Extracellular matrix stiffening of breast tissues has been clinically correlated with malignant transformation and poor prognosis. An increase of collagen fibril diameter and lysyl-oxidase mediated crosslinking has been observed in advanced tumor stages. Many current reports suggest that the local mechanical properties of single fibrillar components dominantly regulate cancer cell behavior. Here, we demonstrate by an independent control of fibril diameter and intrafibrillar crosslinking of threedimensional (3D) collagen matrices that fibril bending stiffness instructs cell behavior of invasive and non-invasive breast cancer cells. Two types of collagen matrices with fibril diameter of either 650 nm or 800 nm at a similar pore size of 10 µm were reconstituted and further modified with the zero-length crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide at concentrations of 0, 20, 100 and 500 mM. This approach yields a set of collagen matrices with overlapping variation of matrix elasticity. Within this set of matrices we could prove the common assumption that matrix elasticity of collagen networks is bending dominated with a linear dependence on fibril bending stiffness. We derive that the measured variation of matrix elasticity is directly correlated to the variation of fibril bending stiffness, being independently controlled either by fibril diameter or by intrafibrillar crosslinking. We use these defined matrices to demonstrate that the adjustment of fibril bending stiffness allows to instruct the behavior of two different breast cancer cell lines, invasive MDA-MB-231 (human breast carcinoma) and non-invasive MCF-7 cells (human breast adenocarcinoma). Invasiveness and spreading of invasive MDA-MB-231 cells as well as clustering of non-invasive MCF-7 cells is thereby investigated over a broad parameter range. Our results demonstrate and quantify the direct dependence of cancer cell phenotypes on the matrix mechanical properties on the scale of single fibrils.

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"Template" and "adder" proteins can be genetically encoded to produce large amyloid fibers when mixed together. Escherichia coli is used to clone a "template" protein, Gd20, which will cooperatively self-assemble with two "adder" proteins, P7Q and P7S, to yield two different large amyloid fibers. Atomic force microscopy (AFM) is used to image the fibers and AFM tip approach/retraction force is used to quantify molecular packing in the fibers. Glutamine (Q)-containing P7Q and serine (S)-containing P7S both have the same hydrophobic core, charge, and hydrogen bonding potential. However, P7Q is highly alpha-helical while P7S contains a beta-sheet core. After 72 hours, the Gd20:P7Q template:adder protein mixture produces tightly packed ~0.3 μm high and ~1.9 μm wide fibers that exhibit a low retraction force of ~44 nN after indentation. The Gd20:P7S mixture produces larger ~1.1 μm high and ~9.7 μm wide fibers exhibiting a much higher retraction force of ~503 nN showing they are much less molecularly packed. These results indicate that the adder protein alpha-helical character is important for self-assembly and molecular packing inside of the large amyloid fiber. The experimental results show that large amyloid fibers with predictable size and mechanical properties can be anticipated and encoded at the genetic level. / Master of Science

Alzheimer’s disease (AD) is the most common cause of senile dementia worldwide. AD is a neurodegenerative disorder characterized by the loss of memory and language skill, collapse of the cognitive function, and distortion of social behavior. As of today, the onset mechanisms of AD and cure are unknown; however, three hallmarks are commonly encountered: extra and intracellular accumulation of amyloid beta (A!) peptide plaques, formation of intracellular neurofibrillary tangles, and inevitable neuronal death. Hypothetically, a possible scenario provoking or involved in the onset of AD is a cascade effect that starts with an imbalance in the production and clearance of Aß peptide that consequently leads to its accumulation, formation of tau protein tangles and neuronal death. This work studied and characterized the mechanisms governing A! peptide aggregation and the effects of using anti-Aß monoclonal antibodies to modify this process. These mechanisms play an important role in the formation of AD plaques and are critical in the search for therapies involving Aß peptide plaque clearance. Yet, antibody-based therapies for plaque clearance are not well understood, adding to the existing concerns about side effects in humans, hence there is a necessity of knowledge in this matter. In this work different Nterminus, C-terminus, and Mid-domain antibodies were used against Aß peptide species (monomers, oligomers, and fibrils) to probe peptide aggregates modification and disruption. Additionally, construction of a soft supported lipid bilayer membrane was proposed to study the adhesion mechanisms of Aß peptide and interactions with antibodies, mimicking the neuronal cell surface. The main characterization techniques used in this work were: atomic force microscopy (AFM) and transmission electron microscopy that allowed the physical exploration and visualization of the different processes of aggregation in terms of adhesion, size evolution, and distribution of the peptide; and attenuated total reflectance Fourier spectroscopy (ATR/FTIR) which allowed monitoring the change of secondary structures for the peptide during the processes studied. It is endeavored that this work will help to elucidate the effects attributed to the molecular interactions between A! peptide species and antibodies to target Aß plaque’s clearance in the brain of AD patients. Ultimately, this study provides novel information critical for the formulation of effective therapies to prevent and treat AD with less collateral effects. It also represents a contribution to the basic scientific knowledge regarding peptide-antibody interactions with application to other diseases related to protein misfolding.

This thesis explores the complex relationship between food protein structure and digestibility. Food proteins are important nutrients that play a central role in controlling the textural properties of many foods. Processing of food proteins may alter the protein aggregate structure and digestibility. The degree of protein aggregation during food processing depends on the denaturing conditions and the presence of other food components. Sugars and lipids may contribute to protein glycation and protein cross-linking via the Maillard reaction. Furthermore, amino acid residues of food proteins may be chemically modified during processing, thereby influencing both the structure and the nutritional value of proteins. An in vitro digestibility assay was used to investigate the relationship between protein aggregate structure and protein digestibility. Raw and boiled egg whites were exposed to a wide range of conditions: pH 2 - 12, in the presence and absence of 200 mM NaCl. It was found that pH and NaCl treatment prior to in vitro digestion resulted in significantly different protein ultrastructures, but did not markedly influence protein digestibility under the tested conditions. Raw egg white was less digestible than boiled egg white under all test conditions. The inclusion of Maillard reaction partners caused protein cross-linking concurrent with a decrease in digestibility. The digestibility decreased with the reactivity of the Maillard reaction partner and with increasing heating time. Proteomic analysis, using tandem mass spectrometry, of raw and heated egg white showed an increase in hydrothermally induced amino acid modifications. In the presence of glucose and methylglyoxal, a Maillard reaction specific increase in arginine modification to hydroimidazolone was observed with increasing heating times. The observed modifications are likely to contribute to a change in the nutritional quality of egg white. Aggregation kinetics of the major egg white protein, ovalbumin, were studied by dynamic light scattering, small angle X-ray scattering, and transmission electron microscopy. Shape determination was only possible for ordered aggregates, but not for disordered aggregates. Prior to heating, ovalbumin molecules in the presence of water and glucose repelled each other in concentrated solution. The presence of NaCl shielded electrostatic repulsion, leading to early onset dimerisation and disordered aggregation upon heating. Methylglyoxal treated ovalbumin formed more ordered aggregates. The scattering of these structures was able to be fitted to cylindrical shape models showing an increase of cylinder length with time while the cylinder diameter remained near constant over 24 hours of heating. In addition, food protein derived amyloid fibril aggregates were characterised. Amyloid fibrils are a common ordered protein fold that has been linked to neurodegenerative diseases. In the recent literature, amyloid fibrils have been proposed as new functional macromolecules in proteinaceous foods because of their desirable textural properties. Food fibrils formed from whey, egg white, soy bean and kidney bean protein were tested to establish whether they are protease resistant or display toxicity to human Caco-2 cells (a model intestinal cell line). The food fibrils were compared to insulin amyloid fibrils, a well characterised amyloid system. It was shown that the food fibrils displayed some resistance towards in vitro hydrolysis and were not found to be toxic. This work contributes to the understanding of food protein aggregation and digestibility under relevant conditions. It highlights the relationship of aggregate structure and digestibility and the particular role of the Maillard reaction. Moreover, evidence is provided that food protein derived amyloid fibrils may be safe ingredients in consumables. These findings may contribute to optimising industrial food processes and creating safe new food products.

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La cellulose étant l'un des biopolymères les plus abondants, elle est employée dans ce travail de thèse sous sa forme nano-fibrille (2 à 5nm de diamètre et plusieurs microns de long) pour préparer des nanomatériaux durables. Les microfibrilles de cellulose (MFC) chargées positivement ou négativement sont assemblées en couches minces dans ces nanomatériaux par la méthode « Layer by Layer » (LbL) par trempage, pulvérisation ou spin assisté. Les différences entre ces films LbL à base de MFC et les films LbL à base de polymères standards sont discutées brièvement et sont reliées à la forme nanofibrillaire de la cellulose. Les MFC réagissent comme des nano-objets anisotropes et rigides. Les films LbL de MFC sont ensuite intégrés à des membranes de séparation, entre la couche polymérique de séparation et le support poreux, pour améliorer le débit à travers ces membranes. Ces films minces sont également déposés sur des aérogels de cellulose pour améliorer la stabilité de ces aérogels en milieu aqueux. Dans les deux applications, les résultats était encouragent et montre une validation de principe. / Cellulose, one of the most abundant biopolymers, is used in this PhD work in its nanofibrillated form, 2-5 nm in diameter and microns long, to prepare sustainable nanomaterials. Both positively and negatively charged microfibrillated celluloses (MFC) are assembled in these nanomaterials using the versatile Layer by Layer (LbL) assembly methods: dipping, spray assisted-deposition and spin-assisted deposition. A brief comparison between the MFC based LbL assembled films and the standard polymeric LbL films is carried out. Thedifferences between the two species are related to the fibrillar form of cellulose. MFC behaves like rigid anisotropic nano-objects. MFC LbL assembled films are then integrated in separation membranes between active polymeric separation layers and a mechanically stable porous support to improve the flux through these membranes. MFC LbL assembled films are also coated on cellulosic aerogels to improve the wet stability of these aerogels. In both cases, results were encouraging and showed a proof of concept.