PhD Studentships available.

1. BBSRC iCASE studentship (with Orla Protein Technologies). Closing date for applications: Monday January 16, 2017.

Developing and optimising defined tissue culture surfaces for cellular differentiation of muscle

Project Description

The study of cardiac and skeletal muscle in culture has the drawback that most commonly used approaches result in poorly differentiated cells, with a great deal of variability in their level of differentiation. This presents significant problems in attempts to quantify effects of disease mutations on sarcomere formation and organisation (myofibrilogenesis). Our goal is to improve in vitro muscle differentiation (cardiac and skeletal) using novel surfaces, developed in collaboration with Orla Technologies. Our recent collaboration with Orla Technologies, was recently published (Parker et al., 2016, Cytotechnology, epub Aug. 2016). In this successful collaboration, we used bio-active peptides derived from extracellular matrix proteins, engineered into the variable loops of the outer membrane protein A (OmpA). The purified protein is then applied stereospecifically onto a gold-coated surface via a cysteine residue in OmpA, generating an oriented monolayer, with peptides all facing upwards, accessible to the cells in a functional conformation. For skeletal muscle myotubes, we tested various combinations of laminin α2 peptides together with FGF, and obtained conditions in which myotubes differentiated on glass coverslips, with much higher levels of fusion and sarcomere formation, compared to controls.

This PhD studentship will now explore and develop this technology further in collaboration with Orla Technologies.

Our overall aims are to develop this approach for
• Patterned 2D surfaces. To better organise myotube formation, and obtain more uniform levels of differentiation across cells.
• Surfaces with variable stiffness. To find conditions that further improve myotube differentiation, known to be influenced by the stiffness of the growing surface and combine this with Orla Peptides. This approach can also be used for patterning (e.g. see Vignaud et al., ’Polyacrylamide Hydrogel Micropatterning’, Methods in Cell Biology, 120, p93: 2014)
• 3D culture. We will develop the techniques to generate ’tubes’ in which to grow myoblasts, that fuse into muscle cells. This approach may also be suitable for live cell imaging of muscle differentiation using light sheet.
• Differentiation of neonatal cardiomyocytes, including those differentiated from iPS cells suitable for the study of heart disease.

The methodology we will use will develop our current work with specific laminin, and other ECM peptides, developed with Orla allowing us to engineer and test new peptides if required.

This is a CASE studentship, and the student will have the opportunity to spend time at Orla Technologies in Newcastle as part of the studentship.

Funding Notes

Fully funded BBSRC iCASE studentship, providing UK/EU level fees plus a stipend (£14,296) for 4 years. EU candidates must have resided in the UK for three years prior to the start of the PhD to be eligible for full support; without evidence of residency the studentship will only provide fees and no stipend. Non-UK/EU candidates are not eligible. Candidates should have or be expecting a 2.1 or above at undergraduate level in a relevant subject. If English is not your first language you will be required to meet our English language requirements. The start date will be Oct 2017.

References

Azioune et al., (2010) Protein Micropatterns: A Direct Printing Protocol Using Deep UVs. Methods in Cell Biology, 97. p133
Vignaud et al., (2014) Polyacrylamide Hydrogel Micropatterning. Methods in Cell Biology. 120. p93
Parker et al., (2016) Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides. Cytotechnology 68(5):2159-69
Clark et al., (1997) Preferential adhesion to and survival on patterned laminin organizes myogenesis in vitro. Exp. Cell. Res. 230: 275-83

 

2. Folding and regulation of non-muscle myosin 2A in vitro and in vivo, a mechanistic understanding. (see FindaPhD.com) BBSRC White Rose DTP studentship (closing date Thursday January 5th 2017)

Project Description

Non-muscle myosin 2A (NM2A) is implicated in a range of diseases from cancer, to defective blood clotting and deafness. Common to all of these diseases is the likelihood that the regulation of NM2A is disrupted, however we only have a poor understanding of the structure and regulation of NM2A.The aim of this project is to use a combination of cell and structural biology, including high resolution cryo-EM and super-resolution light microscopy, to gain a detailed mechanistic insight into how NM2A activity is regulated in cells, and how this regulation is disrupted in disease.
Funding Notes

BBSRC White Rose Mechanistic Biology DTP 4 year studentship.
Studentships covers UK/EU fees and stipend (c.£14,296) for 4 years to start in Oct 2017. Applicants should have/be expecting at least a 2.1 Hons. degree in a relevant subject. EU candidates require 3 years of UK residency in order to receive full studentship.
Not all projects advertised will be funded; the DTP will appoint a limited number of candidates via a competitive process and the projects selected by the successful candidates will be funded.
There are 2 stages to the application process. Please see our website for more information: View Website
References

Wolny, M., Batchelor, M., Knight, P.J., Paci, E., Dougan, L., and Peckham, M. (2014). Stable single alpha-helices are constant force springs in proteins. J Biol Chem 289, 27825-27835.

Samejima, K., Platani, M., Wolny, M., Ogawa, H., Vargiu, G., Knight, P.J., Peckham, M., and Earnshaw, W.C. (2015). The Inner Centromere Protein (INCENP) Coil Is a Single alpha-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis. J Biol Chem 290, 21460-21472.

Brüning-Richardson A, Bond J, Alsiary R, Richardson J, Cairns DA, McCormack L, Hutson R, Burns P, Wilkinson N, Hall GD, Morrison EE and Bell SM. ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival. Br J Cancer 2011 104:1602-10.

Higgins J, Midgley C, Bergh A-M, Bell SM, Askham JM, Roberts E, Sharif SM, Bennett C, Glover DM, Woods CG, Morrison EE, Bond J. Human ASPM participates in spindle organisation, cleavage furrow orientation and cytokinesis. BMC Cell Biology 2010 11:85.

A similar project is also available in the new British Heart Foundation phD studentship scheme: see http://www.cardiovascular.leeds.ac.uk/opportunities/proposals.php

3. Understanding how mutations in myosin 7a contribute to sight and hearing loss. MRC PhD studentship (DiMeN partnership), with Walter Marcotti, University of Sheffield. (closing date Friday January 6th 2017)

Hearing is something we all take for granted, but some people are either born deaf, or lose their hearing abilities early on in life as a result of mutations in the Myo7a gene.  This gene encodes a myosin, Myo7a, a type of myosin important in the ear and the eye.  We think that Myo7a is important for building and the correct function of the stereociliary bundle,. Stereocilia are structures containing actin rich bundles found on the top of the hair cells in the cochlea. Deflection of the stereocilia in response to sound will allow the generation of an electrical signal that can be perceived by the brain. The loss of Myo7a, or single point mutations in this protein are likely to affect the normal functioning of stereocilia, leading to deafness.
The aim of this project is to investigate selective mutations using a battery of techniques, from using ‘super-resolution’ imaging of Myo7a in intact hair cells in the developing cochlea, to investigating how the activity of this protein is regulated in vitro (using motility assays, electron microscopy and so on), and how mutations affect the normal function of this protein.

For more projects: