Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties, and proximal limb weakness. The autosomal dominant form of this disease is caused by the expansion of a polyalanine stretch from 10 to 12--17 alanines in the N-terminus of PABPN1. Mutated PABPN1 (mPABPN1) is able to induce the formation of filamentous intranuclear inclusions that are the pathological hallmark of OPMD. PABPN1 is predominantly localized to the nucleus, binds RNA poly(A) tail, forms oliogmers, and is involved in polyadenylation. In this study we first demonstrated that oligomerization of PABPN1 is mediated by two potential oligomerization domains (OD), while inactivating oligomerization of mPABPN1 by deletions of 6--8 residues in either of the ODs prevents intranuclear protein aggregation. Expression of mPABPN1 in COS-7 cells is associated with cell death, whereas preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. We then identified two PABPN1 interacting proteins, hnRNP A1 and A/B, using a yeast two-hybrid library screen. The interaction between PABPN1 and hnRNP A1 or A/B was confirmed by GST pull-down and co-immunoprecipitation assays. When coexpressed with mPABPN1 in COS-7 cells, predominantly nuclear localized hnRNP A1 and A/B co-localize with mPABPN1 to the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. We finally found a nuclear localization signal (NLS) in PABPN1 that is not homologous to any known NLSs. The 18 amino acids 289RGRVYRGRARATSWYSPY 306 in PABPN1 are necessary and sufficient for nuclear translocation. Attaching this sequence to cytoplasmic protein PKM2 completely re-localizes it to the nucleus. Alanine-scanning mutagenesis analysis showed that the last 9 residues 298RATSWYSPY306 are crucial to the function as an NLS. Our studies showed that mPABPN1 induced intran

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.

Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. As an alternative approach to identifying such factors, a screen was conducted for activities that inhibit the non-specific activity of plant poly(A) polymerases (PAP). One such factor (termed here as Putative Polyadenylation Factor B, or PPF-B) was identified in a screen of DEAE-Sepharose column fractions using a partially purified preparation of a plant nuclear poly(A) polymerase. This factor was purified to near homogeneity. Surprisingly, in addition to being an effective inhibitor of the nuclear PAP, PPF-B inhibited the activity of a chloroplast PAP. In contrast, this factor stimulated the activity of the yeast PAP. Direct assays of ATPase, proteinase, and nuclease activities indicated that inhibition of PAP activity was not due to depletion of substrates or degradation of products of the PAP reaction. The major polypeptide component of PPF-B proved to be a novel linker histone (RSP), which copurified with inhibitory activity by affinity chromatography on DNA-cellulose. The association of inhibitory activity with a linker histone and the spectrum of inhibitory activity, raise interesting possibilities regarding the role of PPF-B in nuclear RNA metabolism. These include a link between DNA damage and polyadenylation, as well as a role for limiting the polyadenylation of stable RNAs in the nucleus and nucleolus. The Arabidopsis genome possesses genes encoding probable homologs of most of the polyadenylation subunits that have been identified in mammals and yeast. Two of these reside on chromosome III and V and have the potential to encode a protein that is related to the yeast and mammalian Fip1 subunit (AtFip1-III and AtFip1-V). These genes are universally expressed in Arabidopsis tissues. AtFip1-V stimulates the non-specific activity of at least one Arabidopsis nuclear PAP, binds RNA, and interacts with other polyadenylation homologs AtCstF77 and AtCPSF30. These studies suggest that AtFip1- V is an authentic polyadenylation factor that coordinates other subunits and plays a role in regulating the activityof PAP in plants.

Protein PEGylation (covalent attachment of poly(ethylene glycol) or PEG to proteins) is an excellent example of a drug delivery system that improves pharmacokinetics and pharmacodynamic properties of therapeutics. However, although PEGylation is clinically proven and attracts both scientific and commercial interest, the technique is associated with many process constraints, in particular related to controlling the number of conjugated PEG chains. A novel, solid-phase PEGylation methodology was attempted to overcome the drawbacks of the commonly used solution-phase methods for preparing PEGylated products. The solid-phase PEGylation methodology involved conjugating protein onto a tethered PEG derivative attached onto a solid matrix, followed by hydrolytic cleavage of the PEG chain from the solid matrix under mild conditions to yield PEGylated protein in free solution. PEGs with molecular weights (MWs) 2000 and 4000 Da were used and a heterobifunctional PEG derivative, α-(β-alanine)-ω-carboxy PEG, with a cleavable β-alanine ester terminal was prepared for surface grafting and protein conjugation. The amine terminal of this PEG derivative was used for grafting PEG onto carboxy functionalized hydrophilic Sephadex and hydrophobic polystyrene derivatives. The free carboxyl terminal was used for protein conjugation via amine coupling. A kinetic study of PEG-surface grafting was performed to understand the influence of a number of parameters on the PEG surface concentration and its conformation, including temperature, reaction time, nature of the matrix, solvent and base, and MW of PEG. PEG grafted matrices were characterized using various surface characterization tools including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Higher PEG grafting was observed with polystyrene matrices (up to 0.3 mmol/g) than either of the Sephadex derivatives (less than 0.15 mmol/g) using both molecular weights. Detailed surface characterization using XPS studies showed a layer thickness of 11.87 nm was achieved with polystyrene matrices using 4000 Da PEG derivatives after a grafting period of 72 hours at 40°C, indicating the presence of brush conformations for the grafted PEGs. In contrast, mushroom conformations were observed for PEG molecules grafted on both carboxymethyl and carboxypentyl Sephadex derivatives after the same reaction period, with a layer thickness of 2.62 nm and 4.14 nm respectively. Optimized PEG grafting and hydrolysis conditions were developed for solid-phase protein PEGylation using Cytochrome c as a model protein. The presence of PEGylated species were detected by size exclusion chromatography (SEC) from Sephadex derivatives but were absent when using polystyrene matrices. Both Sephadex derivatives gave mainly multi-PEGylated species with poor yields, in place of the expected mono-PEGylated products. A solution-phase PEGylation using the same PEG derivatives was performed successfully and various PEGylated species were identified and characterized using SEC and gel electrophoresis, based on their viscosity radius. An examination of the surface characteristics of the PEG-grafted was carried out by XPS, showing that protein conjugation was greatly influenced by surface force interactions, which depended on the PEG grafting densities and the nature of the solid matrices. Finally, fluorescent images obtained using confocal microscope with fluorescein isothiocyanate labelled Cytochrome c provided supporting evidence regarding the factors that constrained the solid-phase PEGylation process.

The viscoelastic relaxation dynamics of a series of poly(methyl methacrylate) [PMMA] based nanocomposites filled with carbon nanotubes have been studied using dynamic mechanical analysis and broadband dielectric spectroscopy. The networks were prepared using four methods: (i) melt mixing, (ii) solution processing, (iii) in-situ polymerization, and (iv) polymer grafting. Nanotube modifications included surface oxidation via acid exposure and surface functionalization for polymer grafting. The effect of variations in processing method and nanotube modification on glass transition temperature (Tg) and relaxation dynamics was investigated. The relaxation behavior of the nanocomposites was sensitive to processing method and nanotube functionalization. Nanotube loading (to 5 wt%) led to a progressive increase in rubbery modulus, with the increase more pronounced in the solution-processed samples owing to enhanced nanotube dispersion. In the case of the oxidized nanotubes, loading led to an increase in modulus, but also a systematic decrease in Tg of ~ 15°C with 3 wt% nanotubes. For in-situ polymerized (PMMA/MWNT-ox) nanocomposites, there was no readily discernable trend in Tg. Composites prepared via in-situ polymerization in the presence of methyl methacrylate functionalized tubes (i.e., polymer grafting) displayed a positive shift in Tg of nearly 20°C at 1 wt% loading. Investigation of the dielectric relaxation of the PMMA/MWNT composites indicated a percolation threshold between 0.3 and 0.4 wt% MWNT.

<p>X-ray computed tomography (CT) is a non-destructive medical imaging technique for assessing the cross-sectional images of an object in terms of attenuation. As it is designed based on the physical processes involved in the x-ray and matter interactions, faithfully modeling the physics in the reconstruction procedure can yield accurate attenuation distribution of the scanned object. Otherwise, unrealistic physical assumptions can result in unwanted artifacts in reconstructed images. For example, the current reconstruction algorithms assume the photons emitted by the x-ray source are mono-energetic. This oversimplified physical model neglects the poly-energetic properties of the x-ray source and the nonlinear attenuations of the scanned materials, and results in the well-known beam-hardening artifacts (BHAs). The purpose of this work was to incorporate the poly-energetic nature of the x-ray spectrum and then to eliminate BHAs. By accomplishing this, I can improve the image quality, enable the quantitative reconstruction ability of the single-spectrum CT scanner, and potentially reduce unnecessary radiation dose to patients.</p><p>In this thesis, in order to obtain accurate spectrum for poly-energetic reconstruction, I first presented a novel spectral estimation technique, with which spectra across a large range of angular trajectories of the imaging field of view can be estimated with a single phantom and a single axial acquisition. The experimental results with a 16 cm diameter cylindrical phantom (composition: ultra-high-molecular-weight polyethylene [UHMWPE]) on a clinical scanner showed that the averaged absolute mean energy differences and the normalized root mean square differences with respect to the actual spectra across kVp settings (i.e., 80, 100, 120, 140) and angular trajectories were less than 0.61 keV and 3.41%, respectively</p><p>With the previous estimation of the x-ray spectra, three poly-energetic reconstruction algorithms are proposed for different clinical applications. The first algorithm (i.e., poly-energetic iterative FBP [piFBP]) can be applied to routine clinical CT exams, as the spectra of the x-ray source and the nonlinear attenuations of diverse body tissues and metal implant materials are incorporated to eliminate BHAs and to reduce metal artifacts. The simulation results showed that the variation range of the relative errors of various tissues across different phantom sizes (i.e., 16, 24, 32, and 40 cm in diameter) and kVp settings (80, 100, 120, 140) were reduced from [-7.5%, 17.5%] for conventional FBP to [-0.1%, 0.1%] for piFBP, while the noise was maintained at the same low level (about [0.3%, 1.7%]).</p><p>When iodinated contrast agents are involved and patient motions are not readily correctable (e.g., in myocardial perfusion exam), a second algorithm (i.e., poly-energetic simultaneous algebraic reconstruction technique [pSART]) can be applied to eliminate BHAs and to quantitatively determine the iodine concentrations of blood-iodine mixtures with our new technique. The phantom experiment on a clinical CT scanner indicated that the maximum absolute relative error across material inserts was reduced from 4.1% for conventional simultaneous algebraic reconstruction technique [SART] to 0.4% for pSART.</p><p>Extending the work beyond minimizing BHAs, if patient motions are correctable or negligible, a third algorithm (i.e., poly-energetic dynamic perfusion algorithm [pDP]) is developed to retrieve iodine maps of any iodine-tissue mixtures in any perfusion exams, such as breast, lung, or brain perfusion exams. The quantitative results of the simulations with a dynamic anthropomorphic thorax phantom indicated that the maximum error of iodine concentrations can be reduced from 1.1 mg/cc for conventional FBP to less than 0.1 mg/cc for pDP.</p><p>Two invention disclosure forms based on the work presented in this thesis have been submitted to Office of Licensing & Ventures of Duke University.</p> / Dissertation

Les nanocomposites à matrice polymère et plus particulièrement les élastomères chargés intéressent depuis de nombreuses années la communauté scientifique du fait notamment de leurs bonnes propriétés mécaniques. Il est établi que l'amélioration des propriétés mécaniques observées dans les élastomères renforcés par des charges nanoscopiques est principalement due à des effets de structure (dispersion des charges) et à des effets d'interface (interactions charges/matrice). Afin d'alimenter la discussion sur l'origine du renforcement dans les élastomères chargés, cette étude s'intéresse à la synthèse et à la caractérisation de nanocomposites modèles pour lesquels les interactions charges/matrice et la microstructure sont maîtrisées. En parallèle, ce travail propose également une analogie entre ces systèmes et les ionomères, matériaux constitués de macromolécules sur lesquelles des groupements ioniques sont greffés. Ces matériaux ionomère présentent des propriétés originales et permettent la création de nœuds de réticulation réversibles avec la température

Poly butylene succinate (PBS) is a biodegradable plastic polymer that has physical and mechanical properties similar to common petroleum plastics like polypropylene (PP) and polyethylene (PE). PBS may be produced from petroleum or bio-based feedstocks, or by a hybrid combination of petroleum and bio-based resources. Producers are reducing content of petroleum components used for the production of PBS, and by doing so are seeking potential environmental performance improvements. In this study, ???hybrid??? PBS refers to the production of PBS polymer from bio-based succinic acid (SAC) sourced from sorghum and petroleum-based 1, 4-butanediol (BDO). Given its biodegradability, PBS is commercially used for compostable bags and agricultural mulching film applications. A recent study in Ontario identified composite materials made with PBS blended with natural fibres like switchgrass (SG) as promising for applications in automotive products. Such novel composite materials are touted as potential bio-based alternatives to conventional petroleum-based plastics. Of the few studies that have considered the environmental performance of PBS materials, none have assessed the potential environmental impacts of a hybrid PBS composite. Therefore, this study undertook a life cycle assessment (LCA) of SG reinforced hybrid PBS composite (hybrid composite). LCA is an environmental management technique that is used to assess environmental aspects (inputs and outputs) and potential environmental impacts of a product or service throughout its life cycle. The analysis considered a cradle-to-gate system boundary and evaluated eleven environmental performance indicators. The environmental performance of the hybrid composite was compared to a conventional glass fibre (GF) reinforced polypropylene (PP) composite (baseline composite), a material that is widely used in automotive components. Results showed that the production of the hybrid composite in comparison to the baseline composite decreased potential impact for most of the assessed indicators: cumulative energy demand by 40%, waste heat by 23%, global warming potential by 35%, smog by 2%, carcinogens by 54%, non-carcinogens by 172%, respiratory effects by 22% and ecotoxicity by 45%. Increases in the values of impact indicators were apparent for ozone depletion, acidification, and eutrophication by 43%, 16%, and 322%, respectively. Analysis revealed that dominant influences on results were not related directly to the bio-based make-up. Rather, the biggest influence on the environmental performance of composite production were the sources of heat used in petroleum-based materials, the energy mix in electricity for bio-based materials, the type of reinforcing fibre and the co-product treatment methodology used. The study helps fill a gap in knowledge regarding bio-based chemicals and hybrid biodegradable plastic composites, and points to opportunities for future research on feedstocks for industrial composite materials. The importance of this study is that it helps to identify the environmental strengths and weaknesses associated with the production of the hybrid composite specifically, and bio-based materials more generally. It points to alternative material substitution options for use in the automotive industry. In this study, life cycle assessment exemplifies multidisciplinary methodologies, which seek to traverse the boundaries between the social and natural sciences and disciplines to support more sustainable policy decisions for a bio-economy. The systematic nature and the widely applicable consequences of this LCA study have the potential to contribute to industrial and business management, and reach the public policy arena in an effort to drive environmental and social change.

As a first project, collision-induced dissociation experiments were carried out using electrospray ionisation mass spectrometry on gas phase complexes involving different poly(methylmetacrylate) oligomers with three amino acids: glycine, leucine, and phenylalanine. After acquiring breakdown diagrams, RRKM modeling was used to fit the experimental data in order to obtain the 0 K activation energy and the entropy of activation. These thermodynamic data were then used to understand the competing dissociation channels observed (except for gas phase complexes involving glycine that had only one dissociation channel). Molecular dynamics simulated annealing calculations were carried on the gas phase complexes to understand further the energetic and entropic effects involved as well as the 3D conformation of these complexes. Valuable insight information was found on the 3D conformations, on a qualitative level. Using rotational constants and vibrational harmonic frequencies, it was possible to evaluate the entropy variation between the experimentally observed competing channels. Reasonable agreement was found between the experimental and theoretical variations of entropies. Finally, the proton affinity of poly(methylmetacrylate) oligomers is being discussed. Even though no absolute values for the proton affinity were found, the experimental and computational results help to understand the variation that accompanies the oligomers length. The second project presents the development an efficient and reproducible screening method for identifying low molecular weight compounds that bind to amyloid beta peptides (Abeta) peptides using electrospray ionization mass spectrometry (ESI-MS). Low molecular weight (LMW) compounds capable of interacting with soluble Abeta may be able to modulate/inhibit the Abeta aggregation process and serve as potential disease-modifying agents for Alzheimer’s disease. The present approach was used to rank the binding affinity of a library of compounds to Abeta1-40 peptide. The results obtained show that low molecular weight compounds bind similarly to Abeta1-42, Abeta1-40, as well as Abeta1-28 peptides and they underline the critical role of Abeta peptide charge motif in binding at physiological pH. Finally, some elements of structure-activity relationship (SAR) involved in the binding affinity of homotaurine to soluble Abeta peptides are discussed. As a third project, the gas phase binding of small molecules to the Abeta1-40 peptide generated by electrospray ionization has been explored with collision-induced dissociation mass spectrometry and kinetic rate theory. This project presents a simple procedure used to theoretically model the experimental breakdown diagrams for the Abeta1-40 peptide complexed with a series of aminosulfonate small molecules, namely homotaurine, 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO), 3-(1,3,4,9-tetrahydro-2H-beta-carbolin-2-yl) propane-1-sulfonic acid, 3-(1,3,4,9-tetrahydro-2H-beta-carbolin-2-yl)butane-1-sulfonic acid, and 3-(cyclohexylamino) propane-1-sulfonic acid. An alternative method employing an extrapolation procedure for the microcanonical rate constant, k(E), is also discussed.