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EnglishIn this work, I applied advanced microscopy techniques to study the molecular mechanisms of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease. The toxicity of α-syn is likely related to its interaction with the neuronal membrane. It is known that α-syn is present in both the cytosolic and extracellular space, and it has been demonstrated that both components have a role in neurodegenerative processes. To study the effect α-syn on lipid membranes, I used two complex lipid mixtures, preparing lipid bilayers that modeled the external and internal leaflets of the neuronal membrane. Exploiting this original approach, I investigated how α-syn may act on membrane structure and stability when it presents in the cytosol or the extracellular space. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM). I used atomic force microscopy (AFM) in different acquisition modes, and I complemented the results with QCM-D, and vesicle leakage experiments. In a second work, I characterized amyloid aggregates formed by α-syn, applying a correlative technique based on the coupling between AFM and stimulated emission depletion (STED) microscopy. In particular, I focused on the definition of the eventual influence that the presence of fluorescent dye molecules can have on the aggregation process of the peptide. I demonstrated that the use of a site-specific labeling method is fundamental to avoid modification of the aggregation process.
| Titolo della tesi: | Advanced microscopy techniques applied to in-vitro study of Alpha synuclein aggregation and toxicity mechanism | |
| Autori: | ||
| Data di discussione: | 4-mag-2022 | |
| Abstract: | In this work, I applied advanced microscopy techniques to study the molecular mechanisms of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease. The toxicity of α-syn is likely related to its interaction with the neuronal membrane. It is known that α-syn is present in both the cytosolic and extracellular space, and it has been demonstrated that both components have a role in neurodegenerative processes. To study the effect α-syn on lipid membranes, I used two complex lipid mixtures, preparing lipid bilayers that modeled the external and internal leaflets of the neuronal membrane. Exploiting this original approach, I investigated how α-syn may act on membrane structure and stability when it presents in the cytosol or the extracellular space. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM). I used atomic force microscopy (AFM) in different acquisition modes, and I complemented the results with QCM-D, and vesicle leakage experiments. In a second work, I characterized amyloid aggregates formed by α-syn, applying a correlative technique based on the coupling between AFM and stimulated emission depletion (STED) microscopy. In particular, I focused on the definition of the eventual influence that the presence of fluorescent dye molecules can have on the aggregation process of the peptide. I demonstrated that the use of a site-specific labeling method is fundamental to avoid modification of the aggregation process. | |
| Handle: | http://hdl.handle.net/11567/1080907 | |
| Appare nelle tipologie: | Tesi di dottorato |
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|---|---|---|---|
| phdunige_4619376.pdf | description of experimental results | Tesi di dottorato | Embargo: 04/05/2023 Richiedi una copia |
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