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Dr E W Hewitt,
Prof S E Radford,
Dr P Actis
Applications accepted all year round
Self-Funded PhD Students Only
Leeds
United Kingdom
Biochemistry
Biophysics
Cell Biology
Molecular Biology
Nanotechnology
Neuroscience
Biological Sciences
Structural Biology
About the Project
Intraneuronal inclusions known as Lewy bodies are a hallmark of Parkinson’s disease. Amyloid fibrils formed by the misfolding of alpha-synuclein are the principal component of Lewy Bodies. However, the role of these fibrils and their oligomeric assembly intermediates in neuronal death is poorly understood. This is because alpha-synuclein misfolds into amyloid fibrils in the cytoplasm, whereas experimental studies have typically involved adding fibrils and oligomers to the cell culture medium. Although this enables biophysical characterisation of the fibrils and oligomers before addition to the cells, their access to the cytoplasm is limited.
This project will use a single molecule nanoinjection platform to deliver alpha-synuclein fibrils and oligomers into the cytoplasm of neurons. Nanoinjection uses quartz needles (≤50nm pore diameter), known as nanopipettes, to deliver molecules into cells. The nanopipettes incorporate electrodes and application of a voltage drives the transport of molecules through the pore. When a protein passes through the nanopipette’s pore there is a corresponding disruption in the ion flow, thus the number of proteins delivered into a cell can be quantified. Thus this project will not only determine whether α-synuclein fibrils or oligomers are toxic in the cytoplasm, but for the first time it will quantify the number of each required to kill a neuron.
Fibrils and oligomers will be made from recombinant α-synuclein, charactersied using an array of biophysical techniques, and nanoinjected into neuronal cell lines and primary neurons. To inject cells the nanopipette will be used as a probe for a scanning ion conductance microscope, which will generate a topographical map of the target cell. This information is used to insert the nanopipette into the cell at a pre-defined depth and location. Application of a voltage will drive the delivery of the fibrils or oligomers into the cell. By monitoring the corresponding disruptions in ion flow, a defined number of oligomers or fibrils will be delivered. The cellular effects of the nanoinjected fibrils and oligomers on cell stress and viability will then be analysed using microscopy-based assays.
This PhD will be a collaborative project in the Astbury Centre for Structural Biology and Bragg Centre at the University of Leeds between the research groups of Dr Eric Hewitt, Dr Paolo Actis and Professor Sheena Radford FRS OBE. The project will provide training in interdisciplinary science, with the PhD student developing expertise at the interface of the nanotechnology, molecular cell biology and structural biology.
Eligibility
Applicants should normally have at least a first class or an upper second class British Bachelors Honours degree (or equivalent) in an appropriate discipline, along with appropriate research experience and/or a Masters degree.
Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The Faculty of Biological Sciences minimum requirements in IELTS and TOEFL tests are:
- British Council IELTS - score of 6.0 overall, with no element less than 5.5
- TOEFL iBT - overall score of 87 with the listening and reading element no less than 20, writing element no less than 21 and the speaking element no less than 22.
How to apply
Please contact Dr Eric Hewitt (E.W.Hewitt@leeds.ac.uk) in the first instance if you have any questions about the project.
To apply for this project applicants should complete an online application form and attach the following documentation to support their application.
- a full academic CV
- degree certificate and transcripts of marks
- Evidence that you meet the University's minimum English language requirements (if applicable)
- Evidence of funding
To help us identify that you are applying for this project please ensure you provide the following information on your application form;
- Select PhD in Biological Sciences as your programme of study
- Give the full project title and name the supervisors listed in this advert
Funding Notes
Self-funded students: International or domestic self-funded or scholarship/fellowship PhD students are always welcome to apply. International students must have a good command of both written and spoken English. In addition to University fees, laboratory fees will be required if you are self-funded. Applications can be made throughout the year.
References
Some of our recent papers:
Jakhria et al (2014). β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes. J Biol Chem 289: 35781-94
Tipping et al (2015). pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers. Proc Natl Acad Sci U S A. 112: 5691-6
Iadanza et al (2018). The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism.Nat Commun. 9: 4517.
Iadanza et al (2018). A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. 19:755-773.
Karamanos et al (2019). Structural mapping of oligomeric intermediates in an amyloid assembly pathway. Elife. 8: e46574.
Chau et al (2020). Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore, Nano Lett. 20, 5553−5561
Chau et al (2020) Methods for protein delivery into cells: from current approaches to future perspectives. Biochem Soc Trans 48:357-365.
Jakhria et al (2014). β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes. J Biol Chem 289: 35781-94
Tipping et al (2015). pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers. Proc Natl Acad Sci U S A. 112: 5691-6
Iadanza et al (2018). The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism.Nat Commun. 9: 4517.
Iadanza et al (2018). A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. 19:755-773.
Karamanos et al (2019). Structural mapping of oligomeric intermediates in an amyloid assembly pathway. Elife. 8: e46574.
Chau et al (2020). Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore, Nano Lett. 20, 5553−5561
Chau et al (2020) Methods for protein delivery into cells: from current approaches to future perspectives. Biochem Soc Trans 48:357-365.
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