The misfolding and aggregation of proteins is closely associated with more than fifty human disorders, including Alzheimer's and Parkinson's diseases, all of which are currently incurable and many represent a major threat to human life. The mechanism of protein aggregation is subject to extensive studies. The damaging effects associated with protein aggregation have been attributed to amyloidogenic species that are present during the misfolding process. In particular, oligomeric species are, however, intrinsically difficult to study as a consequence of their low abundance and highly heterogeneous nature. The first chapter of my thesis gives an introduction into the field of protein folding and misfolding with a focus on the study of protein aggregation, and toxic effects relevant to human disorders. The second chapter of my thesis describes the development of a methodology that enables the study of aggregate induced lipid bilayer permeability, possibly the most general mechanism of protein aggregate toxicity. Surface-tethered lipid vesicles functioning as optochemical probes sensitive to membrane integrity are imaged using total internal reflection microscopy. It is shown that oligomeric species of the 42-residue form of the Aβ peptide (Aβ42) are responsible for the membrane disruption. The methodology can be applied to the study of other proteins such as α-synuclein and tau, and the ability of antibodies and chaperones to counteract the aggregate induced lipid bilayer permeability can be assessed. Furthermore, lipid bilayer permeability induced by aggregates formed in human induced pluripotent stem cells can be studied. The third chapter presents a new approach for the measurement of protein aggregation kinetics by following the development of the lipid bilayer permeability over the course of the aggregation process of Aβ42. The aggregation kinetics can be modulated with molecular chaperones and pre-formed seed fibrils, which allows secondary nucleation to be identified as the process that drives the formation of species responsible for the lipid bilayer permeability. The fourth chapter describes the development of a three-pronged strategy to study the mechanism of α-synuclein amyloid formation. The aggregation is studied in the presence of lipid vesicles or pre-formed fibrils at neutral or acidic pH of the solution. The influence of single-point mutations on the aggregation of α-synuclein is described. Furthermore, the strategy is applied to the characterisation of the ability of antibodies and small molecules to inhibit the aggregation, and thus has the potential for the development of therapeutical agents. The work presented in the fifth chapter characterises the amyloid fibril populations formed by α-synuclein and mutational variants associated with familial Parkinson's disease. X-ray crystallography, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and atomic force microscopy have all been applied to the analysis of these amyloid fibrils. Finally, the sixth chapter summarises the results described in this thesis and points out future opportunities in the context of fundamental and translational studies related to the research area of protein misfolding disorders.

Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines. Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules. The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity. The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5. The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.