Chemoreceptor dimers mediate bacterial chemotaxis through receptor arrays that form complexes with CheW and the kinase CheA. Both kinase activity and the rate of receptor methylation have been observed to change with receptor density in the signaling complexes of an assembled system. Receptor dimers play a structural and functional important role during the signaling process. The dimer is an established unit of a functional receptor array, but the exact packing arrangement of dimers in the signaling array is not known in detail. Two different models of the packing arrangement have been proposed, based on crystal structures of receptor cytoplasmic fragments: the trimer of dimers and hedgerow models. Here, we determine the importance of CF dimer organization on density dependence of kinase activity, receptor methylation, and we probe CF trimer organization by site-directed TMEA cross-linking. CF dimers were prepared by site-directed cysteine disulfide bond formation to test for a possible monomer-dimer equilibrium process in the density-dependence of kinase activity in the vesicle-assembled system. We found that density transition observed for CF4E is similar for both reduced and disulfide-linked CF, indicating that the CF is organized as dimers even at lower densities. CheR-catalyzed methylation experiment revealed that disulfide-linked CF dimers in solution are insufficient for efficient methylation, assembly of the CFs on a vesicle surface is required. CF trimers were trapped by the trifunctional crosslinker TMEA, but only with vesicle assembled CFs. TMEA crosslinking occurred between residues that were expected to form trimer, but also those that were not. The results provide evidence that CF behavior is highly dynamic in maleimide crosslinking reaction which conducted on vesicle assembled system.

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High molecular weight PBTTT-C₁₂ is blended with the pure trimer, BTTT-3, to enhance intergrain connectivity and charge transport. Analysis of the morphology and crystallinity of the blends shows that the polymer and oligomer are well-integrated, leading to high hole mobilities, greater than 0.1 cm² V⁻¹ s⁻¹, in films that contain as much as 83% oligomer.

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.

Cationic photopolymerization of multifunctional epoxides is very useful for efficient cure at room temperature and has been widely used in coatings and adhesives. Despite excellent properties of the final cured polymers, cationic photopolymerizations of epoxides have seen limited application due to slow reactions (relative to acrylates) and brittleness associated with a highly crosslinked, rigid network. To address these issues, two reaction systems were studied in this thesis: photoinitiated cationic copolymerizations of a cycloaliphatic diepoxide with epoxidized elastomers and acrylate/epoxide hybrid photopolymerizations. Oligomer/monomer structures, viscosity, compositions, and photoinitiator system were hypothesized to play important roles in controlling photopolymerizations of the epoxide-based mixtures. A fundamental understanding of the interplay between these variables for the chosen systems will provide comprehensive guidelines for the future development of photopolymerization systems comparable to the epoxide-based mixtures in this research. For diepoxide/oligomer mixtures, the observed overall enhancement in polymerization rate and ultimate conversion of the cycloaliphatic diepoxide was attributed to the activated monomer mechanism associated with hydroxyl terminal groups in the epoxidized oligomers. This enhancement increased with increasing oligomer content. The mixture viscosity influenced the initial reactivity of the diepoxide for oligomer content above 50 wt.%. Real-time consumption of internal epoxides in the oligomers was successfully determined using Raman spectroscopy. Initial reactivity and ultimate conversion of the internal epoxides decreased with increasing the diepoxide content. This trend was more pronounced for the oligomer containing low internal epoxide content. These results indicate that the reactivity of the hydroxyl groups is higher toward cationic active centers of the diepoxide than those of the internal epoxides in the oligomers. These conclusions are consistent with physical property results. The enhanced fracture toughness and impact resistance were attributed to multimodal network chain-length distribution of copolymers containing the oligomer content between 70% and 80%. For acrylate/epoxide hybrid mixtures, diacrylate oligomers significantly suppressed reactivities of cycloaliphatic mono/diepoxides, which was attributed to high mixture viscosity and highly crosslinked acrylate network. In this case, the dual photoinitiator system did not favor the epoxide reaction. Depending on the monovinyl acrylate secondary functionalities, enhanced reactivity and ultimate conversion of the diepoxide were attributed to a combined effect of a reduced viscosity and the radical-promoted cationic polymerization associated with the dual photoinitiator. The retarded and inhibited diepoxide reactivities with ether and urethane secondary groups were attributed to solvation and nucleophilicity/basicity effects, respectively. The influence of the diepoxide on the acrylate reactivity was attributed to dilution and polarity effects. In this case, high concentration of the free-radical photoinitiator is required for the dual photoinitiator system. Physical properties of hybrid polymers also varied with acrylate structures and monomer composition. Dynamic modulation methods were proposed to enhance the diepoxide reactivity and final properties in the presence of urethane acrylates.

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism. Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic Aβ oligomers at physiological concentrations, and monitored in real time the change of size and mole fraction of oligomers in the presence of dissociating agents or conditions. The higher-order dissociation process caused by DHBA isomers produced no transient oligomeric intermediates, a desirable feature for an anti-oligomer therapeutic. Urea and guanidine hydrochloride, in contrast, produced a linear dissociation with a progressive decrease of size and mole fraction of oligomers. FCS allows the facile distinction of small molecule Aβ oligomer dissociators that do not produce stable potentially toxic oligomeric Aβ intermediates.

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. <p>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. <p>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./<p>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.<p>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.<p>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.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

A simple method for increasing the stable temperature range of the liquid crystalline blue phase is demonstrated, by mixing a non-mesogenic polymer of low molecular weight into the blue phase material. In a mixture of cholesteryl benzoate and cholesteryl nonanoate the addition of polystyrene increased the stable blue phase range from 0.5K to 12K. This was measured strictly on heating from the chiral nematic phase through the blue phase in order to minimise non-equilibrium effects, and is one of the largest ranges so measured. The stability range can be closely tuned by changing the polymer concentration and molecular weight. The maximum range found by adding a particular compound seems only to depend on its saturation point in the liquid crystal, and the dependence of the range on concentration is non-linear. These features were explained by a numerical model of a blue phase unit cell incorporating the mean field Flory-Huggins and Maier-Saupe theories where the polymer could fill the high energy defect regions. Two of the oligomers which are shown to stabilise the blue phase are fluorescent, at 450nm and 500nm respectively, and it is proposed that tests on these mixtures could reveal photonic effects caused by the concentration of the fluorophores in the blue phase defect regions. The twist-grain boundary (TGB) phase is present in mixtures of cholesteryl oleyl carbonate and cholesteryl nonanoate over a range of up to 0.3K. The addition of polystyrene has no effect on the stability of the TGB phase. Conventional, in situ UV-initiated polymer stabilisation does not appear to stabilise the TGB phase, but is capable of stabilising over at least 30K the micron-size filaments which appear in the TGB phase when it is heated from the smectic phase in a cell with homeotropic alignment. Some notes are made on the causes and structure of this filament texture, and it is observed that the filaments tend to grow with a characteristic curvature. It is shown theoretically that the correct material could stabilise the TGB phase similarly to the polymers in the blue phase, by extending the previous model to include the Kobayashi-McMillan theory of smectic ordering. A second theoretical model of chirality around the transition to the smectic phase is then presented which takes account of fluctuations, based on an analogy with the state of a smectic-forming material infiltrated into an aerogel. A phase resembling the TGB phase emerges from this model. The model gives two first order transitions in accordance with experiments on the TGB phase, and reflects other experimental pitch and calorimetry measurements too. The electrochemical polymerisation of an acrylate monomer in the nematic and smectic-C* phases is investigated. 30-100V is applied across a cell containing the liquid crystal-monomer mixture, with no additional initiating compound. In both phases, the texture during polymerisation is frozen in by the polymer formed. In a nematic phase in a cell with initially planar alignment, the director in the field off state can be observed to tilt toward the homeotropic over a number of hours. In the ferroelectric case, as well as the textural freezing there is a somewhat reversible agglomeration of polymer strands into micron-scale structures. Scanning electron microscopy reveals a range of structures on both electrode surfaces, including in the nematic case corrugations with a periodicity of 500-750nm. There is no evidence of a polymer network spanning the thickness of the cell - rather the liquid crystal seems to be realigned by a polymer film at the electrode surfaces.

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