linked aromatic rings), were both synthesised and crystals were grown of their tetrabutylammonium (TBA) salts. These crystal structures were then analysed and their structure thoroughly described and compared to previous aromatic Mn-Anderson aromatic hybrids. In the second section, a set of methods are presented for the preparation and isolation of asymmetric Mn-Anderson hybrids, where two different organic moieties are linked to the same POM core. These methods use high pressure liquid chromatography (HPLC) and normal liquid chromatography (LC) techniques to separate the mixtures of symmetric and asymmetric products which inevitably result from the synthesis of the asymmetric compounds, formed via both pre- and post-functionalisation techniques, so long as the two organic moieties have sufficiently different hydrophobicity. In order to by-pass the hydrophobicity requirement, a "Universal" asymmetric Mn-Anderson was developed with a free amine and an FMOC-protected amine. This allows for good separation and also gives a product which can be used to form more complex asymmetric compounds via post-functionalisation reaction without further LC purification, and an asymmetric compound with two hydrophilic substituents was synthesised to prove this strategy. All compounds were then fully analysed to confirm the purity of the asymmetric products. Also included is a means to remedy unwanted protonation of TBA Mn-Anderson salts. In the final section, the easy access to asymmetric Mn-Anderson hybrids provided by the previous project is exploited to produce a series of short-chain POM oligomers. A coupling strategy using alkyne and azide Mn-Anderson hybrids was developed, and a series of symmetric and asymmetric building blocks based on these functional groups were synthesised. These were then strategically combined to form a dimer and trimer of Mn-Anderson cores, which could then be further functionalised with alkyne groups and have an additional two Mn-Anderson units added, to give a tetramer and a pentamer. Methods were then developed to successfully purify these oligomers from the excess starting materials used to make them and the compounds analysed to confirm their identity. Size-exclusion HPLC and ion-mobility mass spectrometry (IM-MS) were then performed to gain more information about the structure of these compounds and how they arrange themselves in solution and in the gas-phase.

This thesis presents the synthesis and characterisation of a range of heterometallated porphyrin oligomers and other novel 3D π-conjugated porphyrin nanostructures. Subsequently, their physical organic properties were evaluated which revealed some fascinating electronic properties. Chapter 1 summarises some of the work done in the Anderson group on porphyrin nanostructures and reviews the literature regarding heterometallated porphyrin oligomers. In addition it introduces the main concepts and techniques used in the remainder of the thesis. In Chapter 2 the stabilities of a family of four linear porphyrin pentamer complexes are determined by UV-vis-NIR titrations and analysed using chemical double-mutant cycles which reveal that the binding energy of the copper centre to an axial pyridine ligand is -6.2 kJ mol^-1. Subsequently, the Zn-Zn-Cu-Zn-Zn pentamer is used in the synthesis of a heterometallated 10-porphyrin nanoring. Chapter 3 will describe the investigation of quantum interference phenomena in a bis-copper six-porphyrin nanoring by using EPR spectroscopy. We show that the exchange coupling between two spin centres is increased by a factor 4.5 in the ring structure with two parallel coupling pathways as compared to an otherwise identical system with just one coupling path. In Chapter 4 the syntheses of three isomers of the bis-copper 6-porphyrin nanoring are described. DFT calculations have indicated potential destructive interference phenomena in one of the isomers which would allow for the formation of a molecular system with behaviour resembling that of a hypothetical molecular interferometer. Chapter 5 reports on the template-directed synthesis of a π-conjugated 14-porphyrin nanoball. This bicyclic structure consists of two intersecting nanorings of 6 and 10 porphyrin units. Fluorescence up-conversion spectroscopy experiments demonstrate that electronic excitation delocalises over the whole 3D π-system within 0.3 ps if the nanoball is bound to its templates or within 5 ps if the nanoball is empty. In Chapter 6 the synthesis and characterisation of a D_4h symmetric analogue of the porphyrin nanoball is described. The structure consists of ten porphyrin units arranged as two perpendicular 6-porphyrin nanorings intersecting at two porphyrins. In the synthesis, a combination of magnesium and zinc porphyrins are used which allows for the introduction of a selective demetallation method crucial for accessing this novel structure.

Laccase-catalysed depolymerisation of lignin offers substantial opportunities to form bio-derived aromatic chemicals. Laccase works with exquisite selectivity to oxidise the 5-5’ and β-5’ linkages in lignin and to cleave the β-O-4’ linkages in the presence of 1-hydroxybenzotriazole (1-HBT). However, the laccase/1-HBT system is inefficient when applied to industrial lignin, and the complexity of the product mixtures hinders rapid optimisation. Therefore, I studied laccase-catalysed oxidation of three synthetic lignin model oligomers, which had well-defined structures, yielded analytically tractable product mixtures, and, thus, permitted faster optimisation. Solvent systems were developed to dissolve the lignin model oligomers and retain laccase activity, which aided mass transfer. Models of “hardwood” (S:G 1:1) and “softwood” (G only) lignin containing nonphenolic β-O-4’ linkages (Angewandte Chemie-International Edition, 54, 258) were oxidised quickly by benzylic carbonyl formation ( > 60% in 24 h), or β-O-4’ bond cleavage. Approximately 25% of the linkages in the “hardwood” (S:G 1:1) oligomer were cleaved in 24 h. A more complex model of “softwood” lignin, containing β-5’, β-O-4’ and 5-5’ phenolic linkages (Green Chemistry, 15, 3031), was oxidised just as rapidly, but reaction products in this case were phenolic dimers that repolymerised. Therefore, I conclude that future bio-production of aromatic chemicals from lignin will depend on the development of improved lignin extraction procedures to allow retention of the β-O-4’ linkages, which will produce more efficient enzymatic depolymerisation.

Duchenne muscular dystrophy (DMD), the most common lethal neuromuscular disease in childhood, arises from protein-truncating mutations in the dystrophin gene. A deficiency in dystrophin leads to loss of the dystrophin associated protein complex (DAPC), which in turn, renders muscle fibres vulnerable to injury, and eventually leads to muscle loss, necrosis and fibrosis. Although, the dystrophin gene was identified nearly two decades ago, and extensive research has been directed at finding a therapy for DMD, to date, there is still no effective treatment available. One promising molecular approach to treat DNID is antisense oligomer (AO) induced splice intervention. AOs were most widely used to induce RNaseH-mediated gene transcript degradation, however, the development of different backbone chemistries heralds a new generation of AOs that can modify gene transcript splicing patterns. Application of AOs to the dystrophin pre-mRNA to influence exon selection and induce shortened, in-frame dystrophin isoforms is being vigorously pursued. The majority of the work presented here explores the concept of personalised therapies for DMD, whereby oligomers are designed to specifically target individual mutations. The importance of AO-optimisation to obtain AOs capable of inducing efficient dual exon skipping in an established animal model of muscular dystrophy (4CV mouse), which carries a DMD-causing mutation in exon 53, is demonstrated. Removal of both exons 52 and 53 was required to by-pass the mutation, maintain the reading frame and restore dystrophin expression. One of the major challenges of AO-induced splice intervention for therapeutic purposes will be the design and development of clinically relevant oligomers for many different mutations. Various models, including cells transfected with artificial constructs and mice carrying a human dystrophin transgene, have been proposed as tools to facilitate oligomer design for splice manipulation. This thesis investigates the relevance of using mouse models to design AOs for human application, and also explores the use of cultured human myoblasts, from both unaffected humans and a DMD patient, as a means of establishing the most effective therapeutic compound. In addition to induction of exon skipping, the applicability of AOs to promote exon inclusion, by masking possible intronic silencing motifs of survival motor neuron (SMN) pre-mRNA in cultured fibroblasts from a spinal muscular atrophy (SMA) patient, is investigated. This study provides additional information about a novel oligomer target site that could be used in combination with previously identified splice silencing motifs for a molecular therapeutic approach to SMA, and may perhaps open up new avenues of treatment for other genetic disorders, where oIigomers could be used to induce exon inclusion.

Au cours de cette thèse, nous avons synthétisé et caractérisé de nouveaux composés en utilisant le macrocycle porphyrine en tant que brique moléculaire. L’objectif, après la synthèse, était d’étudier les propriétés photophysiques de ces nouvelles molécules. Plus précisément, nous avons considérés trois groupes de dendrimères fluorènyles-porphyrines et deux oligomères avec des structures conjuguées. Les corrélations structures et propriétés optiques ont été discutées en détail, ainsi que le transfert d’énergie du dendron conjugué donneur. Dans le premier chapitre, nous avons décrit la chimie des porphyrines en considérant surtout trois aspects: (i) structure, (ii) propriétés optiques et (iii) méthode synthétique. Nous avons également reporté les porphyrines préalablement étudiées dans notre groupe de recherche et proposé de nouveaux modèles lors de cette thèse. Dans le deuxième chapitre, nous avons étudié l’influence de la position de substitution du phényle sur le coeur central qui est la porphyrine TPP. Deux groupes de nouveaux dendrimères ont été synthétisés avec différentes positions de substitution du phényle de la TPP: série parasubstituée TPP1, TPP2 et TPP3, et série méta-substituée TPP4, TPP5 et TPP6. Ces dendrimères ont tous des dendrons conjugués avec des ponts phényl-alcynes et des groupements fluorènyles terminaux. Dans le troisième chapitre, pour étudier l’influence des antennes périphériques, une série de dendrimères de porphyrine à base de TFP avec différentes positions de fluorènyles dans les dendrons conjugués (fluorényle central, fluorényle pontant et fluorényle terminal): TFP1, TFP2 et TFP3 a été synthétisée. Le fluorényle central influence principalement l’émission et le rendement quantique, alors que le fluorényle ponté et le fluorényle terminal ont un plutôt un effet sur l’absorption et l’émission du dendron en raison du transfert d’énergie prenant effet. Pour le plus gros dendrimère TFP3, lors de l’excitation du dendron, une émission résiduelle lors du processus de transfert est observée due à une trop grande distance entre ce chromophore et le coeur de la porphyrine. Dans le quatrième chapitre, afin d’étudier l’influence des connexions dans le dendron, deux nouveaux dendrimères de porphyrine incorporant des ponts vinyles et alcynyles, TPP-D et TFP-D, ont été synthétisés et comparés aux analogues avec des ponts alcynes TPP-T et TFP-T. Ces ponts avec des doubles liaisons influencent dans une certaine mesure les propriétés optiques des dendrimères de porphyrine en fonction des positions occupées. Dans le dernier chapitre, deux oligomères (un dimère linéaire et en un trimère en étoile), ont été synthétisés à partir du même monomère de porphyrine avec TFP comme groupement terminaux
During this thesis, we have worked on the synthesis and characterization of new compounds using the porphyrin macrocycle as a starting material. The aim, after synthesis, was to study the photophysical properties of these new molecules. More precisely, we have synthesized and characterized three groups of fluorenyl-porphyrin dendrimers and two oligomers in conjugated structures. Their correlation on optical property-structure have been discussed in detail, as well as the energy transfer processes from the conjugated dendron donor to porphyrin acceptor. In the first chapter, we introduce the general background of the porphyrin chemistry based on three aspects: (i) structure, (ii) optical properties and (iii) synthetic methods. We further review prior porphyrin studies done in our group and propose new molecular designs based on these results. In the second chapter, we study the influence of substitution position and nature of the antennae on the central core. Two groups of new TPP-cored porphyrin dendrimers were synthesized according to different substituted positions on the peripheral phenyl rings: para-substituted series TPP1, TPP2 and TPP3, and meta-substituted ones TPP4, TPP5 and TPP6. All have conjugated dendrons with bridged phenyl-alkynyl and are terminated by fluorenyl groups. In the third chapter, we study the influence of positions of light absorbers. To this aim, a series of TFP-cored porphyrin dendrimers presenting fluorenyls in conjugated dendrons on different positions is reported (core fluorenyl, bridging fluorenyl and terminal fluorenyl): TFP1, TFP2 and TFP3. The core fluorenyl mainly influences emission peaks and quantum yield, while the bridging fluorenyl and terminal fluorenyl have a large effect on the dendron absorption and on the energy transfer efficiency. Thus, for the largest dendrimer TFP3, dendron emission is not totally quenched by long distance energy transfer process when exciting the peripheral dendrons. In the fourth chapter, we study the influence of linkages in the Dendron. Thus, two new porphyrin dendrimers with bridged vinyl, TPP-D and TFP-D, were synthesized and compared to analogues with alkynyl bridged TPP-T and TFP-T. Vinyl and alkynyl bridges are shown to influence the optical properties of the porphyrin dendrimers to a certain extent depending on the substituted positions. Finally, in the last chapter, two oligomers with n-butyl substituted TFP as terminal group, a linear dimer and a star – shaped trimer, were synthesized from the same porphyrin monomer

This thesis presents a spectroscopic and computational study of a variety of substituted oligothiophenes. The purpose of this study was to elucidate the structural properties of their neutral, oxidised and excited states. This was accomplished using a number of spectroscopic methods, including electronic absorption, resonance Raman and fluorescence spectroscopy. Analysis of the data provided by these methods was aided by the application of density functional theory (DFT) to the neutral and charged species, allowing conclusions regarding bond length changes and molecular orbitals to be drawn. Advancing the understanding of the structural properties of conductive materials such as oligothiophenes is important due to their potential application in plastic electronic devices (organic solar cells, field effect transistors and light emitting diodes, for instance) and the need to clarify the charge transport mechanism. The compounds examined in this thesis are primarily based on 3� styryl-substituted terthiophene. Substituted derivatives of this molecule include those with groups on the para position of the phenyl ring or methyl α,α� end caps. Larger ethenyl-aryl groups were also investigated, as were analogous thiophenes and sexithiophenes. Indeed, the 3�-substituted sexithiophenes formed an integral part of this investigation since they rapidly form in the head-to-head orientation from the oxidation and consequent [sigma]-dimerisation of the substituted terthiophenes. DFT calculations on the sexithiophene charged species have indicated the presence of a full polaronic or bipolaronic structural defect in the centre of the thiophene backbone, as defined by the reversal of the CC bond length alternation to create a domain of quinoidal bond sequence. However, the structural defect of the styryl sexithiophenes is more strongly localised than that observed for unsubstituted sexithiophene; indeed, the charged defect appears to be confined by the positions of the styryl substituents. This defect confinement along the thiophene backbone is particularly apparent in the styryl sexithiophene dications. Electronic absorption and resonance Raman spectroscopy have been applied to the oxidation products of the styryl terthiophenes. Only sexithiophene charged species are observed when the α termini are unsubstituted; these include sexithiophene radical cations, [pi]-dimers (a stabilising face-to-face interaction between two radical cations) and dications. The resonance Raman spectra of these charged species are characterised by a very intense symmetrical CC stretching mode of the thiophene inter-ring bonds, which is also evident in the theoretical spectra. The extensive differences between the styryl and unsubstituted sexithiophene dication Raman spectra have been attributed to defect confinement: the strongest Raman band of the unsubstituted sexithiophene dication is due to a vibrational mode localised over the entire sexithiophene backbone while that of the styryl sexithiophenes is localised over the central two thiophene rings - the same area occupied by the confined defect. A combined steady-state and picosecond time-resolved emission study on the styryl terthiophenes revealed that by judicious choice of the para R group in styryl terthiophenes it is possible to tune the nature of the first excited singlet state from [pi],[pi]* to charge transfer character. Charge transfer states may offer possible strategies for solar cells in which charge separation is a key step.

This thesis deals with the synthesis of new organicmaterials for electronic applications. Several new ring-formingmethods are employed to construct sulphur heterocycles: Tandemelectrophilic aromatic substitution and acid-catalyzedtransetherification of methoxythiophene, double electrophilicaromatic substitutions with ethane-1, 2-disulphenyl chloride,and also, the reaction of dienes with sulphur dichloride. Twonew condensed thiophenes have been incorporated in end-cappedoligothiophenes. An improvement of the synthesis of [3,2-b:2’, 3-d]thiophene is reported, with some attempts toincorporate it in oligomers. A synthesis of substitutednaphthalenes is also described. A new method of producingdisubstituted thiophenes from substituted butadienes anssulphur dichloride is employed in a new route to 3, 4-ethylenedioxythiophene, a very important monomer for conductingpolymers.

Keywords :Organic Semiconductors, Thiophenes,Heterocyclic Synthesis, Dithienothiophene, Naphthalene,Ethylenedioxythiophene

Les organismes vivants produisent des peptides antimicrobiens (PAMs) pour se protéger contre les microbes. La résistance croissante aux antibiotiques nécessite le développement de nouvelles stratégies thérapeutiques et les PAMs sont des candidats prometteurs pour résoudre ce problème. Ils possèdent une activité à large spectre et leur principal mécanisme d'action par perméation de la membrane engendre peu de phénomènes de résistance. Néanmoins, leur faible biodisponibilité empêche leur utilisation. Certaines limitations peuvent être surmontées en développant des mîmes de PAMs qui conservent leur activité mais avec un potentiel thérapeutique accru. Les peptoïdes (oligomères de N-alkylglycine) structurés en hélice cationique amphiphile sont de bons mimes de PAMs. Les peptoïdes sont plus flexibles que les peptides en raison de l'isomérie cis/trans des amides N,N-disubstitués ; cependant la conformation des amides peut être contrôlée par un choix judicieux des chaînes latérales. Le but de cette thèse est d'étudier l'influence de chaînes latérales(hydrophobes ou cationiques) bloquant la conformation des amides en cis et induisant une structure hélicoïdale de type PolyProline I (PPI) robuste, sur l’activité antibactérienne et la sélectivité de peptoïdes. La conception, la synthèse et l’étude conformationnelle de nouveaux oligomères peptoïdes cationiques portant des chaînes latérales de type tert-butyle et/ou triazolium ont été réalisées. Dans un premier temps, la synthèse en solution d'oligomères à base de tert-butyle a été développée puis une stratégie de synthèse en phase solide a été mise en place pour accéder aux oligomères à base de 1,2,3-triazolium. Ensuite, ces nouveaux oligomères ont été évalués pour leur activité vis à vis d’un panel de bactéries Gram-positive et Gram-négative, leur l'activité antibiofilm et leur sélectivité cellulaire. Enfin, pour visualiser les effets des peptoïdes amphiphiles sur les bactéries, une étude de microscopie a été réalisée
Living organisms produce antimicrobial peptides (AMPs) to protect themselves against microbes.The growing problem of antimicrobial resistance calls for new therapeutic strategies and the natural AMPs have shown ground-breaking potential to address that issue. They show broad-spectrum activity and their main mechanism of action by bacterial cell membrane disruption implies low emergence of resistance which makes them potent candidates for replacing conventional antibiotics. Nevertheless, few hurdles are impeding their use, notably poor bioavailability profile. Some of these limitations can be overcome by developing peptidomimetics of AMPs which exhibit antibacterial activities together with enhanced therapeutic potential. Peptoids (i.e. N-alkyl glycine oligomers) adopting cationic amphipathic helical structures are mostly competent AMP mimetics. From a conformational point of view, peptoids are fundamentally more flexible than peptides primarily due to the cis/trans isomerism of N,N-disubstituted amides but studies in this area have shown that cis amide conformation can be controlled by careful choice of side-chain to set a PolyProline I-type helical structure of peptoids. In this thesis, the genesis of novel amphipathic cationic peptoids carrying cis-directing tert-butyl and/or triazolium-type side-chains and their untapped potential to act against bacteria will be discussed comprehensively. First, the solutionphase synthesis of tert-butyl-based oligomers was developed. Second, novel method of solid-phase submonomer synthesis was optimised to access 1,2,3-triazolium-based oligomers. Then, the synthesised cationic oligomers were evaluated for their antibacterial potential, followed by antibiofilm activity and cell selectivity assays. In the end, to have insights on the mode of action of amphipathic peptoids, microscopy was carried out

The work presented in this thesis represents the chemical modifications of unsaturated plant oils to yield oligomeric/polymeric polyols suitable for polyurethane synthesis. Chapter 1 provides the introduction to the chemistry of polyurethanes, plant oils and plant oil based polyols. Chapter 2 focus on making oligomeric polyols from unsaturated plant oils through epoxidation and subsequent epoxide ring opening oligomerization that yielded oligomeric polyols. The properties of these oligomeric polyols were influence by the level of unsaturation of the plant oils. In addition, catalyst loading, monomer concentration and reaction time play vital role in determining the properties of the oligomeric polyols. Plant oil based polyols were also prepared by epoxide ring opening with renewable polyhydric alcohols that provide a variety of plant oil based polyols for polyurethane synthesis. Chapter 3 focus on the synthesis of polyurethanes (PU) from various types of plant oil based polyols as well as the evaluation of the mechanical properties of these synthesized PU. The tensile test of the PU shows that the mechanical properties were related to the structure and functionalities of the plant oil based polyols. The bulk of Chapter 4 discusses the copolymerization of epoxidized plant oils with tetrahydrofuran and the use of these copolymers for the synthesis of PU. The properties of the copolymers were related to the epoxidized oils used in the reaction and therefore influence the mechanical properties of the PU synthesized from them. Finally, Chapter 5 is a collection of work on the one-pot oligomerization of unsaturated fatty acid and plant oils with and without catalyst as well as the synthesis of PU based on these polyols.

This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

In this project it was planned to synthesise and investigate the physical, chemical and electrical properties of the "polyquinoxaline" family of materials. The compounds synthesised and studied included 5,14-dihydro-5, 7,12, 14-tetraazapentacene(L5H[sub]2), dibutyldihydro-5, 7,12, 14-tetraazapentacene (L5Bu[sub]2H[sub]2), 7, 16-dihydro-5, 7,9,14,16,18-hexaazaheptacene(L7H[sub]2), 7,20-dihydro-5, 7, 9, 11, 16, 18, 20, 22-octaazanonacene (L9H[sub]2), dibutyldihydro-5, 7, 9, 11, 16, 18, 20, 22- octaazanonacene (L9Bu[sub]2H[sub]2), "Polyquinoxaline"(poly[ 1, 6-dihydropyrazino(2, 3-g)-quinoxaline-2, 3,8-triyl-7(2H)-ylidene-7,8-dimethylidene) and the dibutyl-substituted (poly[ 1, 6-dihydropyrazino(2,3- g)-quinoxaline-2,3,8-triyl-7(2H)-ylidene-7,8-dimethylidene). Some alkyl-substituted oligomers were also made, so as to increase their solubility in organic solvents. Both liquid phase and solid phase synthesis have been used in preparing these compounds, the latter being done in an attempt to produce the compounds in their purest form. In the case of L7H[sub]2, this method gave the first successful synthesis of the compound. A number of techniques, such as FTIR, solid-state NMR and Visible / Ultraviolet spectroscopy have been used in characterising both the synthesised compounds and the first in the "quinoxaline" series of compounds phenazine (L3), which is commercially available. For solutions of the oligomers in concentrated sulphuric acid, the HOMO-LUMO absorption bands in the UV-visible decreased monotonically with the number of rings. The electrochromic properties of vacuum evaporated thin films of dihydrotetraazapentacene (L5H[sub]2) and dihydrohexaazaheptacene (L7H[sub]2) have been investigated using cyclic voltammetry. These investigations suggest that L7H[sub]2 thin films exhibit similar intermediate redox states to L5H[sub]2 films. Two-point electrical conductivity measurements have been carried out on pressed powder pellets of doped and undoped oligomers, substituted oligomers and ladder polymers. The electrical conductivities showed marked increases of five to six orders of magnitude as a result of doping the materials with hydrogen chloride vapour. The final major part of the project was the first fabrication of metal-semiconductor Schottky diode devices based on vacuum-evaporated thin films of dihydrotetraazapentacene (L5H[sub]2). Schottky diode devices on L5H[sub]2 films were successfully made using a range of metal contacts (Sn, Cu. AI, Pb, Au and Ag). Electrical measurements on various metal-doped L5H[sub]2 films gave I-V characteristics indicative of a Schottky barrier formed at a metal/n-type semiconductor interface. These electrical device measurements were mathematically modelled to determine the magnitude of the Schottky barriers at the various metal-semiconductor interfaces. This information and the electrochromic redox measurements on L5H[sub]2 thin films have helped to characterise the charge-transfer processes in these oligomer thin films.

This thesis presents a combined experimental and computational evaluation of the physical-organic properties of butadiyne-linked porphyrin oligomers. The principal result from the thesis is the synthesis and characterisation of the largest aromatic and antiaromatic systems to date, in the form of an oxidised [6]-porphyrin nanoring, with diameter 2.4 nm. This large electronically coherent system provides insight into the connection between aromatic ring currents and persistent currents in metal and semiconductor mesoscopic rings. Chapter 1 briefly reviews the concepts used in the remainder of the thesis, with a particular focus on aromaticity. In Chapter 2, the barrier to inter-porphyrin torsional rotation in a butadiyne-linked porphyrin dimer is determined computationally and experimentally to be 3 kJ mol^-1. The barrier height is closely related to the resonance delocalisation energy between the porphyrin subunits. In Chapter 3 we show that by oxidising a butadiyne-linked [6]-porphyrin nanoring to its 4+ and 6+ oxidation states, the nanoring becomes antiaromatic and aromatic respectively. In contrast, the neutral oxidation state exhibits only local aromaticity for the six porphyrin units. The 12+ cation can also be generated, and exhibits local antiaromaticity for each porphyrin unit. The characterisation of (anti)aromaticity employs NMR and computational techniques. In Chapter 4, the properties of cation radicals of linear and cyclic porphyrin oligomers are explored. Cations generated by spectroelectrochemistry are measured by optical spectroscopies, and chemically generated radical monocations are examined by cw/pulsed EPR spectroscopies. EPR and optical spectroscopies agree that the dimer monocation radical is fully delocalised, in Robin-Day Class III, whereas the monocations of longer oligomers are localised over 2-3 porphyrin units (Class II). In Chapter 5, photophysical and computational investigations into excited state aromaticity in porphyrin nanorings are presented. The computational results suggest the presence of aromaticity in the triplet excited states, but experiment fails to convincingly demonstrate the effect. Computational results in Chapter 6 show that a butadiyne linked [6]-porphyrin nanoring in which one butadiyne (C≡C-C≡C) is truncated to an alkyne (C≡C) exhibits a reversal of aromaticity and antiaromaticity in its oxidised states, compared to the all-butadiyne linked nanoring, consistent with Hückel's law.