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Homeostasis of metastable proteins in Alzheimer's diseaseKundra, Rishika January 2017 (has links)
Alzheimer’s disease (AD) is the most common cause of dementia, affecting almost 40 million people worldwide, and it is predicted that this number will rise to nearly 150 million by 2050. It results not only in enormous distress for affected individuals and carers but also a substantial economic burden on society. Although more than 100 years have passed since its discovery, no cure for AD exists, despite enormous efforts in basic and clinical research over the past few decades, due to limited understanding of its underlying mechanisms. Neurodegenerative disorders, of which AD is an example, are highly complex disorders characterized by extensive neuronal dysfunction associated with the misfolding and aggregation of a specific set of proteins, including amyloid plaques and neurofibrillary tangles in AD. One promising avenue for progress in the field is to improve our understanding of the mechanisms by which cellular dysfunction arises from the initial protein aggregation events. The studies described in the thesis are based on the recent finding that a large number of proteins are inherently supersaturated, being expressed at concentrations higher than their solubilities, and constituting a metastable subproteome potentially susceptible to aggregation. These studies illustrate the dependence of aggregation prone metastable proteins on the cellular degradation machineries. They also study the role of metastable proteins and their homeostasis complement in the vulnerability of various body and brain tissues to protein aggregation diseases. Using extensive sequencing data and network based systems biology approaches, they elucidate how fundamental physicochemical properties of an individual or group of proteins relate to their biological function or dysfunction.
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Manipulation of Death Pathways in Desmin-Related CardiomyopathyMaloyan, Alina, Sayegh, Jennifer, Osinska, Hanna, Chua, Balvin H., Robbins, Jeffrey 14 May 2010 (has links)
RATIONALE: Transgenic mice with cardiac specific overexpression of mutated αB-crystallin (CryABR120G) display Desmin-related myopathy (DRM) with dilated cardiomyopathy and heart failure. Our previous studies showed the presence of progressive mitochondrial abnormalities and activation of apoptotic cell death in CryABR120G transgenic hearts. However, the role of mitochondrial dysfunction and apoptosis in the overall course of the disease was unclear. OBJECTIVE: We tested the hypothesis that prevention of apoptosis would ameliorate CryABR120G pathology and decrease morbidity. METHODS AND RESULTS: We crossed CryABR120G mice to transgenic mice with cardiac specific overexpression of Bcl-2. Sustained Bcl-2 overexpression in CryABR120G hearts prolonged CryABR120G transgenic mice survival by 20%. This was associated with decreased mitochondrial abnormalities, restoration of cardiac function, prevention of cardiac hypertrophy, and attenuation of apoptosis. CryABR120G misfolded protein aggregation was significantly reduced in the double transgenic. However, inhibition of apoptotic signaling resulted in the upregulation of autophagy and alternative death pathways, the net result being increased necrosis. CONCLUSION: Although Bcl-2 overexpression prolonged life in this DRM model, in the absence of apoptosis, another death pathway was activated.
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An interdisciplinary approach to studying mechanistic, structural and toxic features of protein aggregates associated with neurodegenerative disordersFlagmeier, Patrick January 2018 (has links)
The misfolding and aggregation of proteins is closely associated with more than fifty human disorders, including Alzheimer's and Parkinson's diseases, all of which are currently incurable and many represent a major threat to human life. The mechanism of protein aggregation is subject to extensive studies. The damaging effects associated with protein aggregation have been attributed to amyloidogenic species that are present during the misfolding process. In particular, oligomeric species are, however, intrinsically difficult to study as a consequence of their low abundance and highly heterogeneous nature. The first chapter of my thesis gives an introduction into the field of protein folding and misfolding with a focus on the study of protein aggregation, and toxic effects relevant to human disorders. The second chapter of my thesis describes the development of a methodology that enables the study of aggregate induced lipid bilayer permeability, possibly the most general mechanism of protein aggregate toxicity. Surface-tethered lipid vesicles functioning as optochemical probes sensitive to membrane integrity are imaged using total internal reflection microscopy. It is shown that oligomeric species of the 42-residue form of the Aβ peptide (Aβ42) are responsible for the membrane disruption. The methodology can be applied to the study of other proteins such as α-synuclein and tau, and the ability of antibodies and chaperones to counteract the aggregate induced lipid bilayer permeability can be assessed. Furthermore, lipid bilayer permeability induced by aggregates formed in human induced pluripotent stem cells can be studied. The third chapter presents a new approach for the measurement of protein aggregation kinetics by following the development of the lipid bilayer permeability over the course of the aggregation process of Aβ42. The aggregation kinetics can be modulated with molecular chaperones and pre-formed seed fibrils, which allows secondary nucleation to be identified as the process that drives the formation of species responsible for the lipid bilayer permeability. The fourth chapter describes the development of a three-pronged strategy to study the mechanism of α-synuclein amyloid formation. The aggregation is studied in the presence of lipid vesicles or pre-formed fibrils at neutral or acidic pH of the solution. The influence of single-point mutations on the aggregation of α-synuclein is described. Furthermore, the strategy is applied to the characterisation of the ability of antibodies and small molecules to inhibit the aggregation, and thus has the potential for the development of therapeutical agents. The work presented in the fifth chapter characterises the amyloid fibril populations formed by α-synuclein and mutational variants associated with familial Parkinson's disease. X-ray crystallography, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and atomic force microscopy have all been applied to the analysis of these amyloid fibrils. Finally, the sixth chapter summarises the results described in this thesis and points out future opportunities in the context of fundamental and translational studies related to the research area of protein misfolding disorders.
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Intrinsic Local Balancing of Hydrophobic and Hydrophilic Residues in Folded Protein SequencesBorukhovich, Ian January 2015 (has links)
Protein sequences may evolve to avoid highly hydrophobic local regions of sequence, in part because such sequences promote nonnative aggregation. Hydrophobic local sequences are avoided in proteins even in buried regions, where native structure requirements tend to favor them. In this dissertation, I describe three explorations of this hydrophobic suppression. In Chapter 2, I examine the occurrence of hydrophobic and polar residues in completely buried β-strand elements, and find evidence for hydrophobic suppression that decreases as a β-strand becomes more exposed. In Chapter 3, I present a generalized study of the tendency of local sequences to deviate from the hydropathy (hydrophobicity) expected based on their solvent exposure. First, I examined the hydropathy of local and nonlocal sequence groups over a large range of solvent exposures, within folded protein domains in the ASTRAL Compendium database; second, I calculated the tendency of residues within 10 positions of a nonpolar or polar reference residue to deviate from the hydropathy expected based on their structural environment. Both analyses suggested that protein sequences exhibit 'local hydropathic balance' across a range of 6-7 residues, meaning that polar and nonpolar residues are more dispersed in the sequence than expected based on solvent exposure patterns. This balance occurs in all major fold classes, domain sizes and protein functions. An unexpected finding was that it partly arises from a tendency of buried or exposed residues to be flanked by polar or nonpolar residues, respectively. This relationship may result from evolutionary selection for folding efficiency, which might be enhanced by reduced local competition for buried or exposed sites during folding. Finally, in Chapter 4, I present several exploratory analyses, including a decision-tree approach, to visualize the influence of a large number of sequence-structure properties on residue hydrophobicity. Overall, the work in this dissertation confirms that hydrophobic suppression and local hydropathic balance in general are intrinsic properties of folded proteins. I speculate that local hydropathic balance results from selection for reduced aggregation propensity, increased folding efficiency and increased native state specificity. The concept of local hydropathic balance might be used to improve the properties of designed and engineered proteins.
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Investigating the relationship between abnormal prion protein (PrPSc) and the transmissible spongiform encephalopathy (TSE) infectious agentDobie, Karen Louise January 2013 (has links)
Transmissible spongiform encephalopathies (TSEs) are a group of fatal, neurodegenerative diseases that can affect both humans and animals. TSEs can be sporadic, familial, or acquired diseases. The prion hypothesis states that a misfolded form of the host glycoprotein, PrPC, acts as the infectious agent in TSE disease. The misfolded form, PrPSc, is increased in β-sheet content, detergent insoluble and partially resistant to proteinase K (PK) digestion. Based on the prion hypothesis, most current post-mortem diagnostic tests rely on the presence of PrPSc as indicative of TSE disease. However, recently experimental cases of TSE disease have been identified where no PrPSc deposition is evident. One example of this is a murine transgenic model of Gerstmann Sträussler Scheinker (GSS) disease. GSS is a familial TSE disease, caused by a number of different mutations in human PrP including a point mutation from proline to leucine at residue 102. A murine model of GSS disease, produced through gene-targeting, contains the same point mutation at the equivalent residue, 101, in murine PrP. These mice do not develop spontaneous disease during their lifespan, but when inoculated intra-cerebrally with either human P102L GSS (101LL/GSS) or hamster 263K scrapie (101LL/263K); develop a clinical disease and vacuolar TSE-related pathology. Upon biochemical and immunohistochemical analysis, the brain tissues of these clinically ill mice contain little or no detectable PrPSc. However titration experiments have previously shown infectivity titres of 107-109IU/g of brain tissue. Standard PK digestion (at 37°C), NaPTA precipitation and isolation of PrPSc through detergent insolubility and differential centrifugation all confirmed the observation of little or no detectable PK-resistant PrP (PrP-res) in the 101LL/GSS and 101LL/263K brain tissues, despite the high levels of TSE infectivity. The presence of PrPSc and/or TSE infectivity in the spleen during disease pathogenesis is dependent upon TSE agent strain and host species. Previous studies utilising wild-type mice infected with ME7, have shown that the levels of infectivity observed in spleen tissue are 2- 3log10 lower than those observed in the brain tissue of the same mice. However, experiments conducted as part of this thesis showed that sub-passage of both the brain and spleen tissue from clinically ill 101LL/GSS and 101LL/263K mice into 101LL mice by intra-cerebral inoculation result in short incubation periods, indicating that infectivity levels were similarly high in both tissues. Biochemical analysis of the primary spleen tissue identified the presence of PrP-res, albeit at lower levels than those observed in wild-type spleens infected with a standard laboratory TSE strain, ME7 or 79A. However, the presence of PrP-res indicates that the spleen has a role in disease pathogenesis, which will require further investigation. Additionally, the spleen tissue maintains the discrepancy between PrP-res and TSE infectivity that is observed in the brain tissue of these models and further questions the prion hypothesis. As little or no PrP-res was detectable in the brain tissues of 101LL/GSS and 101LL/263K mice by standard biochemical and immunohistochemical techniques, it was hypothesised that an in vitro amplification technique, protein misfolding cyclic amplification (PMCA) could amplify PrPSc to detectable levels. A series of optimisation experiments were performed to produce a reliable positive control for amplification of mouse PrPSc from a standard laboratory mouse TSE strain, 79A or ME7, with a normal wild-type mouse brain homogenate substrate. While a wide range of technical and experimental conditions were investigated, consistent and reproducible amplification of mouse PrPSc was not achieved and therefore amplification of PrPSc from 101LL/GSS and 101LL/263K tissues could not be performed as interpretation of results would be complicated without the presence of a positive control. Previous research has shown that while other commercial assays, e.g. TeSeE (BioRad), identified tissues from these models as borderline positive or negative for TSE disease, one TSE diagnostic assay, the IDEXX HerdChek kit, that utilises the Seprion ligand, identified both the brain and spleen tissue from 101LL/GSS and 101LL/263K clinical mice as positive for TSE disease. In order to identify if TSE infectivity is associated with the target of the Seprion ligand, brain tissue homogenates from 101LL/GSS, 101LL/263K and a positive control wild-type/79A homogenate were depleted of the Seprion ligand target utilising a PAD-beads kit (Microsens Biotechnologies), which incorporates the Seprion ligand as the capture agent, in combination with magnetic beads. Upon inoculation, a single depletion of the homogenates produced no significant reduction in incubation period to clinical disease in either the depleted homogenates or the wash buffers produced, in comparison to a non-depleted brain homogenate. This result indicates that a single depletion with the Seprion ligand, did not remove enough of the aggregated protein to significantly alter the level of infectivity in the depleted homogenate and that any infectious agent, which was initially bound to the Seprion ligand due to non-specific interactions, was then released during the wash steps of the procedure. Proteomic differences between all components produced during a single depletion of an infected brain homogenate, wild-type/79A, or a normal uninfected brain homogenate were assessed to potentially identify the target of the Seprion ligand. In conclusion, these murine models of TSE disease, 101LL/GSS and 101LL/263K, which contain both high infectivity levels with little or no PrP-res in the brain tissue and similar high levels of infectivity with low levels of PrP-res in the spleen, questions the accepted correlation between levels of infectivity and PrP-res or PrPSc as proposed by the prion hypothesis. It is hypothesised that either an alternative form of PrP, which has not yet been identified is the infectious agent in these disease models, or that the TSE infectious agent is a component which associates with PrPSc rather than being PrPSc itself. The eventual identification of the infectious agent present in these unusual disease models will increase our understanding of these diseases, potentially offer improved diagnostics for infectivity, and perhaps identify novel therapeutic targets.
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Computational Study of Protein-Protein Interactions in Misfolded StatesBastidas, Oscar 01 January 2014 (has links)
Protein-protein interactions (PPI’s) play important roles in biological systems. In particular, intra-protein interactions help create and maintain correctly folded protein states and mutations that result in misfolded states may be associated with significant changes in PPI behavior. Six unrelated protein systems with known structure files, each consisting of a wild-type and mutant strain, were studied using the computational algorithm OpenContact©. OpenContact© is a simple tool that can be used to rapidly identify or map interactions “hot-spots” in a protein and was, consequently, used in this study as a starting point to examine the potential or possible role of PPI’s on the behavior of mutated, misfolded proteins. Specific results include the observations of single chain protein systems exhibiting mutant strains with significantly stronger inter-atomic interactions as well as a surprising gain of secondary structure in the mutant state. These observations stood in contrast to multi-chain systems (proteins with more than two constituent chains) that appeared to display stronger inter-atomic interactions for the wild-type strains. Results also indicated a potential classification scheme for intra-protein interaction behavior in mutated states based on several criteria. It is important to note, however, that observations on PPI behavior presented need to be verified across a greater number of systems than those studied here before any such trends can be concretely established.
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SOD1 Aggregation : Relevance of thermodynamic stabilityLang, Lisa January 2017 (has links)
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur. Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.
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Factors modifying the aggregation of atrophin-1 acting in cis and in transHinz, Justyna January 2012 (has links)
Ten polyQ (polyglutamine) diseases constitute a group of hereditary, neurodegenerative, lethal disorders, characterized by neuronal loss and motor and cognitive impairments. The only common molecular feature of polyQ disease-associated proteins is the homopolymeric polyglutamine repeat. The pathological expansion of polyQ tract invariably leads to protein misfolding and aggregation, resulting in formation of the fibrillar intraneuronal deposits (aggregates) of the disease protein. The polyQ-related cellular toxicity is currently attributed to early, small, soluble aggregate species (oligomers), whereas end-stage, fibrillar, insoluble aggregates are considered to be benign. In the complex cellular environment aggregation and toxicity of mutant polyQ proteins can be affected by both the sequences of the corresponding disease protein (factors acting in cis) and the cellular environment (factors acting in trans). Additionally, the nucleus has been suggested to be the primary site of toxicity in the polyQ-based neurodegeneration.
In this study, the dynamics and structure of nuclear and cytoplasmic inclusions were examined to determine the intrinsic and extrinsic factors influencing the cellular aggregation of atrophin-1, a protein implicated in the pathology of dentatorubral-pallidoluysian atrophy (DRPLA), a polyQ-based disease with complex clinical features. Dynamic imaging, combined with biochemical and biophysical approaches revealed a large heterogeneity in the dynamics of atrophin-1 within the nuclear inclusions compared with the compact and immobile cytoplasmic aggregates. At least two types of inclusions of polyQ-expanded atrophin-1 with different mobility of the molecular species and ability to exchange with the surrounding monomer pool coexist in the nucleus of the model cell system, neuroblastoma N2a cells. Furthermore, our novel cross-seeding approach which allows for monitoring of the architecture of the aggregate core directly in the cell revealed an evolution of the aggregate core of the polyQ-expanded ATN1 from one composed of the sequences flanking the polyQ domain at early aggregation phases to one dominated by the polyQ stretch in the later aggregation phase. Intriguingly, these changes in the aggregate core architecture of nuclear and cytoplasmic inclusions mirrored the changes in the protein dynamics and physico-chemical properties of the aggregates in the aggregation time course. 2D-gel analyses followed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) were used to detect alterations in the interaction partners of the pathological ATN1 variant compared to the non-pathological ATN1. Based on these results, we propose that the observed complexity in the dynamics of the nuclear inclusions provides a molecular explanation for the enhanced cellular toxicity of the nuclear aggregates in polyQ-based neurodegeneration. / Zehn Polyglutamin-basierte (polyQ) Erkrankungen bilden eine Gruppe von erblichen, neurogenerativen, letalen Krankheiten, die durch neuronalen Zellverlust und motorischen und kognitiven Störungen charakterisiert sind. Die mit polyQ Erkrankungen-assoziierten Proteine enthalten eine repetitive Abfolge der Aminosäure Glutamin (den polyQ-Bereich, der die einzige gemeinsame Sequenz aller polyQ Proteine ist). Durch die pathologische Verlängerung des PolyQ-Bereiches bekommen die polyQ Proteine eine Neigung zu aggregieren, und bilden damit unlösliche, fibrilläre Ablagerungen in Neuronen. Es wird vermutet, dass die sich anfangs bildenden kleinen löslichen Ablagerungsvorstufen (Oligomere) toxisch, und die später gebildeten, unlöslichen fibrillären Aggregate jedoch harmlos sind. Im zellulären Milieu werden Aggregations-Prozess und Toxizität durch die polyQ-flankierenden (benachbarten) Sequenzen des jeweiligen Proteins (in cis agierende Faktoren) und unterschiedliche zelluläre Proteine (in trans agierende Faktoren) beeinflusst. Außerdem kann die nukleare Lokalisation der polyQ Spezies mit verlängertem PolyQ-Bereich ihren toxischen Effekt erhöhen.
Die Verlängerung des polyQ-Bereiches im Protein Atrophin-1 (ATN1) über 49 Glutamine hinaus, verursacht Dentatorubro-Pallidoluysische Atrophie (DRPLA), eine progressive Erkrankung, die sich durch Muskelzuckungen, Epilepsie, Ataxie und Demenz äußern kann. In dieser Arbeit wurden die dynamischen Eigenschaften und die Struktur der nuklearen und zytoplasmatischen Aggregate systematisch untersucht, um die Faktoren, die das Aggregations-Verhältnis der Atrophin-1 in cis und in trans beeinflussen zu erkennen. Mittels des mit biochemischen und biophysikalischen Analysen kombinierten Dynamic Imaging, konnte gezeigt werden, dass Aggregate der mutierten ATN1 in vivo, im Säugetier-Zellen Model (Neuroblastoma N2a Zellen), sich von den frühen, löslichen zu später gebildeten unlöslichen Spezies entwickeln. Die Resultate der im Rahmen dieser Arbeit entwickelten Cross-Seeding Methode zeigen, dass das Aggregatcore der früheren Aggregate von den polyQ-Bereich flankierenden Sequenzen kontrolliert wurde, während die Transformation zu unlöslichen Aggregaten von dem expandierten polyQ-Bereich dominiert ist. Außerdem, wie die 2D-Gelelectrophorese und die MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) Analysen beweisen, beeinflusst die Länge des PolyQ-Bereiches die Interaktionen mit zellulären Proteinen. Wir haben auch festgestellt, dass das in nuklearen Aggregaten abgelagerte polyQ-expandierte ATN1 im Vergleich zu den zytoplasmatischen Ablagerungen eine erhöhte Mobilität aufwies. Mindestens zwei Aggregat-Typen mit unterschiedlichen Mobilitäten von mutierten ATN1 koexistieren im Zellkern der N2a Zellen, während im Gegensatz dazu das Protein in den kompakten zytoplasmatischen Aggregaten ausnahmslos immobil erscheint. Dies stellt eine molekulare Erklärung der erhöhten Toxizität der nuklearen ATN1-Aggregate dar.
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Characterization of folding and misfolding of the Tetrahymena thermophila group I ribozymeMitchell, David III 07 November 2013 (has links)
The functions of many cellular RNAs require that they fold into specific three-dimensional native structures, which typically involves arranging secondary structure elements and stabilizing the folded structure with tertiary contacts. However, RNA folding is inherently complex, as most RNAs fold along pathways containing multiple intermediates, including some misfolded intermediates that can accumulate and persist. Our understanding of the origins and structures of misfolded forms and the resolution of misfolding remains limited. Here, we investigate folding of the Tetrahymena intron, an extensively studied RNA folding model system since its initial discovery decades ago. The ribozyme variant predominantly misfolds, and slow refolding to the native state requires extensive structural disruption. Paradoxically, the misfolded conformation contains extensive native structure and lacks incorrect secondary and tertiary contacts despite requiring displacement of a native helix, termed P3, with incorrect secondary structure to misfold. We propose a model for a new origin of RNA misfolding to resolve this paradox, wherein misfolded ribozyme contains within its core incorrect arrangement of two single-stranded segments, i.e. altered topology. This model predicts a requirement for P3 disruption to exchange the misfolded and native topologies. We mutated P3 to modulate its stability and used the ribozyme's catalytic activity to show that P3 is disrupted during the refolding transition. Furthermore, we demonstrate that unfolding of the peripheral tertiary contacts precedes disruption of P3 to allow the necessary structural transitions. We then explored the influence of topology on the pathways leading to the misfolded and native states. Our results suggest that P3 exists in an earlier pathway intermediate that resembles the misfolded conformation, and that P3 unfolds to allow a small yet significant fraction of ribozyme to avoid misfolding. Despite being on a path to misfolding, the decision to misfold depends upon the probability of disrupting P3 and exchanging topology at this intermediate. Additionally, we show that having a stable P3 in the unfolded ribozyme allows almost complete avoidance of misfolding. Together, these studies lead to a physical model for folding and misfolding of a large RNA that is unprecedented in its scope and detail. / text
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Mechanistic insights into alpha-Synuclein neuronal toxicity: misfolding, serine phosphorylation and interactions with Rab GTPasesYin, Guowei 22 November 2013 (has links)
No description available.
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