Diseases associated with amyloid aggregation have been a growing focus of medical research in recent years. Altered conformations of amyloidogenic peptides assemble to form soluble aggregates that deposit into the brain and spleen causing disorders such as Alzheimer's disease and Type II diabetes. Emergent theories predict that fibrils may not be the toxic form of amyloidogenic structures and that smaller oligomer and protofibril aggregates may be the primary source of cellular function damage. Studies show that these amyloidogenic aggregates are characterized by an increased number of poorly dehydrated hydrogen backbones and large surface densities of patches of bulk like water which favor protein association. When proteins aggregate to form larger structures, there is a redistribution of water surrounding these proteins. The water dynamics of amyloidogenic aggregation is different than the monomeric form and has a decrease in the number of patches occupied by molecules with bulk-like water behavior. We demonstrate that the redistribution of water during amyloid aggregation is reflected in a change in the dielectric relaxation signal of protein-solvent mixtures. We use dielectric relaxation spectroscopy (DRS) as a tool for studying the dynamics of amyloidogenic peptides--amyloid beta (Ab 1-42) and human islet amyloid polypeptide (hIAPP)--during self-assembly and aggregation. Non-amyloidogenic analogs-- scrambled (Ab 42-1) and rat islet amyloid polypeptide (rIAPP)--were used as controls. We first present studies of amyloidogenic peptides in a deionized water buffer at room temperature as a function of concentration and incubation time. From this we were able to determine differences in amyloidogenic and non-amyloidogenic peptides through the dielectric modulus. We next present the same analytes in a deionized water-glycerol buffer to facilitate the study of the dielectric permittivity at sub-freezing temperatures and model the kinetics of the alpha- and beta- relaxation processes. We conclude our work by studying the peptides in a bovine serum albumin (BSA) and glycerol buffer to demonstrate dielectric spectroscopy as a sensitive tool for measuring amyloidogenic peptides in an in vivo- like condition.

Many neurodegenerative disorders are characterised by protein misfolding and subsequent amyloid fibrillisation and deposition. Amyloid-beta peptide (Aβ) was found to be the main constituent of the extracellular amyloid plaques of Alzheimer’s disease (AD) in the 1980s. What triggers amyloid formation or inhibits it are of particular interest. This thesis focuses on the effect of endogenous proteins and molecules on amyloid fibrillisation. In Chapter 3, I show that at physiological micromolar levels found in the cerebrospinal fluid, human serum albumin inhibits the rate of Aβ fibrillisation. Indeed in vitro the amount of amyloid fibres generated directly correlates to the proportion of Aβ not competitively bound to albumin. Albumin binds cholesterol and fatty acids in vivo, both of which have been linked with an increased risk of developing AD. In Chapter 4, I show Aβ competes with these molecules for albumin binding, so disrupting albumin’s ability to inhibit Aβ fibrillisation. My observations suggest a significant role for albumin regulating Aβ fibril growth. Albumin also binds Cu2+ in vivo with a tight picomolar affinity. Animal models suggest disrupted Cu2+ homeostasis potentiate AD phenotype. In Chapter 5, I show that regardless of Aβ alloform or fibrillisation stage, the affinity for Cu2+ is in the ~20 picomolar range but weaker than albumin. Circular Dichroism spectroscopy in the visible region (Vis-CD) is a powerful technique to study metal-protein interactions. In Chapter 6, I develop a set of empirical rules that relates the appearance of particular Vis-CD spectral features to the conformation of the complex. These rules are used to gain insight into Cu2+-protein complexes in Prion disease and Parkinson’s disease. I show the N-terminal amino group of cellular prion protein (PrPC) has a tighter affinity for Cu2+ than the individual octa-repeat binding sites present within PrPC and show for the first time that Cu2+ loads on to the N-terminal amino group before the single octa-repeat binding sites. I determine the affinity of Cu2+ for model N-terminal peptides of alpha-synuclein of Parkinson’s disease and show that side- chain coordination stabilises the complex and increases the affinity for copper compared to main-chain coordination only. This thesis highlights the importance of overlapping interactions with endogenous proteins and molecules in the development of neurodegenerative disease. Indeed, the amyloid protein binding partners studied here are all co-localised at the synapse thus future in vitro studies of neurodegenerative disease should consider the complex nature of interactions possible in situ.

The most thermodynamically stable biological structure is the cross-beta secondary structure of the "amyloid"or "prion". As a testament to its stability, the amyloid occurs naturally in 2 rare instances: as a mechanism to protect or destroy life. Pathogenic amyloids are the signature of neurological disorders such as Alzheimer's and Parkinson's disease and bovine spongiform encephalopathy (BSE), which have no effective treatments or known cures. Pathogenic amyloids appear as nanometer sized "plaques" that self-assemble over time. The plaques usually are well-organized crystalline/fibrous structures ~10-20 nm in diameter and >100 nm long. "Functional" amyloids are very rare in nature and serve the direct purpose to proliferate life. Stalks to protect eggs, fibers to coat spores, and adhesive proteins of bacteria, algae, fungi, and mollusks are examples. Functional amyloids can be larger than pathogenic amyloids by 1-2 orders of magnitude. There is a burgeoning research field based on emulating the amyloid. This is because it can be formed from a host of proteins or peptides simply by denaturing them enough to form a cross-beta secondary structure and has a modulus of >10 GPa. As a general reference, "protein" is usually a very high molecular weight, naturally occurring molecule and "peptide" is a much smaller portion of a natural protein or a non-natural molecule synthesized from a few amino acids. Researchers are increasingly attempting to take advantage of the functional amyloid. It is still not understood how the functional amyloid self-assembles or why it can be larger than the pathogenic amyloid. We have identified a potential pathway to large functional amyloids that involves a long alpha-helix containing protein (the "adder") undergoing an alpha to beta transition in the presence of a hydrophobic beta-sheet template. Testing our hypothesis against proteins found in natural large functional amyloids seems to suggest it is a ubiquitous process. The resulting material is a fiber composite similar to the native structures. / Master of Science

Septinas pertencem a uma família de proteínas de ligação a GTP e são encontradas em diversos eucariontes, participando de diferentes processos celulares citoesqueléticos. As septinas apresentam um domínio central de ligação a GTP (domínio G) flanqueado por uma região amino-terminal e outra carboxi-terminal. As septinas se caracterizam por interagirem entre si formando heterocomplexos que se polimerizam, constituindo filamentos. A única estrutura resolvida de um complexo de septinas é de um hexâmero, composto por duas subunidades de três septinas humanas diferentes: SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Esta estrutura revelou que a formação do filamento envolve interações conservadas entre os domínios G, estando o restante da estrutura desordenado no cristal. Além disso, mostrou que dois tipos de interface se alternam ao longo do filamento, as chamadas interfaces G (que incluem a região de ligação do nucleotídeo de duas subunidades) e interfaces NC (que incluem as regiões N e C-terminais do domínio G). Várias evidências sugerem que as regiões C-terminais da proteína sejam as principais responsáveis pela seleção do parceiro correto de interação para montagem dos heterocomplexos. Nesse contexto, buscou-se avaliar a importância das regiões C-terminais na seleção das parceiras SEPT6 e SEPT7 para formar a interface NC, frente ao domínio G. Inicialmente, uma septina quimérica foi produzida de forma a conter o domínio G de SEPT2 e o C-terminal de SEPT6, gerando SEPT2G6C. As proteínas SEPT7GC, SEPT6GC, SEPT2GC e SEPT2G6C foram expressas e purificadas separadamente. Análises de estabilidade térmica e de afinidade proteína-proteína dos pares indicou que a quimera foi capaz de interagir com SEPT7GC, gerando o heterodímero SEPT7GC-SEPT2G6C, mas este não se mostrou tão estável quanto o heterodímero fisiológico. Foi também avaliada a importância da ligação do nucleotídeo para formação da interface G e, para isso, foram construídos os mutantes SEPT2GT78M e SEPT2GD185N, cujos resíduos importantes para hidrólise e ligação do nucleotídeo, respectivamente, foram alterados. A análise de oligomerização por cromatografia de exclusão molecular mostrou deslocamento no volume de eluição das proteínas expressas sozinhas e coexpressas com SEPT6G, indicando que a formação do dímero via interface G depende da ligação do nucleotídeo, mas não da sua hidrólise. Finalmente, foi avaliada a estabilidade térmica e estrutural e a propensão à formação de amilóides do heterodímero SEPT6G-SEPT2G, o qual apresentou maior estabilidade estrutural quando comparado ao homodímero de SEPT2G, mas ainda exibiu alteração de sua estrutura para um estado capaz de ligar Thioflavina-T, sugerindo a formação de amilóides. Entretanto, isso foi observado em temperaturas cerca de 30 ºC acima daquela observada para o homodímero, confirmando a maior estabilidade do heterodímero e sugerindo que a formação da interface G com o parceiro correto pode ser um fator importante na prevenção da formação de estruturas amilóides à temperaturas fisiológicas. / Septins belong to a family of GTP binding proteins and are found in several eucaryotes, participating in different cytoskeletal cell processes. The septins have a central GTP binding domain (G domain) flanked by an amino-terminal and a carboxy-terminal regions. The septins are characterized by the ability to interact with each other forming heterocomplexes which polymerize themselves, forming filaments. The only solved structure of a septin complex is a hexamer, formed by two subunits of three different human septins: SEPT7-SEPT6-SEPT2- SEPT2-SEPT6-SEPT7. This structure revealed that the filament arrangement involves conserved interaction between G domains, being the remainder of the structure disordered in the crystal. Moreover, two types of interface alternate along the filament were shown, socalled G interfaces (which include the nucleotide binding region of the two subunits) and NC interfaces (which include the N- and C- terminal regions of G domain). Plenty of evidences suggest that C-terminal regions of the protein are the main responsible for the selection of the correct interaction partner to assembly of heterocomplexes. In this context, it was sought to evaluate the importance of the C-terminal regions in the selection of the partnerships SEPT6 and SEPT7 to form the NC interface, against the G domain. For this, a chimerical septin was designed so that contains the G-domain of SEPT2 and the C-terminal of SEPT6, creating SEPT2G6C. The SEPT7GC, SEPT6GC, SEPT2GC and SEPT2G6C proteins were expressed and purified individually. Thermal stability and protein-protein affinity analysis of the pairs indicated that the chimera was able to interact with SEPT7GC, forming the heterodimer SEPT7GC-SEPT2G6C, which, however, did not show as stable as the physiological heterodimer. The importance of nucleotide binding to the interaction through G interface was also evaluated and, for that, SEPT2 mutants on GTP-domain were constructed, SEPT2T78M and SEPT2D185N, whose important residues in the hydrolysis and linking of nucleotide, respectively, were changed. Oligomerization analysis by size exclusion chromatography showed a shift in the elution volume of proteins expressed alone and coexpressed with SEPT6, indicating that the complexation of proteins to form G interface depends on the nucleotide binding, but not on its hydrolysis. Finally, the thermal and structural stability and the propensity to amyloid formation of heterodimer SEPT6G-SEPT2G were evaluated, which showed greater structural stability when compared to SEPT2 homodimers, but still exhibited alteration of its structure to a state that was able to bind Thioflavin-T, suggesting amyloid formation. However, this was observed at temperatures around 30 ºC above that observed for the homodimer, confirming the greater conformational stability of the heterodimer and suggesting that the formation of G interface with the right partner can be an important factor of the amyloid filament prevention at physiological temperatures.

Septinas pertencem a uma família de proteínas de ligação a GTP e são encontradas em diversos eucariontes, participando de diferentes processos celulares citoesqueléticos. As septinas apresentam um domínio central de ligação a GTP (domínio G) flanqueado por uma região amino-terminal e outra carboxi-terminal. As septinas se caracterizam por interagirem entre si formando heterocomplexos que se polimerizam, constituindo filamentos. A única estrutura resolvida de um complexo de septinas é de um hexâmero, composto por duas subunidades de três septinas humanas diferentes: SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Esta estrutura revelou que a formação do filamento envolve interações conservadas entre os domínios G, estando o restante da estrutura desordenado no cristal. Além disso, mostrou que dois tipos de interface se alternam ao longo do filamento, as chamadas interfaces G (que incluem a região de ligação do nucleotídeo de duas subunidades) e interfaces NC (que incluem as regiões N e C-terminais do domínio G). Várias evidências sugerem que as regiões C-terminais da proteína sejam as principais responsáveis pela seleção do parceiro correto de interação para montagem dos heterocomplexos. Nesse contexto, buscou-se avaliar a importância das regiões C-terminais na seleção das parceiras SEPT6 e SEPT7 para formar a interface NC, frente ao domínio G. Inicialmente, uma septina quimérica foi produzida de forma a conter o domínio G de SEPT2 e o C-terminal de SEPT6, gerando SEPT2G6C. As proteínas SEPT7GC, SEPT6GC, SEPT2GC e SEPT2G6C foram expressas e purificadas separadamente. Análises de estabilidade térmica e de afinidade proteína-proteína dos pares indicou que a quimera foi capaz de interagir com SEPT7GC, gerando o heterodímero SEPT7GC-SEPT2G6C, mas este não se mostrou tão estável quanto o heterodímero fisiológico. Foi também avaliada a importância da ligação do nucleotídeo para formação da interface G e, para isso, foram construídos os mutantes SEPT2GT78M e SEPT2GD185N, cujos resíduos importantes para hidrólise e ligação do nucleotídeo, respectivamente, foram alterados. A análise de oligomerização por cromatografia de exclusão molecular mostrou deslocamento no volume de eluição das proteínas expressas sozinhas e coexpressas com SEPT6G, indicando que a formação do dímero via interface G depende da ligação do nucleotídeo, mas não da sua hidrólise. Finalmente, foi avaliada a estabilidade térmica e estrutural e a propensão à formação de amilóides do heterodímero SEPT6G-SEPT2G, o qual apresentou maior estabilidade estrutural quando comparado ao homodímero de SEPT2G, mas ainda exibiu alteração de sua estrutura para um estado capaz de ligar Thioflavina-T, sugerindo a formação de amilóides. Entretanto, isso foi observado em temperaturas cerca de 30 ºC acima daquela observada para o homodímero, confirmando a maior estabilidade do heterodímero e sugerindo que a formação da interface G com o parceiro correto pode ser um fator importante na prevenção da formação de estruturas amilóides à temperaturas fisiológicas. / Septins belong to a family of GTP binding proteins and are found in several eucaryotes, participating in different cytoskeletal cell processes. The septins have a central GTP binding domain (G domain) flanked by an amino-terminal and a carboxy-terminal regions. The septins are characterized by the ability to interact with each other forming heterocomplexes which polymerize themselves, forming filaments. The only solved structure of a septin complex is a hexamer, formed by two subunits of three different human septins: SEPT7-SEPT6-SEPT2- SEPT2-SEPT6-SEPT7. This structure revealed that the filament arrangement involves conserved interaction between G domains, being the remainder of the structure disordered in the crystal. Moreover, two types of interface alternate along the filament were shown, socalled G interfaces (which include the nucleotide binding region of the two subunits) and NC interfaces (which include the N- and C- terminal regions of G domain). Plenty of evidences suggest that C-terminal regions of the protein are the main responsible for the selection of the correct interaction partner to assembly of heterocomplexes. In this context, it was sought to evaluate the importance of the C-terminal regions in the selection of the partnerships SEPT6 and SEPT7 to form the NC interface, against the G domain. For this, a chimerical septin was designed so that contains the G-domain of SEPT2 and the C-terminal of SEPT6, creating SEPT2G6C. The SEPT7GC, SEPT6GC, SEPT2GC and SEPT2G6C proteins were expressed and purified individually. Thermal stability and protein-protein affinity analysis of the pairs indicated that the chimera was able to interact with SEPT7GC, forming the heterodimer SEPT7GC-SEPT2G6C, which, however, did not show as stable as the physiological heterodimer. The importance of nucleotide binding to the interaction through G interface was also evaluated and, for that, SEPT2 mutants on GTP-domain were constructed, SEPT2T78M and SEPT2D185N, whose important residues in the hydrolysis and linking of nucleotide, respectively, were changed. Oligomerization analysis by size exclusion chromatography showed a shift in the elution volume of proteins expressed alone and coexpressed with SEPT6, indicating that the complexation of proteins to form G interface depends on the nucleotide binding, but not on its hydrolysis. Finally, the thermal and structural stability and the propensity to amyloid formation of heterodimer SEPT6G-SEPT2G were evaluated, which showed greater structural stability when compared to SEPT2 homodimers, but still exhibited alteration of its structure to a state that was able to bind Thioflavin-T, suggesting amyloid formation. However, this was observed at temperatures around 30 ºC above that observed for the homodimer, confirming the greater conformational stability of the heterodimer and suggesting that the formation of G interface with the right partner can be an important factor of the amyloid filament prevention at physiological temperatures.

De nombreux systèmes biologiques sont intrinsèquement polydispersés, présentant de multiples espèces coexistantes, de taille, de forme ou de conformation différentes (c'est-à-dire, mélanges oligomèriques, des complexes faiblement liés se dissociant en composantes individuelles ou des espèces apparaissant lors de processus amyloïdogéniques). L'étude de tels systèmes complexes est une tâche difficile en raison de l'instabilité des espèces concernées, de leurs concentrations relatives faibles et interdépendantes et des difficultés rencontrées pour l'isolation des composantes pures. Dans cette thèse, j'ai développé des approches méthodologiques pour appliquer la diffusion des rayons X aux petits angles (SAXS), une technique de biologie structurale, à l'étude de systèmes polydispersés. SAXS est une technique additive et par conséquent, le diagramme de diffusion mesuré pour un échantillon polydispersé correspond à la somme pondérée en concentration des contributions de chacune des composantes individuelles du mélange. Cependant, la décomposition des données de SAXS en des spectres spécifiques des espèces et de leurs concentrations relatives est extrêmement laborieuse et ambigue. Dans cette thèse, je présente d'abord une approche objective pour solidement décomposer les jeux de données de SAXS en composantes individuelles. Cette approche adapte la méthode chimiométrique « Multivariable Curve Resolution Alternate Least Squares » (MCR-ALS) aux spécificités des données de SAXS. Notre méthode permet une décomposition rigoureuse et robuste des données de SAXS en introduisant simultanément différentes représentations de ces données et par conséquent, en mettant l'accent sur des changements moléculaires à différentes plages de temps et de résolution structurale. Nous avons appliqué cette approche, que nous appelons COSMiCS (Analyse structurelle objective complexe des systèmes multi-composants) pour étudier deux systèmes polydispersés: la fibrillation des protéines, et les fluctuations conformationnelles de protéines grâce à l'analyse de données obtenues à l'aide d’une technique de couplage de chromatographie d'exclusion de taille (SEC) avec le ligne de SAXS (SEC-SAXS). L'importance d'étudier les processus de fibrillation réside dans leur implication dans des pathologies amyloïdogéniques telles que les maladies de Parkinson ou d'Alzheimer. Il existe de fortes indications que les espèces oligomériques solubles, et non les fibrilles matures, sont la cause principale de la cytotoxicité et des dommages neuronaux. Cette observation souligne l'importance de caractériser les premiers stades des processus de fibrillation. Notre approche COSMiCS a permis d'étudier les processus amyloïdogéniques de l'insuline et du mutant familial E46K de l'α-synucléine, une protéine associée à la maladie de Parkinson. Cette analyse permet la caractérisation structurale des espèces présentes (y compris les espèces oligomériques) et la caractérisation cinétique de leurs transformations.La deuxième partie de la thèse est consacrée à l'utilisation de COSMiCS pour analyser des données de SEC-SAXS. Le SEC-SAXS est extrêmement populaire et a été implémenté sur plusieurs lignes de SAXS à travers le monde. En utilisant des données synthétiques, je démontre la capacité des approches chimiométriques à décomposer des profils chromatographiques complexes. À l'aide de cette approche, j'ai décomposé l’ensemble des données SEC-SAXS mesurés pour la Prolyl OligoPeptidase (POP).En résumé, cette thèse présente une nouvelle approche chimiométrique qui peut être généralement appliquée à tout mélange macromoléculaire pouvant subir une modifacation de son équilibre et pouvant être abordé par SAXS. Les complexes biomoleculaires transitoires, les processus de repliement, les réarrangements structuraux dépendants d’un ligand ou la formation de grands ensembles supramoleculaires peuvent être sondés de façon structurale en utilisant l'approche COSMiCS. / Many biological systems are inherently polydisperse, presenting multiple coexisting species differing in size, shape or conformation (i.e. oligomeric mixtures, weakly bound complexes, and species appearing along amyloidogenic processes). The study of such complex systems is challenging due to the instability of the species involved, their low and interdependent relative concentrations, and the difficulties to isolate the pure components. In this thesis, I have developed methodological approaches to apply Small-Angle X-ray Scattering (SAXS), a low-resolution structural biology technique, to the study of polydisperse systems. As an additive technique, the SAXS pattern measured for a polydisperse sample corresponds to the concentration-weighted sum of the contributions from each of the individual components. However, decomposition of SAXS data into species-specific spectra and relative concentrations is laborious and burdened by ambiguity. In this thesis, I present an approach to decompose SAXS datasets into the individual components. This approach adapts the chemometrics Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method to the specificities of SAXS data. Our method enables the rigorous and robust decomposition of SAXS data by simultaneously introducing different representations of these data and, consequently, emphasizing molecular changes at different time and structural resolution ranges. We have applied this approach, which we name COSMiCS (Complex Objective Structural analysis of Multi-Component Systems), to study two polydisperse systems: amyloid fibrillation by analysing time-dependent SAXSdata, and conformational fluctuations through the analysis of data obtained using on-line size-exclusion chromatography coupled to SAXS (SEC-SAXS). The importance of studying fibrillation processes lies in their implication in amyloidogenic pathologies such as Parkinson’s or Alzheimer’s diseases. There exist strong indications that soluble oligomeric species, and not mature fibrils, are the main cause of cytotoxicity and neuronal damage emphasizing the importance of characterizing early stages of fibrillation. The first application of our COSMiCS approach has allowed the study of the amyloidogenic mechanisms of insulin and the familial mutant E46K of ↵-synuclein, a Parkinson’s disease related protein. The analysis enables the structural characterization of all the species present as well as their kinetic transformations. The second part of the thesis is dedicated to the use of COSMiCS to analyze on-line SEC-SAXS experiments. Using synthetic data, I demonstrate the capacity of chemometric approaches to decompose complex chromatographic profiles. Using this approach, I have studied the conformational fluctuations in prolyl oligopeptidase (POP), a protein related to synaptic functions and neuronal development. In summary, this thesis presents a novel chemometrics approach that can be generally applied to any macromolecular mixture with a tuneable equilibrium that is amenableto SAXS. Transient biomolecular complexes, folding processes, or ligand-dependent structural rearrangements can be probed structurally using COSMiCS.

Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease. / Microbiology and Immunology

Islet amyloid polypeptide (IAPP) is a 37-residue amyloidogenic hormone implicated in the progression of Type II Diabetes (T2D). T2D affects an estimated 422 million people yearly and is a co-morbidity with numerous diseases. IAPP forms toxic oligomers and amyloid fibrils that reduce pancreatic β-cell mass and exacerbate the T2D disease state. Toxic oligomer formation is attributed, in part, to the formation of inter-peptide β-strands comprised of residues 23-27 (FGAIL). Flavonoids, a class of polyphenolic natural products, have been found experimentally to inhibit IAPP aggregate formation. Many of these known IAPP aggregation attenuating small flavonoids differ structurally only slightly; the influence of functional group placement on inhibiting the aggregation of the IAPP(20-29) has yet to be explored. To probe the role of small-molecule structural features that impede IAPP aggregation, molecular dynamics (MD) simulations were performed on a model fragment of IAPP(20-29) in the presence of morin, quercetin, dihydroquercetin, epicatechin, and myricetin. Contacts between Phe23 residues are critical to oligomer formation, and small-molecule contacts with Phe23 are a key predictor of β-strand reduction. Structural properties influencing the ability of compounds to disrupt Phe23-Phe23 contacts include carbonyl and hydroxyl group placement. These structural features influence aromaticity and hydrophobicity, principally affecting ability to disrupt IAPP(20-29) oligomer formation. This work provides key information on design considerations for T2D therapeutics. / Master of Science in Life Sciences / Type II Diabetes (T2D) affects an estimated 422 million people worldwide, with the World Health Organization (WHO) reporting that approximately 1.5 million deaths were directly caused by T2D in 2019. The progression of T2D has been attributed to a protein, called islet amyloid polypeptide (IAPP, or amylin) that is co-secreted with insulin after individuals eat or consumes calories. IAPP has been discovered to form toxic aggregates or clumps of protein material that worsen the disease state and cause a loss of mass of pancreatic cells. There is a large market for therapeutics of T2D and more small molecule drugs are needed to slow progression and severity of T2D. Flavonoids, a class of natural molecules, have been found to inhibit the processes by which IAPP promotes T2D disease progression by stopping the aggregation of IAPP. The structures of these flavonoid compounds differ slightly but show difference in ability to slow IAPP aggregation. By understanding how those differences confer more or less protection against T2D and inhibit IAPP aggregation, we can design more potent and specific drugs to target IAPP. To probe the role of molecular structure in preventing IAPP aggregation, molecular dynamics (MD) simulations — a powerful computational technique — were performed on a model fragment of IAPP in the presence of molecules morin, quercetin, dihydroquercetin, epicatechin, and myricetin. MD simulations provide extremely detailed information about potential drug interactions with a given target, serving as an important tool in the development of new drugs. This work has identified key features and predictors of effective IAPP drugs, providing a framework for the further development of therapeutics against T2D and similar diseases.

La contamination des ressources en eau potable et des terres agricoles par les pesticides qui entrent dans la chaine alimentaire est un problème environnemental et sanitaire majeur. Des études épidémiologiques ont montré un lien entre l’exposition aux pesticides et la maladie de Parkinson, mais peu d’études sont disponibles sur les autres pathologies neurodégénératives. Notre hypothèse est que les pesticides pourraient être des facteurs déclencheurs ou aggravants, communs aux maladies neurodégénératives.Au laboratoire, nous avons identifié par criblage sur la protéine prion, PrP, un composé nommé A6 décrit pour ses propriétés herbicides. A6 est un dérivé de l’α-terthiényl, molécule naturelle extraite des œillets d’inde. Le composé A6 a la capacité de promouvoir des formes oligomériques de la protéine prion sur une lignée cellulaire infectée par des prions. Mon projet de recherche visait à étudier les effets du bio-herbicide A6 sur l’agrégation et la propagation des prions in vivo. Des souris infectées par les prions ont été traitées avec différentes doses de cette molécule (5, 10 et 20 mg/kg). A la dose de 5 mg/kg, le composé A6 diminue le temps de survie des animaux avec une augmentation de la charge amyloïde. Tandis qu’aux plus fortes doses le traitement par A6 augmente la survie des souris avec une diminution de la charge amyloïde. Par un test de centrifugation rapide (RCA), nous avons montré que les faibles concentrations d’A6 favorisent la formation d’oligomères solubles de PrPSc SDS résistants (rSDS-PrPSc), alors que les plus fortes concentrations induisent des formes insolubles. L’analyse des cerveaux montre que seules les souris traitées avec 10 et 20 mg/kg d’A6 présentent des formes dimériques de rSDS-PrPSc. Cette étude montre une dualité d’effet du composé A6 : à de faibles doses, il favorise la pathologie probablement via des formes oligomériques solubles favorisant la réplication des prions. A de plus fortes doses, le composé A6 piègerait une partie de l’infectiosité des prions sous forme d’agrégats amorphes SDS résistants, bloquant la réplication des prions, et ayant un effet « protecteur ».Une recherche d’analogues structurels au composé A6 a permis l’identification d’une classe d’antifongiques commercialisés : les anilinopyrimidines. Cette famille est composée de 3 molécules : cyprodinil, mépanipyrim et pyriméthanil, qui luttent contre des champignons responsables de la pourriture grise des fruits. L’analyse de nombreux rapports a révélé que nous sommes chroniquement exposés à des résidus d’anilinopyrimidines. Dans cette deuxième étude, nous avons étudié l’effet de ces 3 fongicides dans plusieurs modèles de la maladie d’Alzheimer (MA) et les conséquences sur les marqueurs de la pathologie. Des études d’incubation ex vivo associées à des expériences de cinétique d’agrégation de peptides Aβ1-42 ont montré que ces composés interagissent directement avec les peptides et accélèrent sa cinétique d’agrégation. Afin de déterminer leurs effets in vivo, nous avons exposé chroniquement des souris J20 à un cocktail des 3 fongicides via l’eau de boisson. Les souris ont été traitées à 0,1 μg/L (0,44 nM) de chaque composé, correspondant à la concentration maximale autorisée dans l’eau potable. Après 9 mois de traitement, l’analyse des souris J20 a montré une augmentation du nombre et de la surface des plaques amyloïdes au niveau de l’hippocampe et du cortex. Pour déterminer à quel moment se produit l’effet proagrégant du cocktail, une étude longitudinale d’apparition des agrégats à 3, 6 et 9 mois par microscopie biphotonique, a montré que les plaques amyloïdes augmentent entre 6 et 9 mois et que les pesticides exacerbent les agrégats vasculaires. Les anilinopyrimidines modifient la production et la clairance des peptides Aβ en augmentant l’expression de BACE1 et en diminuant l’expression de la néprilysine. Nos travaux montrent que les antifongiques aggravent la MA. / Contamination of the drinking water and agricultural lands by the use of pesticides entering into the food chain is a major environmental and health problem. Epidemiological studies have shown a link between pesticide exposure and Parkinson's disease, but few studies are available on other neurodegenerative disorders. Our hypothesis is that pesticides may be triggering or aggravating factors common to neurodegenerative diseases.In the laboratory, we identified by a screening on the prion protein, PrP, a compound named A6 described for its herbicidal properties. A6 is a derivative of α-terthienyl, a natural molecule extracted from marigolds. A6 compound has the ability to promote oligomeric forms of the prion protein on a prion-infected cell line. My research project aimed to study the effects of the bioherbicide A6 on prion aggregation and propagation in vivo. Mice infected with prions were treated with different doses of this molecule (5, 10 and 20 mg/kg). At the dose of 5 mg/kg, A6 compound decreases the survival time of animals with an increase of the amyloid load. While at the higher doses, A6 treatment increases survival of mice with a decreased amyloid burden. Using a rapid centrifugation assay (RCA), we have shown that low concentrations of A6 promote soluble SDS-resistant oligomers of PrPSc (rSDS-PrPSc), while higher concentrations favour insoluble forms. Brain analysis shows that only mice treated with 10 and 20 mg/kg of A6 exhibit dimeric forms of rSDS-PrPSc. This study shows a dual effect of A6 compound: at low doses, it strenghtens the pathology probably via soluble oligomeric forms favouring the replication of prions. At higher doses, A6 compound would trap part of the infectivity of prions as SDS resistant amorphous aggregates, blocking prion replication, and thus having a “protective” effectA search for structural analogs of A6 compound allowed the identification of a class of commercial antifungals: anilinopyrimidines. This family is composed of 3 molecules: cyprodinil, mepanipyrim and pyrimethanil, used to fight against fungi responsible for the gray mold of fruits. Analysis of many reports have revealed that we are chronically exposed to residues of anilinopyrimidines. In this second study, we evaluated the impact of the 3 fungicides in several models of Alzheimer’s disease (AD) and their consequences on pathological markers. Ex vivo incubation studies associated to kinetics of fibril formation of Aβ1-42 peptides have shown that these compounds interact directly with Aβ peptides and accelerate its kinetics of aggregation. To determine their effects in vivo, we chronically exposed J20 mice to a cocktail of the 3 fungicides through drinking water. Mice were treated with 0.1 μg/L (0.44 nM) of each compound, corresponding to the maximal concentration allowed in the tap water. After 9 months of treatment, analysis of J20 mice showed an increase in the number and surface of plaques in the hippocampus and cortex. To determine the moment when the pro-aggregative effect occurs, a longitudinal study of appearance of aggregates at 3, 6 and 9 months by 2-photon microscopy was done. Our results showed that amyloid plaques increase between 6 and 9 months, and exacerbate vascular amyloid aggregates. Anilinopyrimidines modified the production and clearance of Aβ peptides by increasing BACE1 expression and by decreasing neprilysin expression. Our researches show a role of fungicides in the aggravation of AD.

Neste trabalho estudamos por espalhamento de raios-X a baixos ângulos (SAXS) quatro diferentes sistemas de interesse biológico. Visamos investigar a auto-agregação de proteínas e de complexos proteicos que darão origem a fibras amilóides, interação proteína-proteína, simulando ambientes altamente concentrados, interação proteína-membrana simulando vesículas de matriz extracelular (MVs) de sistemas de biomineralização e interações proteína-agentes desnaturantes. No caso de formação de amilóides, investigamos a agregação do domínio GTPase da septina 6 (SEPT6G) e do complexo formado com o domínio GTPase da septina 2 (SEPT2G-SEPT6G). A temperaturas de até 15°C, tanto SEPT6G quanto SEPT2G-SEPT6G apresentam-se predominantemente diméricas em solução. Já a 25°C, o heterodímero SEPT2G-SEPT6G permanece estável enquanto agregados maiores de SEPT6G evoluem e coexistem em solução com SEPT6G-SEPT6G dimérica, sendo que a proporção de dímeros diminui com a temperatura. No estudo das MVs, mostramos que miméticos lipossomais de DPPC e DPPC:DPPS (9:1) possuem as mesmas características estruturais na ausência e presença de cálcio na solução. A interação da proteína anexina V humana (A5), envolvida em processos de biomineralização, impacta na membrana modelo induzindo a formação de nanoporos. A adição da fosfatase alcalina tecido não-específico (TNAP) não altera as propriedades estruturais do proteolipossomo na presença de A5. A ação do surfactante dodecil sulfato de sódio (SDS) a 30 mM não altera a conformação da albumina soro bovina (BSA), de maneira que é observada a formação de micelas de SDS coexistindo com a proteína livre em solução. Já a adição de 50 mM de SDS induz um desenovelamento parcial da proteína, identificado pela análise das curvas de SAXS via modelo de \"colar de pérolas\". A ação de uréia a 3 M e 8 M promove um desenovelamento parcial e total da BSA, respectivamente, com subsequente agregação de proteína dependente da temperatura (T > 30°C). A adição de 6 mM de SDS em proteínas parcialmente desenoveladas pela ação da uréia promove um desenovelamento mais acentuado. O potencial efetivo resultante da interação entre duas proteínas distintas, BSA e lisozima a concentração total de 100 mg/mL em solução, pH 7.0, foi obtido da análise de curvas de SAXS. Para isto, utilizou-se uma análise simplificada (em primeira aproximação) considerando um potencial efetivo de interação entre BSA-BSA, lisozima-lisozima e lisozima-BSA. Variamos a razão molar BSA:LISO até 1:42. No pH estudado, BSA tem uma carga residual superficial de -11e, enquanto a lisozima possui +9e. Conforme variamos a razão molar BSA:LISO, observamos dois regimes para o potencial efetivo resultante: i) até BSA:LISO 1:2, a carga efetiva do sistema é praticamente nula com um potencial resultante de caráter atrativo e ii) para razões entre BSA:LISO 1:3 a 1:42, a carga efetiva aumenta e o potencial resultante tem caráter repulsivo. Assim, lisozima e BSA coexistem sem agregar, através de um delicado balanço de forças atrativas e repulsivas no sistema. / In this work we have used small-angle x-ray scattering (SAXS) to study four systems of biological interest. We aim to investigate the self aggregation of proteins and protein complexes that would form amyloid fibers; protein/protein interaction, simulating high concentrations; protein/cell-membrane interaction, simulating extracellular matrix vesicles (MVs) from biomineralizing systems; and protein/denaturating-agents interactions. On the case of amyloid formation, we have investigated the aggregation of G-domain of septin-6 (SEPT6G) and the protein complex formed with G-domain of septin-2 (SEPT2G-SEPT6G). At temperatures lower than 15°C, both SEPT6G and SEPT2G-SEPT6G were found predominantly as dimers. At 25°C, SEPT2G-SEPT6G heterodimer is still stable while aggregates of SEPT6G grow. Both coexist in solutions of SEPT2G-SEPT6G dimers, with the percentage of dimers decreasing the higher the temperature. As for the study of MVs, we have shown that DPPC and DPPC:DPPS (9:1) liposomal mimetics have the same structural characteristics at the absence or presence of Calcium. The interaction with human annexin V protein (A5), related to biomineralization processes, affects the model membrane by the creation of nanopores. The addition of tissue-nonspecific alkaline phosphatase (TNAP) does not change the structural properties of the proteoliposome when A5 is present. The addition of SDS surfactant (30 mM) does not alters the conformation of bovine serum albumin (BSA), and we have observed the formation of SDS micelles coexisting with free protein in solution. The addition of 50 mM of SDS, on the other hand, induces the partial unraveling of the protein, as seen by the analysis of SAXS data via the pearl necklace\'\' model. The effect of adding 3M and 8M urea is, respectively, the partial and total unraveling of BSA, with ensuing aggregation of the protein dependent on the temperature (T > 30°C). The introduction of SDS 6mM promotes further unraveling in proteins that were previously partially unraveled by urea. The resulting effective potential for the interaction between BSA and lysozyme at total concentration of 100mg/ml and 7.0 pH has been obtained from the analysis of SAXS curves. In order to obtain this result we have used a simplified analysis (first order approximation) in which were considered the effective potentials for the interactions between BSA-BSA, lysozyme-lysozyme and lysozyme-BSA. We have varied the BSA:LISO molar ratio up to 1:42. At the studied pH, BSA has a surface residual charge of -11e, and lysozyme has +9e. As we changed the BSA:LISO molar ratio, we have found two regimens for the resulting effective potential: i) up to BSA:LISO 1:2, the effective charge of the system is virtually zero and the resulting potential is attractive; and ii) for BSA:LISO between 1:3 and 1:42 the effective charge increases, and the resulting potential is repulsive. Therefore, both lysozyme and BSA coexist without forming aggregates, by a delicate balance of attractive and repulsive forces.