| Supervisor Organisation | PhD Awarding Entity: | phd location |
|---|---|---|
ALBA & CNB-CSIC | Universitat Autònoma de Barcelona | ALBA Synchrotron, Barcelona, Spain |
Research Focus
The project will focus on locating specific viral proteins within infected cells by Hepatitis C virus (HCV) and West Nile virus using a cryo correlative imaging approach, building on previous studies performed at the Alba synchrotron to investigate by cryo Soft X-ray Tomograghy (cryo-SXT) the cell ultrastructural changes caused by HCV infection.
By establishing protocols and correlative workflows using the cryo 3D structured illumination microscope currently in development (cryo-3D-SIM), specific viral proteins conveniently tagged will be imaged. Afterwards, cryo Soft X-ray Tomograghy will allow revealing the ultrastructure of the infected cells in close-to-native conditions, therefore putting in context the protein location within the cellular environment.
The resolution of cryo-3D-SIM is 2X better than conventional visible light fluorescence microscopy, thus narrowing the resolution gap and allowing more accurate correlation between the two imaging techniques.
Methodology
Cryo-soft X-ray tomography (cryo-SXT) is the only imaging technology that can provide nanoscale 3D information from cryo-preserved, unstained whole cells. This technique provides unique capabilities to capture high resolution (30nm) images of the cellular architecture maintaining the native state of the samples, enabling investigating the viral replication in unprecedented detail.
Cryo-3D-SIM allows fast 3D imaging of volumetric specimens at resolutions beyond those achievable with the confocal microscope, making it an excellent complement to SXT at cryogenic temperatures, matching its penetration depth and detecting fluorescent molecules with wavelength-dependent resolutions of up to 150nm, therefore allowing correlation with the imaged 3D ultrastructure.
This Correlative Light X-ray Tomography approach (CLXT) enables new insights into viral infection by providing complementary morphological, specific localization of proteins and chemical information beyond what is achievable through the use of any individual technique alone. CLXT can provide state-of-art information for viral infection research.
Aim 1
Cryo-SXT to study the morphology of membranous rearrangements during the infection of West Nile virus (WNV)
Evaluate reversion of morphological changes after treatment with antiviral drugs by cryo-SXT and deep UV fluorescence microspectroscopy (secondment at SOLEIL)
Aim 2
Characterization and optimization of the cryo-3D- SIM to establish workflows to locate HCV ns5a in the native cellular environment by correlative cryo-3D-SIM and cryo-SXT on the same infected cells.
Report describing the results of testing cryo-cell preparation on micropatterned grids (project deliverable).
Aim 3
Locate ns5a-cherry and sigma-GFP in HCV replicated cells by cryo-3D-SIM/SXT to understand their role in the formation of the viral replication sites, virion morphogenesis and viral egress
Pictures Attached
Role/Focus of PhD:
ALBA is the Spanish synchrotron light source, is a large infrastructure of excellence with international teams representing a broad range of disciplines, with first class core technologies to support the research projects, operating ten beamlines, complementary facilities, and an Electron Microscopy Center. Currently constructing multiple more beamlines, expanding the Electron Microscopy Center, and integrating advanced data analytics, it sums up a wide range of infrastructures geared toward finding solutions to societal challenges.
Our highly motivated staff works in a multidisciplinary work environment with an atmosphere formed by lived diversity, inclusion and respect for our colleagues. Located close to Barcelona in a natural park and well connected by car and public transportation, with excellent connectivity to the world and an employer who understands the importance of life-work balance.
We are interested in hiring a highly motivated student with a background in cell biology, biochemistry, biophysics, bioengineering, or related field, having a strong interest in fluorescence microscopy, image analysis and structural biology. The ideal candidate has a strong interest in understanding biological questions at a cellular level and enjoys developing new experimental approaches.
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Application Deadline: 18th August 2023 (Closed)