Protein deposition in the form of amyloid fibrils is the hallmark of more than 40 human pathologies, including Alzheimer's disease (AD) and Parkinson's disease (PD). Misfolded protein oligomers formed as intermediates during the aggregation process have been strongly implicated in the onset and progression of these diseases. In this thesis, I describe our efforts to uncover molecular agents that can reduce the toxicity caused by protein aggregation via targeting the generation, the physiochemical properties or the membrane affinity of oligomeric species. We employed an integrative approach combining in vitro techniques, including chemical kinetics, atomic force microscopy, and biophysical measurements, and in vivo methods, including neuroblastoma cells and C. elegans models of AD and PD, to identify a range of small molecules and antibodies that can suppress the toxicity related to protein aggregation through a variety of mechanisms. In Chapter 3, we show that the deleterious effects of protein aggregation can be suppressed in AD and PD worms by interfering with the aggregation rates of the amyloid-β peptide (Aβ) and the α-synuclein protein (αS). In Chapter 4, we resolve the mechanism of action for a molecule that enhances the rate of Aβ42 aggregation in AD worms with the result that toxicity is reduced, and find that it potentiates the secondary nucleation microscopic step in vitro. In Chapter 5, we characterize molecules and antibodies that modify the physiochemical properties and self-association of oligomers comprised of several proteins into clusters with reduced diffusibility. In Chapter 6, we classify a family of molecules that protect the cell by displacing several types of oligomeric species from the membrane through a generic mechanism. These results demonstrate strategies by which one can target the aggregation process to alter its resulting toxicity, provide insight into modifying the properties of the most deleterious species associated with protein aggregation and suggest that the protection of the cell from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases is a promising strategy to combat protein misfolding diseases.

Vanadium compounds have been reported to cause numerous toxicological effects including NAD(P)H oxidation and lipid peroxidation. The purpose of this thesis is to determine the active form of vanadium in causing these effects, and to determine any possible free radical involvement in these processes. Vanadium-stimulated oxidation of NADH was studied spectrophotometrically and by electron spin resonance spectroscopy. In 25 mM sodium phosphate buffer at pH 7. 4, vanadyl , (V(IV)), is slightly more effective in stimulating NADH oxidation than was vanadate (V(V)). Addition of a superoxide generating system, xanthine/xanthine oxidase, results in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. In contrast, addition of hydrogen peroxide did not change the NADH oxidation by vanadate, but greatly enhanced NADH oxidation by vanadyl. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl radical. Hydroxyl radical was also detected in the system containing vanadate plus superoxide. It was found that superoxide is capable of reducing vanadate to vanadyl, and that vanadyl is capable of reaction with hydrogen peroxide in a Fenton-like mechanism to produce hydroxyl radical. Hydroxyl radical is suggested to be the active species involved in NADH oxidation. Other reductants, such as thiols, are also capable of supporting vanadate-stimulated NADH oxidation . The above results indicat that ability of vanadium to act in a Fenton-like mechanism is an important process in the vanadium-stimulated oxidation of NADH. Vanadyl was found to be the active form of vanadium involved in initiating and stimulating lipid peroxidation in purified and partially purified fatty acid micelle preparations. Hydroxyl radicals were shown to be involved in initiating diene conjugation when vanadyl and hydrogen peroxide were added together in the reaction mixture. Furthermore, hydroxyl radicals were shown to be generated in the vanadyl-catalyzed decomposition of fatty acid lipoperoxides. The results of this study indicat that the ability of vanadium compounds to oxidize NADH and to stimulate lipid peroxidation are related by the common mechanism of hydroxyl radical production from the reaction of vanadyl with hydrogen peroxide.

Les nanoparticules (NPs) sont définies comme des particules ayant au moins une dimension comprise entre 1 à 100 nanomètres. Plusieurs études in vitro et in vivo indiquent que les NPs pourraient constituer un risque potentiel pour la santé des personnes les synthétisant ou les manipulant lors de leur incorporation dans d’autres matériaux. La nanotoxicologie est un domaine de recherche émergeant. Les propriétés physico-chimiques particulières des NPs sont responsables d’interférences non spécifiques entre les nanomatériaux et certains des composants des essais in vitro pouvant mener à de faux résultats. L’inhalation a été identifiée comme une voie d’exposition présentant un risque important de toxicité. Dans le cadre de ce projet, nous avons utilisé la lignée de cellules épithéliales alvéolaires humaines, A549. Nous avons étudié chez cette lignée les conséquences de l’exposition aux points quantiques (PQs), NPs d’intérêt pour leurs applications potentielles en médecine (nanovecteur ou nanosonde). La mise au point des conditions expérimentales (interférence entre l’essai LDH et le milieu de culture) a permis de valider les essais de cytotoxicité MTS et LDH en présence des PQs. Nous avons montré que les PQs présentaient une cytotoxicité à court et long terme, et nous avons par la suite étudié un des mécanismes de toxicité potentielle, la mesure du cadmium (Cd2+) libéré des PQs. Nous avons déterminé que la mesure du Cd2+ comportait plusieurs interférences qui invalident cet essai. En conclusion, notre étude a permis d’identifier des interférences qui remettent en question plusieurs conclusions d’études publiées qui n’ont pas vérifié l’existence de telles interférences. / Nanoparticles (NPs) are defined as particles with a diameter ranging from 1 to 100 nm. Several studies have suggested or demonstrated that NPs are a health risk factor for workers handling nanomaterials or for the general population using products containing NPs. Nanotoxicology is a new field of research. The particular physicochemical properties of NPs have revealed unexpected interferences of these nanomaterials with components of cytotoxic assays leading to false positive or negative interpretations. Inhalation being one of the most potential risk of NPs exposures, in this project, in vitro experiments were performed with the human alveolar epithelial cell line A549. Quantum dots (QDs) have been selected over other NPs because of their potential applications in medicine as nanovectors or nanoprobes. We have first validated the use of nanocytotoxic assays (LDH and MTS with QDs), and found that LDH assay interacts with components of the cell culture medium. A protocol established to counteract this interference has allowed demonstrating that both short and long term exposures to QDs are cytotoxic. We next addressed the underlying mechanisms of QDs nanotoxicity by determining concentrations of free cadmium potentially released from QDs when interacting with cells. We found that the cadmium assay has several interferences which invalidated the use of this assay. In conclusion, our study has allowed to identify several interferences with that call into question conclusions of published studies which have not examined these potential interferences.