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Systematic study of amyloid beta peptide conformations

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Systematic study of amyloid beta peptide conformations implications for alzheimer's disease
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Jimenez, Jeffy Pilar
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Atr-ftir
Biomimetic membrane
Fibril
Lipid bilayer
Oligomer
Sfa
Soft supported
Dissertations, Academic -- Chemical Engineering -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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ABSTRACT: The amyloid beta peptide particularly the 40 and 42 amino acid residues are the responsible for plaque formation in Alzheimer's disease (AD) patients. Extra cellular plaque formation has been recognized after incessant investigations along with the formation of intracellular tau protein tangles as the hallmarks of AD. Furthermore, the plaque formation has been linked mostly as a cause of the disease and the tangles mostly as a consequence. Our investigation is focused on studying the formation of AD plaques. The amyloid beta (Abeta) is a physiological peptide secreted from neurons under normal conditions, along with other soluble forms cleaved from the amyloid precursor protein (APP). These soluble forms of APP have neuroprotective and neurotrophic functions, while the Abeta is considered an unwanted by-product of the APP processing.Under normal conditions there is an anabolic/catabolic equilibrium of the Abeta peptide; therefore, it is believed that the formation of the plaque does not take place. On the other hand, the neurons' surface may play an important role in the adhesion mechanisms of the Abeta peptide. Our experiments show that the neuron surfaces along with the media conditions may be the most important causes for progressive formation of plaques. We have incubated rigid supports (mica) and soft biomimetic substrates (lipid bilayers on top of a PEG cushion layer drafted onto a silica surface) with the three different conformations of the Abeta peptide (monomeric, oligomeric and fibrils structures) to determine the adhesion mechanisms associated with in situ plaque formation. The soft biomimetic substrates have been assembled first by depositing and activating a thin film of silica (i.e., to create surface silanol groups).This film is then reacted with polyethylene glycol (PEG), which is a biocompatible polymer, to create a cushion-like layer that supports and allows the lipid bilayer to have high mobility. A lipid bilayer is then deposited on this soft support to reproduce a cell membrane using the Langmuir Blodgett deposition technique. The characterization of such biomimetic membranes has been studied by using Atomic Force Microscopy (AFM) in liquid environments. Our results show that these lipid bilayers are highly mobile. Additionally the structure and topography characteristics of the Abeta conformations have been followed with atomic force microscopy (AFM). The kinetics and rates of adhesion have been measured with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Our results show the progress of the plaques' formation with time where simple monomers deposit on the substrates and allow the development of oligomeric species.
Thesis:
Thesis (M.S.Ch.)--University of South Florida, 2005.
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Includes bibliographical references.
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by Jeffy Pilar Jimenez.
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Systematic Study of Amyloid Beta Pep tide Conformations: Implications for Alzheimer’s Disease by Jeffy Pilar Jimnez A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Norma Alcantar, Ph.D. David Morgan, Ph.D. Ryan Toomey, Ph.D. Date of Approval: July 27, 2005 Keywords: atr-ftir, biomimetic membrane, fi bril, lipid bilayer, oligomer, sfa, soft supported Copyright 2005, Jeffy P. Jimnez

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Dedication I entirely dedicate this new achievement to my beloved: mom, dad, sister and brothers, for their support, love, advice, and words of encouragement.

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Acknowledgments Completion of this thesis involved guidance, dedicati on, and patience for which I am in debt to my major professor, Dr. Norma Alcantar. I would like to thank my committee members, Dr. David Morgan and Dr. Ryan Toomey for their words of encouragement and support. My sincere gratitude goes to: The Chemical Engineering Faculty a nd Staff for their help during the completion of my master The National Science Foundation Inte grative Graduate Education and Research Training (NSF-IGERT) program for their support and their concern on my educational and professional enhan cement, and my special gratitude to Mr. Bernard Batson, Coordinator of the USF-IGERT program My peer graduate students Dr. Alessandro Anzalone, Mr. Jose I. Rey, and Mrs. Zoe Seda for their advice and support

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i Table of Contents List of Tables v List of Figures vi Abstract viii Chapter One: Introduction 1 1.1. Alzheimer’s Disease 1 1.2. Significance of this Work 5 1.3. Research Aims 6 1.3.1. Aim 1 6 1.3.2. Aim 2 7 1.3.3. Aim 3 7 1.4. Structure of this Thesis 7 Chapter Two: Fundamentals in Bi ochemistry and Cell Biology 9 2.1. Amino Acids 9 2.2. Peptide 10 2.3. The Peptide Bond 10 2.4. Proteins 11 2.5. Architecture of Protein Molecules 12 2.5.1. Protein Shape 12 2.5.2. Levels of Protein Structure 13

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ii 2.6. AmyloidPeptide and Protein Denature Process 15 2.7. Amphiphilic Molecules 16 2.8. Biomolecular Interactions 18 2.8.1. Van der Waals Attractive Forces 18 2.8.2. Hydrogen Bonds 18 2.8.3. Ionic Bonds 18 2.8.4. Hydrophobic Interactions 19 2.9. The Neuron Cell Membrane 20 Chapter Three: Chemistry and Conformations : Amyloid Peptide and Cell Membrane 22 3.1. AmyloidPeptide Conformations 22 3.1.1. Monomers 22 3.1.2. Oligomers 23 3.1.3. Fibrils 23 3.2. Common Pathways of Aggregation AmyloidPeptide 24 3.3. Biomimetic Cell Membrane 26 Chapter Four: Experimental a nd Characterization Techniques 29 4.1. Surface Deposition Techniques 29 4.1.1. Plasma Enhanced Ch emical Vapor Deposition 29 4.1.2. Langmuir-Blodgett Deposition 31 4.2. Physical and Chemical Characterization Techniques 32 4.2.1. Atomic Force Microscopy (AFM) 32 4.2.2. Attenuated Total Reflectance Fourier Transform Infrared 33 (ATR-FTIR) Spectroscopy

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iii Chapter Five: Experimental Design and Procedures 38 5.1. Reagents and Materials 38 5.2. Experimental Procedures 40 5.2.1. Conditioning of A Peptides and Synthesis of Structures 40 5.2.1.1. A 1-40 and A 1-42 Peptides 40 5.2.1.2. AmyloidConformations 40 5.2.2. Surface Characterization of the Amyloid Peptides Topology 42 5.2.3. Chemical Characterization of the Amyloid Peptides 42 5.2.4. Biomimetic Cell Membrane Constr uction and Studies of Interaction with AmyloidPeptides 43 5.2.4.1. Soft-Support Layer 43 5.2.4.2. Lipid Deposition 44 5.2.4.3. Surface Topology 47 Chapter Six: Results and Discussion 48 6.1. Reaching the Overall Goal 49 6.2. A (1-40) and A (1-42): Study and Comparison of their Structure and Kinetics 49 6.2.1 A (1-40) vs. A (1-42): Comparison of their Structure 49 6.3. Chemical Characterization of A (1-42) Peptide Conformations 54 6.3.1. Monomeric Conformation 56 6.3.2. Oligomeric Conformations 59 6.3.3. Fibril Conformation 61 6.4. Membrane Construction 64

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iv Chapter Seven: Conclusions and Future Work 68 7.1. General Findings and Conclusions 68 7.2. Final Remarks and Future Work 70 References 72 Appendices 76 Appendix A. Milestones Toward Formulating and Testing the A Hypothesis 77 Appendix B. The 20 Protein Amino Acids 78 Appendix C. Chemical Forces and Thei r Relative Strengths and Distances 79 Appendix D. Infrared Spectra 80 Appendix D.1. Characteristic Infrared Bands of Proteins 80 Appendix D.2. Characteristic Infrared Bands of Amino Acid Side Chains 80 Appendix E. Material Safety Data Sheets 81 Appendix E.1. A 1-40 81 Appendix E.2. A 1-42 84 Appendix F. One Example on AFM data processing 87 Appendix G. Transferred Area Calculation 89

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v List of Tables Table 1. Comparison of Biomolecular Interactions 20 Table 2. Primary Reagents Used 38 Table 3. Instrument Description and Miscellaneous Materials 39 Table 4. Amyloid Peptides 40 and 42, Monomeric Stability Comparison 50 Table 5. Amyloid (1-40) and (1-42), Oligomer s Formation and Stability 51 Table 6. Amyloid (1-40) and (1-42), Fibrils Form ation and Stability 52 Table 7. Amide I and II IR Regi ons for Peptide Molecules 53 Table 8 Evolution of Indivi dual Conformations for Monomeric Precursors 58 Table 9 Evolution of Individual Conformations for Oligomeric Precursors 60 Table 10. Evolution of Individual Conf ormations for Fibrillar Precursors 62

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vi List of Figures Figure 1. Two of the Alzheimer’s Disease Hallmarks 2 Figure 2. Regions in the Brain Affected by AD 3 Figure 3. Synthesis of A in the Cell 4 Figure 4. L and D Isomers of Amino Acids 9 Figure 5. The Peptide Bond 11 Figure 6. Amyloid (1-42) Peptide 13 Figure 7. Two Structural Motifs Arrange 14 Figure 8. Representation of an Amphiphilic Molecule 16 Figure 9. Micelle Formation by Amphiphi lic Molecules in Aqueous Solution 17 Figure 10. Schematic Representation of a Neuron Picturing the Cell Membrane 21 Figure 11. Alzheimer’s Disease AmyloidPeptide Monomer 22 Figure 12. Pathway of A Aggregation and Fibril Formation 24 Figure 13. Model of a Prototypical Cell Membrane 27 Figure 14. General Schematic Diagram of a PECVD Reactor 30 Figure 15. Water Plasma is Induced over the Silica Surface 30 Figure 16. Types of Deposition using LB Based on the Order of Layers 31 Figure 17. Atomic Force Microscope 32 Figure 18. IR Spectrum 35 Figure 19. Schematic Diagram ATR FTIR Spectrometer 36

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vii Figure 20. Langmuir-Blodgett Trough 45 Figure 21. Schematic of Lipid Bilayer Construction 46 Figure 22. Picture of the Wafer Contented in the Petri Dish 47 Figure 23. Average Structural Ratio (Height vs. Width) Comparison 54 Figure 24. Example of the Deconvolution for Fibrils at Initial Conditions 56 Figure 25. FTIR Spectra of Amyloid (1-42) Peptide Monomeric Precursors 57 Figure 26. Structures Evolution with Ti me of Oligomeric Precursors 59 Figure 27. Time Evolution Spectra of Fibril Structures of A (1-42) 61 Figure 28. Comparison of Surface Dynamics for the Three Peptide Structures 63 Figure 29. Surface Tension of DSPE as a Function of Lipid Concentration 64 Figure 30. Loading/Unloading Cycle fo r DSPE at 2.45mg/mL and 30mN/m 65 Figure 31. Deposition and Transfer of the Lipid DSPE Bilayer 66 Figure 32. Interacting Mechanisms Between Lipid Bilayers and A (1-42) 66 Figure 33. Section Profile of Biomimetic Membrane in Contact with Fibrils 67

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viii Systematic Study of Amyloid Beta Peptides: Implications for Alzheimer’s Disease Jeffy Jimnez ABSTRACT The amyloid beta peptide particularly the 40 and 42 amino acid residues are the responsible for plaque formation in Alzheime r's disease (AD) patie nts. Extra cellular plaque formation has been recognized afte r incessant investigations along with the formation of intracellular tau protein tangles as the hallmarks of AD. Furthermore, the plaque formation has been linked mostly as a cause of the disease and the tangles mostly as a consequence. Our investigation is focu sed on studying the form ation of AD plaques. The amyloid beta (A) is a physiological peptide secret ed from neurons under normal conditions, along with other soluble forms cl eaved from the amyloid precursor protein (APP). These soluble forms of APP have neuroprotective and ne urotrophic functions, while the A is considered an unwanted byproduct of the APP processing. Under normal conditions there is an anabolic/catabolic equi librium of the A pep tide; therefore, it is believed that the formation of the plaque does not take place. On the other hand, the neurons' surface may play an important role in the adhesion mechanisms of the A peptide. Our experiments show that the ne uron surfaces along with the media conditions may be the most important causes for progr essive formation of plaques. We have

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ix incubated rigid supports (mica) and soft biomim etic substrates (lipid bilayers on top of a PEG cushion layer drafted onto a silica surface) with the three different conformations of the A peptide (monomeric, oligomeric and fi brils structures) to de termine the adhesion mechanisms associated with in situ plaque formation. The soft biomimetic substrates have been assembled first by depositing and acti vating a thin film of silica (i.e., to create surface silanol groups). This film is then reacted with polye thylene glycol (PEG), which is a biocompatible polymer, to create a cush ion-like layer that supports and allows the lipid bilayer to have high mobility. A lipid bila yer is then deposited on this soft support to reproduce a cell membrane using the Langm uir Blodgett deposition technique. The characterization of such biomimetic membra nes has been studied by using Atomic Force Microscopy (AFM) in liquid envi ronments. Our results show th at these lipid bilayers are highly mobile. Additionally th e structure and topography characteristics of the A conformations have been followed with at omic force microscopy (AFM). The kinetics and rates of adhesion have been measured with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Our results show the progress of the plaques' formation with time where simple monomers deposit on the substrates and allow the development of oligomeric species. Th e oligomers then grow into fibril-like structures leading finally to th e plaques that eventually are s een to insulate real neurons and stop them from the synapse process. The ultimate outcome of this investigation will contribute to understand, prev ent and determine possible m echanisms for removing AD plaques.

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1 Chapter One Introduction 1.1. Alzheimer’s Disease Worldwide the most common cause of seni le dementia is Alzheimer’s disease (AD), an age-associated neur odegenerative disease character ized by loss of memory and language skills, damage cognitive function, a nd altered behavior. The first AD’s clinical symptoms are typically seen afte r the age of 65 years old [1]. After the age of 85, one out of every tw o people is affected either by AD or by what is called a mild cognitive impairment (M CI) condition [2]. MCI is recognized by a significantly reduced memory with cogniti on being within a normal range and is considered as a symptom indicating the onset of dementing disorders primarily represented by AD [2]. More than 15% of the cases of MCI turn into Alzheimer’s disease [3]. The estimated number of AD’s cases is over 20 million worldwide and is expected to keep growing as the world population ages [2]. The pathologic mechanisms of Alzheimer’s disease are still uncertain, however two hallmarks are recognized : intracellular neurofibri llary tangles (NFTs) and extracellular senile plaques [4]. The NFTs ar e structures composed of paired helical filaments mainly formed of abnormally hyperphosphorylated tau protein (P-TAU) [5] which is mainly but not exclusively neurona l microtubule-associate d protein (MAP) [6].

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2 Some of the roles of the tau protein are: st abilization of axonal microtubules, signaling transduction, interaction w ith the actin cytoskeleton, a nd neurite outgrowing. The formation of this NFT is believed to be a consequence of the amyloid plaque formation [2]. Figure 1 shows both deposit of plaques an d NFT. The extracellular senile plaques are composed by amyloid peptide deposits of mainly 40and 42-amino acids long (A 1-40 and A 1-42) [5]. Amyloidpeptides do not appear to pl ay a major physiological role indeed the based on the original amyloid ca scade hypothesis amyloid plaque depositions or partially aggregated soluble A trigger a neurotoxic cascade, thereby causing neurodegeneration and fina lly pathology of AD [7]. Figure 1. Two of the Alzheimer’s Disease Hallmarks: (left) A High-power photomicrograph of an amyloid plaque; (right) A photomicrograph of silver stained (black) neurofibrillary tangl es in the cell bodies [7] The plaques and tangles are accumulated in neurons throughout the cortical and limbic brain regions [5] (regions shown in Figure 2). The cort ical region is responsible for the abilities and activities related to our thinking, some examples are language and abstract thinking, it is also involved in basic aspects of perception, movement, and adaptive response to the outside world [8]. The limbic region is prim arily responsible for our emotional life and the formation of memories [8].

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3 Based on the studies over the years a hypothesis involvi ng plaque formation has been proposed and studied. The amyloidhypothesis sustains that the main cause of AD is the presence of this protein as the main component of the plaques that block the synapses in the neuronal cells [9 ]. A historical report about th e discoveries that relate AD with amyloidpeptides is shown in Appendix A. Figure 2. Regions in the Brain Affected by AD The amyloidis a physiological peptide is secreted from the neurons under normal conditions in addition with other solu ble peptides that are cleavaged from the transmembrane amyloid precursor protein (APP) APP consists of a large extracellular Nterminal domain and a smaller intracellular ta il (Figure 3). Apparen tly the soluble forms of APP have neuroprotective and ne urotrophic functions. However, A is considered as an unwanted by-product of the APP processing [2]. The A peptides are found as globular and non-fibrillar forms in small concentrations of picoto nano-molar, and are found in the extrace llular and cytoplasmic

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4 (inside the cell) regions in both normal as we ll as AD tissues. Nevertheless, significantly small oligomeric molecules, and non-fibrillar A peptides, are sufficient to cause profound cytoskeletal degenera tion and cell death through mechanisms of plaque’s formation still not completely understood. Figure 3. Synthesis of A in the Cell. The -amyloid domain is partly embedded in the plasma membrane. To generate A APP is first cleaved by -secretase, resulting in the release of -secretase-cleaved soluble APP ( -sAPP). In the second step, the 99 amino acid C-terminal fragment (C99 CTF) is cleaved by the -secretase complex releasing free -amyloid[3] The A peptides are found as globular and non-fibrillar forms in small concentrations of picoto nano-molar, and are present in both extracellular and cytoplasmic (inside the cell) regions in both normal as well as AD tissues. Nevertheless, significantly small oligomeric molecules, and non-fibrillar A peptides, are sufficient to cause profound cytoskeletal degeneration and cell death through mechanisms of plaque’s formation still not completely understood. Under normal conditions, in the brain there is an anabolic/catabolic equilibrium of the amyloidpeptide; that prevents the formation of AD plaques [2]. However, once this

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5 equilibrium is broken, the prot ein progressively aggregates causing the plaque formation [2]. There are more than 13,000 publications [10] concerning A peptide have shown different approaches to treat and preven t AD. These studies involve genetics and molecular biology, among other approaches. Ho wever, among all thes e publications little has been done about the interacting mechanisms of the proteins structures with the cell membrane. In this thesis, we conduct syst ematic investigations to elucidate the mechanisms between protein and membrane structures under di fferent physiological conditions (i.e., varying the pH, temperature of the media, and the composition of the membrane). The surface of the neurons pl ays an important role in the adhesion mechanisms of A ; mechanisms associated with a cascad e of events that may begin with the aggregation of monomer (seed) as bigger mo lecules (oligomers) that eventually result in larger structures (fibril) that accu mulates on the cell membrane forming plaques blocking synaptic processes. It is believed th at this surface coating leads to intracellular tangles formation and finally to neuron death. Our approach is to mimic cell conditions changing the media and characterizing the structure formation and the interaction mechanisms of these forms using a biomimetic cell membrane. 1.2. Significance of this Work This work represents a contribution to the “amyloid hypothesi s” investigation, that not only states that AD is characteri zed by the reduction and increase levels of sAPP and A respectively when the APP metabolism is altered, but also views other abnormalities in AD as either leading to A plaque formation/depos ition [11]. We are mainly focused in answering two questions:

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6 a. What are the intermolecular forces an d kinetic mechanisms involved in the formation of the different amyloid(A 40 and A 42) peptide conformations (i.e., monomeric, oligomeric and fibril)? b. What are the surface interactions between amyloidpeptide conformations and cell membranes, and their role in plaque formation? Therefore, this work will elucidate wa ys that may lead to plaque prevention and/or removal, based on the understanding of neurotoxic structures assemble, and surface energy features, in connection with the medium and the interacting substrate conditions. 1.3. Research Aims This work has been divided in three main aims: 1.3.1. First Aim To study and compare conformation assembly for A (1-40) and A (1-42): This will be done by looking at the stability of the monomeric state and the formation and stability of oligomers and fibrils. Many studi es have proven the medium influence on the amyloid peptide conformations [12-16]. Ther efore, we need to find a reproducible conformational assembly scheme based on the medium conditions employed, these are controlling pH, temperature, and incubati on time. This study is done for the two characteristic peptides found in the Alzheimer’s disease patients, A (1-40) and A (142). We are using the Atomic Force Micros copy (AFM), which allo ws one to visualize the structures with a resolu tion within nanometers, giving topological details of the structures.

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7 1.3.2. Second Aim To study and understanding of oli gomeric and fibrillar kinetics: In order to properly understand the adhesion mechanisms of A conformations to the cell membrane that drive to plaque formati on, it is needed to understand first their kinetics of its formation. This study is conducted using the Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy. This technique allo ws one to recognize the structural chemistry of the mo lecules under study in real time. 1.3.3. Third Aim To study the adhesion mechanisms: This aim is subdivide d in two stages: a. The study of adhesion behavior of monomeric, oligomeric and fibrillar forms with variable subs trates looking at the substrate rigidity or softness dependenc y. This study is done by coupling AFM and ATR-FTIR analysis. b. To analyze the dominant processe s during the aggregation of the three A conformations by deconvolution of the ATR-FTIR spectra and the relationship of the topography ratios of the AFM scans. For this specific aim diff erent substrates have been used such as mica, and biomimetic cell membranes constructed by assembling lipid bilayers. 1.3. Structure of this Thesis For a systematic study of the interactions between A and membrane surfaces it is needed to know some basic concepts concerning molecular biology, biological

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8 interaction forces, and cell membrane (interfa ce properties), which will be described in Chapter two among other basic concepts. A brief description of the systems that ha ve been studied are presented in Chapter three. The interest on looking at the monomeric, oligomeric and fribrillar conformations and their relationship to AD are also explained. Additionally, a concise explanation involving biomimetic cell membrane structural design is described. Chapter five illustrates the technical details of this investigation and all the protocols followed in the preparation and characterization of the systems. The results and discussion of this work are presented in chapter six, as well as a comprehensive analysis of the data. The conclusions for this work along with the final suggestion and future work are presented in Chapter seven.

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9 Figure 4. L and D Isomers of Amino Acids ( R refers to the side chain). The L and D isomers are mirror images of each other Chapter Two Fundamentals in Biochemistry and Cell Biology 2.1. Amino Acids Amino acids are the building blocks of proteins. A so-called -amino acid consists of a central carbon atom, called the carbon, linked to an amino group, a carboxylic acid group, a hydrogen atom, and a distinctive R group. The R group is often referred to as the side chai n. Because of the four differe nt groups connected to the tetrahedral -carbon atom, -amino acids are chiral; the two isomers are called the L isomer and the D isomer (Figure 4). Only L amino acids are constituents of proteins [17]. Essentially, 20 amino acids are components of protein in living organisms. The properties displayed by proteins, which in one way or another mark the path of protein aggregation, come from amino acid features such as th e capacity to polymerize, novel acid-based properties, varied structure and chemical func tionality in the amino acid side chains, and chirality [18].

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10 2.2. Peptide Peptide is the name assigned to short chains of amino acids with less than fifty amino acid residues [17]. A comm on classification for peptides uses the number of amino acid units in the chain. That is, each unit in the protein chain is called an amino acid residue, whereas a chain of amino acids is called a sequence. The word residue comes from the act of residing, after an ami no acid forms a peptide linkage upon joining a peptide chain. For instance, a peptide is a short chain with two amino acid residues, tripeptides are formed by three residues and so forth. Since the terminology becomes burdensome after 12 residues, th e peptide chains with more th an 12 and less than about 20 amino acid sequences are referred to as oligopeptides. According to this terminology, only when the chain exceeds 20 amino acids in length, the term polypeptide is then used [18]. 2. 3. The Peptide Bond Peptides and proteins are sequential and unbranched polymers of amino acids linked head to tail. In every instance, a carboxyl gr oup is linked to an amino group through a type of covalent amide linkage re ferred to as peptide bonds [18]. When a peptide bond is formed, the react ion releases water molecules. The peptide “backbone” of a protein follows a repeated sequence –N-C-C-, where the N represents the amide nitrogen, and the C is the -carbon atom of an amino acid in the polymer chain. A schematic of the peptide backbone is shown in Figure 5 [18].

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11 The geometry of the protein backbone is somewhat responsible for the unique qualities that proteins attain. For instance, the peptide bond is essentially planar, and has what it is considered a d ouble-bond strength (although it is not a double-bond in essence) that prevents free rotation around it. Also, th e peptide bond is uncha rged, the absence of charge allows tightly packing of ami no acid chains, linked by peptide bonds, into globular structures [17]. Even though peptide bo nds only lost or gain protons at extreme pH conditions, the overall charge of a prot ein can be induced by changing the pH of the media [18]. In contrast with the peptide bond, th e bonds between the amino group and the carbon atom, and between the -carbon atom and the carbonyl group are mainly single. This causes two adjacent rigid peptide units to rotate about these bonds only and allows the residues to take various orientations with respect to each other in the chain. Consequently, the proteins can fold in scores of different ways owi ng to the free rotation between two each amino acid groups in the chain [17]. Figure 5. The Peptide Bond Shown in its Usual Trans-Formation of Carbonyl O and Amide H. The C atoms are the -carbon of two adjacent amino acids joined in a peptide linkage. The dimensions and angles are average values observed by crystallography analysis of amino acids and small peptides. The peptide bond is stippled

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12 2.4. Proteins In brief, the term protein defines molecu les composed of one or more polypeptide chains. Proteins having only one polypeptid e chain are monomeric proteins. Proteins composed of more than one polypeptide chain are multimeric proteins or oligomeric. Amyloidand other fibril-like proteins are call ed homomultimeric proteins, since they only contain one kind of polypeptide. Heteromultimeric proteins are composed of several different kinds of polypeptide chains. Mul timeric proteins are usually designated by Greek letters and subscripts to denote their polypeptide composition such as in the case of -amyloid [18]. 2.5. Architecture of Protein Molecules The architecture of proteins is usually arranged as follows: 2.5.1. Protein Shape Proteins can be organized according to thei r basic of shape. That is, they can be fibrous or globular. Fibrous proteins tend to have relativ ely simple, regular linear structures, such as -amyloid fibrils. These proteins of ten have structural roles in cells (e.g., collagen and fibrinogen). Typically, they are insoluble in water or in dilute salt solutions. In contrast, globular proteins are roughly spherical. The polypeptide chain is compactly folded in globular proteins, so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are at the out side of the molecule. Hence, globular proteins are very soluble in aqueous solutions.[18]

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13 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Val Gln Lys Leu Ser Glu His His Val Phe Phe Ala Glu Asp Val Gly Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Figure 6. Amyloid (1-42) Peptide Visualized as a Simple Amino Acid Sequence of 42 Residues 2.5.2. Levels of Protein Structure To cope with the complex architecture of protein molecules, they have been classified in several levels dependi ng on their structural conformation. a. Primary Structure The amino acid sequence is the primary structure of a protein. A schematic representation of this conformation is shown in Figure 6 [18]. b. Secondary Structure Secondary structures are one level higher than primary structures and represent a three-dimensional arrangement of the polypept ide in space [18]. Polypeptide chains can arrange themselves into characterist ic patterns by means of hydrogen bonding interactions between adjacent am ino acid residues. These patterns can appear as helical or pleated segments as shown in Figure 7. These segments constitute regular structures that extend along one dimension, like th e coils of a spring. Most prot eins exhibit this type of structures, and depending on the medium conditions they arranged themselves as -helix or -sheet structures. Amyloid peptide stru ctures are usually described in this classification.

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14 N C C N C N C C N C C C N C C C C C N CCC N CC N N CN CC C N N C C C C C C C CN CCO C N H C CCN O C CCC H N CCC N O CC “Shorthand” -helix “Shorthand” -strand Figure 7. Two Structural Motifs Arrange th e Amino Acid Sequence into a Higher Level of Organization Predominantly: The -helix and the -pleated strand. Atomic representations of these sec ondary structures are seeing. Th e flat, helical ribbon is used for the -helix and the flat, wide arrow is used for -structures. Both of these structures own their stability to the formation of hydrogen bonds between the N-H groups and the O=C functions within the polypeptide backbone c. Tertiary Structure A higher level of structure is generated when polypeptide chains assume a more compact three-dimensional shap e by bending and folding. For instance, ternary structure allows proteins to adopt a gl obular. Ternary structures are us ually seen in those proteins commonly existing in cells. The globular c onformation promotes a lower surface-tovolume ratio and shields the protein from interacting with the solvent [18].

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15 d. Quaternary Structure Many proteins consist of two or more ter tiary structures. Each of these tertiary structures is commonly referred to as a subunit of a protein. This subunit organization in proteins constitutes yet another level in the hierarchy of protein structure [18]. 2.6. AmyloidPeptide and Protein Denature Process The term amyloid was introduced in 1854 by the German physician scientist Rudolph Virchow. In his studies, he analyzed cerebral corpora amylocea that had an unusual and abnormal appearance when seen under the microscope. Virchow concluded that the substance responsible for such macroscopic abnormality was cellulose and named it “amyloid”, derived from the Lati n amylum and the Greek amylon that means “starch”. Later on, Friedreich and Kekule demonstrated th e presence of protein in a “mass” of amyloid and pointed the high nitr ogen content and absence of carbohydrates. Several events have led to the study of amyloi d from a single type of protein to a class of proteins with propensity to undergo distinct changes in conformation (denature), hence resulting in fibril formation .[19] For instance amyloidoses are fundament ally diseases related to denatured proteins. The propensity to de nature of such proteins may be enhanced by mutation, environmental factors or posttranslational m odifications. Additionally, some proteins are “natively denatured” or conf ormationally mutable. Cells co ntain elaborate machinery to ensure that proteins fold properly, such mach inery includes proteins such as chaperones and processes such as selective degradati on systems. The proteasome is a degradation system that disposes of misfolded proteins. A characteristic that distinguishes amyloids

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16 NonpolartailPolar head NonpolartailPolar headFigure 8. Representation of an Amphiphilic Molecule: Sodium Palmitate from other misfolded or denatu red proteins is thei r ability to aggregat e into higher order oligomeric and fibrillar structures that allows them to evade quality control systems such as the proteasome. Inhibition of the proteasome or lysosomal1 degradation systems can lead to the accumulation of misfolded amyloidogenic proteins and peptides. Amyloid aggregates also inhibit the proteasome2, most likely by adopting st ably folded structures that cannot be unfolded and therefore cannot be transported into the central catalytic pore of the proteasome enzyme complex. It is thought that the critical initiation or nucleating event in the formation of amyloi d is the aggregation of stable -sheet structures that may form a seed. This system grows and propa gates owing to the amyloid structure’s relatively resistance to degradation in vivo and the concentration of aggr egation of misfolded proteins. Thus, a sm all increase in the concentration of misf olded proteins can dramatically accelerate the onset of AD. [20] 2.7. Amphiphilic Molecules The concept of amphiphilic character is very important because the amyloidpeptide and the lipids forming the cell me mbrane show this behavior. Amphiphilic molecules are compounds that consist of both strongly polar and strongly nonpolar groups. The name amphiphilic comes from the Greek amphi meaning “both”, and philos meaning “loving. They are also referred to as amphipathic molecules from the Greek 1 Lysosomal: a membrane-bound cavity in living cells that contains enzymes that are responsible for degrading and recycling molecules. 2 Proteasome: a cluster of proteins found in the cytoplasm of living cells that degrades damaged or redundant proteins.

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17 Figure 9. Micelle Formation by Amphiphili c Molecules in Aqueous Solution pathos meaning “passion, suffering”. Salts of fatty acids are typical examples of amphiphilic molecules that have special biological relevance. They have a nonpolar hydrocarbon tail and a strongly polar carboxyl head group, such as sodium salt palmitic acid (Figure 8). Their behavior in aqueous solution reflects the combination of the polar and nonpolar nature of these substances. The ionic carboxylate component hydrates readily, whereas the lo ng hydrophobic tail is in trinsically insoluble. Because of this, sodium palmitate, a soap, shows little tendency to form a true ionic solution in water. Nevertheless, sodium palmitate and other amphiphilic molecules readily disperse in water because the hydrocarbon tails of these substances are joined together in hydrophobic interactions as their polar carbox ylate functions are hydrated in typical hydrophilic fashion. The term micelle refers to a cluster of amphiphatic molecules (Figure 9). The amphipathic behavior of biomolecules has an enormous biological significance. The polar ends express their hydrophi licity by means of ionic intera ctions with the solvent, whereas the nonpolar ends are excluded from water and clustere d into a hydrophobic domain constituted by the hydrocarbon tails of many of these molecules. Membranes are structures that define the limits and comportments of cells. [18]

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18 2.8. Biomolecular Interactions Molecules are formed by covalent bonds th at allow atoms to hold together and thus to form molecules. Weak chemical for ces work mainly in intramolecular or intermolecular attractions be tween atoms, such as hydrogen bonds, Van der Waals forces, ionic bonds, and hydrophobic interactions [18] Each force is described below. 2.8.1. Van der Waals Attractive Forces are the result of induced electrical interactions between closely appro aching atoms or molecules as their negatively charged electron clouds fluctu ate instantaneously in time. These fluctuations allow attractions to occur between positively charged nuclei and the electron density of the incoming atom [18]. 2.8.2. Hydrogen Bonds are formed between a hydrogen atom covalently bonded to an electronegative atom (such as oxygen or nitrogen) and a second electronegative atom that serves as the hydrogen bond acceptor. Hydrogen bonds are stronger than Van der Waals fo rces (as Table 1 shows) and have an additional property. That is, H bonds tend to be highly directional, forming straight bonds between donor hydrogen, and accep tor atoms[18]. Hydrogen bonds are also more specific than van der Waal s interactions because they require the presence of complementary hydrogen donor and acceptor groups. This type of bond has a remarkable influence in the possible structures that a molecule can adopt. 2.8.3. Ionic Bonds are the result of attractive fo rces between oppositely charged polar molecules, such as negative carboxyl groups and positive amino

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19 groups. Since the opposite charges are restricted to sterically defined positions, ionic bonds can impart a high degree of structural specificity.[18] 2.8.4. Hydrophobic Interactions are due to the strong tendency of water to exclude nonpolar groups or molecules. Hydrophobic intera ctions arise from water molecules which prefer and share stronger interactions with one another. Since the strongest chemical interaction that is possible between two molecules actually determines its properties, the preferential hydrogen bonding interactions between polar water molecules excludes nonpolar groups. It is this exclusion of nonpolar groups that drives their tendency to cluster in aqueous solution. Thus, nonpolar regions of biological macromolecules are often in the molecule ’s interior of the system, so that they are excluded from the aqueous environment. [18] One of the reasons why living systems are restricted to a narrow range of environmental conditions is due to the fact th at weak forces are present in biomolecular interactions. Biomacromolecules are functionally active only within a narrow range of media conditions, such as temperature, ionic strength, and alkalinity. Extremes situations of these conditions disrupt these weak for ces, which are essential to maintaining the intricate structure of macromolecules. The loss of structural order in these complex macromolecules is called denaturation, and it is inevitably accompanied by loss of functionality. [18]

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20 Table 1. Comparison of Biomolecular Interactions Force Strength (kJ/mol) Distance (nm) Description Van der Waals interactions 0.4 4.0 0.2 Strength depends on the relative size of the atoms or molecules and the distance between them. The size factor determines the area of contact between two molecules. That is, the greater the area, the st ronger the interaction. Attractive VDW forces are inversely proportional to the sixth power of the distance, r, separating two atoms or molecules: F 1/r6. Hydrogen bonds 12 – 30 0.3 Relative strength is proportional to the polarity of the H bond donor and H bond acceptor. More polar atoms form stronger H bonds. Ionic bonds 20 0.25 Strength also depe nds on the relative polarity of the interacting ch arged species. Some ionic bonds are also H bonds: OOC NH ...3 Hydrophobic interactions < 40 Force is a complex phenomenon determined by the degree to which the structure of water is disordered as discrete hydrophobic molecules or molecular regions coalesce. 2.9. The Neuron Cell Membrane As it has been explained so far, the A plaques are found on the surface of the neurons, hence it is important to identify what else is present on thei r surface. Therefore, the neuron cell membrane is the outer most surface facing the plaques and defines the boundaries that separates the inside and outside of the neuron cell. A lipid bilayer makes the backbone of cell membranes.

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21 Membranes resemble supramolecular comple xes in their construction since they are complexes of proteins and lipids main tained by covalent forces (Figure 8). Some proteins traverse the lipid layer a nd influence the cell membrane structure. Some are for structural support, whereas othe rs are receptors, channe ls or tunnels that allow passage of certain molecules into and ou t of the cell. Thousands of these channels populate the cell membrane and allow passage of the ions. For instance, sodium is a major player to the neuron cell because of its positive charge. This ion flows across the membrane in different ways depending on th e exact status of excitation of the neuron. Additionally, in the cas e of cell membranes, the hydrophobic interactions ar e particularly important in maintaining the membrane structure. These interactions reflect the tendency of nonpolar molecules to come together as they are excluded by a polar solvent. [18] Direction of impulse To next neuron To next neuron Dendrites Nucleus Axon Direction of impulseCell membrane Lipid bilayer Protein Figure 10 Schematic Representation of a Neuron Picturing the Cell Membrane

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22 A (1-40) A (1-42) Figure 11. Alzheimer’s Disease AmyloidPeptide Monomer Theoretical Confi g uration Chapter Three Chemistry and Conformations: Am yloid Peptide and Cell Membrane 3.1. AmyloidPeptide Conformations 3.1.1. Monomers Monomers are about 1.0 0.3 nm in size [16], with a molecular weight of: 4329.9 Da for A (1-40) and 4514.1 Da for A (1-42) (Appendix E). Monomers present mostly random coils and -helix secondary structures (Figure 7). The conformation of A (1-40) and A (1-42) single monomers can be visualized in Figure 11. These models have been ge nerated using a comparative modeling online resource called 3D-JIGSAW (version 2.0). For this program, the source protein sequence is entered and then a mathematical model is developed based upon the potentials data that its bank holds assuming that th ere are not internal or solven t interactions whatsoever. The complete sequences for the A (1-40) and A (1-42) are provided in appendix E.

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23 Monomers are highly stable in fl uorinated alcoho ls including hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE ). These alcohols disrupt hydrophobic forces in aggregated am yloid preparations, and promote -helical secondary structures [16]. By dissolving A peptides in 100% HFIP, the resulting structures for A (1-40) and A (1-42) solutions are mostly -helical (~50-70%) and random coil (~3050%), with a little -sheet secondary stru ctures (<1%) [16]. 3.1.2. Oligomers The oligomers also known as protofibrils are considered by several authors [2123] as intermediate configurations in the fibrillogenic process. Oligomers are soluble spherical aggregates of 2-20 nm and have been study for many different types of amyloids with electron and atomic fo rce microscopy [16, 20]. These spherical conformations appear at intermediate times of monomeric incuba tion and disappear as fibrils appear, suggesting that they are an intermediate step in the pathway of fibril formation. Their sizes vary depending on the number of monomer a ggregates. The size of this quasi-stable intermediate aggregate ranges from dimmers up to particles of a million Da or greater and have been observed by a several techniques including SDS gel electrophoresis, fluorescence resonance ener gy transfer, light sc attering, and atomic force microscopy [16, 20]. 3.1.2. Fibrils Many proteins assemble into amyloid fi brils, even those that are not disease related. Amyloid fibrils are approximately 6-10nm in diameter. The unbranched fibrils are characterized by a “crossed -sheet” structure (Figure 7) in which the polypeptide

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24 Figure 12. Pathway of A Aggregation and Fibril Formation. chain is oriented perpendicula rly to the fiber axis with th e hydrogen bond parallel to the axis. The ability to form amyloids is not li mited to disease relate d proteins. Rather, it appears to be a fundamental polymer motif of the polypeptide backbone, although it is not commonly observed in native proteins. Specific sequences or side chain interactions are not required for amyloid formation, since many different polyamino acids form crossed -sheet amyloid fibers. This also suggest s that the fundamental tendency to form amyloids is overcome by the fact that specific sequences tend to adop t stable structures with time. 3.2. Common Pathways of Aggregation amyloidpeptide One particular hypothetical pathway for A fibril assembly is depicted in Figure 12. The earliest event in the process of aggregation is the formation of dimmers. A derived from a segment of APP is believed to be predominantly -helical in its native state. On the other hand, monomeric A is largely conformed as random coil in solution.

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25 The formation of A dimmers coincides with the adoption of partial structure. The A dimmers assemble into higher order aggregates or oligomers, since there is a remarkable increase in the amounts of higher order aggregates and a decrease in the amount of dimmers. These higher order A aggregates are protein mi celles with a hydrophobic core and a polar exterior that appear as 3 nm sphe rical particles determined by atomic force or electron microscopy [20]. At later times, these spherical shape partic les appear to form cu rvilinear strings or “protofibrils”. The protofibr ils undergo a conformation cha nge to form the straight, unbranched mature fibrils. Once the amyloid fi bril lattice has been established, the fibril can grow by the addition of A monomer or dimmer at the ends of the fibril. The higher order aggregates have molecular we ights ranging from approximately 105 to 106 Da and with an average size corresponding to approxi mately 24 monomers. A oligomers that appear as spherical partic les are approximately 3 nm in diameter and have the characteristics of a protein amphipathic, sin ce it lowers the surface tension of water. Formation of high molecular weight aggregat es displays a critical concentration of approximately 25 M [20] and it is correlated with the formation of a new hydrophobic environment. At longer incubation times, the oligomers also appear to co-aggregate to form curvilinear fibrils with a characteristic beaded appearance. The spherical oligomers and protofibrils appear to be intermediates in the pathway of fiber formation because they disappear as mature fibrils accumulate and the rate of monomer dissoci ation from them is too slow to account for fibril growth. Th e transition appears to involve a major

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26 conformational change, because fluorescence quenching analysis indicates that the carboxyl terminus is highly shielded from the aqueous solvent in th e soluble oligomeric state, whereas it is exposed to the solvent in the fibrillar st ate. Once the amyloid fibril lattice has been established, it can grow by the addition of monomers onto the ends of the fibrils. [20] Additionally, numerous in vitro studies have indicated that A oligomers are toxic to cells or interfere with the normal f unction of neurons [2123]. Mounting evidence indicates that soluble amyloid oligomers are ge nerally toxic for a wide variety of disease related amyloids. Moreover, soluble oligomer s are intrinsically t oxic even though they are formed by proteins that are not dis ease related. The idea that soluble A oligomers may play a primary role in AD pathogens is attractive, however, the evidence to support it is largely correlative and derived from in vitro toxicity studies. In order to evaluate the role of soluble A oligomers in diseased human brain, there needs to be a way to distinguish them from soluble A monomer, APP and A fibrils. All of these species contain the same amino acid sequence, but differ in confor mation [20]. These conformations are analyzed se parately in this work. 3.3. Biomimetic Cell Membrane Biomimetic cell membranes are used to represent the neuronal surface. This cell membrane model interacts in vitro with the three conformations of the amyloidexplained above and is built with features of real cell membranes. A double layer of lipids molecules makes up the main substance of the cell membrane. Lipids containing phosphorus in th eir head group are cal led phospholipids. In

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27 the cell membrane, some molecules such as c holesterol (there are mainly three types of cholesterol lipids: phosphoglycer ides, sphingolipids, and sterol s), and proteins are located between the phospholipids. Cholesterol is known to strengthen the membrane and promotes rigidity. A variety of different pr oteins float within th e phospholipids. Some proteins act as receptors in the cell, that is, as points of attachment for materials coming to the cell in the blood or tissue fluid. Additionally, enzymes are also participating in reactions occurring at the plasma membrane. Some are transporters, shuttling materials into or out of the cell, and some form channels through which only selected substances can pass. Carbohydrates are present in small am ounts, combined either with proteins or with lipids. These carbohydrates cells help to recognize each other and to adhere to oneanother. A representation of a model cell me mbrane is shown in Figure 13 [24]. Lipid bilayers are represented by outer, hydrophilic (exposed to an aqueous environment) and inner, hydrophobic (maintains a barrier to so lute movement) domains. Inserted through the membrane or in either inner or outer plan es are various proteins that permit transport, Figure 13. Model of a Prototypical Cell Membrane.

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28 cell-to-cell and signal molecule interactio ns, and linkage of the membrane to the intracellular cytoskeleton along with c holesterol and carbohydrate molecules. Additionally, most biological membrane lipids are composed by double-chained phospholipids or glycolipids with 16 to 18 carbons per chain, one of which is unsaturated or branched. These properties are not accid entally but carefully design by nature to ensure: a. Biological lipids will self-assemble into thin bilayer membranes that can compartmentalize different regions with in a cell, as well as protect the inside of the cell from the outside. b. Because of their extremely low criti cal micelle concentration (CMC) the membranes remain intact even when the bathing medium is grossly depleted of lipids. c. Due to unsaturation or branching, the membranes are in the fluid state at physiological temperatures which le t them deliver various molecules through them. The construction of the membrane for this work will be done on a substrate such that it allows the lipid bilaye r to be in a mobile state, s upported by a soft cushion. Such conditions simulate the real conditions of cel l membranes. Preliminary studies involving just a simple lipid bilayer were performed. Ho wever, future work involves the addition of a new component to the bilayer to tune its mobility and surface conformation. We will also study the response of these model membra nes the three different conformation of the amyloid peptide (i.e., monomers, oligomers and fibrils).

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29 Chapter Four 4. Experimental Methodology and Characterization Techniques 4.1. Surface Deposition Techniques Two deposition techniques are mainly used in this work, one is a chemical vapor deposition based on oxidation reaction and the other is a physical deposition based on interacting forces. Both deposition techniques are described below. 4.1.1. Plasma Enhanced Chemical Vapor Deposition Plasma enhanced chemical vapor deposition (PECVD) is a thin film deposition method that is used to deposit thin film on metals and other materials that cannot sustain high temperature. It takes pla ce at relatively low temperatur es (< 300 C) due to the use of a radio frequencies (RF) source that induces a plasma discharge to transfer energy into the reactant gases. The ionized gas (plasm a) breaks down the gase s and the activation energy barrier is lowered forming radicals (decomposition). These radicals produce many instable and stable species in the vapor phase which then react with each other to form heavy stable molecules a nd deposit into a film. In this work, PECVD is utilized to de posit a thin layer of silica (i.e., silicon dioxide (SiO2)) that is later use as the substrate for a reaction with polyethylene glycol (PEG) that ultimately serves as the cushion support for the biomimetic model membranes.

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30 Figure 14. General Schematic Diagram of a Plasma Enhanced Chemical Vapor Deposition Reactor Figure 15. Water Plasma is Induced over the Silica Surface to Create Active SiOH Species that React with PEG to Produce a Soft Polymeric Support for the Model Membranes The silica films are produced by reacting SiH4 and O2 gases in this reactor (Figure 14). The plasma is produced by a helical reso nator discharge source using RF powered copper coil at 13.56 MHz [25]. PECVD is also used to produce water plasma (see Figure 15). Water plasma treatments are used to increase the hydroxyl group concentration on the silica surface controllably and reproducibly. The conditions for this process were established previously, so that the silica surface is completely saturated the surface with hydroxyl groups, without roughening the surface [25].

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31 X-type Y-type Ztype Figure 16. Types of Deposition Using LB Based on the Order of Layers. 4.1.2. Langmuir-Blodgett Deposition The Langmuir Blodgett deposition technique is used to deposit compact and fine organized monolayers. The molecules that are deposited can be organi c or inorganic, but with amphiphilic nature. The monolayer is fo rmed on the surface of the subphase (water) by dissolving the amphiphilic substance in a suitable solvent. In this investigation chloroform is used to dissolve the lipids th at form the monolayer on the water surface. Once the monolayer is formed the depos ition takes place upward or downward depending on the nature of the substrate and on the type of deposition that is been pursued (See Figure 16). The deposition is expl ained in more detail in the next Chapter. Bilayers or multilayer can be deposited with this sophisticated technique but simple. The deposition of the biomimetic cel l membrane is constructed as the type Y (Figure 16). However, we have a PEG monol ayer instead of an initial monolayer of lipids.

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32 4.2. Physical and Chemical Characterization techniques 4.2.1. Atomic Force Microscopy (AFM) The Atomic Force Microscope (AFM) also known as Scanning Force Microscope (SFM) or Scanning Probe Microscope (SPM). This technique allows one to visual ize topographies of samples in very small ranges with resolution of about 10 pm [26]. The AFM technique operates by measuring attractive or repulsive forces between a tip and the sample. As the tip is drag thr ough the sample surface a profile of heights is constructed as function of the cantilever deflection lectures that are translate into a real image of the surface under study [26]. There ar e two types of operati onal modes: contact and non contact. In its repulsive "contact" mode, the inst rument lightly touches a tip at the end of a leaf spring or "cantilever" to the sample, moreover in contact mode the AFM measures hard-sphere repulsion forces between the tip and sample; this mode is mostly use when working in solid-liquid interf aces. In non contact mode, the AFM derives topographic images from measurements of attrac tive forces; but the tip does not touch the sample. This is also referred as tapping mode and it is mostly used for solid-gas interfaces. In this investiga tion where used both modes, due to the characteristic of the Z Piezo Cantiliver Sample Segmented Photodiode Detector X-Y Piezo Stage Figure 17. Atomic Force Microscope

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33 medium as will be explained in the follo wing chapter. Figure 17 shows a schematic representation of a common AFM. [26] 4.2.2. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy Infrared spectroscopy is being widely use for the analysis of peptides and proteins due to its reliab ility of probing the unive rsally available amide (peptide) bonds, which show a distinct IR signals for differen tly folded peptides and proteins [27]. Fourier transform infrared (FTIR) spect roscopy has being predominantly utilized to study the secondary structure ( -helix and -sheets) of proteins in recent years. Proteins or polypeptides have a continuous chain of ami no acids connected via amide bonds also known as the “peptide bond”. Th e frequency at which amide bond vibrations occur can be attributed to different seconda ry structures in which the amide bonds are present. The differences in vibration of the amide bonds are due to different hydrogen bonding among amino acid residues (see Appendix D.2). For instance, -helix and sheet folding have ordered hydrogen bonding, although differing in their patterns. The differential pattern in H-bonding, along with ge ometric orientations of amide bonds in helix, -sheet, and random coil structures allo ws the different vibration frequencies associated with individual s econdary structural folding. Am ide vibrations (see Appendix D.1) in-plane mode involve C=O stretchi ng, C-N stretching, N-H stretching and O-C-N bending, while an out-of-plane mode is due to C-N torsion [28]. These vibrations result in characteristic spectral features of proteins. Three major spectral regions (amide I, amide II, and amide III) have been id entified based on theoretical a nd experimental studies [27].

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34 The amide I vibration region, (1700-1600 cm-1), has been widely used due to its strong signal exhibited by proteins in this region. Howeve r, there are several difficulties encountered in this region when analyz ing protein spectra. The first difficulty encountered in this region is the OH vibra tions caused by liquid water and water vapor that need to be subtracted out of the prot ein solution spectrum. Another problem is the difficulty of peak assignments. Since there is a greater deal of overlapping of peaks representative of the different secondary struct ures, it is difficult to assign bands to their correct structure. For ex ample, bands at 1650-1655 cm-1 could be assigned either to helix or random coil. Using the amide III region (1220-1320 cm-1), many of these problems are resolved. In the amide III region, OH vibrations due to water do not interfere with spectrum in the amide III region as much as in the amide I region. The overlapping of bands arising from different secondary stru cture of a pr otein is not significantly encountered in th is region. In the amide I re gion, the frequencies at which the different amide bond vibrations occur are not as localized as they are in amide III. In the amide III region, various spectral bands are more resolved in the original protein spectrum than they are in the amide I spectra. This fact allows a greater ease in peak refinement, as well as in peak assignment. The only drawback in us ing amide III is that the protein signal is significantly weaker th an the signal obtained in the amide I region [27] as is shown in Figure 18, where two proteins has been superimposed: chymotrypsin, and fibrils of A 1-42 (this was a sample taking after 10 hours of preparation).

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351637 1548 Amide II Amide I Amide III 2.5 2 1.5 1 0.5 -0 200019001800 1600 170015001400 1300120011 Wavenumber(cm-1)Absorbance. (a.u) Figure 18. IR Spectrum of -Chymotrypsin (black lin e), and Fibrils of A 1-42 (in blue). The amide I region (1600-1700cm-1) corresponds to the C=O stretch weakly coupled with C-N stretch and N-H be nding. The amide II region (1500-1600 cm-1) represents C-N stretch strongly coupled with N-H bending. The amide III region (1200-1350cm-1) is N-H in-plane bending c oupled with C-N stretching and also includes C-N and N-H de formation vibrations Because IR spectroscopy does not affect protein and peptide samples either in solid or liquid form, it can be used for data collection. Two types of sample accessories are typically used: Windows such as calciu m fluoride, and attenuated total reflectance (ATR) accessory, generally made of zinc seleni de or germanium. For protein absorption studies, or for studies with solid powder and thin films, ATR accessory is most commonly used for sampling. One of the a dvantages of ATR technique in recording protein spectrum is the avoidance of solvent in terference in IR spectr a, because it limits the effective sample thickness to a thin layer near the surface of an internal reflection crystal (Figure 19) [27].

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36 IR -OH CH2-C=O NHX C=CSample In Out ZnSeCrystal Flow cell Figure 19. Schematic Diagram of Light Undergoing Multiple Reflections in an ATR FTIR Spectrometer Once the spectra are obtained, protein band positions are identified after spectral processing by Fourier self-dec onvolution and/or second-order derivatization. A variety of software packages are available commercially. The data obtained from the deconvolved and secondderivative spectra are used to determine the number of bands and their positions in order to resolve the protein spectrum into their components. This is accomplished with a curve-fitti ng process employing computer software commercially available from the manufacturers of IR instruments. The software resolves the original protein spectrum to individua l bands that fit the spectrum. Two main parameters control the fitting process: (1 ) the individual bands, and (2) the baseline

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37 position. Each individual band in turn is controlled by three parameters: a. The height of the band. b. The position of the band (wavenumber). c. The bandwidth at half-height. The program iterates the curve-fitting proces s, and each iteration flows (increases or decreases) each parameter (height, bandwidth, position and baseline) to determine individual parameters in orde r to achieve the best Gaussian Lorentzian, or a mixture of Gaussian/Lorentzian-shaped curves that fit th e original protein spectrum. A best fit is determined by the root mean square (rms) of differences between the original protein spectrum and the strength of individual bands. In order to estimate the strength of individual band, the band area or intensity is used to calculate the relative contribution of each band(s) to a particular sec ondary structure of the protein. Additionally the fractional areas of th e fitted component bands are directly proportional to the relative proportions of struct ure that they represent. The percentage of helices, -structures and turns may be estimated by addition of the areas of all of the components bands assigned (after deconvolution) to each of these structure (based on the specific wavelengths this structures are) and expressing the sum as a fraction of the total area [28].

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38 Chapter Five Experimental Design and Procedures Regarding the reagents used in the expe riments, section 5.1 describes most of these in Table 2. Details about peptide com position can be found in Appendix E. Table 3 describes the instrumentation and related materials. 5.1. Reagents and Materials Table 2. Primary Reagents Used Reagents Acquired From Appearance Amyloidpeptide (1-40) A (1-40) American Peptide INC White powder Amyloidpeptide (1-42) A (1-42) American Peptide INC White powder 1,1,1,3,3,3-Hexafluro-2Propanol (pa: >99%) HFIP Fisher Sci. ACROS Org. ViscousTransparent Liq. Methyl Sulfoxide Anhydrous (p:>99%) Me2SO or DMSO Fisher Sci. ACROS Or g. Transparent Liq. Dulbecco's Phosphate Buffered Saline powder PBS MP.Biomedicals White powder Tris (Crystallized) p:>99%) Tris Fisher Sci. ACROS Org. White powder Polyethylene glycol (400Da) PEG Sigma Transparent Liq. 1,2-Dipalmitoyl-snGlycero-3-Phosphoethanolamine-NDSPE (lipid) Avanti Polar Lipids, INC. Powder partially dissolve in chloroform Ammonium hydroxide NH3OH Fisher Sci. ACROS Or g. Transparent Liq. Hydrochloric acid (HPLC) HCl Fisher Sci. ACROS Org. Transparent Liq. Sodium chloride NaCl Fisher Sci. ACROS Org. White fine granules Sodium hydroxide NaOH Fisher Sci. ACROS Org. White pellets a purity

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39 The water used for all the experiments is ultra purified and obtained from a Millipore Water (Milli-Q) st ation with a resistance <18M and organics content <5ppb. Table 3. Instrument Description and Miscellaneous Materials Material / instrument Description Centrifuge Fisher Sci. model AccuSpin-400 Laminar flood cabinet Form a Sci., 95 fpm & 98 fpm Vortex Fisher Sci., Volts 120 Vac, Watts 150, 50/60 Hz Atomic Force Microscope (AFM) for gas/solid Int. DI Dimension 3000. Tapping Mode. Cantilevers Silicon. Nanosensors INC. R = 0.01-0.025 Ohm/cm, L=132 m, diam.=15nm, fo=281-339 kHz, k =28-54 N/m. AFM liquid/solid Int. Asylum Res. Corp. Contact mode. The cantilever used is special for contact mode, and is made of Cantilevers SiN. L=60m, diam.=>80nm, fo=37-13 kHz, k=0.027-0.006 N/m FTIR Spectrometer Nicolett, FT-IR 850 Bench PECVD Reactor Six-way stainless steel cross vacuum chamber with a 16-in long, 2-in-diam Pyrex cylinder connected to the feedthrough port at the top. With a helical resonator discharge source using a radio-frequency (rf) powered copper coil at 13.56 MHz, and surrounded by a grounded cylindric al copper shield enclosing the Pyrex tube. The rf power, provided by a RF Plasma products model RF5S power supply L-B Trough Nima Technology, Model 611D Sonicator Fisher Sci. Model FS30 pH meter Fisher Sci., Accumet 1003 Water Bath Precision 180 series water bath, T range: RT + 5 to 100 C Miscellaneous All gas tight Hamilton syringes, Pipettes, Adjustable micro Ependorf pipetters; Autoclaved Pipette tips, Ependorf Micro Tubes (1000 L). Dessicator. Mica. Silicon/Silica wafer.

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40 5.2. Experimental Procedures 5.2.1. Conditioning of A Peptides and Synthesis of Structures 5.2.1.1. A 1-40 and A 1-42 Peptides Lyophilized peptide (1mg) kept at -20C was removed from the freezer and let equilibrated for 30 minutes at room temperat ure (RT). The peptide is then dissolved in HFIP following the procedure desc ribed by W. Blaine Stine et al [29]. HFIP is used to break any residual tertiary and -sheet secondary structure of the peptide. The peptide is composed by -helix (~50-70%) and random coil (~3050%) structures mainly [29]. The HFIP is then injected into the peptide flas k with a gas-tight Ham ilton 0.5ml glass syringe until a concentration of 3.33 g/ l is obtained. Peptide dissolu tion takes not longer than five minutes. Such stock solution is aliquote d into 25 parts and storage in sterile micro centrifuge tubes of 12.5 l, containing 40 g of A each approximately. Thus, each micro tube contains 9.238 nmol and 8.861 nmol for A (1-40) and A (1-42), respectively. Aliquoted samples are left under the laminar-f low hood to allow the HFIP to evaporate. The peptides are then placed for 12 hours in a desiccator at a vacuum of 27 in Hg. The final peptide samples have an even appearance (i.e., transparent film), which is appreciated at the botto m of the Eppendorf tubes. The tube s with the peptide are flushed and closed with nitrogen, wrapped with alumi num foil and parafilm, and enclosed in a jar at -20C for later used. 5.2.1.2. AmyloidConformations a. Monomers The following procedures are the same for both 1-40 and 1-42. 40 g of pretreated

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41 A peptide is removed from the freezer and le t to equilibrate at RT. The pretreated peptide is dissolved in 2 L of DMSO, which is a polar, water-soluble organic solvent commonly used to solubilize hydrophobic peptides. After a minute of sonication with DMSO, 88 L of milli-Q water are added to the sample. Finally, it is incubated at RT [29]. b. Oligomers First, a tris/NaCl/HCl buffer solution is pr epared. This buffer serves as incubation media for the oligomer’s synthesis. The buf fer is prepared in an Eppendorf of 50ml, where 0.3028g of tris and 0.2922g of NaCl are dissolv ed in DI water until the pH is fixed to 7.4. Sometimes a few drops of concentrated HCl need to be added. The pretreated peptide film kept at -20 C is then dissolved in 2 L of DMSO, sonicated for 1 min, and then incubated at 4 C in 88 L of Tris/NaCl/HCl. This proced ure is described in detail by Kayed et al. [30]. c. Fibrils The first step to incubate A fibril structures is to prepare a solution of ammonium hydroxide at pH=8.5. Th e next step is to prepar e a PBS buffer. Fibrils are prepared by dissolving 40 g of the pretreated peptide (at RT) in 20 L of NH3OH and later adding 20 L of PBS. The sample is gently vortexed and finally incubated at a concentration of 1 g/ L at 37 C. This procedure was suggested by Dr. David Morgan (PhD) from the Department of Pharmacology & Therapeutics in the College of Medicine at the University of South Florida (USF), and director of the Alzheimer Research Laboratory at USF.

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42 5.2.2. Surface Characterization of the Amyloid Peptides Topology The sample deposition procedure is c onducted under laminar flow cabinet to avoid contamination by undesirable particles. The samples to be analyzed with the AFM are deposited on mica, which is an inert, smoot h, and easily to clean mineral, and widely used for biological samples in AFM imaging. Mica sheets are freshly cleaved prior to each deposition. A small Teflon O-rings (external 4.45mm, internal 1mm) is used as an incubation chamber placed on top of the mica which ensure the maintenance of the desired location of fibril formation. 3 L of each incubated sample are dissolved with ultrapure water until a concentration of 20 M is reached. This c oncentration has been tested as the optimum to produce fibrils in a control manner. A small drop of such solution is then deposited on mica and incubate d for 5 min, flushed w ith 3mL of ultrapure water, dry with ultrapure nitrogen, and stored in a desiccator under vacuum (30 torr) for later AFM study. 5.2.3. Chemical Characterization of Amyloid Peptides No fibrils were observed when A 40 peptide was incubated. Therefore, the chemical characterization was done only for A 42 peptide conformations. The chemical analysis is done using an ATR-FTIR spectrome ter, and a zinc selenide ATR crystal. The ATR cell allows a maximum volume of 62 L solutions (Figure 19). The crystal was first cleaned with isopropanol, and then a fresh media solution was flushed before peptide contact. The background for th e IR data was taken when the ATR crystal was in contact with non-peptide media. Spectra were take n at about 10 min intervals for about 3 hours for monomers, 10 hours for oligomers, and 12 hours for fibrils, which is the time that it

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43 took for the samples to reach saturation and steady state conditions. This part of the study was conducted in the Materials Research Laborat ory at the University of California-Santa Barbara (UCSB). 5.2.4. Biomimetic Cell Membrane Construc tion and Studies of Interaction with AmyloidPeptide 5.2.4.1. Soft –Support Layer Polyethylene glycol (PEG) is used to build the soft support layer. PEG is a linear or branched neutral polyether soluble in water and most organic solvents. Its chemical formula is: HO-(CH2CH2O)n-CH2CHOH. PEG is utilized in this study because its biotechnical applications. In fact, Norma Alcantar and colla borator have studied this polymer widely [25] including its use as cushion substrate or coating polymer. PEG is unusually effective at excluding other polymers from its presence in aqueous solution, such as lipids. This property is directly rela ted to the fact that PEG rejects proteins and forms two-phase systems with other polymer s; it is nontoxic; it ha s immunogenicity and nonantigenicity; it does not harm active proteins or cells even if it interacts with cell membranes; it attaches to other surfaces with very little effect on their chemistry; and it increases the solubility of large molecules despite thei r size. In addition, PEG coated surfaces become hydrophilic and protein rejectin g. Therefore in our study we are insuring there is no absorption of the A to the silica surface, if there is any adhesion, it is due to the interaction of the lipids themselves with this peptide. The inert character of PEG is based on its molecular conformation in aque ous solution, where PEG exposes uncharged hydrophilic groups and shows very high surface m obility (steric exclusion). It is worth

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44 mentioning that in order to maintain its biol ogical and biocompatible properties, the PEG has to be chemically attached to the surface. PEG is grafted to silica films deposited on silicon wafers creating a thin hydrophilic film The silica deposition takes place reacting silane (SiH4) and O2 gases in a PECVD reactor (Fi gure 14). The samples are then exposed to a water plasma treatment in order to increase the hydroxyl group concentration on the surface controllably and reproducibly [25]. Finally, the surfaces are storage on a dry and free of particles container. Concentrated sulfuric acid is used fo r cleaning the surfaces by submerging them overnight, and then are rinsed with DI water, dried with ultrapure nitrogen, and exposed to UV-light for about 5 minutes. The reactiva tion is done by submerging the surfaces in a NaOH solution at 10 %w for about 5 minutes. The surfaces are then rinsed with water and dried very well, since any traces of wate r could interfere with the PEG reaction. In the meantime, a solution of PEG (of wt ~ 400Da) is heated until it reaches 100 C maintaining constant agitation. The cleaned an d activated surfaces are placed into the PEG for one hour. These parameters have been determined previously by Alcantar et. al. [25]. Once the PEG grafting reac tion is done, the surfaces ar e retrieved from the hot PEG and gently rinsed with water (avoiding dire ct contact with the flux o water), and dried with ultrapure nitrogen. Sometimes the su rfaces with PEG that are not used at the immediately after the reaction are storage in PEG media. 5.2.4.2. Lipid Deposition The lipid bilayers are deposited onto th e soft-support PEG layer using a Langmuir Blodgett (LB) trough (Figure 20). The first step is to ensure that the LB trough is

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45 impeccable, since it is very important to avoi d particles clinging to the surface. These particles can contaminate and perturb the de position quality. To clean the trough, we use DI water and sometimes organic solvents. We look at the quality of the water by watching for bubble formation. Bubbles are a sign of contamination. A suction tube is used to eliminate these bubbles out of th e water without touchi ng the surface of the trough avoiding damage to the trough surface. The water is suctioned several times until non-bubbles are observed. Figure 20. Langmuir-Blodgett Trough. The prin cipal components the trough are highlighted in this photo The LB trough is plugged to a computer that allows one to control parameters such as surface area, superficial tension, and substrate deepness necessary for the actual deposition. The trough is located inside of a laminar flow cabinet, which turn on when the trough is being cleaned. However, the air flow is off, while the lipids are being compressed or deposited. Once the water surface is clean, the surface pressure sensor is calibrated to 0 N/m. An isotherm is recorded using a velocity of 50 cm2/min. The trough

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46 is clean and ready to used, if the isotherm is a straight line. The target surface is submerged for the deposition in the back so cket of the trough. The trough is cleaned once more to avoid any possible particles introduced by the surface. Another isotherm is run to ensure cleanliness. The pressure is set to zero and the surface area is set to 300cm2. The area, deposition pressure, and lipid concentration used for the depositions were chosen based on previous experiments. T ypical lipid concentrations are 15 g/ L. A total volume of 100 L is gently spread on the air/water interface using a syringe (giving it about 5 minutes for the solvent to evaporate). During the de position, the surf ace pressure is kept constant at 30 mN/m. The deposition takes place at constant pressure and using a barrier velocity of 50 cm2/min (Figure 21). Figure 21. Schematic of Lipid Bilayer Construction. (upper left) PEG grafting reaction (upper right) bilayer deposition, and (bottom) soft supported bilayer 5.2.4.3. Surface Topography The soft-supported membranes constructe d above have also been scanned by AFM in solution. They are kept in contact wi th saturated lipid solution at all times to

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47 avoid air exposed and consequently, damage to the sample (Figure 22). The biomimetic cell membranes were also scanned while in contact with A fibrils (0.933 M). Scans are shown and explained in the next chapter. Figure 22. Picture of the Wafer Contented in the Pe tri Dish. This is were the biomimetic membrane has been deposited

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48 Chapter Six Results and Discussion This work is based on the fact that real interactions of A peptides, with both 40 and 42 amino acid sequences, could be controlled and studied by following a comprehensive approach. The co ntrol on the structure of the A peptides was done by optimizing the incubation conditions previously studied by Stine et al. [29] and Kayed et al. [30]. Depending on the conformation th at is being pursued (i.e., monomeric, oligomeric and fibrillar), the peptide molecules will adopt diverse structures that defined their physical and chemical pathways of aggr egation, which evolve with time. By tuning and promoting specific conformations, we were able to explicitly dist inguish the response of the peptide in terms of their assembl y, surface adhesion, chemical composition and kinetics. Explicitly, our re sults are three-fold: a. We resolved the differences of behavi or depending on the number of amino acid residues contained in each peptide molecule (i.e., A -40 and A -42) b. We promoted structural rearrangement depending on the incubation media and substrate character c. We were able to correlate peptide structures (i.e., -helix, -sheet, -turns, or random) with the adhesion dynamics and ultimately, to determine the role of molecular stability of the pe ptide that leads to the fo rmation of the AD plaques.

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49 6.1 Reaching the Overall Goal According to the three aims that we set from the beginning: a -To study and compare conformation assembly for A (1-40) and A (1-42). b -To study the adhesion mechanisms: c -To study and understanding of fibril lar kinetics with model membranes: We have been able to achieve our goal of elucidating, from the physicochemical point of view, the implications of the st ability and conformations of A in the formation of AD plaques. 6.2 A (1-40) and A (1-42): Study and Comparison of their Structure and Kinetics The three A peptide conformations that were considered in this work are: monomeric, oligomeric and fibrillar. From the AFM data, we were not only able to directly measure the height and width that co rresponds to each of thes e structures on rigid (mica) and soft supported (model membrane) su bstrates, but also, to understand how their assembly occur with time and how their he ight/width ratio relates to their kinetic mechanisms for both peptides. 6.2.1 A (1-40) vs. A (1-42): Comparison of their Structure The A conformations were prepared on mica and analyzed by AFM. The samples were incubated as a function of ti me to observe the following processes: solubility, stability and dynamics of forma tion. The evolution of the monomers is shown below from their initial pr eparation until 72 hours of in cubation in solution. The A 40 compared to A 42 shows very little absorption with time. In addition, the A 40 aggregates slowly into large clusters, with no indication of fibrillar structures being ever

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50 formed (Table 4). On the contrary, the A 42 evolve in two differe nt stages where an initial effect took place dominated by dissolu tion of the molecule, and the formation of thin large fibrils resulted betw een 9 and 20 hours of incubation. Table 4. AmyloidPeptides 40 and 42, Monomeric Stability Comparison Peptide EVOLUTION TIME [hours] A 0 5 9 40 42 20 48 72 40 42 The scan size for all the images is 2 m and th e height scale is indica ted by the bar at the right hand side of each picture.

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51 Likewise, oligomeric structures show similar results, where the A 40 did not form fibrillar conformations but clusters. Again, the A 42 formed fibrillar structures after 9 hours of incubation (Table 5). Table 5. Amyloid(1-40) and (1-42), Oligomers Formation and Stability Peptide EVOLUTION TIME [hours] A 0 5 9 40 42 15 25 > 25 40 They did no form fibrils 42 More and longer fibrilliar conformations were observed Similarly, scan size for all the images is 2 m and the height scale varies from each picture and is indicated by the bar at the right hand side of each picture.

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52 In the case of fibrilliar structures, the A 40 behaves in the same way as for monomers and oligomers. It was very surpri sing that almost no fibrils were detected, although the incubation conditions should have produced them. On the contrary, fibrils were observed on the A 42 after 10 hours of incubation (Table 6). Table 6. Amyloid(1-40) and (1-42), Fibrils Formation and Stability Peptide EVOLUTION TIME [hours] A >5 10 15 40 42 20 48 91 40 42 Scan size is 2 m and the variable hei ght scale as indicated on each picture.

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53 A very interesting result display by the A 42 fibrils is that they are intrinsically different for each structure. In other words, if we look at the scans after 20 hours of incubation time, the fibrils formed by the oligom ers are thicker than that formed by fibrils or monomeric precursors. In addition, the fi brils formed at longer times seem to be similar in thickness and shape. This indicates that the preferential structure independently of the precursor molecules for the A 42 is fibril conformation. In order to compare the evolution of the topography of A structures quantitatively, we have measured and compar ed the average ratios between height and width for each scan, which relates to the affinity of adhesion and surface density. Details about how these values were calculate d are shown in the Appendix F section. This analysis indicates that two of th e three structures undergo a conformation change at 11 hrs for monomers and 18 hr s of incubation for oligomers for both A 40 and A 41, which corresponds to taller fibrils while the thickness remains low. This conformation change (maximum point) determines a saturation point, where the fibrils seem to pile up instead of spreading on the s ubstrate. After that, both molecules show that the fibrils continue spreading onto the surf ace again. However, for fibril structures the behavior is very different. The A 40 and A 42 show opposite performance. In addition, we observed three interesting stages, one at 4, one at 10 and one at ~48 hours. The one at 4 and 50 hours correspond to a maximum in the Ratio value for A 40, but a minimum values for A 42. This is also related to the satura tion of the surface as explained above. At 10 hours both peptides show similar surface density and topography.

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54 6.3. Chemical Characterization of A (1-42) Peptide Conformations By using the ATR-FTIR technique, we are able to rec ognize the type of conformation that each precursor molecule ha s as a function of time. This study shows the evolution and dynamics of each chemical structure as a function of incubation conditions. We were able to distinguish specif ic structures depending on the IR vibration modes of the peptides. These structures show well defined processes indicating its dilution, molecular arrangement, a nd a steady state (equilibrium). 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 01020304050607080Height/Width RatioA 1-40 A 1-42 Incubation Time (hours)Figure 23. Average Structural Ratio (Height vs. Width) Comparison as a Function of Time Between A 40 and A 42 Conformations as Scanned by AFM (top left) Monomeric precursors (bottom left) Oligomeric precursors bottom right) Fibrillar precursors 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 051015202530Height/Width RatioA 1-40 A 1-42 Incubation Time (hours) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 020406080Height/Width Ratio Incubation Time (hours) A 1-40 A 1-42

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55 Prior to present this analysis; let’s define the ranges of wavenumber used to classify the existence of determined structure (Table 7). Table 7. Amide I and II IR Regions for Peptide Molecules Structure Wavenumber (cm-1) Amide I region3 Beta turn 1695-1670 Intermolecular -structure 1690-1680 Intramolecular -structure Random coil 1645-1648 Alpha helix 1657-1648 Beta sheet 1640-1630 Intramolecular -structure 1625-1610 Intermolecular -structure Amide II region4 NH2 deformations 1567 N-H bending (60%); C-N stretching (40%) Related to proteins absorptio n/desorption centered at 1550 From the FTIR spectra, a deconvolution of the absorbance data was performed to differentiate from all the structures pres ent in each conformation. The deconvolution analysis is done with Origin version 6.1. A sa mple of how this analysis was done is presented in Figure 24. This corresponds to a sample of fibrillar st ructures at initial conditions. 3 Amide I region is between 1600 and 1700 cm-1 4 Amide I region is between 1600 and 1700 cm-1

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56 Chi^2/DoF 9.69E-07 R^2 0.99992 ID Theoretical Value Error y0 00 xc1 1645.83 1.31298 w1 68.005130.93894 A1 22.117640.87222 xc2 1631.015 0.46575 w2 30.446021.25849 A2 3.010950.48154 xc3 1595.676 2.49805 w3 28.668215.90861 A3 0.345830.25173 xc4 1558.676 2.86705 w4 59.331132.82359 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 150015501600165017001750Absorbance (a.u.)Wavenumber (cm-1) 1645cm-1 1631cm-1 1595cm-1 1558cm-1 A4 2.515920.25166 Figure 24. Example of the Deconvolution for Fibrils at Initia l Conditions. The black line represents original data; the re d line one represents the outcome of the deconvolution fitting. The resulti ng peaks are shown in green 6.3.1. Monomeric Conformation Figure 25, shows the time evolution spectra for monomeric conformation of A (1-42) peptides. Spectra were taken from its initial preparati on until about 150min of incubation time. This time interval was c hosen based on the AFM analysis. Iit was observed that fibrils did not form during this time (Table 4). Two particular regions are observed corresponding to the Amide I and II. The amide I band, between 1600 and 1700 cm-1, is associated with the backbone conformation of the peptide (-C =O). Amide II results from the N-H bending vibrati on (40-60%) and from the C-N stretching vibration (18-40%), and is sensitive to the structural configurations.

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57 0 0.002 0.004 0.006 0.008 0.01 1550 1600 1650 1700 1750 Wavenumber(cm-1) N-H bending and C-N stretching Beta Sheet (~1630cm-1) Intra molecular beta Turns 0min 150min Intermolecular beta turns Alpha helix Figure 25. FTIR Spectra of Amyloid(1-42) Peptide Monomeric Precursors The deconvolution analysis of these peak s based in the assignment from Table 6 appears in the next Table, which the evol ution of the various species from initial conditions until 150 min of incubation time. The N-H bending and C-N stretching (Not-s hown), progressively decrease with time until they equilibrate after the transition process (>100 min).

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58 Table 8. Evolution of Individual Conforma tions for Monomeric Precursors 00.020.040.060.080.1 0 15 30 45 60 75 100 105 120 135 150 Integrated absorbance (a.u)Time (min)Steady state Peptide rearrangement Dilution 00.10.20.30.40.50.6 0 15 30 45 60 75 100 105 120 135 150Integrated absorbance (a.u.)Time (min) Peptide rearrangement & steady state Dilution Intermolecular -turns: Three stages can be seen: 1) dilution process, from 0-30 min., 2) peptide rearrangement, after 45 minutes there is a steady increase of this structure, and 3) equilibrium process, from 100 min to 150 Intramolecular -turns: Notice a decrease in the amount of this type of structure from 0 to 30min, which is associated with the dilution zone. > 45 min, the protein steadily reaches a stable concentration. The amount of turns decays during monomeric evolution 00.020.040.060.080.10.120.14 0 15 30 45 60 75 100 105 120 135 150 Integrated absorbance (a.u.)Time (min)Peptide rearrangement 00.050.10.150.20.250.30.350.4 0 15 30 45 60 75 100 105 120 135 150 Integrated absorbance (a.u.)Time (min) Dilution Peptide rearrangment Steady state -helix structure: After dissolution stage, these structures are suddenly visible. They appear in the transition process, and disappear again coin ciding with the formation of sheets sheets structures: Increase continuously until they reaches an equilibrium state (> 105 min). The sudden shift corresponds to the lost of -helix structure

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59 6.3.2. Oligomeric Conformations The oligomeric precursors were studied from initial conditions until after 9 hours (Figure 26). Remember that between 10 to 15 hours, the oligomers started producing fibrillar conformations as shown Table 5. Ov erall, the IR spectra clearly demonstrated that the oligomeric species are centered in the Amide I (1685-1670 cm-1). This is region was deconvoluted into two regions that correspond to turns both interand intramolecular (Table 9). In addition, there is a well defined region that can be assigned to Amide II (1567-1500 cm-1), and corresponds to the absorption process of oligomeric structures onto a substrate. Similarly to th e monomeric species, these molecules undergo a dissolution process in itially (0-3 hours). The absorption process accelerates with time until it reaches equilibrium from about 3 hours until after 9 hours of incubation. 0 0.05 0.1 0.15 0.2 0.25 0.3 1500 1550 1600 1650 1700 1750Absorbance (a.u.)Wavenumber (cm-1) 0min 60min 120min 180min 540min Figure 26. Structures Evolution with Time of Oligomeric Precursors of A (1-42) Peptides

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60 Table 9. Evolution of Individual Conformati ons for Oligomeric Precursors 02468101214 0 60 120 180 240 300 360 420 480 540 Integrated absorbance (cm-1)Time (min)Steady state Dilution and peptide rearrangement 02468100 60 120 180 240 300 360 420 480 540 Integrated absorbance (cm-1)Time (min)Steady state Dilution and peptide rearrangement Intermolecular -turns: Two stages can be seen: 1) dilution process, which also includes the rearrangement of the peptide molecules and, 2) steady state equilibrium Intramolecular turns: Similar behavior as for intermolecular turns. It is amazing how both structures follow parallel paths 051015 0 60 120 180 240 300 360 420 480 540 Integrated Absorbance (cm-1)Time (min)Steady state Dilution and peptide rearrangement 05101520 0 60 120 180 240 300 360 420 480 540 Integrated absorbance (cm-1)Time (min)Steady state Dilution and peptide rearrangement NH2 deformations: Taken from the Amide II region, two stages are seen as function of time, where they follow similar formation and transition paths Absorption/desorption process: It is clear that oligomeric sp ecies deposit on the substrate surface. Very good agreement with AFM data (Table 5).

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61 6.3.3. Fibril Conformation The time evolution of fibrillar species is a little bit more co mplicated than for that for other configurations, because th ey show a longer dissolution processes, a transition process, an a state where some chemical conformation reach equilibrium but other molecules keep changing and do not reach a constant dynamics. The IR spectra show a very interesting beha vior. First, the regions assigned to -sheet decays with time, where as the Amide II region decreases and reaches an equilibrium state. This indicates two processes, one, the sheets are evolving and are been redistributed or transformed into other chemical c onformations. Second, the fibrils form and absorbs onto the surface in the first 3 hours (Table 10). After that they rearrange or chemical alter while being attached to the su bstrate. This is a very important finding since indicates that the fibrils may form in solution, but they form, react and transform even after being ab sorb on a surface (Figure 27). Figure 27. Time Evolution Spectra of Fibril Structures of A (1-42)

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62 Table 10. Evolution of Individual Conforma tions for Fibrillar Precursors 0246810 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 526 553 578 610 640 670 700 725 Integrated absorbance (cm-1)Time (min)Dilution and peptide rearrangement Steady state Disappearance 01234567 30 60 90 120 150 180 210 240 270 300 330 360 390 420 526 553 578 610 640 670 700 725 Integrated absorbance (a.u.)Time (min)Transition to Steady State Peptide rearrangement Dilution Intermolecular -turns: Three stages can be seen: 1) diluti on process & peptide rearrangement where they decreased (<0210), 2) steady state formation (270610min), and >620 disappearance. -helix: Similarly, 3 stages are observed but they inversely proportional to the turns. 0510152025 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 526 553 578 610 640 670 700 725 Integrated absorbance (a.u.)Time (min) Dilution and rearrangement Intramolecular beta sheet structures Steady state Intermolecular beta sheet structures 00.511.522.533.54 0 30 60 90 120 150 180 210 240 270 300 330 578 610 640 670 700 725 Integrated absorbance (a.u.)Time (min) Dilution Peptide rearrangment Steady state sheets: Two types of behavior are visible, 1) during the dissolution, the conformations are mainly intramolecular structures (1640-30cm-1), and 2) steadystate existence of intermolecular conformations (1625-10cm-1) Absorption/desorption process: As explained before, the fibrils absorb during the first 3 hours and after that, they stop absorbing but they change conformation while being adhered to the substrate

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63 The N-H bending and C-N stretching beha vior shows a constant dynamics that varies with respect to the ot her species. The interactions come and go according to the dissolution and transition processes. We found an astonishing behavior when we determine the integrated absorbance for each structure in time. The fibrils and o ligomers go from a maximum energetic level, when they just started dissolving, to a lower region when formation/absorption mechanisms are competing with each other. Th is change of energy takes place in the first three hours of formation. On the other hand, monomeric precursors do not change with time but after 3 hours, they start degenerating. Figure 28. Comparison of Surface Dynamics for the Three Peptide Structures: Integrated Absorbance Evolution with Time

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64 6.4. Membrane Construction One of the goals of this research is to study the interactions of fibril structures with model membranes. The membranes are prepared by using L-B deposition techniques on rigid and soft supported surfaces. The lipid membranes were constructed with DSPE and its surface stability was studied as explained next. At the air/water interface, we observed the behavior of DSPE layers as a function of lipid concentration. We found that the optim um concentration for the deposition of the layers was 2.45mg/mL, and the optimum surface pressure should be between 20-40 mN/m. Other concentrations resulted on weaker layers or limited the areas to perform the transfer of the lipid layers to a substrate (Figure 29). 0 10 20 30 40 50 60 050100150200250300350400Pressure (mN/m)Area (cm2) 2.45mg 1.4mg 0.8mg 0.84mg 0.35 0.4mg Figure 29. Surface Tension of DSPE as a F unction of Lipid Concentration. Low concentrations resulted in low ordering, and high concentrations re sulted in rigid and fragile layers

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65 Once the optimum concentration was determined, we performed loading/unloading cycle to test the respons e of the layer to dynamic changes. We observed that this concentration also promote stable layers (Figure 30). 0 5 10 15 20 25 30 35 240260280300320Pressure (mN/m)Area (cm2) Compression Decompression Figure 30. Loading/Unloading Cycle for DSPE at 2.45mg/mL and 30mN/m The layers were transferred to the PEG/ silica substrates with a transferred ration of 99.94%. The pressure-area ( -A) isotherm is shown in Figure 31 for our system. Once the layers were deposited we look at them using AFM in lipid rich-aqueous solutions. The images of the membranes show a layer that has high mobility (Figure 32a). This layer was then exposed to a very high concentr ation of fibrillar solution and then scanned in situ. We found that initially, some fibr il structures deposited on the surface of the membrane (Figure 32b). However, they did not stay absorbed for very long. We believe

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66 that the mobility of the layers influence th e absorption mechanism to which this fibrils attach to surfaces, since the fibrils did not a ppear in further scans of the model membrane surface (Figure 32c). 0 5 10 15 20 25 30 35 40 150200250300350400Pressure (mN/m)Area (cm2) Compression until 30 mN/m Deposition of first monolayer Deposition of second monolayer Jump ~ 204 cm2 Figure 31. Deposition and Transfer of the Lipi d DSPE Bilayer onto the PEG/silica Substrates to Create the Model Membranes Figure 32. Interacting Mechanisms Be tween Lipid Bilayers and A (1-42) Peptide Fibrils Using AFM Liquid Imaging. (a) 10 by 10 m scan of the model membrane surface as deposited, (b) Scan after fibrils have been added to the membrane, and (c) Scan soon after fibril exposure. The fibr il structures refused to adhere on the surface possibly owing to the surface m obility of the soft-supported membranes

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67 One of the questions is if the fibrils rea lly are influenced by the mobility of the membrane. Let’s look at the se ction scan from Figure 32b (F igure 33) to analyze such behavior. The height of the fibrils corr esponds to 6nm, which happens to match the height of the fibers adhered to rigid supports also detect ed by AFM (Table 4, 5, and 6). Therefore, it is reasonable to assume that the fibers indeed adhere to cell membranes, but the surface mobility makes such adhesion very weak. This is an important finding since the implications of finding the molecular mechanism of A peptides to membranes may lead us to determine the processes that cont rol fibril adhesion and plaque formation. That is, these findings might lead us in a direction to find release mechanisms that will prevent or stop adhesion all together. Figure 33. Section Profile of Biomimetic Membrane Surfaces in Contac t with Fibrils. The height of the fibrils average 6nm, whic h corresponds to the va lues report elsewhere and reported early in this ch apter (See Table 6) [23, 26]

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68 Chapter Seven Conclusions and Future Work 7.1 General Findings and Conclusions This project had a very well defined goal from the beginning, which was to study, control and determine the implications of ha ving three different structures of the two peptides that are most influential in AD brain’s dysfunction, the A -40 and A -42. We were able to establish the procedures to prepare stable and reproducible configurations of A peptides, so that we could study their in teractions with biomimetic membranes. The most important findings in terms of amyloid beta peptide adhesion to surfaces are as follow: a. There is a distinct trend between the A 1-40 and A 1-42 in terms of adhesion to rigid surfaces. A 1-40 exhibits a poor adhesion to mica, whereas A 1-42 shows a strong adhesion to rigid suppor ts. This difference in adhesion behavior may be correlated to the toxi city and propensity to form l onger aggregates that ultimately form fibrils and plaques in normal condi tions, since our ATR data shows that fibrillar configurations have a very complex adhesion mechanism to surfaces. b. The A 1-40 show to be more stable in terms of aggregation. In most cases, this peptide did not form fibrillar structures even though the conditions to form them were heavily induced.

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69 c. There is a strong correlation between data from the two surface analytical techniques used in this project. AFM and FTIR data show the effect of a dilution process. This mechanism takes place in the first 30 min. of precursor incubation. However, it has a great impact in the evol ving time to achieve equilibrium state. There was a transition zone where the stru ctures of the peptides rearrange and most of the conformations decrease or increase at this time (between 1-3 hours). d. In the case of the A 1-42, the formation of bigger aggregates into fibrillar configurations started to take place > 9 hours of incubation for most cases. e. We were able to correlate the individual configurations of th ese molecules with kinetics and dynamics of adhesion. f. We were able to control fibril formation or fibrilogenesis, since it was shown that nonfibrillar assemblies were maintained and studied in vitro g. The adhesion process of fibrillar structur es and aggregates is very complex and depends on the preparation conditions and th e affinity of the peptide to surface structure. h. We were able to distinguish between intermolecular turn structures (1695-1670 cm-1), intramolecular turn structures (1690-1680 cm-1). Typically, these structures are not seen in protein conformation studies. i. AFM studies illustrate how oligomeric and monomeric configurations of A -40 and 42 follow a general trend in terms of their ratio of height and width. That is, they absorbed onto themselves instead of the substrate until saturation is reached. Only after that the peptide will continue absorbing onto the substrate.

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70 j. For fibril configurations, the ratio of height/width of A -40 and A -42 follows an inversely proportional trend. k. Biomimetic membranes can be built onto PEG layers to mimic real surface mobility. l. The interactions between biomimetic memb ranes and fibrils show that the fibrils initially absorb, but that the absorption is very weak. This may be a function of surface flexibility and softness created by the PEG underneath layer. m. By comparing the adhesion behavior of th e fibril to the mica with the adhesion behavior to biomimetic membranes, we conc luded that the rigidity of the substrate indeed is plays an important role in the adhesion mechanisms of the fibrils. n. These findings led us to hypothesize that normal fibrils attach to real cell membranes because they have lost mobility somehow. It has been discussed that cholesterol may have an important role in AD plaque formation. This hypothesis agrees with the literature studies were St atins have helped to reduce the effects of AD symptoms [31-33]. 7.2 Final Remarks and Future Work a. This work has advanced our ability to master a systematic way to prepare the three different conformations of A peptides that may play an important role in AD plaque formation. b. If these three conformations may influen ce plaque formation, we may be able to understand the molecular mechanisms asso ciated with their adhesion, surface kinetics and assembly processes by usi ng the Surface Forces Apparatus and the

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71 ATR-FTIR technique co ncurrently (IR-SFA). c. Further investigation developing essays with the two peptides together is needed to elucidate why only A 42 segments cause adhesion, and how they influence A 40 peptide dynamic processes. d. Additional studies to corrobor ate our initial findings on the effect of substrate mobility will be performed. For instance, we would like to vary systematically the rigidity of model membranes by introduc ing cholesterol in their structure.

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72 References [1] John H. Growdon, S.C., Eva Ritter-Walker, and Richard J. Wurtman, Aging And Alzheimer's Disease. Sensory syst ems, nueronal growth, and neuronal metabolism ed. T.N.Y.A.o. Sciences. Vol. 640. 1991. [2] Saido, T.C., A metabolism and alzheimer's disease ed. T.L.B. Georgetown, Inc.,. 2003. [3] Blennow, K., CSF Biomarkers in Alzheimer's Disease. Research Advances in Alzheimer Disease and Related Disorders (Alzheimer's Association), 2004: p. 3645. [4] Serpell, L.C., Alzheimer's amyloid fibrils : structure and assembly. Biochimica et Biophysica Acta 1502, 2000: p. 16-30. [5] Annika Olsson, H.V., Niels Andreasen, Geert De Meyer, Anders Wallin, Bjorn Holmberg, Lars Rosengren, Eugeen Vanmechelen, and Kay Blennow., Simultaneous Measurement of B-amyloi d (1-42), Total Tau, and Phosphorylated Tau (Thr181) in Cerebrospinal Fluid by xMap Technology. Clinical Chemistry, 2005. 51 (2): p. 336-345. [6] Peter Friedhoff, M.v.B., Eva-Ma ria Mandelkow, Eckhard Mandelkow, Structure of tau protein and assembly into paired helical filaments. biochimica et Biophysica Acta, 2000. 1502 : p. 122-132. [7] Wang, J., The Role of Abeta-Peptide on Spatia l Memory, EEG, Auditory Evoked Potentials And Nicotinic Cholinergic Receptors in A/P Transgenic Mice in Department of Neurology 2003, University of Kuopio: Kuopio. p.76. [8] Nolte, J., Human Brain, an introduction to its functional anatomy Fourth Edition ed, ed. Mosby. 1998, New York.

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73 [9] K. Beyreuther, Y.C., C. L. Masters, Neurodegenerative Disorders: Loss of Function Through Gain of Function ed. Springer. 2001. [10] William Klein, P.D. Soluble Abeta Oligomers (ADDLs) in Alzheimer's Disease Memory Loss, Therapeutics and Diagnostics in Alzheimer's Association International Conference on Prevention of Dementia 2005. Washington, DC. [11] Welsh, E.M., Focus on Alzheimer's Desease Research ed. N. Biomedical. 2003, New York. [12] Teplow, Y.F.a.D.B., Kinetic Studies of Amyloid Be ta-Protein Fibril Assembly. Journal of Biological Chemistry, 2002. 277 (October 4): p. 36948-36954. [13] Lars O. Tjernberg, D.J.E.C., Agne ta Tjernberg, Solveig Hahne, Christina Lilliehook, Lars Terenius, Johan T hyberg, and Christer Nordstedt, A Molecular Model of Alzheimer Amyloid Beta-Peptide Fibril Formation. The Journal of Biological Chemistry, 1999. 274 (April 30): p. 12619-12625. [14] Thunecke M, L.A., Kosciessa U, Dyrks T, Oakley AE, Turner J, Saenger W, Georgalis Y, Aggregation of A Beta Alzheimer' s disease-related peptide studied by dynamic light scattering. The Journal of peptide rese arch: official journal of the American Peptide Society, 1998. 52 (6): p. 509-517. [15] T.H. Jackson Huang, D.-S.Y., Paul E. Fraser, and Avijit Chakrabartty, Alternate Aggregation Pathways of the Alzheimer Beta-Amyloid Peptide. The Journal of Biological Chemistry, 2000. 275 (Nov. 17): p. 36436-36440. [16] W. Blaine Stine, J., Karie N. Dahl gren, Grant A. Krafft, and Mary Jo LaDu, In Vitro Characterization of Conditions for AmyloidPeptide Oligomerization and Fibrillogenesis. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2003. 278 (13): p. 11612–11622. [17] Jeremy M. Berg, J.L.T., Lubert Stryer, Biochemistry Fifth Edition ed. 2001, New York. [18] Grisham, G.a., Biochemistry 1995: Saunders College Publishing.

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74 [19] Sipe, J.D. and A.S. Cohen, Review: History of Amyloid Fibril. Journal of Structural Biology, 2000. 130 : p. 88-98. [20] Fahrendholz, R.H.a.F., Alzheimer's Disease Cellula r and Molecular Aspects of amyloid B ed. Springer. Vol. 38. 2005: Subcellular Biochemistry. [21] Michael R. Nichols, M.A., Amyloid-Beta Protofibrils Di ffer from Amyloid-Beta Aggregates Induced in Dilute H exafluroroisopropanol in Stability and Morphology. The Journal of Biology Chemistry. 280 (January 28): p. 2471-2480. [22] S. Y. Fung, C.K., J. Duhamel, and P. Chen, Concetration Effect on the Aggregation of a Self-Assembling Oligopeptide. Biophysical Journal, 2003. 85 (July 2003): p. 537-548. [23] Ashok Parbhu, H.L., Julian Thimm, Ratneshwar Lal, Imaging real-time aggretation of amyloid beta protein (1 -42) by atomic force microscopy. Peptides, 2002. 23 : p. 1265-1270. [24] Wood, B.J.C.a.D.L., Memmler's The Human Body in Health and Disease Ninth Edition ed, ed. L.W.a. Wilkins. 2000. [25] Alcantar, N.A., Plasma Assisted Synthesis and C haracterization of Biocompatible Surface coatings in Chemical Engineering 2000, University of California Santa Barbara: Santa Barbara. p. 237. [26] Samuel H. Cohen, M.T.B.a.M.L.L., Atomic Force Microscopy/Scanning Tunneling Microscopy 1994, New York: Plenum. [27] Singh, B.R., Infrared Analysis of Peptides and Proteins, Principles and Applications ed. A.S.S. 750. 2000, Washington, DC. [28] Stuart, B., Infrared Spectroscopy: F undamentals and Applications 2004, Sydney, Australia: Wiley.

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75 [29] W. Blaine Stine, J., Karie N. Dahl gren, Grant A. Krafft, and Mary Jo LaDu, In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis. J of Biol Chem, 2003. 278 (13): p. 11612– 11622. [30] Kayed, R.S., Yuri; Edmonds, Brian; McIntire, Theresa; Milton, Saskia; Hall, James, E; and Glabe, Charles,G, Permeabilization of Lipid Bilayers Is a Common Conformation-dependent Activity of So luble Amyloid Oligomers in Protein Misfolding Diseases. J of Biol Chem, 2004. 279 (45): p. 46363–46366. [31] Kirsch, C., et al., Brain cholesterol, statins and Alzheimer's disease. Pharmacopsychiatry, 2003. 36 : p. S113-S119. [32] Mills, J.K., G. Eich enbaum, and D. Needham, Effect of bilayer cholesterol and surface grafted poly(ethylene glycol) on pH-induced release of contents from liposomes by poly(2-ethylacrylic acid). Journal of Liposome Research, 1999. 9 (2): p. 275-290. [33] Rockwood, K. and S. Darvesh, The risk of dementia in relation to statins and other lipid lowering agents. Neurological Research, 2003. 25 (6): p. 601-604.

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76 Appendices

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77 Appendix A. Milestones Toward Formulating and Testing the A Hypothesis of AD[9] 1892-1907 Lesions associated with Alzheimer’s neurodegeneration described and categorized (Alzheimer 1907; Blocq and Ma rinesco 1892; Redlich 1898) 1920-40 Amyloid plaques and perivascular deposits described and proposed to adversely affect cerebral function (Divry 1927; Scholz 1938) 1970-86 Isolation (Nikaido et al. 1971), ami no acid composition (Allsop et al. 1983; Roher et al. 1986, Selkoe et al. 1986) and N-terminal sequences of perivascular (Glenner and Wong 1984) and pa renchymal plaque A (Masters et al. 1985) 1987-88 Amyloid precursor protein (APP) gene cloned, located on chromosome 21 and A recognized as putative proteolytic produc t (Kang et al. 1987; Tanzi et al. 1987, 1988; Goldgaber et al. 1987; Robakis et al. 1987; Kitaguchi et al. 1988; Ponte et al. 1988) 1989-99 Toxicity of A in cell culture (Whitson et al. 1989; Yankner et al. 1989), mediated by oxidative processes (Behl et al. 1992, 1994; Dyrks et al. 1992; Butterfield et al. 1994) and metals (Bush et al. 1994; Schubert and Chevion 1995; Huang et al. 1999; Cherny et al. 1999) 1990-92 Mutations in proximity to A secretase sites cause AD (Levy et al. 1990; ChartierHarlin et al. 1991; Goate et al. 1991; Murrell et al. 1991; Citron et al. 1992; Hendriks et al. 1992) and confirm centrality of A hypothesis 1992-93 A and p3 identified as definite proteolytic products from APP (Seubert et al. 1992, 1993; Shoji et al. 1992; Busciglio et al. 1993) 1992-93 ApoE identified as an A -interacting protein (Wisniewki and Frangione 1992; Strittmatter et al. 1993) and its alleles iden tified as genetic risk factors in sporadic forms of AD (Coder et al. 1993) 1991-97 Transgenic mouse models of A deposition (Quon et al. 1991; Borchelt et al. 1997; Sturchler-Pierrat et al. 1997; Games et al. 1995; Hsiao et al. 1996) 1995-2000 Presenilins recognized as major com ponents in the amyloidogenic pathway (Levy-Lahad 1995; Borchelt et al. 1996; Sherrington et al. 1995; Rogaev et al. 1995; Scheuner et al. 1996), probably acting as -secretases (Ray et al. 1999; Capell et al. 2000; Kimberly et al. 2000; Jacobsen et al 1999; Octave et al. 2000; De Strooper et al. 1998, 1999; Wolfe et al. 1999a, b; Steiner et al. 1999. 1998-99 -secretases identified (Buxbaum et al. 1998; Lammich et al. 1999) 1999-2000 Major A degradative and clearance pathway identified (Iwata et al 2000; Qiu et al. 1999; Vekrellis et al. 2000) and improved clearance of A from the brain by immunization with A (Schenk et al. 1999) 1999-2000 -secretases identified (Vassar et al. 1999; Yan et al. 1999; Sinha et al. 1999; Lin et al. 2000; Hussain et al. 1999) 1995-2000 First rational anti-amyloid therapeutic strategies (Higaki et al. 1995) and commencement of early phase human trials (Bristol Myers Squibb, Elan Pharmaceuticals, Prana Biotechnology)

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78 Appendix B. The 20 Protein Amino Acids

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79 Appendix C. Chemical Forces and their Rela tive Strengths and Distances Force Strength (kJ/mol) Distance (nm) Description Van der Waals interactions 0.4 4.0 0.2 Strength depends on the relative size of the atoms or molecules and the distance between them. The size factor determines the area of contact between two molecules: the greater the area, the stronger th e interaction. Attractive force is inversely proportional to the sixth power of the distance, r, separating two atoms or molecules: F 1/r6. Hydrogen bonds 12 – 30 0.3 Relative strength is proportional to the polarity of the H bond donor and H bond acceptor. Mo re polar atoms form stronger H bonds. Ionic bonds 20 0.25 Strength also depends on the relative polarity of the interacting charged species. Some ionic bonds are also H bonds: OOC NH ...3 Hydrophobic interactions < 40 Force is a complex phenomenon determined by the degree to which the structure of water is disordered as discrete hydrophobic molecules or mo lecular regions coalesce.

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80 Appendix D. Infrared Spectra Appendix D.1. Characteristic Infrared Ba nds of Proteins [28] Amino acid Wavenumber (cm-1)Assignment A 3300 N-H stretching in resonance with overtone B 3110 (2 x amide II) I 1653 80% C=O stretching; 10 % C-N stretching; 10% NH bending II 1567 60% N-H bending; 40% C-N stretching III 1299 30% C-N stretching; 30% N-H bending; 10% C=O stretching; 10% O=C-N bending; 20% other IV 627 40% O=C bending; 60% other V 725 N-H bending VI 600 C=O bending VII 200 C-N torsion Appendix D.2. Characteristic Infrared Bands of Amino Acid Side Chains [28] Amino acid Wavenumber (cm-1) Assignment Alanine 1465 CH2 bending Valine 1450 CH3 asymmetric bending Leucine 1375 CH3 symmetric bending Serine 1350-1250 OH bending Aspartic acid 1720 C=O stretching Glutamic acid 1560 1415 CO 2 asymmetric stretching CO 2 symmetric stretching Asparagine 1650 C=O stretching Glutamine 1615 NH2 bending Lysine 1640-1610,1550-1485 1160,1100 NH 3 bending NH 3 rocking Phenylalanine 1602,1450,760,700 Benzene ring vibrations Tyrosine 1600,1450 Benzene ring vibrations Arginine 1608,1586 Benzene ring vibrations

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81 Appendix E. Material Safety Data Sheets. Appendix E.1. A 1-40

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82 Appendix E.1. (Continued)

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83 Appendix E.1. (Continued)

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84 Appendix E.2. A 1-42

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85 Appendix E.2. (Continued)

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86 Appendix E.2. (Continued)

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87 Appendix F. One Example on AFM Data Processing I. Amyloid (1-40) Monomer 0 hours (F ile: ab40-monomer-0h-2um) a. Group A: H1 H2 W1 W2 A B C A B C

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88 b. Group B: c. Group C: H1 H2 W1 W2 H1 W1

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89 Appendix G. Transferred Area Calculation Transferred Rate Area (ATR) : ATR = data) from (read ed transferr area alculated) tweezer(c and wafers the to area ed transferr total 100% From Figure 31, one can see the transferred area, this is Transferred area= (the initial area at which 30mN/m is reach – final area after deposition) Hence, Transferred area= 230.37 cm2 –177.32 cm2 = 53.05 cm2 Now the wafer’s dimension were 1.912cm with a thickness of .05 cm 1.912cm The total area for the wafer is Awafer = 2*(1.912cm)2 + (0.05cm*1.912cm)*4 = 7.7cm2 The deposition was done to two wafers at the same time with the same dimension, therefore the total area transfer to the wafe r twice because we are building a bilayer, is; Atransfer to the wafer s (bilayer) = 7.7cm2 4 = 30.8cm2

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90 Appendix G. (Continued) Next we need to calculate the area transferre d to the tweezers use to hold the wafers, and the dimensions were: 1.31 cm 1.31 cm 1.896cm 1.189 cm Thickness= 0.5 Thickness= 0.2 Area of 1: A1 = (1.31 cm 1.896 cm) 2 = 4.97 cm2 + (0.5 cm 1.31 cm)* 2 = 1.31 cm2 (0.5cm 1.896) = 0.948 cm2 Total area 1: A1 = 7.23 cm2 Area of 2: A2 = (1.31 cm 1.189 cm) 2 = 3.12 cm2 + (0.2 cm 1.31 cm)* 2 = 0.524cm2 (0.2cm 1.189) = 0.2378 cm2 Total area 1: A2 = 3.88 cm2 2 1

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91 Appendix G. (Continued) Total area transferred to the tweezers= 2* A1 + A2 = 22.22cm2 Total transferred area is: Area transferred to th e wafers + area transferred to the tweezers= Atotal transferred = 30.8 cm2 + 22.22cm2 = 53.02 cm2 Hence the transferred rate is: ATR = 2 2c m 52.03 cm 52.02 100% = 99.94%


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ABSTRACT: The amyloid beta peptide particularly the 40 and 42 amino acid residues are the responsible for plaque formation in Alzheimer's disease (AD) patients. Extra cellular plaque formation has been recognized after incessant investigations along with the formation of intracellular tau protein tangles as the hallmarks of AD. Furthermore, the plaque formation has been linked mostly as a cause of the disease and the tangles mostly as a consequence. Our investigation is focused on studying the formation of AD plaques. The amyloid beta (A[beta]) is a physiological peptide secreted from neurons under normal conditions, along with other soluble forms cleaved from the amyloid precursor protein (APP). These soluble forms of APP have neuroprotective and neurotrophic functions, while the A[beta] is considered an unwanted by-product of the APP processing.Under normal conditions there is an anabolic/catabolic equilibrium of the A[beta] peptide; therefore, it is believed that the formation of the plaque does not take place. On the other hand, the neurons' surface may play an important role in the adhesion mechanisms of the A[beta] peptide. Our experiments show that the neuron surfaces along with the media conditions may be the most important causes for progressive formation of plaques. We have incubated rigid supports (mica) and soft biomimetic substrates (lipid bilayers on top of a PEG cushion layer drafted onto a silica surface) with the three different conformations of the A[beta] peptide (monomeric, oligomeric and fibrils structures) to determine the adhesion mechanisms associated with in situ plaque formation. The soft biomimetic substrates have been assembled first by depositing and activating a thin film of silica (i.e., to create surface silanol groups).This film is then reacted with polyethylene glycol (PEG), which is a biocompatible polymer, to create a cushion-like layer that supports and allows the lipid bilayer to have high mobility. A lipid bilayer is then deposited on this soft support to reproduce a cell membrane using the Langmuir Blodgett deposition technique. The characterization of such biomimetic membranes has been studied by using Atomic Force Microscopy (AFM) in liquid environments. Our results show that these lipid bilayers are highly mobile. Additionally the structure and topography characteristics of the A[beta] conformations have been followed with atomic force microscopy (AFM). The kinetics and rates of adhesion have been measured with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Our results show the progress of the plaques' formation with time where simple monomers deposit on the substrates and allow the development of oligomeric species.
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