Tubulins are conserved cytoskeletal proteins with essential roles in intracellular transport, cell division, cell migration and neuronal synapse connectivity.Humans have multiple tubulin genes, and different isotypes. Microtubules are assembled from dimers of α- and β-tubulin. Although proteins within the tubulin superfamily show a high degree of similarity, the phenotypic differences associated with variants in the various tubulin isotypes suggests that each tubulin has a distinctive function. Mutations in human tubuins have been found in patients with a range of brain malformations and other deficits, and are collectively known as tubulinopathies. De novo mutaions in TubulinA3D were found in patients with the degenerative eye disease, Keratoconus.How TubA3D functions in the eye is not understood.In this project, the student will generate corneal cell culture, organoid and animal models of TubA3D mutations and study progression of the disease through high resolution live imaging. The findings from this project have the potential to lead to early diagnostic tests which can aid the development of new therapeutic interventions for the disease.

• Genome editing in zebrafish and cell cultur
• Analysis of zebrafish mutant embryos and cultured cells/organoid
• High resolution in vivo time lapse imagin
• Computational analysis of imaging data

Understanding cell biology means exploring subcellular organization in 3D and locating important proteins at high resolution. Correlative light-electron microscopy (CLEM) is a powerful technique to do this, since we can combine the specificity and dynamics of fluorescence light microscopy (LM) with the high resolution and cellular context of electron microscopy (EM). We are developing genetically encoded tools that allow us to track intracellular events using CLEM. This project will apply our existing tags to new cell biological questions and develop the next generation of CLEM tools. Our lab is interested in several cell biological processes and the probes are useful for all of them. These processes include: cell division, membrane trafficking and cell migration and invasion in cancer.

During your PhD you would:

1.Apply FerriTagging technology to track cellular events by live-cell fluorescence microscopy and visualisation by electron microscopy.

2.Make cell biological discoveries. Our primary interests are in microtubule-associated proteins during mitosis and proteins involved in membrane trafficking.

3.Develop new genetically encoded probes for CLEM that are fluorescent and electron-dense.

In this project you will primarily use multi-modal imaging (correlating light microscopy and electron microscopy images) and cell culture. The development of the probes will require molecular biology and cell manipulation methods. All projects in the lab involve quantitative unbiased analysis using automated computational methods.

The vertebrate skeletal muscle forms in distinct segments during development. Each segment consists of striated and aligned muscle fibres. The muscle fibres themselves are differentiated into fast and slow muscle populations. Defects in formation of the skeletal muscle results in a plethora of human diseases. Yet, our understanding of how muscle forms into a very robust structure remains poorly understood.

In this project, we will take advantage of the imaging capabilities of zebrafish to generate live movies of skeletal muscle formation. We will focus on two important questions: (1) what role does actin play in formation of skeletal muscle; and (2) how do the different cell populations interact to ensure robust spatial organisation of the tissue?

For the first part, we have exciting provisional data on how actin dynamics evolve during the process of skeletal muscle formation. There appear to be distinct modes of actin action, that combined ensure precise tissue formation. The student will build on these results in a range of suitable mutant backgrounds. In the second part, we have recently published single cell tracks of how the cell fibres move during skeletal muscle formation. However, how the different fibre types interact mechanically remains poorly understood. We will dissect the 3D cell shape changes during the muscle fibre rearrangements to build up a more thorough understanding of how the cellular mechanical

environment adapts during muscle formation. Relatedly, we will quantify changes in nucleus shape and correlate this with cell behaviours.

This PhD project offers an exciting opportunity to leverage recent advances in live imaging and genetics to gain unprecedented insights into how a critical organ forms during development, with potential impact on human development.

This project is a truly interdisciplinary project, ideally suited for either (a) a trained biologist interested in developing their expertise in quantitative approaches or (b) a physicist or engineer (preferably with some optics experience) interested in applying the latest microscopy approaches to important biological systems.

Methodologies required as part of the project:

1)Zebrafish genetics. This will include learning to perform crossing and injections.

2)Live imaging. We will utilise spinning disc, multiphoton microscopy and lattice light-sheet microscopy to gain a subcellular view of how the muscle initially forms

3)Image analysis. Taking data from our microscopy and generating quantitative data is essential. In particular, we want to understand the specific developmental time when morphological changes occur

4)Mechanobiology approaches to understanding cell function and morphology

The Saunders lab is a highly interdisciplinary environment, with biologists and physicists working together to tackle major questions concerning how organs form. This project offers a motivated student the opportunity to learn a breadth of techniques in quantitative biology that have broad applicability.

Microtubules are one of the key components of the cellular skeleton, providing both structural integrity and serving as the tracks for intracellular long-distance transport. Microtubules also form the mitotic spindle and their dynamic assembly and disassembly drives chromosome segregation. Thus, microtubule are dynamically rearranging polymers that form different arrays to fulfil vitally important tasks in the cell. My lab studies how microtubule arrays self-organise with the help of motorised and passive crosslinking proteins in the context of motile cells and differentiating skeletal muscle cells. We use a combination of quantitative live cell imaging approaches and biochemical experiments to directly test mechanistic hypotheses.

Your PhD project could address one of the following questions:

(1)How is the number and position of microtubule minus ends controlled? This is an open question in skeletal muscle cells, which do no longer have a centrosome and nucleation of new microtubules plays an important role in rearranging microtubule arrays.

(2)How are actin and microtubule arrays integrated? In skeletal muscle cells both cytoskeletal filament systems form paraxial arrays, how they influence each other is largely unknown. Testable hypotheses are that microtubule assembly could follow actin bundles or that actin could be transported and aligned along microtubules.

(3)How is microtubule dynamics regulated at the cell cortex? Selective stabilisation of microtubule ends at certain regions of the cell cortex – such as at the protruding edge in migrating cells or the tips in skeletal muscle cells – is thought to contribute to the formation of polarised microtubule arrays. Careful quantitative observation and identification of the cellular machinery controlling this process is required to understand both microtubule array organisation itself and its interdependence with cell shape.

Any of the three projects will enable the better understanding of how microtubule arrays are formed and maintained in healthy cells and allow us to identify what goes wrong in patients with muscular dystrophies that show abnormal microtubule density and organisation.

You will culture human cells and generate genome-edited cell lines either to tag endogenous proteins with fluorescent markers or to manipulate protein expression (i.e. generate knockouts or make mutations). You will use a variety of live cell imaging techniques from widefield fluorescence to lattice light sheet microscopy to record multi-colour timelapse images and use image analysis tools to segment, track and quantify imaging data. Depending on the project, you might also do biochemical and biophysical assays using purified proteins.

For students with a background in mathematics or physical sciences there are many opportunities to apply their quantitative skills to these research projects and develop mathematical models and/or new analysis tools.

Ehlers-Danlos Syndromes are a group of 13 hereditary connective tissue disorders characterized by tissue fragility and laxity Hypermobile Ehlers-Danlos Syndrome (hEDS) is the most common form the EDS with an approximate incidence of 1-500. It primarily affects the joints causing joint instability and chronic pain. Little is known about the pathogenesis of hEDS. Fibroblast cells derived from patients with hEDS show an abnormal integrin expression. a class of transmembrane proteins that link the cell cytoskeleton to the extracellular matrix (ECM). This alters the way fibroblasts bind to the ECM, how mechanical forces are transmitted and the mechano-signalling of cells. Interestingly, hEDS fibroblasts were shown to express elevated levels of α-smooth muscle actin (α-SMA) compared to normal fibroblasts which is associated with a transition to the more contractile myofibroblasts. While trans-differentiation of fibroblast to myofibroblast is a complex process the mechanical properties of the ECM are an important influence on it.

We will test how the mechanical properties of the wildtype and hEDS ECM affect fibroblast contractility and vice versa, how wildtype or hEDS fibroblasts affect and remodel the ECM.Using protocols developed in the Koester lab, we will derive ECM from wildtype and hEDS fibroblasts and study their ultrastructure using electron microscopy as well as test its mechanical properties using AFM.

To study the dynamic interplay of cells and ECM using our state-of-the-art confocal microscope combined with an atomic force microscope (AFM). We will seed (wildtype or hEDS) fibroblasts within ECM (wildtype or hEDS) and visualise dynamics of cell cytoskeletal components (fluorescently labelled actin, microtubules, integins) using confocal fluorescence microscopy while testing the mechanical properties of the ECM-cell composite using AFM.

In a second part, we will test the idea that changes in the mechanical properties of the ECM-cell composite in hEDS causes mast cell activation syndrome, due to mechanical stimulation of mast cells leading to degranulation.Mast cells will be seeded together with wildtype and hEDS fibroblast ECM composites, and their activation monitored over time or before and after global mechanical stimulation by stretching the cell-ECM composite.

• Culture of fibroblast cell lines is established in the lab, and ECM can be derived from these cell lines by simply adding ascorbic acid to the medium
• For fluorescence microscopy, cell components are either labelled by fixation and immunofluorescence or by expressing fluorescently labelled marker proteins (e.g. lifeact-mRuby, integrin-alpha1-GFP…
• An important element will be atomic force microscopy combined with confocal fluorescence microscopy on our new BioAFM
• For the measurement of cell contractility, we will perform traction force microscopy by seeding cells on soft, PDMS substrates containing tracer particles
• To generate patterned substrate with defined regions of cell adhesion sites we will use the new Primo2 micropatterning device.

The vaginal microbiota plays a key role in women’s health. Low diversity microbiota dominated by Lactobacillus crispatus is considered as the hallmark of health. Conversely, a high diversity microbiota, leading to inflammation, is associated with adverse women’s reproductive health outcomes such as bacterial vaginosis, increase susceptibility to sexually transmitted infections and adverse pregnancy outcomes such as infertility and preterm birth. Inflammation is a key driver of women’s health and disease. Interestingly, we identified a subset of anti-inflammatory lectins, innate immune receptors specialised in detecting glycan motifs, interacting specifically with the commensal bacterium L. crispatus. However, the consequences of these interactions remain unknown.

The aim of this project is to identify the bacterial ligands of these anti-inflammatory receptors, characterise the immune response triggered and develop chemical mimetics with improved anti-inflammatory properties.

The results of these project will not only help to better understand how the beneficial vs detrimental bacteria modulate the maternal immune system but will also open new ways to develop synthetic glyco-molecules mimicking the anti-inflammatory effect of the lactobacilli to prevent a broad range of women’s health diseases.

The immunomodulatory glycans will be purified from Lactobacillus crispatus according to protocols established in the host laboratory. Briefly, L. crispatus will be grown and the glycans will be extracted and purified by size exclusion and affinity chromatography. The isolated glycans will then be analysed for binding to plant lectins as a proxy to determine glycan composition and by mass spectrometry.

The glycans identified as ligands of will then be tested for their ability to activate HEK reporter cell lines and monocyte derived dendritic cells. Signalling will be assessed by ELISA, Western Blot, flow cytometry and microscopy.

Finally, the bacterial glycans will be coupled to multivalent scaffolds and their biological activity will be assessed.

Neurogenic claudication is a condition causing backpain among older people. It can impact on quality of life, ability to engage socially and, limit the potential for successful aging. Jointly with University of Oxford we have undertaken a longitudinal qualitative study with people diagnosed with neurogenic claudication. Interviews were undertaken at three time points over 12 months. Interviews explored the experience of living with the condition and how this changed over time. Data was collected from 60 participants resulting in 163 interviews. Initial data analysis has been undertaken to understand patterns of change in 30 participants. This rich data set has potential for further analysis as a PhD. This secondary data analysis can be tailored to the interests of the PhD candidate and would be complemented by a systematic review of published evidence on a topic relevant to back pain/neurogenic claudication/successful aging.

Example research question: When social interaction is limited by pain and mobility problems, how do individuals respond as the pain/mobility changes over time and what influences this?

Aim: to understand how pain and mobility problems interact with the goal of successful aging

Anticipated outcomes: social/behavioural understanding of living with and rehabilitating from back pain in older age

Secondary analysis of longitudinal data set

Systematic evidence review – methodology chosen depending on interest of PhD candidate (e.g. realist review/narrative review)

We have a keen interest in research relevant to nursing and healthcare. We have experience of patient and public involvement, patient and staff experiences, patient reported outcomes and getting evidence into practice.We have experience of quantitative, qualitative and mixed methodologies.

Student are welcome to approach us for PhD supervision in any of these areas. Please email to make an appointment to discuss your interests.

Kate.seers@warwick.ac.uk (qualitative, mixed methods, pain, getting evidence into practice, patient/staff experiences)

Sophie.Staniszewska@warwick.ac.uk (patient and public involvement, patient experiences)

Kirstie.haywood@warwick.ac.uk (patient reported outcomes)

Qualitative, quantitative and mixed methods