La formation d'agrégats protéiques insolubles et fibreux, appelés fibrilles amyloïdes, est impliquée dans une large variété de maladies humaines. Parmi elles, figurent entre autres, le diabète de type II, l'arthrite rhumatoïde et, notamment, les atteintes neurodégénératives débilitantes, telles que les maladies d'Alzheimer, de Parkinson ou encore de Huntington. Actuellement, il n'existe ni traitement, ni diagnostic précoce pour aucune de ces maladies.De nombreuses études ont montré que la capacité à former des fibrilles amyloïdes est une propriété inhérente à la chaîne polypeptidique. Ce constat a conduit au développement d'un certain nombre d'approches computationnelles permettant de prédire les propriétés amyloïdogéniques à partir de séquences d'amino-acides. Si ces méthodes s'avèrent très performantes vis à vis de courts peptides (~ 6 résidus), leur application à des séquences plus longues correspondant aux peptides et protéines en lien avec les maladies, engendre un nombre trop élevé de faux positifs. Le principal objectif de cette thèse consiste à développer une meilleure approche bioinformatique, capable de prédire les régions amyloïdogéniques à partir d'une séquence protéique. Récemment, l'utilisation de nouvelles techniques expérimentales a permis de mieux appréhender la structure des amyloïdes. Il est ainsi apparu que l'élément caractéristique de la majorité des fibrilles amyloïdes impliquées dans les maladies, était constitué d'une structure étagée (β-arcade), résultant de l'empilement de motifs « feuillet β – coude – feuillet b » appelés « β-arches ». Nous avons mis à profit cette particularité structurale pour créer une approche bioinformatique permettant de prédire les régions amyloïdogéniques d'une protéine à partir de l'information contenue dans sa séquence. Les résultats provenant de l'analyse des structures de type β-arcade, connues et modélisées, ont été compilés et traités à l'aide d'un algorithme écrit en langage Java, afin de créer le programme ArchCandy.L'application de ce programme à une sélection de séquences protéiques et peptidiques, connues pour leur lien avec les maladies, a permis de démontrer qu'il était en mesure de prédire correctement la majorité de ces séquences, de même que les séquences mutées impliquées dans les maladies familiales. Outre la prédiction de régions à haut potentiel amyloïde, ce programme suggère la conformation structurale adoptée par les fibrilles amyloïdes. Le séquençage de génomes entiers devenant toujours plus abordable, notre méthode offre une perspective de détermination individuelle des profils à risque, vis à vis de maladies neurodégénératives, liées à l'âge ou autres. Elle s'inscrit ainsi pleinement dans l'ère de la médecine personnalisée. / A broad range of human diseases are linked to the formation of insoluble, fibrous, protein aggregates called amyloid fibrils. They include, but are not limited to, type II diabetes, rheumatoid arthritis, and perhaps most importantly, debilitating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. There currently exists no cure, and no means of early diagnosis for any of these diseases. Numerous studies have shown that the ability to form amyloid fibrils is an inherent property of the polypeptide chain. This has lead to the development of a number of computational approaches to predict amyloidogenicity by amino acid sequences. Although these methods perform well against short peptides (about 6 residues), they generate an unsatisfactory high number of false positives when tested against longer sequences of the disease-related peptides and proteins. The main objective of this thesis was to develop an improved bioinformatics based approach to predict amyloidogenic regions from protein sequence.Recently new experimental techniques have shed light on the structure of amyloids showing that the core element of a majority of disease-related amyloid fibrils is a columnar structure (β—arcade) produced by stacking of β-strand-loop-β-strand motifs called “β-arches”. Using this structural insight, we have created a bioinformatics based approach to predict amyloidogenic regions from protein sequence information. Data from the analysis of the known and modeled β-arcade structures was incorporated into a rule based algorithm implemented in the Java programming language to create the ArchCandy program. Testing it against a set of protein and peptide sequences known to be related to diseases has shown that it correctly predicts most of these sequences and a number of mutated sequences related to the familial diseases. In addition to the prediction of regions with high amyloidogenic potential, a structural arrangement of the amyloid fibril is also suggested for each prediction. As whole genome sequencing becomes cheaper, our method provides opportunity to create individual risk profiles for the neurodegenerative, age-related and other diseases ushering in an era of personalized medicine.

Alzheimer’s disease (AD) is a severe age-related neurodegenerative disorder and is the most common form of dementia. Although the pathogenesis of AD remains unknown, the deterioration of the cerebrovascular system constitutes a risk factor associated with the development of the disease. Notably, brain hypoperfusion, a feature of healthy ageing brain and AD, occurs prior to the onset of cognitive decline in AD and correlates with the severity of dementia. Although there is a clear link between hypoperfusion and cognitive alterations in AD, a causal relationship remains to be established. It was hypothesised that chronic cerebral hypoperfusion leads to the accumulation of parenchymal and vascular amyloid-β (Aβ), triggering the development of vascular lesion (microinfarcts (MIs) and haemorrhages) and altering the neurovascular unit (NVU) integrity. Second to this, it was hypothesised that reductions in Aβ levels by immunotherapy targeted to amyloid in young mice, reduce amyloid levels, and prevent vascular lesions improving cognitive performance. Three studies were conducted to test these hypotheses. In the first study, the aim was to characterise age-dependent changes in amyloidrelated pathology in a transgenic mouse model (Tg-SwDI). The temporal amyloid precursor protein (APP) expression, accumulation of parenchymal and cerebrovascular Aβ and Aβ-related microglial and astrocytic activation in the cortex, hippocampus and thalamus of the Tg-SwDI mice at 3, 6 and 9 months of age was compared to wild-type controls. Significantly higher APP expression (p < 0.05), as well as Aβ aggregation (p < 0.001) as the animals aged was found in the Tg-SwDI mice in all the brain regions analysed, which was accompanied by extensive and progressive activation of microglial (p < 0.001) and astrocytic (p < 0.01) cells. These data provided a basis to design the next studies, as it was planned to induce hypoperfusion in these mice before significant Aβ deposition occurs. In the second study, the aim was to investigate the effect of hypoperfusion on Aβ dynamics and subsequently, to study the contribution of hypoperfusion and Aβ pathology to the development of MIs and haemorrhages, and to the potential alteration of astrocyte and tight junction (TJ) integrity. To address this, mild chronic cerebral hypoperfusion was induced in Tg-SwDI and wild-type mice by bilateral common carotid stenosis for 1 and 3 months. A significant increase in soluble Aβ40/42 levels was initially found after 1 month of hypoperfusion in the parenchyma (Aβ40, p = 0.0239; Aβ42 p = 0.0198) in parallel with elevated APP levels and APP proteolytic cleavage products (p < 0.05). Thereafter, following 3 months, a significant increase in insoluble Aβ40/42 levels was determined in the parenchyma (Aβ40, p = 0.0024; Aβ42 p = 0.008) and vasculature (Aβ40, p = 0.0046; Aβ42 p = 0.0118) of Tg-SwDI mice. There was no change in the levels of Aβ co-localised to vessels following 1 month of hypoperfusion; however Aβ levels were significantly increased in cerebral vessels after 3 months (p = 0.0483). The proportion of Aβ containing vessels was significantly higher in the small vessels of the hypoperfused animals compared to sham mice (p < 0.05). MIs associated with microglial proliferation were present in the Tg-SwDI mice and the burden was exacerbated by hypoperfusion at 1 and 3 months (p < 0.05). Significantly higher levels of NADPH Oxidase-2 (NOX2) were found in the transgenic mice compared to the wild-type controls at both time-points analysed (p < 0.05), and this was exacerbated after 1 month of hypoperfusion in the Tg-SwDI mice (p < 0.05). There was a positive correlation between NOX2 and soluble parenchymal Aβ levels (r = 0.6643, p = 0.0019). A minimal effect on the development of haemorrhages at these time-points was observed. In parallel to this, astrocyte activation was significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points studied (p < 0.05); however, no effect of hypoperfusion was observed. Also, significantly higher levels of aquaporin-4 (AQP4) in the Tg-SwDI mice compared to the wild-type controls following 1 month of hypoperfusion were found (p < 0.001). There was a positive correlation between AQP4 and soluble parenchymal Aβ levels (r = 0.4735, p = 0.0095). Claudin-5 levels were significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points analysed (p < 0.0001), and this was exacerbated following 1 month of hypoperfusion in the transgenic model (p < 0.05). A positive correlation between claudin-5 and vascular Aβ levels was observed (r = 0.6113, p = 0.0004). Together, these data suggest a synergistic contribution of amyloid and hypoperfusion pathologies to the tissue damage and implicate a role of oxidative stress and inflammation. In the third study, the aim was to determine the effects of passive amyloid immunisation on Aβ levels, development of MIs and haemorrhages and behavioural performance in the Tg-SwDI mice. To address this, the mice underwent weekly intraperitoneal injections with either 3D6 or 10D5 antibodies during 3 months. Although there were no significant changes between control and 10D5/3D6 treated mice in amyloid levels, appearance of MIs and cognitive performance, it was noted that there was a trend towards a reduction in amyloid levels and MI area in the 10D5/3D6 treated mice compared to the control animals. Furthermore, there was no evidence of microhaemorrhages in response to the immunisation. These results demonstrate that Aβ immunotherapy with the antibodies 3D6 and 10D5 may potentially decrease parenchymal and vascular amyloid accumulation, reducing the appearance of MIs and notably without triggering the development of microhaemorrhages. Collectively, the findings presented in the current thesis demonstrate that chronic cerebral hypoperfusion increases parenchymal and vascular Aβ levels and point towards a mechanism in which the cascade of events including inflammation and oxidative stress, triggered synergistically by hypoperfusion and Aβ, resulted in the widespread development of MIs and NVU changes which may further induce the alteration of cognition networks. A mixed therapy, aimed at improving cerebrovascular health and targeting the accumulation of Aβ, represents a promising strategy to prevent neurodegenerative processes and further cognitive decline in AD.

Dementia, the most common cause of which is Alzheimer's disease (AD), currently affects 850,000 people in the UK, a figure set to rise to over 1 million by 2025. There is currently no disease modifying therapy available to slow or halt this progressive disease. Current understanding of AD implicates the neurotoxic amyloid-β (Aβ) peptide as the primary initiator in a cascade of events leading to the neuronal cell death and brain atrophy associated with the disease. Therefore, inhibiting the production or enhancing the clearance of Aβ within the brain has become a major target for the production of disease modifying therapeutics. Aβ is produced by brain cells through the sequential proteolytic cleavage of a larger transmembrane protein known as the amyloid precursor protein (APP) by β- and γ-secretases. Several aspects of APP physiology can influence its proteolysis, and thus Aβ production, including the isoform of APP which is expressed, its trafficking and subcellular location and its physical interactions with other proteins in the cellular environment. Here we have investigated the influence of subcellular trafficking and location and protein-protein interactions on the differential proteolysis of two APP isoforms, APP695 and APP751 in a neuroblastoma cell line. We have shown that APP751 undergoes less amyloidogenic proteolysis than APP695 and that retention within the early secretory pathway may contribute to this difference. APP751 shows higher co-localisation to the trans-Golgi network than APP695 in immunofluorescence microscopy studies, while addition of a mutation which causes APP proteolysis in the secretory pathway reduces the large difference in amyloidogenic proteolysis of these two isoforms. Targeting APP endocytosis from the cell surface, thought to be a key determinant in Aβ generation, effects APP isoform proteolysis and Aβ production to a similar extent in both the APP isoforms suggesting differences in proteolysis occur before this trafficking event. We also show by immunoblot analysis that the APP isoforms may be differentially cleaved by proteases other than β- and γ-secretase to produce recently identified proteolytic fragments. Using a liquid chromatography - tandem mass spectrometry approach coupled to prior stable isotope labelling of amino acids in cell culture (SILAC), we have identified the interactomes of the two APP isoforms in our model system. Gene ontology analysis identified enrichment of nuclear and mitochondrial proteins specifically in the APP695 interactome. Using siRNA mediated protein knockdown, we have shown interactions with Fe65 and ataxin-10 specifically influence Aβ generation from the APP695 isoform. Fe65 alters proteolysis at the rate limiting β-secretase cleavage step, while ataxin-10 alters proteolysis by γ-secretase. Interaction with growth-associated protein 43 specifically influences Aβ generation from the APP751 isoform, altering proteolysis at the γ-secretase step. Finally we have shown that recently discovered familial AD-linked mutation and protective mutation within the Aβ region of the APP protein have consistent effects on APP proteolysis in both the APP isoforms.

Cognitive impairment after stroke or transient ischemic attack (TIA) is a prototype of vascular cognitive impairment (VCI). 30% of subjects with poststroke cognitive impairment are detected Alzheimer’s disease (AD)-like amyloid-beta (Aβ) retention with reference to ¹¹C-Pittsburgh compound B (PiB) positron emission tomography (PET). Therefore, poststroke cognitive impairment provides a good clinical context for the study about contribution of comorbid Alzheimer’s and cerebrovascular disease (CVD) pathologies to cognition. In this thesis, there are four studies addressing the effect of Aβ on poststroke cognitive impairment. / The first study investigated the accuracy of diagnosing cognitive impairment subtype in subjects with stroke/TIA using current clinical diagnostic criteria with reference to ¹¹C-PiB PET. We found the agreement between the pre and the post-PET diagnoses was poor (Kappa=0.194). The overall accuracy of clinical diagnosis of pure VCI (pVCI) was 66.7%, while that of mixed (i.e., AD with CVD) VCI (mVCI) was 68.0%. / Dementia may occur after stroke/TIA within 3-6 months (early poststroke dementia [PSD]) or from 3-6 months onward (delayed PSD). In the second study, apart from age and history of diabetes mellitus, chronic small vessel disease (SVD) lesions including lacunes and white matter changes (WMC) predicted delayed PSD as they did for early PSD. With comparable levels of SVD, the presence of acute infarcts and AD-like Aβ retention were associated with the early dementia after stroke/ TIA. / So far, there is a lack of research on the long-term effect of Alzheimer’s pathology on cognitive impairment in the context of stroke/TIA. We hypothesized that comorbid AD-like Aβ deposition played a key role in progressive cognitive decline after stroke/TIA. To test this hypothesis, we conducted a 3-year longitudinal study as study 3. Over 3 years, there was significant difference between mVCI and pVCI on the changes of the Mini-Mental State Examination (MMSE) score over time. We observed a significant decline in MMSE in the mVCI group but not the pVCI group. The annual rates of decline in MMSE and Montreal Cognitive Assessment (MoCA) score were greater in the mVCI group compared to the pVCI group. Of all MoCA domains measured, memory, executive and visuospatial functions were related to Aβ deposition. / In study 4, we investigated the relative contribution of Aβ deposition and CVD lesions to neuropsychological profiles in subjects with cognitive impairment after stroke/TIA. We found that in mVCI, Aβ retention in deep region or parietal lobe was predominantly associated with memory or executive function, respectively. In pVCI, frontal WMC and global large acute infarcts could affect memory or executive function via brain atrophy. / The conclusion of these studies reported herein can be summarized as follows: First, the overall accuracy of clinical diagnosis for cognitive impairment subtypes after stroke/TIA was low. Second, subjects with AD-like Aβ deposition tended to have dementia early after stroke/TIA, and they were more likely to experience a continuous and more severe cognitive decline 3 years later. Finally, Aβ deposition could affect both memory and executive function directly as a predominant factor in subjects with mixed Alzheimer’s and CVD pathologies. / 中風或短暫性腦缺血發作後的認知障礙被普遍視為血管性認知障礙的一種原型。通過澱粉樣蛋白正電子發射計算機斷層掃描技術（¹¹C-PiB PET），30%的中風或短暫性腦缺血發作後認知障礙患者具有阿爾茲海默氏病型的澱粉樣蛋白(Aβ)沈積。因此，中風或短暫性腦缺血發作後認知障礙是一種研究共存的阿爾茲海默氏病和腦血管疾病對認知功能的影響的良好模型。該論文通過四個研究，闡述了Aβ對中風或短暫性腦缺血發作後認知功能的影響。 / 第一個研究通過藉助¹¹C-PiB PET,調查了臨床診斷中對中風或短暫性腦缺血發作後認知障礙的分型的準確性。我們發現，對不同認知障礙類型的臨床診斷準確率較低（Kappa=0.194）。其中，對血管性認知障礙的臨床診斷準確率為66.7％，對混合性(阿爾茲海默氏病和腦血管疾病混合型)認知障礙的臨床診斷準確率為68.0％。 / 通常，我們把於中風或短暫性腦缺血發作後3至6個月內發生的癡呆定義為早髮型中風或短暫性腦缺血發作後癡呆，3至6個月后發生的癡呆定義為晚髮型中風或短暫性腦缺血發作後癡呆。在第二個研究中，我們發現，慢性小血管病（腔隙性梗塞和腦白質病變）不僅可以導致早髮型中風或短暫性腦缺血發作後癡呆，而且和晚髮型中風或短暫性腦缺血發作後癡呆也有關聯。然而，如果在相同程度的小血管病損傷的情況下，具有急性缺血性損傷和阿爾茲海默氏型Aβ沈積的患者更易提早發生中風或短暫性腦缺血發作後癡呆。 / 迄今，尚無關於共存的阿爾茲海默氏病和腦血管疾病對認知功能的長期影響的研究。我們假設合併的阿爾茲海默氏病可以導致患者中風或短暫性腦缺血發作後認知功能持續下降。為了驗證這一假設，我們進行了一個為期3年的長期隨訪研究（研究三）。在三年的隨訪中，混合性認知障礙患者和血管性認知障礙患者的簡短認知檢測（MMSE）評分變化有著顯著不同：混合性認知障礙患者的MMSE評分顯著下降，而血管性認知障礙患者的MMSE評分則無明顯改變。而且，混合性認知障礙患者的MMSE和蒙特利爾認知評估量表（MoCA）評分每年下降的平均速度皆高於血管性認知障礙患者。此外，藉助MoCA，我們發現中風或短暫性腦缺血發作後認知障礙患者的記憶、執行能力和視覺空間能力的損傷都和Aβ沉積有關。 / 在第四個研究中，我們研究了Aβ和腦血管病損傷對中風或短暫性腦缺血發作後患者不同認知功能的影響。我們發現，在混合性認知障礙患者中，腦深部的Aβ沉積和記憶功能損害直接相關，腦頂葉的Aβ沉積則和執行功能損害直接相關。在血管性認知障礙患者中，額葉腦白質病變和全腦大型腦梗病灶則可通過腦萎縮的介導，影響記憶或執行功能。 / 總之，我們的研究發現: 1.目前關於中風或短暫性腦缺血發作後患者認知障礙分型的臨床診斷的準確性較低。2.具有阿爾茲海默氏型Aβ沉積的患者不僅易於在中風或短暫性腦缺血發作後早期發生認知障礙，而且其認知水平在長期隨訪中也會不斷下降。3. Aβ沉積可以作為主導因素直接影響混合性認知障礙患者的記憶和執行功能。 / Liu, Wenyan. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 164-187). / Abstracts also in Chinese; appendixes in Chinese. / Title from PDF title page (viewed on 12, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

A common theme in amyloid disorders is the deposition of disease-specific protein aggregates in tissues. Amyloid proteins bind to heparan sulfate (HS), a sulfated glycosaminoglycan, and HS has been found to promote the aggregation process. The present work relates to HS mediated mechanisms of amyloidosis, particularly transthyretin (TTR) amyloidosis, AA-amyloidosis and Alzheimer’s disease (AD). TTR is a transport protein present in the blood and cerebrospinal fluid, which under unclear circumstances can deposit as amyloid in the myocardium of elderly individuals. Examination of cardiac tissue from a 70 year old patient with reported cardiomyopathy reveald co-deposition of TTR amyloid and HS. Studies revealed that HS promotes TTR fibrillization through interaction with a basic motif in the protein. Empolyment of a cell model demonstrated that cell surface HS mediates internalization of TTR, an effect likely facilitated by HS-binding to the basic motif on TTR. Collectively, HS-TTR interactions at the cell surface may have dual outcomes, resulting in either fibrillization or internalization, respectively. During inflammatory conditions, serum amyloid A (SAA), an acute-phase protein associated with the high-density lipoprotein (HDL), can assemble into insoluble amyloid fibrils, causing AA-amyloidosis. We found that HS structures exceeding 12-14 sugar units in length separates SAA from HDL and induces subsequent aggregation of the polypeptide. Our result proposes a novel role for HS in AA-amyloidosis in which a critical length of HS is required for separation of SAA from HDL. Late-onset AD patients show reduced ability to clear cerebral amyloid-β (Aβ) aggregates, a pathological hallmark of the disease. Althought the pathway of Aβ clearance is still unclear, several cell-surface receptors are implicated in Aβ internalization. We found that ApoE facilitated Aβ uptake through interactions with HS-proteoglycans and low-density lipoprotein receptor-related protein 1. The ApoE interaction with Aβ likely promotes Aβ clearance in the brain, but, if unbalanced, may contribute to the pathology of AD.     These findings are in accord with the concept of HS as a promoter of amyloid protein aggregation, but also point to more complex relationship.

Amyloid diseases are a world-wide problem causing great human suffer and large economical costs. Although amyloid deposits, a common denominator in all amyloid disorders, are detrimental to the surrounding tissue, there is a poor correlation between total amyloid burden and clinical symptoms. Soluble oligomers are much more potent to exert a tissue damaging effect.  Alzheimer’s disease (AD) is strongly linked to self-assembly of the amyloid-β (Aβ) peptide. Antibodies selectively targeting cytotoxic Aβ-species are useful both for understanding oligomer formation and for their therapeutic abilities. We hypothesized that the effect of avidity would compensate for a low single site affinity and be enough to selectively target oligomers. To evaluate this hypothesis, we focused on the IgM isotype having ten antigen-binding sites. In accordance with the hypothesis, the IgM isotype effectively bound oligomeric Aβ also in presence of a vast excess of its monomeric counterpart, clearly illustrating the potentiating effect of avidity. As a continuation of this work, we have shown that the avidity effect from a bivalent binding is enough to induce oligomer specificity. This finding facilitates a direct application on the clinically more useful IgG isotype, where the binding properties now can be controlled in detail. The method is general and we have, using this technique, also designed oligomer specific antibodies targeting α-synuclein. Transthyretin (TTR) is an amyloidogenic protein involved in both hereditary and sporadic amyloidosis. The cytotoxicity of TTR is intriguing since studies have shown cytotoxic potential from oligomers, tetramers and even monomers. Elucidation of the molecular properties associated with TTR cytotoxicity is hence of interest. By preventing tetramer dissociation, TTR aggregation and TTR-induced cytotoxicity is abolished. Based on this rationale, a current therapeutic strategy is to stabilize the TTR tetramer with small molecules. The kinetic stability within the spectra of known TTR mutations spans more than three orders of magnitude. However, although the most stable mutants are inert, a poor correlation within the group of cytotoxic variants exists where the cytotoxic effect is not potentiated in proportion to their kinetic stability. Through analysis of a large spectra of TTR variants, our results indicate that TTR induced cytotoxicity requires an intermediate stability of the TTR molecule. The kinetic stability should be low enough to permit tetramer dissociation and the thermodynamic stability high enough to prevent instant aggregation and to allow formation of the cytotoxic fold.

Frankfurt (Main), Universität, Thesis (doctoral), 2002.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

München, Techn. University, Diss., 2004.

abstract: Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable &beta;-turn fibers. These non-amyloid fibers are present in the 10 &mu;M concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid &#64257;brils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP &#64257;bril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs. / Dissertation/Thesis / Ph.D. Physics 2013