De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes. / Many proteins associated with human neurodegenerative diseases are intrinsically disordered. They are proteins which lack stable tertiary or secondary structure under physiological conditions. More specifically, intrinsically disordered proteins (IDPs) undergo various structural conversions between random coil, helical conformations and β-strand structures, these two latter being generally involved in protein-protein recognition. Among about twenty known amyloidogenic peptides related to human degenerative diseases, we focus our study on two disordered proteins: the Amyloid-β peptide (Aβ) associated to the Alzheimer’s disease and the Islet Amyloid Polypeptide (IAPP) involved in type II diabetes. Aβ has two common alloforms of 40 and 42 residues in length, meanwhile IAPP is a 37-residues peptide hormone. Aggregates of Aβ are toxic to the brain cells, meanwhile IAPP fibrillization affects the pancreatic β-cells. The aggregation mechanism of these two peptides is not known in detail, but it was proposed that in solution, these peptides visit various conformations, one of them being rich in β-strands. This would lead to peptide oligomerization, through β-strand / β-strand interactions and eventually to the fibril formation. The aim of our study is to provide insights into the conformational dynamics of these two peptides in monomeric and oligomeric forms. Understanding the early steps of their aggregation is crucial for the development of new effective therapeutic molecules against these amyloid proteins.De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

<p>The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.</p>

Protein nanofibres, commonly known as amyloid fibrils, are emerging as potential biological nanomaterials in a number of applications. Protein nanofibres are a highly ordered insoluble form of protein, which results when a normally soluble protein aggregates via a self-association process. However, researchers are currently faced with several challenges such as finding a cheap source of proteins that can be obtained without expensive purification and optimizing a scalable method of the manufacturing of protein nanofibres. This thesis has identified crude mixtures of fish lens crystallins as a cheap protein source and has optimized methods for large scale production of protein nanofibres of varying morphologies. Results show that by varying the conditions of fibre formation, individual protein fibres can be used as building blocks to form higher order structures. This ability to control the morphology and form higher ordered structures is a crucial step in bottom up assembly of bionanomaterials and opens possibilities for applications of protein nanofibres. The method of formation of protein nanofibres was optimized on a bench scale (1.5 mL Eppendorf tubes) and successfully scaled-up to 1 L volume. For larger scale-up volume (i.e. greater than 10 ml), internal surface area was important for the formation of protein nanofibres. The crude crystallin mixture prepared at 10 mg/mL was heated at 80oC in the presence of 10% v/v TFE at pH 3.8 for 24 hours and stored for an additional of 24 hours at room temperature for storage process. Aggregation and precipitation of proteins were observed as the protein solution was added to the pre-heated TFE. The resulting protein nanofibres were characterised using ThT dye binding, TEM and SEM. The TEM images show a network of long and criss-crossing protein nanofibres with individual fibres of approximately 10 to 20 nm in diameter and 0.5 to 1 μm long. These protein nanofibres were prepared in 1 mL centrifuge tubes and were left on the laboratory bench at room temperature. After 5 months, fresh TEM grids of the sample were prepared and visualized using TEM. Interestingly, TEM images show that a number of individual fibres had self-assembled in an intertwining fashion to form large bundles and higher order structures containing bundles of nanofibres up to 200 nm thick.

No description available.

Senile plaques (Aβ) and neurofibrillary tangles (tau) are pathological hallmarks of Alzheimer’s disease (AD). If and how the formation of these deposits are mechanistically linked remains mainly unknown. In recent years, the focus has shifted from insoluble protein deposits to soluble aggregates of Aβ and tau. Protofibrils are large soluble Aβ oligomers which were linked to AD by the discovery of the Arctic AβPP mutation. Treatment of young tg-ArcSwe mice with an Aβ protofibril-selective antibody, mAb158, cleared protofibrils, prevented amyloid plaque deposition and protected cultured cells from protofibril-mediated toxicity. This suggests that Aβ protofibrils are necessary for the formation of Aβ deposits. Functional assessment of tg-ArcSwe mice in IntelliCage demonstrated hippocampal-dependent behavioral deficits such as memory/learning impairments, hyperactivity and perseverance behavior. Learning impairments did not correlate to Aβ-measures but to calbindin, which might be a good marker for Aβ-mediated neuronal dysfunction. Splicing of exon 10 in the tau gene differs between human and mouse brain. Exon 10 is part of the microtubule-binding domains which helps to maintain microtubule stability and axonal transport, functions vital to neuronal viability. Axonal transport dysfunction has been proposed as a common pathway of Aβ and tau pathogenesis in AD. Generation of a novel tau mouse model with absence of exon 10 led to age-dependent sensorimotor impairments which may relate to dysfunctions in cerebellum. No tau pathology was evident suggesting that a trigger of tau fibrillization e.g. a human Aβ or tau aggregate is needed. Generation of AβPPxE10 bitransgenic mice with no exon 10 showed lower Aβ plaque burden. Possibly changes in microtubule function lead to altered intracellular AβPP transport and Aβ production. Initiation of tau pathology in AβPPxE10 mice might require a certain type of Aβ-aggregates which is not produced or exist at too low concentration in transgenic mouse brain. In summary, the Aβ protofibril-selective antibody was found to be a promising treatment for AD. The IntelliCage system was proven to be useful for functional evaluation of AβPP mice. Exon 10 in tau was shown to affect sensorimotor functions and Aβ pathology in bitransgenic mice by mechanisms that deserve further investigation.

Background Traumatic brain injury (TBI) is a complex disease with a spectrum of symptoms and disabilities. Over the past decade TBI has become the focus of research due to growing epidemiological and clinical evidences that TBI incidences are strong risk factors for Alzheimer’s disease (AD). Major pathological hallmarks of AD are massive accumulations of amyloid-β peptide (Aβ) toxic oligomers and plaques. Neuroinflammation is also considered as a common denominator in AD and aging. The epidemiological and experimental studies have supported that non-steroidal anti-inflammatory drugs markedly reduce the age-related prevalence of AD and can slow amyloid deposition by mechanisms that still remain elusive. S100A9 is a multifunctional cytokine with diverse roles in the cell signaling pathways associated with inflammation and cancers. A widespread expression of S100A9 was also reported in many other ailments involving inflammatory processes, such as AD, malaria, cerebral ischemia and TBI, implying that S100A9 may be a universal biomarker of inflammation. The distinctive feature of S100A9 compared to other pro-inflammatory cytokines is its ability to self-assemble into amyloids, which may lead to the loss of its signaling functions and acquired amyloid cytotoxicity, exceeding that of Aβ. Methods S100A9 properties was studied under various ex vivo and in vitro conditions. First, human and mouse tissues with TBI and AD were subjected to microscopic, immunohistochemical and immunofluorescent techniques. Then, aged mouse treated with native, oligomeric and fibrillary S100A9 was also studied by using behavioral and neurochemical analysis. Moreover, S100A9 was established as a biomarker of dementia progression and compared with others such as Aβ42 and tau proteins, by studying cerebrospinal fluid (CSF) samples from different stages of dementia. Finally, in vitro experiments on S100A9 amyloidogenesis, co-aggregation with Aβ40 and Aβ42, digestion and cytotoxicity were also performed by using spectroscopic, atomic force microscopy and cell biology methods. Results S100A9-driven amyloid-neuroinflammatory cascade serves as a link between TBI and AD. We have found that S100A9 contributes to the plaque formation and intraneuronal responses in AD, being a part of the amyloid-neuroinflammatory cascade. In TBI we have found that extensive S100A9 neuronal production and amyloid self-assembly is triggered immediately after injury, leading to apoptotic pathways and neuronal loss. S100A9 is an integral component of both TBI precursor-plaques, formed prior to Aβ deposition, and AD plaques, characterized by different degree of amyloid maturation, indicating that all plaques are associated with inflammation. Both intra- and extracellular amyloid-neuroinflammatory cascades are intertwined and showed similar tendencies in human and mouse tissues in TBI and AD. Ex vivo findings are further supported by in vitro experiments on S100A9 amyloidogenesis, digestion and cytotoxicity. Importantly, being highly amyloidogenic itself, S100A9 can trigger and aggravate Aβ amyloid self-assembly and significantly contribute to amyloid cytotoxicity. Moreover, the CSF dynamics of S100A9 levels matches very closely the content of Aβ42 in AD, vascular dementia and mild cognitive impairment due to AD, emphasizing the involvement of S100A9 together with Aβ in the amyloid-neuroinflammatory cascade in these ailments. Conclusions The conclusions of this thesis is that the inflammatory pathways and S100A9 specifically represent a potential target for the therapeutic interventions during various post-TBI stages and far prior AD development to halt and reverse these damaging processes. / Role of pro-inflammatory S100A9 protein in amyloid-neuroinflammatory cascade in Alzheimer’s disease and traumatic brain injury

Compositions are vectors of nonnegative numbers that sum to a constant, usually one or 100%. They arise in a wide array of fields: geological sampling, budgets,fat/protein/carbohydrate in foods, percentage of the vote acquired by each political party, and more. The usual candidate distributions for modeling compositions -- the Dirichlet and the logistic normal distribution -- have density zero if any component is zero. While statistical methods have been developed for "rounded" zeros, zeros stemming from values below a detection level, and zeros arising from count data, there remain problems with essential zeros, i.e. cases in continuous compositions where a component is truly absent. We develop a model for compositions with essential zeros based on an approach by Aitchison and Kay (2003). It uses a mixture of additive logistic normal distributions of different dimension, related by common parameters. With the requirement of an additional constraint, we develop a likelihood and methods estimating parameters for location and dispersion. We also develop a permutation test for a two-group comparison, and demonstrate the model and test using data from a diabetes study. These results provide the first use of the additive logistic normal distribution formodeling and testing compositional data in the presence of essential zeros.

The pathology of Alzheimer's disease (AD) remains elusive. Competing evidence links amylois \U+fffd\-peptide (A\U+fffd\) amyloid formation to the phenotype of AD (1). The mechanism of amyloid fibril formation has been an ongoing investigation for many years. A\U+fffd\10-23 peptide, a fragment of A\U+fffd\1-42 peptide, contained crucial hydrophobic core residues (2). In this study, an investigation was launched to study the aggreagation process of A\U+fffd\1023 peptide and its ability to form amyloid fibrils. Furthermore, the presence of its hydrophobic core showed importance for its ability to aggregate and form amyloid fibrils. Thereafter, the inhibition of A\U+fffd\1-42 peptide aggregation was studied by using pyrimidine-based compounds. A\U+fffd\1-42 peptides, known to be neurotoxic, aggregate to form amyloid fibrils (3). This investigation may provide insight into the development of novel small molecular candidates to treat AD. / by Ahmad Alex Hijazi. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Este trabalho de mestrado teve como objetivo o desenvolvimento de uma metodologia analítica focada no preparo de amostra protéica. O objeto de estudo foram os fragmentos solúveis das isoformas da proteína precursora do amilóide (APPs) presentes no plasma rico em plaquetas. As APPs têm sido amplamente estudadas em diversos grupos de pesquisa no Brasil e em outros no mundo como possíveis biomarcadores para a doença de Alzheimer. O preparo de amostra é a etapa fundamental que influencia significativamente nos resultados seguintes, especialmente quando se trata de amostras protéicas que exigem maiores cuidados. Para a avaliação dos melhores preparos de amostra para as APPs, foi utilizado SDS-PAGE e eletrotransferências de proteínas por Western Blotting. A eficiência dos preparos foi avaliada baseando-se nos resultados de revelação com anticorpos específicos para APP e medidas de densitometria de bandas. Após a escolha do melhor preparo de amostra utilizando SDS-PAGE e Western Blotting, as isoformas da APP foram separadas por eletroforese bidimensional (2DE). Durante a etapa de preparo de amostra, os resultados inesperados de massa molecular, o que indicou possível biodegradação das APPs. A identificação da fonte de interferência foi realizada estudando as variáveis dos preparos de amostra. Com isso foi possível determinar a fonte de interferência, mas uma avaliação mais detalhada das isoformas (como utilização de espectrometria de massas) não foi possível. / The goal of this Master\'s work was to develop an analytical methodology focused on protein sample preparation. The analyte studied were soluble amyloid precursor protein isoforms (APPs) which has been studied in many groups in Brazil and around the world as a possible biomarker for Alzheimer\'s disease. Sample preparation is a crucial step that influence significantly on next results, especially about biological samples which require more attention. For the best sample preparation for APPs, was used SDS-PAGE and protein electrotransference by Western Blotting techniques. The efficiency of the sample preparations was evaluated based on specific antibody reactions and densitometry measures of these interactions. After that, the APP isoforms were analyzed by two dimensional electrophoresis (2DE). During the sample preparation, were obtained unexpected molecular mass results, which indicated some APPs biodegradation. For the determination of the interference source, the variants steps of the sample preparation were analyzed. The sample preparation interference source was identified, but a more detailed study of the isoforms (by mass spectrometry) was not possible as well as the analysis of the identity of the possible fragmented isoforms.