Thesis advisor: Jianmin Gao / Protein aggregation can be highly detrimental to organisms, and has been associated with diseases including Alzheimer's, Huntington's, type II diabetes, and transmissible spongiform encephalopathies such as Mad Cow disease. There is no single amino acid sequence responsible for aggregation into amyloid-like structures, but rather a large range of amyloidogenic peptides have been discovered. A fragment of the yeast Sup35 prion, GNNQQNY, has been found to aggregate using a "dry, steric zipper" structure. This study looks at mutants of GNNQQNY in order to elucidate the exact contributions of various amino acids to the aggregation process. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Protein aggregation, oligomer and fibril formation is one of the dominant characteristics in the pathogenesis of a number of neurodegenerative diseases, such as Alzheimer’s disease (AD). Inhibition of toxic oligomer and fibril formation is one of the approaches to find potential drug candidates for AD. Additionally, early diagnosis of these amyloid species can provide mechanistic understanding of protein aggregation and thus can pave the way for preventing the onset of AD. The aim of this dissertation was 1) to explore the effects of charged cholesterol derivatives on the aggregation kinetic behavior of Amyloid-β40 (Aβ40), 2) to probe Aβ40 oligomer and amyloid formation in vitro using gold nanoparticles (AuNPs), and 3) to monitor the kinetic effect of various natural product molecules on Aβ40 aggregation in vitro. In the first chapter, a general introduction about AD as an amyloidogenic disease, amyloid cascade hypothesis, and the manipulation of Aβ peptides aggregation kinetics using different approaches was presented. In the second chapter, we studied the effects of oppositely charged cholesterol derivatives on the aggregation kinetics of Aβ. In the third chapter, we developed a gold nanoparticles (AuNPs) assay to probe Aβ40 oligomers and amyloid formation. In chapter IV, we monitored the effects of various small molecules on the aggregation kinetics of Aβ40. In chapter V, we discussed the methods and experimental details. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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.

Septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina que atuam no ciclo de divisão celular e também são amplamente encontradas em doenças neurodegenerativas tais como mal de Parkinson e Alzheimer e em alguns tipos de câncer como leucemia, linfoma e tumores sólidos. Neste trabalho investigamos como a temperatura e a concentração impactam na agregação do domínio GTPase da septina 2 (SEPT2G), podendo levar a formação de bras amilóides, por espalhamento de luz (DLS) e Raios-X a baixos ângulos (SAXS). Resultados de DLS revelaram que a cinética de agregação da proteína é da ordem de segundos para temperaturas maiores que 25ºC. Os dados de SAXS da proteina a 0,5 mg/ml mostraram que a SETP2G é um dímero em solução aquosa a 4ºC e esta conguração se mantém estável por cerca de 1 hora de observação experimental. A 15ºC, os resultados de SAXS revelaram uma coexistência de três populações em solução compostas por 88% de dímeros, 10% de agregados pequenos tipo-cilindros (protobrilas), e 2% de agregados grandes maiores que a resolução da técnica. Após cerca de 30 minutos existe um rearranjo preferencial de dímeros em favor de agregados muito grandes cuja contribuição à curva de espalhamento torna-se 8%. A 25ºC, a porcentagem de dímeros decresce para 70% com uma contribuição de cerca de 30% de agregados grandes já no início das medidas experimentais. Nas temperaturas de 37ºC e 45ºC, dímeros e agregados muito grandes coexistem em solução desde o início das medidas experimentais, cujo equilíbrio se desloca rapidamente tal que após 20 minutos de observação a solução é composta majoritariamente por agregados muito grandes, identicados como estruturas amilóides pela técnica de uorescência da tioavina, que se intercala em estruturas cross-. A 1 mg/mL e temperatura de 4ºC, a proteína permaneceu estável durante cerca de 1 hora de observação sendo que existe um equilíbrio de dímeros (93%) com agregados alongados (contendo cerca de 80 monômeros) em solução. Com o aumento da temperatura para 15ºC, a maioria da população ainda é dimérica. Já a 25ºC, a presença de agregados muito grandes é bem significativa (da ordem de 30% coexistindo com dímeros e oligômeros). A 37ºC e 45ºC existe a formação de grandes agregados similar ao observado para a SEPT2G a 0,5 mg/mL. Em suma, os resultados de SAXS demonstraram que a SEPT2G tem uma cinética muito rápida de agregação a temperatura siológica, acentuada com o aumento de concentração da proteína em solução. / Septins are proteins from the GTP-binding family and participate in cell division cycle performing functions such as secretion and cytoskeletal division. They can also be found in neurodegenerative conditions as Alzheimer\'s and Parkinson\'s diseases and some kinds of cancer as leukemia, lymphoma and solid tumors. In this work, we investigated the influence of temperature and concentration on the septin 2 GTPase domain (SEPT2G) aggregation using dynamic light scattering (DLS) and small angle x-ray scattering (SAXS). DLS results revealed the protein aggregation kinetic is around seconds for temperatures above 25ºC. SAXS data of the protein at 0.5 mg/mL showed that SEPT2G is a dimer in aqueous solution at 4_C and this condition is kept stable for approximately one hour of experimental observation. At 15ºC, SAXS results revealed the coexistence of three populations in solution composed by 88% of dimers, 10% of cylinder-like smaller aggregates (protofibrils) and 2% of aggregates bigger than the technique detection. After 30 minutes there is a preferential rearrangement of dimers into very large aggregates which contribution on the scattering curve becomes 8%. At 25ºC, the dimers percentage decreases to 70% with a contribution of circa 30% of bigger aggregates, even at the beginning of data acquisition. At temperatures of 37ºC and 45ºC, dimers and very large aggregates coexist in solution since the beginning of data acquisition, which equilibrium quickly shifts in such a way that after 20 minutes of observation the solution is mostly composed by very large aggregates, indented as amyloid structures by the thioflavine fluorescence technique, which intercalates in the cross- structures. At 1 mg/mL and 4ºC, the protein was stable over 1 hour of observation where an equilibrium of dimers (93%) and elongated structures (composed by approximately 80 monomers) in solution takes place. Increasing the temperature to 15ºC, most of the protein remains dimeric. On the other hand, at 25ºC the very large aggregates contribution is around 30% coexisting with dimers and oligomers. At 37ºC and 45ºC there is the formation of large aggregates, similar to what was observed at 0.5 mg/mL. In conclusion, our SAXS results indicated that the aggregation process of SEPT2G in solution may follow different pathways depending on concentration and temperature.

Nicotinic acetylcholine receptor (nAChR) activation is neuroprotective and nicotine is a cognitive enhancer. Loss of nAChRs, deposition of tau neurofibrillary tangles, cleavage of amyloid precursor protein (APP) and inflammation are well documented in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β- and γ-secretase enzymes generates soluble Aβ peptides, with oligomeric forms of Aβ implicated in both the control of synaptic excitability and dysregulation of synaptic transmission and induction of neuronal death in AD. Aβ production is inhibited by calcium-dependent recruitment of α-secretase, as exemplified by activation of N-methyl-D-aspartate receptors (NMDAR). All neurodegenerative diseases are associated with inflammation, arising from altered homeostasis of the innate immune system, resulting in heightened activation of immune cells and induction of a pro-inflammatory environment. Stimulation of the α7 subtype of nAChR is anti-inflammatory and also enhances cognition and promotes neuronal survival. This work addressed the hypotheses that stimulation of highly calcium-permeable α7nAChR inhibits Aβ production by promoting α-secretase-mediated processing of APP and also modulates inflammatory cellular behaviour of microglia. Thus, this study assessed the role of α7nAChR at glutamatergic synapses, through probing effects on APP processing and phagocytosis in primary cortical neurons and microglia, respectively. Primary cortical neurons expressed functional α7nAChR and glutamate receptors, and through a number of experimental approaches, including immunoblotting and a cleavage reporter assay, results indicated α7nAChR activation with the α7nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596 had no effect on APP and Tau, in contrast to NMDAR activation that significantly modulated these proteins. Data suggest low expression of α7nAChR, coupled with distinct localisation of presynaptic α7nAChR and postsynaptic APP could explain the lack of effect. In addition, primary microglia were highly responsive to lipopolysaccharide and possessed functional α7nAChR that coupled to ERK phosphorylation. Microglial α7nAChR activation promoted neuroprotective phagocytic behaviour, in agreement with the ‘cholinergic anti-inflammatory pathway’. This study supports the hypothesis that α7nAChR are modulators of anti-inflammatory behaviour, thus α7nAChR-selective ligands are viable candidates for the treatment of AD and promoting cognitive enhancement.

Supported lipid bilayers (SLBs) are one of the most common model membranes used in the field of cell membrane biology as they provide a well-defined model membrane platform for determination of molecular-level interactions between different biomolecules (e.g. proteins, peptides) and lipid membrane. Compared to model organisms, the use of SLB is preferable since it mimics cell plasma membrane in a very simple and well-controlled way. Therefore, molecular structure of membrane and experimental conditions (e.g. solution chemistry, temperature, and pH) can be easily adjusted to the required conditions of any systematic research. In addition, SLBs are typically easy to form, cheap and very reproducible and they are compatible with different surface characterization techniques, such as quartz crystal microbalance with dissipation (QCM-D), ellipsometry and atomic force microscopy (AFM). This study demonstrates that QCM-D analysis of SLBs serve as powerful tool to investigate and characterize the mechanisms of interactions between lipid membrane and gold nanoparticles (NPs), environmentally relevant polymers, and disease-inducing peptides. Due to many critical applications of gold NPs in drug delivery and diagnostics, understanding of membrane-NP interactions is crucial especially for determination of NPs cytotoxicity. In this study we focus on membrane disruption as one of the different mechanisms by which metal NPs induce cytotoxicity. The use of SLB is beneficial for this goal as it elucidates the unique mechanism of membrane disruption without interference of other mechanisms taking place simultaneously in biological cells. For NP-membrane interaction studies, a SLB composed of L-α-phosphatidylcholine (egg PC) was formed on a SiO2-coated crystal and QCM-D analysis was performed to obtain information about mass and viscoelastic changes of SLB resulting from interactions with gold NPs. For better understanding of the mechanisms of NP-membrane interactions, we systematically changed the NPs properties and the experimental conditions. In order to understand the effect of NP size, gold NPs with diameters of 2,5,10, and 40 nm were tested and compared to each other. NPs were tested in their citric acid-stabilized state as well as in the presence of poly (methacrylic acid) (PMAA), representing an organic coating that could become associated with NPs in the environment. The results indicated that when dissolved in water, gold NPs with the dimeters of 2, 5, 10, and 40 nm did not perturb the membrane, but in the presence of environmentally relevant polymer, the larger nanoparticles were found to disrupt the membrane. In order to elucidate the effect of surface chemistry, 10 nm - gold NPs with various functionalizations (i.e. anionic, cationic and non-ionic ligands) were tested. Control experiments were designed to test the effect of NPs in the absence of humic substances which means the NPs were dissolved in water. In these cases, regardless of the type of NP functionalization, no substantial bilayer mass changes were observed. This suggests that the charge and chemistry of the ligands had a minor effect on NP-membrane interactions. Furthermore, in both the control and humic acid experiments, there were small dissipation changes (less than 1 unit) indicating that the overall membrane structure was not perturbed. In order to mimic environmentally-relevant conditions, mass and viscoelasticity of SLB was characterized in the presence of four different natural polymers, also known as natural organic materials (NOMs): Fulvic and humic acids extracted from Suwannee River (SRFA and SRHA), which had relatively lower molecular weights and a commercial humic acid (HA) and the humic acid extracted from Elliott soil (ESHA) with higher molecular weight. The results showed that NOMs with lower molecular weights, adsorbed to the bilayer, while higher molecular weight components, did not induce any changes to the bilayers. In addition, the NPs in SRFA and SRHA increased the mass of the bilayer by 20-30 ng, while the NPs in HA and ESHA changed the mass of the bilayer by < 10 ng. It was concluded that the presence of humic substances as well as their physical and chemical properties exert a direct impact on the interactions between cell membrane and the nanoparticles. In addition to the field of NP toxicity, SLBs play a pivotal role in the field of neurodegenerative diseases, such as Alzheimer’s disease (AD), in which the pathological cascade of events starts from interactions of a misfolded peptide with cell membrane. In this thesis, we confirm the validity of QCM-D analysis of SLB as an important platform for investigation of amyloid β (the peptide associated with AD) interactions with lipid membrane. Adsorption of Aβ peptide to cell membrane is known to take place on the so-called “lipid raftâ€� which are membrane microdomains enriched with cholesterol, sphingomyelin and ganglioside. The formation of SLBs containing lipid rafts is not only important for the field of AD research, but also it is important for other in vitro studies of cell biology as the lipid rafts are responsible for a variety of biological functions such as association of some membrane proteins and cellular signaling. However, the presence of lipid raft components such as sphingomyelin and cholesterol makes the formation of the bilayer more challenging which leads to adsorption of intact vesicles on the substrate without formation of the bilayer. In this study, the formation of lipid bilayer composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM) was investigated using QCM-D. A challenge was that the raft-containing vesicles remained intact on the SiO2 crystal. Therefore, different experimental conditions were tested to induce vesicle fusion, such as pH, temperature, osmotic pressure, and vesicle size. The key parameter in forming the bilayer was found to be applying osmotic pressure to the vesicles by having the vesicles exterior concentration of NaCl higher than interior concentration. When this concentration gradient was applied to the vesicles before flowing them on the substrate, vesicle rupture was favored and formation of a complete bilayer could occur. Here, we report the effects of each tested variable on the adsorption and fusion of the raft-containing vesicles, and the results are discussed based on the mechanisms of vesicle-vesicle and vesicle-substrate interactions.After developing the robust method for formation of SLB with lipid rafts, we used that as a template to characterize the mechanism of interactions between Aβ peptide and cell membrane which leads to onset of AD. The mechanism of Aβ toxicity leading to AD has not fully discovered yet, due to the complexity of the process including several steps of Aβ peptide adsorption on membrane, conformational change from disordered in solution to a membrane-bound α-helix structure and then formation of β-sheet aggregates that serve as fibrillation seeds. In this study, we showed that QCM-D technique as a promising tool to conduct systematic studies on the mechanism of interactions between Aβ peptide with lipid membrane. To our knowledge, this was the first time QCM-D was utilized for characterization of Aβ fibrillation starting from monomer states until formation of mature fibrils. The data indicated that peptide-membrane interactions follow a two-step kinetic pathway starting with the adsorption of small (low-n) oligomers until covering all the adsorption sites on the surface. In the second step, the membrane structure is destabilized as the result of interaction with oligomers which leads to lipid loss from the surface. Consistency of the results with the data obtained via other techniques substantiates QCM-D technique as a robust approach to answer the remaining unanswered questions in the field of Alzheimer’s disease.

Transthyretin (TTR)-associated amyloidoses are diseases wherein wild-type or mutant TTR forms amyloid fibrils that infiltrate multiple organs. Wild-type TTR amyloidosis, ATTRwt, is a sporadic disease characterized by deposits that occur mainly in the heart. Alternatively, >100 TTR mutants cause inherited forms, ATTRm, frequently featuring cardiac amyloid deposits. The goals of this research were to create a cell-based model of ATTR amyloidosis, to define the mechanism of cardiac TTR-associated amyloid at the cellular level, and to study several agents that could interrupt the amyloid process. We hypothesized that TTR oligomers were cardiotoxic and played a role in the mechanism of ATTR amyloidosis, and that cytotoxicity could be inhibited by diflunisal, doxycycline, and Kiacta®. Focusing on TTR proteins associated with cardiac amyloidosis (wild-type, L55P, V30A, and V122), we developed a thermal denaturation method for creating TTR oligomers that allowed us to study the direct effect of oligomers on cells. Congo red and thioflavin T analyses confirmed that the oligomers were on pathway to amyloid fibril formation. We tested the effect of TTR oligomers on rat and human cardiac cells by measuring cell viability and stress response (through live protease activity and qPCR). TTR-L55P oligomers elicited a cytotoxic effect; fluorescent microscopy indicated cellular uptake of the oligomers and continued intra-cellular aggregation. Cytotoxicity was blocked when TTR was heated in the presence of doxycycline; the drug appeared to dissociate TTR aggregates or stabilize the monomeric forms. We also investigated retinol-binding protein (RBP), a natural binding partner of TTR. By immuno-histochemistry, RBP was demonstrated in ATTRwt and ATTRm `non-amyloid' transplant heart tissues, localized to areas containing amyloid or in the case of the transplant tissue, regions that appeared to display ischemic damage. Serum RBP levels were significantly different in ATTR vs. age-matched controls (p = 0.03), and in ATTRwt vs. ATTRm (p <0.0001) by ELISA. These data provide evidence that TTR oligomers are cardiotoxic, possibly due to cellular internalization and progressive intracellular aggregation. Furthermore, our results support the use of doxycycline as a therapeutic in ATTR to target these amyloidogenic oligomers, and suggest that RBP may have potential as a disease biomarker.

No description available.

Mass Spectrometry (MS) and Ion Mobility - Mass Spectrometry (IM-MS) can be used to investigate protein structure and dynamics and are ideally positioned to study intrinsically disordered and amyloidogenic proteins, whose diverse conformational space and/or oligomeric state is hard to track accurately. This thesis uses hybrid MS approaches including IM-MS, Cross-linking IM-MS and ECD-FT-ICR MS to probe the structure of alpha-Synuclein and Amyloid-beta (Abeta). For alpha-Synuclein, the effect of solution pH and ionisation polarity on the species observed by MS and IM-MS is investigated. Conformational families observed by Cross-linking IM-MS provides a link between the solution and gas phase structures of alpha-Synuclein observed here and our data correlates with that reported by other groups. MS, IM-MS and HDX-MS are used to probe alpha-Synuclein during the early stages of aggregation. A specific aggregation competent conformer is not observed suggesting that the solution constituents remain conformationally dynamic. We observe shifts in the species observed by MS and IM-MS between samples and our data contributes to an array of conflicting structural studies indicating that alpha-Synuclein adopts a diverse range of species with significant variation. For Abeta(1-42) and Abeta(1-40) Collision Induced Unfolding and ETD/ETcaD demonstrate that Abeta(1-42) adopts a compact conformation bound by intramolecular interactions. Changes to the Abeta(1-42) and Abeta(1-40) ATDs following SID are correlated to known structure influencing intermolecular interactions and demonstrate the large structural difference between Abeta(1-42) and Abeta(1-40) despite differing by only two C-terminal amino acids. IM-MS is used to classify the mode of action of anti-aggregation drug candidates on Abeta(1-42). The anti-aggregation capacity of the retro-inverso peptide, RI-OR2 is shown to result from inducing the compaction or extension of Abeta(1-42), preventing the adoption of an aggregation competent structure. In contrast, the flavonoid Rutin is shown to act solely through inducing Abeta(1-42) compaction. This thesis demonstrates the power of MS based methods to investigate the diverse range of structures of intrinsically disordered aggregating proteins implicated in disease.

The aim of the thesis project was to examine what form(s) of Amyloid beta (Aβ) (25-­‐35) peptide interact with phospholipids in vitro and the implications of this for the mechanism of Alzheimer’s Diseases (AD). The mechanism of AD is thought to involve protein folding and misfolding. An increasing amount of evidence has shown that protein misfolding plays an important role in the biological and pathological processes of AD. Although seen as the biomedical markers of those diseases, the roles of amyloid aggregates themselves are still not fully understood. Whether the aggregates, or the monomer, or some other intermediates of Aβ cause AD is still unknown. In order to investigate the membrane-­‐interaction of Aβ and its implications for AD, two forms of Aβ, namely levorotary and dextrorotary (L-­‐ and D-­‐) Aβ isomers were used. Evidence has shown that L-­‐ and D-­‐ peptide can each form aggregates in a humid environment. However, when mixed together, L-­‐ and D-­‐ peptides tend not to form any aggregates. Using the mixtures of L-­‐ and D-­‐ peptides at different proportions and as well as using L-­‐ and D-­‐ alone can help us to determine the toxic form of Aβ. Phospholipids have been used to mimic membrane bilayers. Biological membranes in vivo are a complicated system. They contain three types of lipids, namely phospholipids, glycolipids, and steroids. Different types of cells and different membranes have different proportions of those lipids. Studying the interaction between Aβ and membranes in vivo can be extremely difficult. Artificial membranes, which only contains one kind of lipids, on the other hand, are a useful tool for the study of molecular interactions. Phospholipids are the most abundant type of membrane lipid and thus that can be seen as representative of cell membranes. The interactions of Aβ and different kinds of phospholipids have been investigated in this project. This thesis discusses the secondary structure of Aβ in different environment, the interaction between Aβ and phospholipids at the air-­‐water surface, and the location of Aβ in membranes during the interaction. The study provides useful information of the mechanisms and the origin of AD. At the end of the thesis, a discussion chapter analyses the difficulties of studying Aβ and AD and the potentials and inadequacies of this research.