Investigating the function of mechanotransduction pathways in the zebrafish tailbud
Supervisor: Dr. Ben Steventon Assistant Professor Department of Genetics and co-supervisor: Dr. Kate McDole MRC Investigator MRC Laboratory of Molecular Biology Cell Biology Division
Project Description:
During vertebrate body axis elongation, populations of progenitor cells in the posterior-most tailbud region of the embryo continually make choices about which cell type they should differentiate into. These decisions must be carefully balanced against the rates of expansion of anterior structures such as the spinal cord, notochord and somites so that a well-proportioned body axis is generated. We hypothesise that anterior tissue expansion generates force production in the tailbud that is sensed by the progenitors to regulate their rates of differentiation and movement. We have preliminary data showing the activation of key mechano-transduction pathways within the tailbud, and mutant zebrafish lines where regulators of this pathway are disrupted. The project will characterise these mutants using light-sheet imaging. We will collaborate with the lab of Kate McDole to apply their machine learning approaches to track and analyse cell behaviours, comparing wild-type and mutant conditions.
Specific aim:
Characterising the consequence of disrupted mechanotransduction on the cell movements in the zebrafish tailbud using light-sheet microscopy and 4D image analysis.
- Duration: 6 - 8 weeks
- Essential knowledge, skills and attributes that would be advantageous: Light-sheet imaging, machine learning, image analysis, zebrafish genetics
Supporting information:
McLaren, S., and Steventon, B. (2021). Anterior expansion and posterior addition to the notochord mechanically coordinate embryo axis elongation. Development. 2021 Sep 15;148(18):dev199459
Attardi, A., Fulton, T., Florescu, F., Shah, G., Muresan, L., Lenz, M., Lancaster, C., Huisken, J., van Oudenaarden., A and Steventon, B (2018). Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics Development: dev.166728 doi: 10.1242/dev.166728
A single-molecule perspective of mechanical signalling during stem cell differentiation
Supervisor: Dr Srinjan Basu Wellcome-MRC Cambridge Stem Cell Institute
Project description:
During tissue development, cells encounter and respond to forces (1). These forces can be transduced through the cell surface and cell adhesions from extracellular cues, or these forces can arise as a result of shape changes and cytoskeletal reorganisation due to alterations in cell function such as cell differentiation. Regardless of the origin of the force, the cytoskeleton propagates these stresses to the nucleus through a process called nuclear mechanotransduction (2). Nuclear mechanotransduction affects the molecular composition and physical arrangement (3, 4) of the nucleus, the structural organization and apico-basal polarity of the nuclear lamina (5), intracellular signalling (6, 7), and molecular traffic across the nuclear envelope (8).
My lab has recently shown with Dr Kevin Chalut that mechanical signals are required for the differentiation of pluripotent cells, cells that emerge in the mammalian embryo with the ability to generate all the lineages that form the organism. Specifically, inactivation of the mechanical transducer Nesprin blocks differentiation of pluripotent cells into neuroectoderm lineages by altering 3D genome folding within the nucleus. Nesprin connects the nuclear envelope to the actin cytoskeleton to allow nuclear mechanotransduction but how it alters the 3D genome remains a mystery.
Understanding what governs this process will shed light on how mechanical forces influence tissue growth or disease, but also on how to unlock the potential of pluripotent cells for regenerative medicine.
The aim of this project is therefore to provide insight into how the mechanical sensor Nesprin alters 3D genome folding during pluripotent cell differentiation.
Objective 1: Based on preliminary findings that Nesprin inactivation changes the 3D genome at a neuroectoderm gene called Sox1, we will determine how Nesprin alters the 3D genome at 3 genes required for neuroectoderm differentiation. To achieve this, we will use multi-colour 3D DNA FISH, an imaging approach we recently established in the lab (9).
Objective 2: Based on preliminary findings that Nesprin inactivation alters the levels of a genome mark called H3K27me3, we will determine whether Nesprin alters the binding of two proteins responsible for deposition/removal of this mark (MLL4, Polycomb). To achieve this, we will use live-cell single-molecule tracking, an imaging approach we recently established in the lab (9).
During the project, the student will also gain literature review skills through journal clubs, presentation skills through a final talk to the lab. They will receive technical training from a postdoc supervisor Dr Agsu:
- 2D pluripotent stem cell culture;
- Super-resolution imaging;
- Computational analysis.
Contingency plans: My lab has established cell lines, stem cell differentiation and imaging pipelines. If DNA FISH fails, we have pipelines for labelling DNA using inactive dCas9 proteins. If chromatin binding kinetics fail, we have expertise in orthogonal approaches e.g. ChIP-seq.
Outlook: We plan to use these approaches to link other mechanical sensors (e.g. Emerin) to enhancer-promoter communication, transcription and cell fate in future experiments. Mutations in mechanical sensors lead to premature ageing and neuromuscular diseases (e.g. Emery–Dreifuss muscular dystrophy). We therefore have collaborations (Jim Holaska, Mark Kotter) to translate our findings to disease.
- Duration: 8 weeks
- No essential skills needed but students will be prioritised if they have:
- knowledge of stem cell/developmental biology, mechanical signalling or single-molecule biophysics
- cell culture, imaging and data presentation skills
Supporting information:
1. E. Hannezo, C. P. Heisenberg, Mechanochemical Feedback Loops in Development and Disease. Cell 178, 12-25 (2019).
2. M. S. Hamouda, C. Labouesse, K. J. Chalut, Nuclear mechanotransduction in stem cells. Curr Opin Cell Biol 64, 97-104 (2020).
3. S. Talwar, A. Kumar, M. Rao, G. I. Menon, G. V. Shivashankar, Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells. Biophys J 104, 553-564 (2013).
4. S. Biedzinski et al., Microtubules deform the nucleus and force chromatin reorganization during early differentiation of human hematopoietic stem cells. bioRxiv, 763326 (2019).
5. T. O. Ihalainen et al., Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension. Nat Mater 14, 1252-1261 (2015).
6. G. Uzer et al., Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates betacatenin nuclear access. J Biomech 74, 32-40 (2018).
7. Q. Zhang et al., N-terminal nesprin-2 variants regulate beta-catenin signalling. Exp Cell Res 345, 168-179 (2016).
8. E. Kassianidou, J. Kalita, R. Y. H. Lim, The role of nucleocytoplasmic transport in mechanotransduction. Exp Cell Res 377, 86-93 (2019).
9. S. Basu et al., Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics. bioRxiv, 2020.2004.2003.003178 (2020).
Mechanical interactions between multiple tissues in a developing chick embryo
Supervisor: Dr. Fengzhu Xiong Wellcome Trust / CRUK Gurdon Institute
Project Description
A developing embryo is like a dynamic jigsaw puzzle where distinct growing pieces (tissues) must fit closely to ensure an overall correct structure. What mechanisms coordinate the sizes and shapes of different tissues as they develop their own identities are unclear. Our recent work with chicken embryos shows that tissues in the elongating body axis shape each other through mechanical forces and such interactions may enable tissues to detect / measure their morphogenetic progress. In this way, morphogenesis can be more robust and coordinated. In this summer the student will have the opportunity to uncover novel connections between tissues that relay mechanical forces, to observe the cellular dynamics responsible for and responding to mechanical changes, and to assess the underlying molecular genetic mechanisms. In the interdisciplinary environment of the lab, the student will also have the opportunity to learn embryology, modeling and tool development.
Specific aims:
1. Quantifying tissue deformation and cell dynamics of normal and mechanically loaded embryos;
2. Constructing a model to predict the strengths and patterns of tissue connections;
3. Testing the predictions in live avian embryos with mechanical and surgical tools.
- Duration: 8 weeks
- Essential knowledge, skills and attributes: Biologists, engineers and physicists with an interest in embryo development are all encouraged to apply.
Supporting information:
Lab info: https://www.gurdon.cam.ac.uk/people/fengzhu-xiong/; https://scholar.harvard.edu/fengzhu_xiong/research;
Note: This project will be under CPB support for 8 weeks. The host lab will support further flexible extensions should the student or project require.
Polymer characterization for whole-brain Imaging
Supervisor: Dr. Elizabeth Barsotti Physiology, Development and Neuroscience and co-supervisor: Prof. Albert Cardona Physiology, Development and Neuroscience
Project Description
We are seeking a student interested in highly collaborative and interdisciplinary research for characterizing the composition of a polymer film for use in whole-brain imaging. During this studentship, the student will learn transmission electron microscopy and a variety of spectroscopy techniques. The student will work within an interdisciplinary team of biologists, engineers, and materials scientists to determine the chemical composition of an unknown polymer and identify possible methods for synthesizing it at the bench scale. The student’s work will have real world implications for improving electron microscopy protocols for imaging the entire brains of vertebrates.
Imaging the human brain in three dimensions at nanometre resolution would resolve the structure of every neuron and synapse. No such map, or connectome, for the human brain exists, but if it did, it would impact every aspect of life as we know it. For example, pinpointing the neural networks responsible for consciousness would revolutionize artificial intelligence. Likewise, understanding the recurrency of the hippocampus would reveal how memories are made and stored, leading to insights into neurodegenerative diseases. Consequently, the human connectome is the Holy Grail of neuroscience. However, current electron microscope sample preparation techniques are too expensive to realize it.
To image the brain at synaptic resolution, it would need to be sliced into 2mmx3mmx40nm sections. Each of these sections needs to be placed onto a slot supported by a mechanically strong, electron-transparent, radiation-resistant film. Due to the robustness of imaging and sample preparation protocols for brains, typical films used on TEM grids, such as formvar, are unusable because they are too fragile. For this reason, a company, Luxel, has developed a proprietary film. So far, the Luxel film has been successfully used to image the brains of invertebrates and very small (i.e., on the order of 1 cubic millimetre) segments of vertebrate brains. Because the brain volumes are small, these projects only require a few thousand film-coated slots.
The human brain, which has a volume of approximately 1.3 litres, would require 5.5 billion film-coated slots. Film-coated slots purchased commercially are approximately £5/slot. 5.5 billion slots would cost £30,000,000,000! Considering the cost of the microscope is only £50,000, the cost of the slots is exorbitant and unreasonable, stemming solely from the fact that Luxel monopolises their production. A cost of £30,000,000,000 means that if the human brain were ever to be imaged, it could only be achieved by the richest lab in the world. Thus, not only is film-coated slot production monopolized by a single company, but also whole-brain imaging is monopolized by only the wealthiest laboratories.
We seek to democratize whole-brain imaging so that not only can our lab afford it but also other labs. In this effort, we have procured a polyimide film that can support 2mmx3mm slots. The composition of this film is unknown. We seek to determine its composition using a combination of spectroscopy techniques. Once the composition is known, we will attempt to synthesize it.
- Duration: 8 weeks
- Essential knowledge, skills and attributes: Enthusiasm for and a willingness to learn a variety of materials characterization techniques is essential but no prior experience with any is required. An understanding and/or interest in synthetic chemistry or polymeric materials would be beneficial. The student will learn or be exposed to a multitude of imaging and characterization techniques including transmission electron microscopy, microtomy, energy dispersive X-ray spectroscopy, fourier transform infrared spectroscopy, and nuclear magnetic resonance.
Supporting information:
High-throughput transmission electron microscopy with automated serial sectioning | bioRxivA petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy | Nature Communications
Thin film coating for brain sample support
Supervisor: Dr. Elizabeth Barsotti Physiology, Development and Neuroscience and co-supervisor: Prof. Albert Cardona Physiology, Development and Neuroscience
Project Description
We are seeking a student interested in highly collaborative and interdisciplinary research for developing new techniques to prepare polymer substrates for mounting brain tissue for three-dimensional imaging. During this studentship, the student will learn transmission electron microscopy, microtomy, and mechanical engineering design. The student will work within an interdisciplinary team of biologists, engineers, and materials scientists. The student’s work will have real world implications for improving electron microscopy protocols for imaging the entire brains of vertebrates.
Imaging the human brain in three dimensions at nanometre resolution would resolve the structure of every neuron and synapse. No such map, or connectome, for the human brain exists, but if it did, it would impact every aspect of life as we know it. For example, pinpointing the neural networks responsible for consciousness would revolutionize artificial intelligence. Likewise, understanding the recurrency of the hippocampus would reveal how memories are made and stored, leading to insights into neurodegenerative diseases. Consequently, the human connectome is the Holy Grail of neuroscience. However, current electron microscope sample preparation techniques are too expensive to realize it.
To image the brain at synaptic resolution, it would need to be sliced into 2mmx3mmx40nm sections. Each of these sections needs to be placed onto a slot supported by a mechanically strong, electron-transparent, radiation-resistant film. Due to the robustness of imaging and sample preparation protocols for brains, typical films used on TEM grids, such as formvar, are unusable because they are too fragile. For this reason, a company, Luxel, has developed a proprietary film. So far, the Luxel film has been successfully used to image the brains of invertebrates and very small (i.e., on the order of 1 cubic millimetre) segments of vertebrate brains. Because the brain volumes are small, these projects only require a few thousand film-coated slots.
The human brain, which has a volume of approximately 1.3 litres, would require 5.5 billion film-coated slots. Film-coated slots purchased commercially are approximately £5/slot. 5.5 billion slots would cost £30,000,000,000! Considering the cost of the microscope is only £50,000, the cost of the slots is exorbitant and unreasonable, stemming solely from the fact that Luxel monopolises their production. A cost of £30,000,000,000 means that if the human brain were ever to be imaged, it could only be achieved by the richest lab in the world. Thus, not only is film-coated slot production monopolized by a single company, but also whole-brain imaging is monopolized by only the wealthiest laboratories.
We seek to democratize whole-brain imaging so that not only can our lab afford it but also other labs. In this effort, we will develop our own cost-effective method of coating slots with polymer films.
- Duration: 8 weeks
- Essential knowledge, skills and attributes: An understanding and/or interest in synthetic chemistry or polymeric materials is required. A passion for mechanical design is necessary but no previous experience is required. The student will learn or be exposed to a variety of imaging and characterization techniques including transmission electron microscopy and microtomy.
Supporting information:
High-throughput transmission electron microscopy with automated serial sectioning | bioRxiv
Spray coating methods for polymer solar cells fabrication: A review - ScienceDirect
The mechanics of spin coating of polymer films (scitation.org)
Comparative analysis of polymers for whole-brain imaging
Supervisor: Dr. Elizabeth Barsotti Physiology, Development and Neuroscience and co-supervisor: Prof. Albert Cardona Physiology, Development and Neuroscience
Project Description
We are seeking a student interested in highly collaborative and interdisciplinary research for testing and comparing polymer substrates for use in whole-brain imaging. During this studentship, the student will learn transmission electron microscopy and microtomy. The student will work within an interdisciplinary team of biologists and engineers to assess and compare a variety of polymers for use in whole-brain imaging. The student’s work will have real world implications for improving electron microscopy protocols for imaging the entire brains of vertebrates.
Imaging the human brain in three dimensions at nanometre resolution would resolve the structure of every neuron and synapse. No such map, or connectome, for the human brain exists, but if it did, it would impact every aspect of life as we know it. For example, pinpointing the neural networks responsible for consciousness would revolutionize artificial intelligence. Likewise, understanding the recurrency of the hippocampus would reveal how memories are made and stored, leading to insights into neurodegenerative diseases. Consequently, the human connectome is the Holy Grail of neuroscience. However, current electron microscope sample preparation techniques are too expensive to realize it.
To image the brain at synaptic resolution, it would need to be sliced into 2mmx3mmx40nm sections. Each of these sections needs to be placed onto a slot supported by a mechanically strong, electron-transparent, radiation-resistant film. Due to the robustness of imaging and sample preparation protocols for brains, typical films used on TEM grids, such as formvar, are unusable because they are too fragile. For this reason, a company, Luxel, has developed a proprietary film. So far, the Luxel film has been successfully used to image the brains of invertebrates and very small (i.e., on the order of 1 cubic millimetre) segments of vertebrate brains. Because the brain volumes are small, these projects only require a few thousand film-coated slots.
The human brain, which has a volume of approximately 1.3 litres, would require 5.5 billion film-coated slots. Film-coated slots purchased commercially are approximately £5/slot. 5.5 billion slots would cost £30,000,000,000! Considering the cost of the microscope is only £50,000, the cost of the slots is exorbitant and unreasonable, stemming solely from the fact that Luxel monopolises their production. A cost of £30,000,000,000 means that if the human brain were ever to be imaged, it could only be achieved by the richest lab in the world. Thus, not only is film-coated slot production monopolized by a single company, but also whole-brain imaging is monopolized by only the wealthiest laboratories.
We seek to democratize whole-brain imaging so that not only can our lab afford it but also other labs. In this effort, we will test a variety of cheaper polymers to find one that can be used to prepare samples cost-effectively.
- Duration: 8 weeks
- Essential knowledge, skills and attributes: Enthusiasm for and a willingness to learn electron microscopy is essential but no prior experience is required. An understanding and/or interest in synthetic chemistry or polymeric materials would be beneficial.
Supporting information:
High-throughput transmission electron microscopy with automated serial sectioning | bioRxiv
Characterisation of the biophysical properties of nanobodies obtained from integrating discovery of in silico design and in vitro screening
Supervisor: Dr Pietro Sormanni Yusuf Hamied Department of Chemistry and co-supervisor: Dr Xing Xu
Project Description
Nanobodies are powerful tools in research, diagnosis, and therapeutics. They derive from the heavy-chain antibodies produced by camelids and few other animals. The conventional way to discover nanobodies requires the immunisation of camelids and a series of screening steps to isolate those that bind the target of interest. The whole process is labour intensive and time consuming. Thanks to recent advances, we are now able to design the complementary-determining regions (CDRs) of nanobodies fully in silico to target specific epitopes of interest. The designed CDR fragments can be readily grafted to the scaffold of natural nanobodies to generate nanobodies binding their targets within high nanomolar range affinity values (see Ref). However, comparing to the approved therapeutic antibodies, which have low nanomolar or even picomolar binding affinity, the designed nanobodies are not optimal for applications in the clinic. Further optimisations, such as affinity maturation, of these nanobodies are required.
Therefore, we sought to build a pipeline which combines in silico design and in vitro library screening to achieve a platform for the fast and reliable discovery of nanobodies binding to predetermined epitopes of interest.
An essential component of the development of this platform consists in characterising the biophysical properties of nanobodies selected from the screening, which will provide the crucial evidence showing that our approach for epitope-specific nanobody discovery is working properly. The biophysical properties need to be characterised include the binding affinity of the selected nanobodies to the target antigen, thermostability, stability during storage in different buffering conditions, etc. Therefore, the project will be focused on obtaining high purity nanobodies and characterising their biophysical properties.
- Duration: 8 weeks
- Essential knowledge, skills and attributes: Some previous exposure to molecular biology techniques, and protein production and purification is beneficial. Biophysical laboratory skills are also a plus
Supporting information
Link Group website: https://www-sormanni.ch.cam.ac.uk/
Reference: Rangel, Mauricio Aguilar, et al. "Fragment-based computational design of antibodies targeting structured epitopes." Science Advances (2022)
Identifying highly fluctuating methylation sites in the human genome for lineage tracing applications
Supervisor: Dr. Jamie Blundell Early Cancer Institute, Department of Oncology and co-supervisor: Dr. Diana Fusco Department of Physics
Project Description
Improving early detection of cancers requires the ability to sensitively detect, and quantify the growth rates of, clonal expansions in human tissue samples (e.g. in blood or oesophagus). Genomic techniques based on sequencing the bases of DNA and their modifications offer an attractive approach for doing this.
One such approach involves using sequencing technology to measure the status of methylation marks on DNA (methyl groups added to a cytosine in a CpG context). At some sites in the genome the methylation status (methylated / unmethylated) fluctuates randomly on short timescales. These fluctuating “epimutations” act like a molecular clock which can be used to lineage trace and date clonal expansions that occurred in tissue. sites
The first step in developing such a technology is to robustly quantify the methylation mutation rate in human tissues (blood in this case) and then identify regions where these methylation mutation rates are high. In this project the student will analyse whole genome methylation sequencing datasets generated from human blood samples. The student will learn how to analyse large genomic data sets, how to develop computational approaches to correct sequencing errors and how to interact and plot quantitative data.
The central aim of this project is to use whole genome sequencing data from human blood samples to identify regions of the human genome where the methylation status (a chemical mark added to Cs in DNA) fluctuates. The key aims are:
- Use whole genome bisulphite sequencing data from the blood of 30 healthy individuals (which has been generated already) to identify CpG sites which are highly variable across people
- Use whole genome data generated from serial blood samples collected from the same individuals over a 10-year period to understand how often these highly variable sites are fluctuating within a person through time.
- Duration: 8 weeks This project is expected to run for most of the summer but exact dates are flexible
- Essential knowledge, skills and attributes: It would be advantageous for the student to have some programming skills in python and some mathematical background (e.g. A-level of undergraduate courses in maths).
Investigating biomolecular condensates underlying segmented viral genome replication
Supervisor: Dr Alex Borodavka Department of Biochemistry and co-supervisor Julia Acker Department of Biochemistry
Project Description
We have recently discovered that rotaviruses, a large and evolving class of multi-segmented RNA viruses, assemble their eleven distinct RNA transcript and replicate their genomes within multi-component biomolecular condensates. Recently, we have successfully reconstituted a two-component viral condensate system using recombinantly expressed viral proteins NSP5 and NSP2, and quantitively characterised its phase boundaries by employing high-throughput microfluidics approach. We have shown that in vivo, such condensates accumulate cognate viraltranscripts, as well as the viral RNA-dependent RNA polymerase. We have shown that liquid-like viral condensates change their material properties during infection, resulting in their resistance to aliphatic diol treatments. This, combined with the established in vitro and in vivo tools available in Borodavka’s group, makes RVs an ideal model system to gain insights into the role of biomolecular condensates in viral replication. We will investigate several key properties of viral condensates (e.g., RNA selectivity, liquid-gel/solid transitions), as well as RNA conformational dynamics within such condensates, in order to dissect the mechanistic role of condensate formation in supporting viral genome replication. These findings will be used to identify new targets for future therapeutic intervention.
The key aims of the proposal are:
i) To reconstitute viral biomolecular condensates in vitro, and
ii) to explore their molecular selectivity and conformational dynamics of RNAs within NSP2/NSP5 droplets using fluorescently labelled RNA stem-loops.
- Duration: 8 weeks
- Desired knowledge, skills and attributes: some experience in recombinant protein production and purification or experience with fluorescence microscopic techniques.
- More information: https://www.borodavkalab.org/ or contact Dr Alex Borodavka
Introducing physical factors for bioengineering human embryonic lung organoids
Supervisor: Dr Emma Rawlins Gurdon Institute and PDN and co-supervisor: Dr Hannah Woodcock
Project Description
During human lung development, airway epithelial morphogenesis is driven by branching of the distal-most tip which comprises SOX9+ multipotent progenitor cells. As their descendants exit the tip into the stalk region of the airway, they initiate airway epithelial differentiation. By 17 post-conception weeks (pcw) the complete structure of the human airway tree has been laid down.
The adult airway epithelium is complex and is composed of multiple distinct mature cell types: basal, secretory, mucin-rich goblet cells, multi-ciliated cells, and rarer cell types such as pulmonary neuroendocrine cells (NE), tuft cells, and ionocytes. It is not clear how fetal tip progenitor cells differentiate into all these different airway cell fates. Currently,our understanding of the lineage hierarchy and mechanisms regulating human airway development is limited and inferred indirectly through murine models or fate trajectories from singlecell RNA sequencing data of human fetal lungs.
One of the aims of the Rawlins group is to define epithelial progenitor-airway lineage hierarchy in the developing human lung in an ex vivo epithelial organoid system derived from human embryonic lung tissue. Simultaneously, we are interested in the extrinsic signalling mechanisms in the human fetal airway niche influencing cell fate decisions.
In collaboration with the Hagiwara group from RIKEN, Japan, we would like to examine how different growth factors and matrix stiffness influence human fetal tip progenitor cell fate. The Hagiwara group use bioengineering approaches to improve 3D organoid culture. They have developed a unit-based scaffold with a unique cubic unit design (CUBE) to trap hydrogel solutions in their designated units, which can be customized into many shapes and arrangements to allow various hydrogel configurations. MultiCUBE can be used to examine the effect of localised growth factors and hydrogel stiffness on cellular phenotypes and improve spatial control of morphogenesis. The aim of this project would be to culture early tip progenitor organoids (7-15pcw) in the MultiCUBE to examine the effect of candidate growth factors that are hypothesised to influence airway cell fate e.g FGF, Wnt, EGF on differentiation and branching of tip progenitor cells. Tip progenitor cells will be seeded as single organoids into each CUBE containing different growth factor conditions or seeded into a channel across different CUBEs to form a tubular structure. At specific timepoints, CUBEs will be sectioned and then immunostained to examine for airway fate markers. Another approach will be to use the CUBE to examine the effect of morphogen gradients on differentiation. Tip organoids will be embedded in hydrogels in a CUBE and placed into a two-compartment gradient chip device and cultured with separate differentiation media on two opposing ends of the CUBE to recreate spatial gradients seen in vivo. Finally, the effects of altering the crosslinking and stiffness of hydrogels on tip progenitor cell fate and branching will be examined.
This novel platform will elucidate how extrinsic biochemical and mechanical signalling influence fetal tip progenitor cell fate and contribute to the understanding of fetal airway development in vivo.
Key aims:
- Culture organoids constrained to a cylinder (airway-like) shape in multi-cube device.
- Apply biochemical gradient.
- Apply mechanical stiffness gradient.
Investigate the biochemical and mechanical effects on cell differentiation.
- Duration: 6-8 weeks
Supporting information:
Extracting metabolism with the next generation of wearable brain imaging devices
Supervisor: Dr Gemma Bale Department of Physics and Department of Engineering and co-supervisor: Prof Sir John Aston Department of Pure Maths and Mathematical Statistics.
Project description:
Wearable brain imaging is possible using an optical technique called near-infrared spectroscopy (NIRS). It uses radiative transfer theory, applied to the brain modelled as a diffuse medium, to estimate changes in concentrations of optical absorbers within the brain.
The attenuation of infrared light, as a function of wavelength and time, is measured between source and detector locations on the head. Fitting spatiotemporal spectral data to the theory provides information about endogenous chromophores of interest, such as haemoglobin (Hb) and cytochrome c-oxidase (CCO), which have infrared absorption properties that depend on their oxidation states.
The concentrations of these chromophores are valuable medical signals. For instance, changes in the concentrations of oxygenated or deoxygenated Hb are used in blood oximeters to infer heart rate and blood oxygenation level. CCO’s oxidation states are alternated between during oxidative metabolism where CCO acts as the terminal electron acceptor in the electron transport chain. Hence, measurements of the changes in the relative amount of reduced/oxidised CCO provide a signal directly correlated with the level of metabolism.
In situations where other functional imaging methods are not suitable, such as when the patient may not be moved, NIRS provides an alternative which benefits from portability and a relatively low cost. Bedside NIRS devices bring diagnostic systems directly to vulnerable patients, however efforts to further reduce their footprint are continual.
The current generation of wearable devices make use of two or three wavelengths to extract relative changes in oxygenated and deoxygenated Hb concentrations. With an array of sources and detectors across the head, a functional activation map may be constructed. In moving from a broadband source to three discrete wavelengths, the CCO signal is lost and the SNR of Hb signals increases. There is a great deal of interest in developing wearable NIRS devices which retain a large degree of the accuracy of benchtop devices.
Making use of miniature spectrometers and a broadband source or a larger number of discrete wavelengths are both promising avenues. However, they present problems of statistical analysis due to the low-resolution of smaller spectrometers and finite extent of source bandwidths respectively.
In these situations, the data recorded is blurred by a point-spread function which must be taken into account for robust data analysis.
This project will involve developing the statistical tools to establish how well specific medical signals may be extracted from blurred, noisy, spectra. These statistical methods are then to be tested on some combination of simulated and experimental data, including from biologically-mimicking phantoms and human volunteers
If this project is successful, future wearable devices will be able to accurately report chromophore concentration changes. Such devices are increasingly being used in studies of dementia, neonatal and traumatic brain injury. Making the CCO signal readily accessible will provide additional functional information to the advantage of medical understanding in a range of different research and clinical applications, including traumatic brain injury and dementia.
- Duration: 8 weeks
- Desired knowledge, skills and attributes:
- Comfortable using a scientific programming language
- Experience with diffusion equations (heat equation, etc.)
- Understanding of statistical methods
Exploring prion domain phase behaviour through evolutionary tools
Supervisor: Prof. Rosana Collepardo Guevara Yusuf Hamied Department of Chemistry and co-supervisor Maria Julia Maristany
Project Description
Prion proteins are known for their ability to misfold and aggregate, leading to neurodegenerative diseases such as Alzheimer's and Creutzfeldt-Jakob disease. Recent studies have shown that prion domains exhibit liquid-liquid phase separation, which may be involved in their pathological behaviour. However, the factors that regulate prion domain phase behaviour are not well understood. In this project, we aim to use evolutionary tools such as phylogenetic analysis and sequence alignment to explore the sequence space of prion domains and identify key determinants of phase behaviour through pathogenic mutations. Specifically, we will use computational methods to analyse a large dataset of prion domain sequences and correlate sequence features with phase behaviour. This exciting project will provide insights into the fundamental mechanisms of prion domain behaviour and may lead to the development of new therapeutic strategies for prion-related diseases.
Short summary: This project will use evolutionary tools to explore the sequence space of prion domains and identify key determinants of phase behaviour. The goal is to gain insights into the fundamental mechanisms of prion domain behaviour and develop new therapeutic strategies for prion-related diseases.
Key aims/tasks:
- Analyse large datasets of prion domain sequences
- Correlate sequence features with phase behaviour
- Identify key determinants of prion domain phase behaviour
- Provide insights into the fundamental mechanisms of prion domain phase behaviour
- Identify evolutionary mutation effects in prion domain phase behaviour
Specific details:
- Essential requirements: Basic understanding of molecular biology and protein behaviour is assumed, familiarity with computational tools for sequence analysis and modelling would be advantageous, and a good handle on a computational programming language is required (such as Python or C++).
- Duration: 6 to 8 weeks (August-September).